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Endocytosis in elongating root tip cells of Lobelia erinus Samuels, Anne Lacey 1989

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ENDOCYTOSIS IN ELONGATING ROOT TIP CELLS OF LOBELIA ERINUS By ANNE LACEY SAMUELS B.Sc, McGill University, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Botany We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH May 1989 © A n n e Lacey Samuels COLUMBIA In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) i i ABSTRACT E n d o c y t o s i s was measured a l o n g the a x i s of d i f f e r e n t i a t i o n of e p i d e r m a l and c o r t i c a l r o o t c e l l s of L o b e l i a e r i n u s from m e r i s t e m a t i c t o f u l l y expanded v a c u o l a t e c e l l s . Both lanthanum and l e a d were t e s t e d as markers of e n d o c y t o s i s ; lanthanum p r o v e d t o be more e f f e c t i v e . Lanthanum t r e a t m e n t produced e l e c t r o n dense d e p o s i t s i n the a p o p l a s t of the r o o t , as w e l l as c o a t e d p i t s , c o a t e d v e s i c l e s , smooth v e s i c l e s and m u l t i v e s i c u l a r b o d i e s w i t h i n the c e l l s . X-ray m i c r o a n a l y s i s was used t o c o n f i r m the l a n t h a n i d e n a t u r e of the d e p o s i t s . In b oth s e c r e t o r y ( m e r i s t e m a t i c and e l o n g a t i n g c e l l s a c t i v e l y d e p o s i t i n g new c e l l w a l l m a t e r i a l ) and non s e c r e t o r y (mature v a c u o l a t e ) c e l l s the amount of e n d o c y t o s i s o c c u r r i n g was measured by c o u n t i n g the' number of lanthanum l a b e l l e d v e s i c l e s / u m / c e l l . The amount of e n d o c y t o s i s c o r r e l a t e d v e r y w e l l w i t h the c e l l w a l l s e c r e t o r y a c t i v i t y . The h i g h e s t amount of e n d o c y t o s i s was found i n the e l o n g a t i n g c e l l s , w i t h m e r i s t e m a t i c h a v i n g an i n t e r m e d i a t e v a l u e . Mature, v a c u o l a t e c e l l s had the l e a s t e n d o c y t o s i s . The r e l a t i o n s h i p between e n d o c y t o s i s and s e c r e t o r y a c t i v i t y s u g g e s t s t h a t e n d o c y t o s i s may be a c t i n g t o remove exce s s membrane m a t e r i a l added d u r i n g e x o c y t o s i s of s e c r e t o r y v e s i c l e s . C y t o c h e m i c a l t e s t s f o r p o l y s a c c h a r i d e s were performed on both c o n v e n t i o n a l t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y p r e p a r a t i o n s and u l t r a r a p i d l y f r o z e n , f r e e z e s u b s t i t u t e d p r e p a r a t i o n s . The u l t r a s t r u c t u r a l p r e s e r v a t i o n was s u p e r i o r u s i n g the c r y o t e c h n i q u e . The o r g a n e l l e s i n v o l v e d i n s e c r e t i o n of c e l l w a l l components were compared w i t h the o r g a n e l l e s a s s o c i a t e d w i t h e n d o c y t o s i s i n c h a p t e r 1. The G o l g i showed d i s t i n c t d i c t y o s o m e p o l a r i t y , s m a l l p e r i p h e r a l v e s i c l e s and an e l a b o r a t e t r a n s G o l g i network. V e s i c l e s i n the c y t o p l a s m d i s p l a y e d d i v e r s e s t a i n i n g p r o p e r t i e s . One p o p u l a t i o n of l a r g e r , d e n s e l y s t a i n i n g v e s i c l e s l o c a t e d on the t r a n s G o l g i and i n the c o r t i c a l c y t o p l a s m was i n t e r p r e t e d t o be s e c r e t o r y v e s i c l e s . M i c r o t u b u l e s were d i s r u p t e d w i t h c o l c h i c i n e t o t e s t the importance of t h e s e c y t o s k e l e t a l elements i n e n d o c y t o s i s . Immunofluorescence was used t o det e r m i n e the c o n c e n t r a t i o n of c o l c h i c i n e which d i s r u p t e d c o r t i c a l m i c r o t u b u l e s i n t h e s e c e l l s . A f t e r c o l c h i c i n e t r e a t m e n t , t h e r e were fewer e n d o c y t o t i c v e s i c l e s and l e s s lanthanum l a b e l i n the m u l t i v e s i c u l a r body, s u g g e s t i n g m i c r o t u b u l e s a r e i n v o l v e d i n e n d o c y t o s i s d u r i n g e l o n g a t i o n . i v C e l l w a l l pore s i z e was t e s t e d u s i n g e l e c t r o n dense a p o p l a s t markers. P a r t i a l enzyme d i g e s t i o n s were used t o f i n d which components of the c e l l w a l l d e t e r m i n e p o r o s i t y . P e c t i n was found t o be the most i m p o r t a n t component, w h i l e c e l l u l o s e and p r o t e i n d i d not seem t o e f f e c t p o r o s i t y i n th e s e p r i m a r y r o o t s . The p o r o s i t y of the w a l l was i n t e r p r e t e d i n terms of the i n t a c t w a l l u l t r a s t r u c t u r e . U l t r a r a p i d f r e e z i n g , f r e e z e s u b s t i t u t i o n were used t o reexamine e n d o c y t o s i s i n e l o n g a t i n g c e l l s . U s i n g lanthanum as a marker f o r e n d o c y t o s i s , the r e s u l t s of the e a r l i e r , c o n v e n t i o n a l TEM s t u d y were s u p p o r t e d and extended. In a d d i t i o n t o c o a t e d and smooth v e s i c l e s , m u l t i v e s i c u l a r b o d i e s were l a b e l l e d . The i n s t a n t a n e o u s p r e s e r v a t i o n of the endomembrane system a l l o w e d the l o c a l i z a t i o n of l a b e l i n the p a r t i a l l y c o a t e d r e t i c u l u m and i n v e s i c l e s a s s o c i a t e d w i t h t h e G o l g i as w e l l . The p a r t i a l l y c o a t e d r e t i c u l u m and m u l t i v e s i c u l a r body are proposed t o r e p r e s e n t p l a n t endosomes. When i n t e r p r e t e d i n c o n j u n c t i o n w i t h the s e c r e t i o n s t u d y , i t can be suggested t h a t the heterogenous p o p u l a t i o n s of v e s i c l e s o c c u r i n the c y t o p l a s m of the e l o n g a t i n g L o b e l i a e r i n u s r o o t c e l l s . TABLE OF CONTENTS A b s t r a c t i i L i s t of T a b l e s v i i L i s t of F i g u r e s v i i i Acknowledgement x i G e n e r a l I n t r o d u c t i o n 1 Chapter 1 r E n d o c y t o s i s i n e l o n g a t i n g c e l l s 8 i n t r o d u c t i o n 9 methods and m a t e r i a l s 14 r e s u l t s 17 d i s c u s s i o n 32 Chapter 2: S e c r e t i o n of c e l l w a l l 41 components i n t r o d u c t i o n 42 methods and m a t e r i a l s 52 r e s u l t s 56 d i s c u s s i o n 71 Chapter 3: M i c r o t u b u l e s : e f f e c t of 80 of c o l c h i c i n e on e n d o c y t o s i s i n t r o d u c t i o n 81 methods and m a t e r i a l s 86 r e s u l t s 92 d i s c u s s i o n 104 TABLE OF CONTENTS ( c o n t i n u e d ) Chapter 4 : C e l l w a l l p o r o s i t y i n t r o d u c t i o n methods and m a t e r i a l s r e s u l t s d i s c u s s i o n Chapter 5 : R e - e x a m i n a t i o n of e n d o c y t o s i s u s i n g u l t r a r a p i d f r e e z i n g i n t r o d u c t i o n methods and m a t e r i a l s r e s u l t s d i s c u s s i o n G e n e r a l C o n c l u s i o n Appendix 1 Appendix 2 B i b l i o g r a p h y V I 1 LIST OF TABLES TABLE TITLE PAGE Tab l e 1 C e l l l e n g t h s and c e l l w a l l w i d t h s of d i f f e r e n t i a t i n g p r i m a r y r o o t c e l l s of L o b e l i a e r i n u s 18 T a b l e 2 Lanthanum l a b e l l e d v e s i c l e d i a m e t e r s 20 T a b l e 3 T o t a l number of c o a t e d v s . smooth lanthanum l a b e l l e d v e s i c l e s . 20 Ta b l e 4 Comparison of c y t o c h e m i c a l s t a i n s . 57 Ta b l e 5 E f f e c t of p e c t i n a s e on c e l l w a l l 124 p o r o s i t y . 1 V I 1 1 LIST OF FIGURES FIGURES TITLE PAGE 1-3 D i f f e r e n t i a t i o n of p r i m a r y 24 r o o t c e l l s from meristem r e g i o n t o mature v a c u o l a t e c e l l s 4-8 Comparison of d i f f e r e n t heavy 26 met a l t r e a t m e n t s (La,Pb) v s . c o n t r o l ( d i s t i l l e d water) 9-14 Coated and smooth v e s i c l e s 28 found i n r o o t c e l l s of L o b e l i a e r i n u s 15-19 I n t r a c e l l u l a r s t r u c t u r e s 30 l a b e l l e d w i t h lanthanum 20 D e n s i t y of e n d o c y t o t i c 32 v e s i c l e s a l o n g a x i s of d i f f e r -e n t i a t i o n 21 Model of membrane r e c y c l i n g 40 d u r i n g c e l l w a l l s e c r e t i o n 22-25 Endogenous p e r o x i d a s e a c t i v i t y 64 i n e l o n g a t i n g L o b e l i a r o o t c e l l s . 26-31 A l k a l i n e b i s m u t h s t a i n i n g of 66 c o n v e n t i o n a l TEM p r e p a r a t i o n s LIST OF FIGURES ( c o n t i n u e d ) FIGURES TITLE PAGE 32-35 A l k a l i n e b i smuth s t a i n i n g of 68 c o n v e n t i o n a l TEM p r e p a r a t i o n s 36-38 A l k a l i n e b i smuth s t a i n i n g of 70 u l t r a r a p i d l y f r o z e n , f r e e z e s u b s t i t u t e d m a t e r i a l . 39-42 M i c r o t u b u l e s i n e l o n g a t i n g 97 c e l l s 43-45 A n t i - t u b u l i n Immunofluore- 99 scence 46-49 E f f e c t of c o l c h i c i n e on 101 e n d o c y t o s i s i n e l o n g a t i n g r o o t c e l l s 50 Q u a n t i f i c a t i o n of a n t i - 103 t u b u l i n immunofluorescence 51-53 I n f l u e n c e of w a l l components 125 on c e l l w a l l p o r o s i t y 54-56 C e l l w a l l u l t r a s t r u c t u r e 127 57-58 F r o z e n h y d r a t e d samples of 129 L o b e l i a e r i n u s , viewed on c o l d s t a g e of SEM 59 E f f e c t of p e c t i n a s e on c e l l w a l l p o r o s i t y . LIST OF FIGURES ( c o n t i n u e d ) FIGURES TITLE PAGE 60 G r a d i e n t of f r e e z i n g . 157 61-63 Lanthanum l a b e l l e d v e s i c l e s . 157 64-66 M u l t i v e s i c u l a r b o d i e s 159 l a b e l l e d w i t h lanthanum. 68-70 G o l g i w i t h p e r i p h e r a l 161 lanthanum l a b e l l e d v e s i c l e s . 71 R e v i s e d model of L o b e l i a 174 e r i n u s endomembranes i n v o l v e d i n e n d o c y t o s i s . x i ACKNOWLEDGEMENTS T h i s t h e s i s i s d e d i c a t e d t o my s u p e r v i s o r Dr. Thana B i s a l p u t r a , on the o c c a s i o n of h i s r e t i r e m e n t , December 31, 1988, f o r h i s i n s p i r a t i o n and t i m e l y a d v i c e . The t e c h n i c a l and mora l s u p p o r t of M i c h a e l Weis i s g r a t e f u l l y acknowledged. The e x p e r t computer h e l p of Donald Wong i s a p p r e c i a t e d . Thanks t o a l l the p a s t and p r e s e n t g r a d s t u d e n t s and b r o t h e r s i n arms f o r s t i m u l a t i n g d i s c u s s i o n s . My g r a t i t u d e a l s o goes t o Rob DeWreede and B r i a n N i c h o l f o r t h e i r c a l m i n g i n f l u e n c e s . F i n a l l y , thanks t o B r i a n Samuels f o r i n f i n i t e p a t i e n c e . 1 GENERAL INTRODUCTION The q u e s t i o n a d d r e s s e d i n t h i s t h e s i s i s whether e n d o c y t o s i s o c c u r s i n the r o o t c e l l s of a g e r m i n a t i n g h i g h e r p l a n t , L o b e l i a e r i n u s . E n d o c y t o s i s has been d e f i n e d by deDuve (1963) as " i n v a g i n a t i o n of plasmalemma, g i v i n g r i s e t o an independent, c y t o p l a s m i c v e s i c l e " . The r e s u l t i n g c y t o p l a s m i c v e s i c l e c a r r i e s i n t o the c e l l p a r t of the e x t r a c e l l u l a r e n vironment. In h i g h e r p l a n t c e l l s , t h e p o r o s i t y b a r r i e r of t h e c e l l w a l l p r e v e n t s l a r g e p a r t i c l e s from i n t e r a c t i n g w i t h the s u r f a c e membrane of h e a l t h y p l a n t c e l l s . P h a g o c y t o s i s , d e f i n e d as uptake of p a r t i c l e s >0.5 urn, i s g e n e r a l l y r u l e d o u t , a l t h o u g h i t may s t i l l o c c u r i n ca s e s of s p e c i a l i z e d c e l l - c e l l i n t e r a c t i o n s , such as Rhizobiurn-legume n o d u l a t i o n ( D j o r d j e v i c e t a l . 1987). P i n o c y t o s i s can be d e f i n e d as uptake of s o l u t i o n , p r o t e i n s ( c a l l e d l i g a n d s ) , and/ or v i r u s e s . P i n o c y t o s i s of m o l e c u l e s t h a t a r e s m a l l e r than the pore s i z e of the c e l l w a l l c o u l d occur i n h i g h e r p l a n t c e l l s . For the purpose of t h i s s t u d y , the more g e n e r a l term e n d o c y t o s i s w i l l be used t o d e s c r i b e t h i s p r o c e s s . T h i s term has become more w i d e l y used i n the l i t e r a t u r e as e n d o c y t o s i s has been r e c o g n i z e d as a mechanism f o r plasma membrane 2 t u r n o v e r and ho m e o s t a s i s i n a wide v a r i e t y of a n i m a l c e l l s (Besterman 1985). The s p e c i f i c b i n d i n g of l i g a n d s by r e c e p t o r s on the c e l l s u r f a c e and i n t e r n a l i z a t i o n of r e c e p t o r - l i g a n d complexes by c o a t e d v e s i c l e s i s now c a l l e d r e c e p t o r mediated e n d o c y t o s i s (RME) ( G o l d s t e i n e t a l . 1985). The p r e l y s o s o m a l o r g a n e l l e s t h a t r e c e i v e membrane from the plasma membrane f o l l o w i n g e n d o c y t o s i s a r e c a l l e d endosomes ( H e l e n i u s e t a_l. 1983, Schmid e t a l . 1988). E n d o c y t o s i s i n h i g h e r p l a n t s has been p o s t u l a t e d s i n c e the f i r s t t r a n s m i s s i o n e l e c t r o n m i c r o s c o p e (TEM) o b s e r v a t i o n s of v e s i c l e s near the h i g h e r p l a n t plasma membrane (Buvat 1963, Mahlberg e t a l . 1971, Rol a n d and V i a n 1971). The d i f f i c u l t y of i n t e r p r e t i n g t h e s e images of v e s i c l e s i s t h a t " [ t h e ] d i r e c t i o n of movement of t h i s v e s i c u l a t i o n i s not apparent from e l e c t r o n m i c r o s c o p e s t u d i e s " (Baker and H a l l 1 973) . The e x i s t e n c e of v e s i c l e s near the plasma membrane has been i n t e r p r e t e d as s t r i c t l y e n d o c y t o s i s (Nassery and Jones 1976, N i s h i z a w a and M o r i 1977, 1978, 1984) or as s t r i c t l y e x o c y t o s i s (Robinson 1985, S t e e r 1985). These i n t e r p r e t a t i o n s a r e b oth s p e c u l a t i v e i n the absence of markers f o r 3 e n d o c y t o s i s . One of t h e g o a l s of the p r e s e n t s t u d y i s t o compare the pathways of e n d o c y t o s i s and e x o c y t o s i s i n a c e l l w a l l s e c r e t i n g c e l l . W h i l e the e x o c y t o s i s of m a t e r i a l from the G o l g i t o the plasma membrane has been w e l l documented, e n d o c y t o s i s has remained a c o n t r o v e r s i a l s u b j e c t i n p l a n t c e l l b i o l o g y . In the 1970's e n d o c y t o s i s was suggested as a mechanism of i o n uptake (Baker and H a l l 1973). Models were g e n e r a t e d i n which i o n s , bound t o the e x t r a c e l l u l a r s u r f a c e of the plasma membrane, were i n t e r n a l i z e d and c a r r i e d t o the c e n t r a l v a c u o l e (MacRobbie 1970). T h i s t h e o r y has been d i s c o u n t e d on t h e o r e t i c a l grounds (Cram 1980) and on f u r t h e r c h a r a c t e r i z a t i o n of i o n t r a n s p o r t e r s (MacRobbie 1982). However, t h e r e a r e some s p e c i a l i z e d c e l l t y p e s where e n d o c y t o s i s may be a mechanism f o r i o n or s u c r o s e uptake (Rozema e t a_l. 1977, B r o s s a r d -C h r i q u i and Is k a n d e r 1982, A u r i a c and T o r t 1985). E n d o c y t o s i s was s u g g e s t e d t o be the mechanism of p r o t e i n uptake i n t o r o o t s ( N i s h i z a w a and M o r i 1977, 1978). I t must be c o n s i d e r e d t h a t the p r o t e i n s may have been p a r t i a l l y degraded, or taken up i n t o the a p o p l a s t or dead c e l l s ( U l r i c h and MacLaren 1965, Drew e t a l . 1970). The m o r p h o l o g i c a l 4 e v i d e n c e f o r p r o t e i n uptake ( N i s h i z a w a and M o r i 1977, 1978) was the presence of l a r g e r (about 0.5 um) v e s i c l e s f o r m i n g from the plasma membrane, which resembled t h e intramembrane p a r t i c l e (IMP) f r e e b l e b a r t i f a c t s i n d u c e d by g l u t a r a l d e h y d e f i x a t i o n (Hasty and Hays 1978). The uptake of p r o t e i n s or i o n s by e n d o c y t o s i s i s p r o b a b l y not s i g n i f i c a n t i n the h i g h e r p l a n t c e l l , but e n d o c y t o s i s c o u l d s t i l l p l a y an i m p o r t a n t r o l e i n the t u r n o v e r of plasma membrane components. One example of e n d o c y t o s i s as a mechanism f o r membrane hom e o s t a s i s i s t h e i n t e r n a l i z a t i o n of exces s plasma membrane added d u r i n g e x o c y o s i s . The r e s u l t of an e x o c y t o t i c v e s i c l e f u s i n g w i t h the plasma membrane i s a net i n c r e a s e i n the c e l l s u r f a c e a r e a ; an e n d o c y t o t i c v e s i c l e budding from the plasma membrane would produce a net d e c r e a s e i n the c e l l s u r f a c e a r e a . The study of e n d o c y t o s i s i n h i g h e r p l a n t s has been slow as i t i s d i f f i c u l t t o a s s a y f o r e n d o c y t o s i s i n the i n t a c t p l a n t system. In l i g h t m i c r o s c o p e s t u d i e s of P i sum s a t i v u m l i q u i d endosperm c e l l s , t h e c y t o p l a s m showed e x t e n s i v e v e s i c u l a t i o n t h a t resembled p i n o c y t o s i s over a s h o r t time c o u r s e . However, t h i s v e s i c u l a t i o n 5 was shown t o be an a r t i f a c t of sample p r e p a r a t i o n ( B r a d f u t e e t a l . 1964). E n d o c y t o t i c v e s i c l e s a r e s m a l l e r than the l i m i t of r e s o l u t i o n of the l i g h t m i c r o s c o p e ; t r a c e r s f o r e n d o c y t o s i s must be s m a l l t o f i t t h r o u g h the c e l l w a l l . For t h e s e reasons e l e c t r o n m i c r o s c o p y has been used t o study e n d o c y t o s i s i n h i g h e r p l a n t c e l l s . There a r e r e p o r t s of e n d o c y t o s i s i n i n t a c t h i g h e r p l a n t s u s i n g u r a n y l a c e t a t e as an e l e c t r o n dense marker. T h i s marker s e v e r e l y damaged e x t e r i o r r o o t cap c e l l s , w h i l e i n t e r i o r r o o t cap c e l l s showed e v i d e n c e of e n d o c y t o s i s (Wheeler e_t a l . 1972). R e s u l t s o b t a i n e d u s i n g t h i s t o x i c marker must be viewed w i t h c a u t i o n , s i n c e u r a n y l a c e t a t e has been shown t o induce v e s i c u l a t i o n of b o th l i p o s o m e s and plasma membranes of i n t a c t c e l l s (Romanenko et a_l. 1986). In the presence of u r a n y l a c e t a t e , t h e r e were 15-20 t i m e s more membrane i n v a g i n a t i o n s and v e s i c l e s formed. In c o n t r a s t , i n c u b a t i o n of v e s i c l e s w i t h lanthanum n i t r a t e produced the same e f f e c t as i n c u b a t i o n i n sodium c h l o r i d e : l i t t l e v e s i c u l a t i o n was i n d u c e d i n the p r e s e n c e of c a l c i u m and no v e s i c u l a t i o n i n the absence of c a l c i u m . T h e r e f o r e , lanthanum n i t r a t e seems t o be a more r e l i a b l e marker f o r e n d o c y t o s i s 6 than u r a n y l a c e t a t e . Both lanthanum and l e a d have been used t o show e n d o c y t o s i s i n r o o t cap c e l l s of maize (Hubner et a l . 1985). T h i s i s t h e s i n g l e r e p o r t of e n d o c y t o s i s i n an i n t a c t t i s s u e u s i n g a r e l a t i v e l y r e l i a b l e marker. When h i g h e r p l a n t c e l l s have t h e i r c e l l w a l l s e n z y m a t i c a l l y removed ( p r o t o p l a s t s ) , e l e c t r o n dense markers f o r e n d o c y t o s i s can i n t e r a c t f r e e l y w i t h the plasma membrane. P r o t o p l a s t s can e n g u l f p o l y s t y r e n e beads or b a c t e r i a l s p h e r o p l a s t s (Mayo and C o c k i n g 1969, H a r d i n g and C o c k i n g 1986, S u z u k i e t a l . 1977). A d s o r p t i v e p i n o c y t o s i s , uptake of m o l e c u l e s bound t o the plasma membrane, has been demonstrated u s i n g c a t i o n i z e d f e r r i t i n and c o l l o i d a l g o l d - l e c t i n (Joachim and Robinson 1984, Tanchak e t a l . 1984, H i l l m e r e t a l . 1986). P r o t o p l a s t s t u d i e s have r e v e a l e d the i n t r a c e l l u l a r l o c a t i o n s of membrane ta k e n up by e n d o c y t o s i s . T h i s i n c l u d e s c o a t e d and smooth c o r t i c a l v e s i c l e s , m u l t i v e s i c u l a r b o d i e s , p a r t i a l l y c o a t e d r e t i c u l u m . T h e r e f o r e , the p r o t o p l a s t s t u d i e s show t h a t the o r g a n e l l e s n e c e s s a r y f o r e n d o c y t o s i s a r e p r e s e n t i n the h i g h e r p l a n t c e l l . Whether t h i s p r o c e s s o c c u r s i n the i n t a c t c e l l where c e l l s a r e under t u r g o r i s not a d d r e s s e d . 7 In t h i s s t u d y , the p r o c e s s of e n d o c y t o s i s i n i n t a c t e l o n g a t i n g e p i d e r m a l and c o r t i c a l r o o t c e l l s i s examined u s i n g lanthanum n i t r a t e as a marker of the e x t r a c e l l u l a r medium. The h y p o t h e s i s t h a t v e s i c u l a r membrane r e c y c l i n g o c c u r s i n s e c r e t o r y c e l l s i s t e s t e d by comparing e n d o c y t o s i s i n s e c r e t o r y c e l l s ( e l o n g a t i n g w a l l s e c r e t i n g c e l l s ) and n o n - s e c r e t o r y c e l l s (mature, v a c u o l a t e c e l l s ) . The p r o c e s s of s e c r e t i o n i s examined u s i n g c y t o c h e m i s t r y f o r p o l y s a c c h a r i d e s on c o n v e n t i o n a l TEM p r e p a r a t i o n s compared w i t h u l t r a r a p i d l y f r o z e n / f r e e z e s u b s t i t u t e d m a t e r i a l . The p o r o s i t y of the p r i m a r y c e l l w a l l surrounded th e s e e l o n g a t i n g r o o t c e l l s i s measured u s i n g image a n a l y s i s of c o l l o i d a l g o l d p a r t i c l e s . F i n a l l y , e n d o c y t o s i s i s reexamined u s i n g c r y o t e c h n i q u e s t o i n s t a n t a n e o u s l y i m m o b i l i z e the endomembranes. Chapter 1: ENDOCYTOSIS IN ELONGATING ROOT TIP CELLS OF LOBELIA ERINUS INTRODUCTION 9 E n d o c y t o s i s has been demonstrated i n h i g h e r p l a n t p r o t o p l a s t s (Joachim and Robinson 1984, Tanchak et a l . 1984, Tanchak et a l . 1988, H i l l m e r e t a l . 1986, Record and G r i f f i n g 1988), and i n w a l l e d r o o t cap c e l l s (Hubner et a l . 1985). In t h i s p r o c e s s , c o a t e d or smooth v e s i c l e s bud o f f from the plasma membrane, i n t e r n a l i z i n g plasma membrane m a t e r i a l as w e l l as an a l i q u o t of e x t r a c e l l u l a r medium. The e n d o c y t o t i c v e s i c l e s a r e b e l i e v e d t o c a r r y membrane m a t e r i a l t o o t h e r o r g a n e l l e s such as the . m u l t i v e s i c u l a r body ( a l s o known as m u l t i v e s i c u l a r endosome, Tanchak and Fowke 1987), p a r t i a l l y c o a t e d r e t i c u l u m , the G o l g i and the c e n t r a l v a c u o l e (Record and G r i f f i n g 1988). I t i s g e n e r a l l y agreed t h a t e n d o c y t o s i s and e x o c y t o s i s ( g r a n u l o c r i n e s e c r e t i o n ) a r e o p p o s i n g a c t i v i t i e s of the o v e r a l l i n t r a c e l l u l a r t r a n s p o r t system i n most e u k a r y o t i c c e l l s (see r e v i e w s by Farquhar 1985, Besterman 1985, Steinman e_t a l . 1983). T h i s has been e s t a b l i s h e d by the d e m o n s t r a t i o n of e n d o c y t o s i s f o l l o w i n g s e c r e t o r y v e s i c l e d i s c h a r g e i n a wide v a r i e t y of a n i m a l c e l l s (Farquhar 1981). I t appears t h a t e n d o c y t o s i s r e p r e s e n t s a mechanism f o r membrane r e t r i e v a l d u r i n g the p e r i o d of a c t i v e s e c r e t i o n . 10 A l t h o u g h such a f u n c t i o n a l r o l e f o r e n d o c y t o s i s has not been e l u c i d a t e d i n p l a n t c e l l s , i t has o f t e n been suggested t h a t e n d o c y t o s i s i s a f a c t o r i n membrane r e c y c l i n g (Joachim and Robinson 1984, Hubner e t a l . 1985, S t e e r 1985). The most abundant s e c r e t o r y p r o d u c t of p l a n t s i s p o l y s a c c h a r i d e s , u n l i k e most a n i m a l c e l l s which s e c r e t e p r i m a r i l y p r o t e i n s (Robinson 1984). D u r i n g the development of each p l a n t c e l l , p o l y s a c c h a r i d e c e l l w a l l components must be s e c r e t e d t o m a i n t a i n w a l l t h i c k n e s s as the c e l l grows (Dauwalder and Whaley 1982, F i n c h e r and Stone 1981, D i x o n and N o r t h c o t e 1985); i t i s g e n e r a l l y agreed t h a t the w a l l does not " t h i n " or s t r e t c h over th e c e l l s u r f a c e d u r i n g c e l l w a l l growth ( M c N e i l e t a_l. 1984 V a r n e r and L i a n g - S h i o u 1989). The p r i m a r y c e l l w a l l c o n s i s t s of about 20 % c e l l u l o s e and 80 % m a t r i x m a t e r i a l ( M c N e i l e t a l . 1984). There a r e s e v e r a l l i n e s of e v i d e n c e t h a t suggest t h a t new c e l l w a l l ' m a t r i x i s added by the e x o c y t o s i s of G o l g i d e r i v e d v e s i c l e s . M o r p h o l o g i c a l s t u d i e s show v e s i c l e a g g r e g a t i o n d u r i n g c e l l p l a t e f o r m a t i o n ( H e p l e r and Newcomb 1967, Whaley and M o l l e n h a u e r 1966). 11 A u t o r a d i o g r a p h y of r a d i o a c t i v e monosaccharides t r a c e t h e r o u t e of p o l y s a c c h a r i d e s y n t h e s i s from G o l g i c i s t e r n a e t o v e s i c l e s and t o the plasma membrane and c e l l w a l l ( P i c k e t t - H e a p s 1966). C e l l f r a c t i o n a t i o n and i s o l a t i o n of endomembrane components have been used t o show t h a t the p o l y s a c c h a r i d e s l o c a t e d i n the G o l g i have the same c o m p o s i t i o n as those of the c e l l w a l l and t h a t the enzymes of g l y c o s y l a t i o n a r e l o c a l i z e d i n the G o l g i (Bowles and N o r t h c o t e 1976). The k i n e t i c s of l a b e l l e d p o l y s a c c h a r i d e t r a n s p o r t t h r o u g h the f r a c t i o n a t e d o r g a n e l l e s suggest an i m p o r t a n t r o l e f o r the d ictyosome i n c e l l w a l l s y n t h e s i s (Robinson et a l . 1976) . By measuring the c e l l l e n g t h and c e l l w a l l w i d t h b e f o r e and a f t e r e l o n g a t i o n , i t i s p o s s i b l e t o c a l c u l a t e the s u r f a c e a r e a of plasma membrane m a t e r i a l t h a t must be added onto the c e l l s u r f a c e by the e x o c y t o s i s of G o l g i d e r i v e d v e s i c l e s (see Appendix 1) . For example, i n the p r i m a r y r o o t of L o b e l i a e r i n u s s e e d l i n g s , the c e l l s i n c r e a s e i n s i z e from 20 urn t o 80 urn i n l e n g t h . D u r i n g e l o n g a t i o n , most of the change i n c e l l shape i s due t o i n c r e a s e d c e l l l e n g t h a l o n g the a x i s of the r o o t . The s i d e s of the expanding c e l l s change from squares (meristem) t o r e c t a n g l e s ( v a c u o l a t e ) . 12 The volume of c e l l w a l l m a t e r i a l s e c r e t e d d u r i n g the e l o n g a t i o n of the r o o t c e l l can be c a l c u l a t e d from the observ e d t h i c k n e s s of the f i n a l c e l l w a l l minus the volume of the o r i g i n a l c e l l w a l l . When t h i s volume i s d i v i d e d by t h e volume of s e c r e t o r y v e s i c l e the t o t a l number of v e s i c l e s needed t o s e c r e t e the observed c e l l w a l l t h i c k n e s s can be found. The s u r f a c e a r e a of t h i s number of v e s i c l e s can be compared t o the f i n a l c e l l s u r f a c e a r e a . When thes e c a l c u l a t i o n s a r e performed f o r t h i s t i s s u e , we f i n d t h a t about e i g h t t i m e s the f i n a l c e l l s u r f a c e a r e a i s r e q u i r e d t o s e c r e t e the c e l l w a l l o b s e r v e d . T h i s c a l c u l a t i o n a g r e e s w i t h s i m i l a r c o m p u t a t i o n s (Raven 1987, S t e e r 1985) and su g g e s t s t h a t membrane r e c y c l i n g s h o u l d occur i n s e c r e t o r y p l a n t c e l l s . The mechanism of membrane r e t r i e v a l i n p l a n t s i s not w e l l u n d e r s t o o d . By ana l o g y w i t h the a n i m a l systems, e n d o c y t o s i s i s one p o s s i b l e mechanism. In t h i s s t u d y , the d i f f e r e n t i a t i n g p r i m a r y r o o t c e l l s of L o b e l i a e r i n u s were used t o compare the amount of e n d o c y t o s i s a l o n g the a x i s of d i f f e r e n t i a t i o n of the r o o t c e l l s as they go from m e r i s t e m a t i c t h r o u g h d i f f e r e n t i a t i o n t o the mature, 13 v a c u o l a t e s t a t e . E n d o c y t o s i s was demonstrated u s i n g heavy metal s a l t s as a marker f o r the e x t r a c e l l u l a r medium ( p r o c e d u r e of Hubner e_t a l . 1985). The amounts of e n d o c y t o s i s o c c u r r i n g i n each c e l l t y p e were then measured. T h i s i s the f i r s t t i me q u a n t i t a t i v e methods have been used t o compare e n d o c y t o s i s i n d i f f e r e n t c e l l t y p e s and d i r e c t l y a d d r e s s the q u e s t i o n of whether v e s i c u l a r membrane r e c y c l i n g o c c u r s i n a c t i v e l y s e c r e t i n g h i g h e r p l a n t c e l l s . L o b e l i a e r i n u s r o o t s were used f o r t h i s s t u d y because the e l o n g a t i o n phase a l o n g a f i l e of c e l l s i s as few as t h r e e t o f o u r c e l l s , a l l o w i n g easy c o m p a r i s i o n between a c t i v e l y s e c r e t i n g c e l l s ( e l o n g a t i n g ) and n o n s e c r e t i n g c e l l s ( v a c u o l a t e ) . For t h i s r e a s o n , L o b e l i a e r i n u s p r o v i d e s an i d e a l m a t e r i a l f o r s t u d y i n g e l o n g a t i o n by t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y w i t h accompanying image a n a l y s i s . 14 METHODS AND MATERIALS  e l e c t r o n m i c r o s c o p y L o b e l i a e r i n u s seeds were donated by B u c k e r f i e l d ' s Seed Co., Vancouver, B.C. The seeds were g e r m i n a t e d on f i l t e r paper moistened w i t h d e i o n i z e d d i s t i l l e d water f o r 7 days. The s e e d l i n g s were i n c u b a t e d i n heavy m e t a l s a l t s o l u t i o n s f o l l o w i n g a m o d i f i c a t i o n of the pro c e d u r e of Hubner e t a l . (1985). The s o l u t i o n s used were aqueous 5 mM L a ( N 0 3 ) 3 (JBS #104, s u p p l i e d by JBEM, D o r v a l , P.Q.), w i t h pH a d j u s t e d t o 7.6 u s i n g 1 N NaOH or aqueous 5 mM P b ( N 0 3 ) 2 ( A l l i e d C h e m i c a l s , #1838), pH 5.6. The r o o t s were immersed i n e i t h e r of the s o l u t i o n s or d i s t i l l e d water ( c o n t r o l ) f o r 1 hour a t room t e m p e r a t u r e . They were then f i x e d w i t h o u t r i n s i n g i n a d i l u t e K a r n o v s k y ' s f i x a t i v e : 1.5% formaldehyde, 1% g l u t a r a l d e h y d e , b u f f e r e d i n 50 mM PIPES pH 7.4, f o r 1 hour. A f t e r r i n s i n g i n b u f f e r f o r 15 m i n u t e s , t w i c e , the r o o t s were p o s t f i x e d i n 1% OsO^, b u f f e r e d as above, f o r 2 hours a t room temperature or o v e r n i g h t a t 4°. D e h y d r a t i o n was performed u s i n g a graded s e r i e s of methanol s o l u t i o n s ; p r o p y l e n e o x i d e was the s o l v e n t f o r t h e subsequent Epon r e s i n (JEMBED 812) i n f i l t r a t i o n and embedding. 15 L i g h t m i c r o s c o p e s e c t i o n s (0.5 urn) were c u t w i t h g l a s s k n i v e s and s t a i n e d w i t h 1 % t o l u i d i n e b l u e i n 1 % sodium b o r a t e f o r 45 seconds. S i l v e r s e c t i o n s (60-90 nm) were o b t a i n e d u s i n g a R e i c h e r t OMU3 u l t r a m i c r o t o m e and mounted on copper g r i d s . The s e c t i o n s were p o s t s t a i n e d i n s a t u r a t e d u r a n y l a c e t a t e i n 70% methanol f o r 25 m i n u t e s . The s e c t i o n s were examined u s i n g a Z e i s s EM10C. X-ray m i c r o a n a l y s i s For x - r a y m i c r o a n a l y s i s , samples were p r e p a r e d as above. U n s t a i n e d s e c t i o n s of 250 nm i n t h i c k n e s s were mounted on formvar c o a t e d , carbon s t a b i l i z e d b e r y l i u m g r i d s . S e c t i o n s were examined u s i n g a Z e i s s EM10C, adapted f o r s c a n n i n g t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y (STEM) and energy d i s p e r s i v e x - r a y a n a l y s i s (EDX). Spot s i z e of 15 nm was r o u t i n e l y used. A spot or r e g i o n scan was used t o o b t a i n a c h a r a c t e r i s t i c x - r a y spectrum. X-ray mapping was performed by i n t e r f a c i n g the STEM w i t h a Kontron Image P r o c e s s i n g System ( I P S ) . A 128x128 p i x e l r e s o l u t i o n and a d w e l l time of 2 seconds were used. 1 6 Q u a n t i f i c a t i o n of E n d o c y t o s i s F i f t e e n i n d i v i d u a l r o o t s were trimmed and s e c t i o n e d f o r each t r e a t m e n t . From each i n d i v i d u a l r o o t t i p , t h r e e c e l l s from each the t h r e e d e v e l o p m e n t a l s t a g e s (meristem, e l o n g a t i n g , and v a c u o l a t e ) were measured. T h i s gave a sample s i z e of 45 c e l l s / d e v e l o p m e n t a l s t a g e . The v e s i c l e d i a m e t e r , number of v e s i c l e s / c e l l , and c y t o p l a s m i c a r e a ( e x c l u d i n g v a c u o l e s and n u c l e i ) were measured u s i n g the Kontron IPS i n t e r a c t i v e l y w i t h the TEM. The number of v e s i c l e s / c e l l was d i v i d e d by the c y t o p l a s m i c a r e a / c e l l t o get v e s i c l e s / u m / c e l l ; t h i s a l l o w s comparison between c e l l s of d i f f e r e n t s i z e s . Frequency d i s t r i b u t i o n s ' w e r e g e n e r a t e d and n o n - p a r a m e t r i c s t a t i s t i c a l t e s t s performed ( K r u s k e l - W a l l a c e and Mann-Whitney U t e s t ) . Measurements of c e l l d i m e n s i o n s F i v e t o t e n i n d i v i d u a l r o o t s were trimmed and s e c t i o n e d f o r each t r e a t m e n t . From each r o o t f o u r t o f i v e c e l l s from each d e v e l o p m e n t a l s t a g e were measured u s i n g a Z e i s s p h o t o m i c r o s c o p e w i t h a v i d e o attachment. The v i d e o s i g n a l was a n a l y z e d u s i n g t h e Kontron IPS. 17 RESULTS Three s t a g e s of c e l l development were d i s t i n g u i s h e d f o r the purpose of t h i s s t u d y : m e r i s t e m a t i c , e l o n g a t i n g and v a c u o l a t e ( F i g . 1-3). M e r i s t e m a t i c c e l l s a r e a p i c a l c e l l s p r i o r t o d i f f e r e n t i a t i o n ; f o r the l a s t two s t a g e s , the e p i d e r m a l and u n d e r l y i n g two t o t h r e e l a y e r s of c o r t i c a l c e l l s were s t u d i e d . The c e l l s i n c r e a s e i n l e n g t h as they mature, w h i l e the c e l l w a l l w i d t h s t a y s the same or i n c r e a s e s s l i g h t l y ( T a ble 1 ) . Both lanthanum and l e a d were t e s t e d as markers f o r e n d o c y t o s i s ( F i g s . 4-6); lanthanum p r o v e d t o be more e f f e c t i v e and was used i n subsequent e x p e r i m e n t s . To c o n f i r m the i d e n t i t y of the heavy e l e c t r o n dense d e p o s i t s i n the t i s s u e s as lanthanum, x-ray m i c r o a n a l y s i s was performed. When the e l e c t r o n beam was scanned over the d e p o s i t s i n the c e l l w a l l , a spectrum w i t h c h a r a c t e r i s t i c lanthanum peaks was c o l l e c t e d ( F i g . 7 ) . Mapping the l o c a t i o n of the x - r a y s i n d i c a t e d the m a j o r i t y of the s i g n a l emanated from the heavy d e p o s i t s i n the c e l l w a l l ( F i g . 8 ) . In a d d i t i o n t o a p o p l a s t i c l a b e l l i n g , e l e c t r o n dense l a b e l appeared a l o n g the c e l l s u r f a c e , i n c o a t e d p i t s , i n membrane i n v a g i n a t i o n s , and i n T a b l e 1: C e l l l e n g t h s and c e l l w a l l w i d t h s of d i f f e r e n t i a t i n g p r i m a r y r o o t c e l l s of L o b e l i a e r i n u s . c e l l l e n g t h s t a n d a r d sample d e v e l o p m e n t a l stage mean d e v i a t i o n s i z e m eristem 18 urn + 4 urn 44 e l o n g a t i n g 30 urn + 1 4 urn 44 v a c u o l a t e 80 urn + 1 4 urn 22 c e l l w a l l w i d t h d e v e l o p m e n t a l stage meristem e l o n g a t i n g v a c u o l a t e s t a n d a r d sample mean d e v i a t i o n s i z e 0.15 urn + 0.3 urn 38 0.21 um + 0.7 urn 50 0.40 urn + 0.2 um 50 19 c o a t e d and smooth v e s i c l e s of a l l c e l l t y p e s . The smooth membrane i n f o l d i n g s were o f t e n l o n g necked s t r u c t u r e s ( F i g . 18) s i m i l a r t o those d e s c r i b e d by S t a e h e l i n and Chapman (1987). The c r i t e r i o n f o r c o n s i d e r i n g a s t r u c t u r e as l a b e l l e d was the p r e s e n c e of dark e l e c t r o n dense d e p o s i t s w i t h d e n s i t i e s s i m i l a r t o d e p o s i t s i n the c e l l w a l l which were i d e n t i f i e d by x - r a y m i c r o a n a l y s i s as lanthanum. These c o u l d be d i s t i n g u i s h e d from the f i b r i l l a r , l e s s e l e c t r o n dense d e p o s i t s seen i n the s t r u c t u r e s of ' the d i s t i l l e d water t r e a t e d c o n t r o l specimens ( F i g . 10). U r a n y l a c e t a t e p o s t s t a i n i n g was n e c e s s a r y t o v i s u a l i z e c o a t e d membranes d u r i n g c o u n t i n g , and comparisons w i t h u n s t a i n e d lanthanum s e c t i o n s p r o v i d e d a c o n t r o l f o r t h i s s t e p . Lanthanum t r e a t m e n t d i d not r e s u l t i n c l e a r l a b e l l i n g of a l l v e s i c l e s ( F i g . 9 ) . The a p p a r e n t l y u n l a b e l l e d v e s i c l e s c o u l d be G o l g i d e r i v e d s e c r e t o r y v e s i c l e s or endosomes whose d e p o s i t s a r e not i n the p l a n e of s e c t i o n . Lanthanum d e p o s i t s were d e t e c t e d i n c o a t e d v e s i c l e s which d i s p l a y e d the c h a r a c t e r i s t i c b r i s t l e s on t h e i r c y t o p l a s m i c s u r f a c e ( F i g . 12, 14; T a b l e 2 ) . Smooth v e s i c l e s , i . e . l a c k i n g a 20 T a b l e 2: Lanthanum l a b e l l e d v e s i c l e d i a m e t e r s s t a n d a r d sample type of v e s i c l e mean d i a m e t e r d e v i a t i o n s i z e c o a t e d v e s i c l e 101 nm + 14 nm 129 smooth v e s i c l e 106 nm + 34 nm 1037 T a b l e 3: T o t a l number of c o a t e d v s . smooth lanthanum l a b e l l e d v e s i c l e s . T o t a l v e s i c l e s found i n 27 c e l l s c o u n t e d f o r each s t a g e of development. d e v e l o p m e n t a l c o a t e d smooth r a t i o s t a g e v e s i c l e s v e s i c l e s c v ; sv meristem 16 143 1:11 e l o n g a t i n g 42 358 1:12 v a c u o l a t e 17 188 1:9 21 c y t o p l a s m i c c o a t were a l s o l a b e l l e d ( F i g . 11, 13, 15, 17; T a b l e 2 ) . The r a t i o of smooth: c o a t e d v e s i c l e s was a p p r o x i m a t e l y 10:1 f o r a l l c e l l t y p e s ( T a b l e 3 ) . Both c o a t e d and smooth v e s i c l e s were o f t e n a s s o c i a t e d w i t h c o r t i c a l m i c r o t u b u l e s which u n d e r l i e the plasma membrane ( F i g . 13, 15). In some ca s e s f i b r i l l a r c o n n e c t i o n s between the m i c r o t u b u l e and v e s i c l e c o u l d be seen ( F i g . 17). The amount of e n d o c y t o s i s o c c u r r i n g i n each d e v e l o p m e n t a l s t a g e i n terms of the number of 2 endosomes/um / c e l l a r e p r e s e n t e d i n F i g . 20. The e l o n g a t i n g c e l l s had the most l a b e l l e d v e s i c l e s / u m / c e l l and the v a c u o l a t e c e l l s had the l o w e s t number. D e s p i t e an adequate sample s i z e , t h e r e were a few c e l l s which c o n t a i n e d u n u s u a l l y 2 h i g h v a l u e s of l a b e l l e d v e s i c l e s / u m , and t h i s i s r e f l e c t e d by the h i g h s t a n d a r d d e v i a t i o n s . A c c o r d i n g t o the n o n p a r a m e t r i c s t a t i s t i c a l t e s t s , the t h r e e d e v e l o p m e n t a l s t a g e s have s i g n i f i c a n t l y d i f f e r e n t amounts of e n d o c y t o s i s . S m a l l v a c u o l e s (0.2-0.5 urn i n d i a m e t e r ) c o n t a i n i n g numerous v e s i c l e s i n t h e i r l u m i n a were l a b e l l e d w i t h the lanthanum t r e a t m e n t ( F i g . 19). 22 These can be i n t e r p r e t e d t o be the m u l t i v e s i c u l a r b o d i e s (MVB) d e s c r i b e d by Tanchak and Fowke (1987), i n t h e i r d e s c r i p t i o n of e n d o c y t o s i s i n the soybean p r o t o p l a s t . The d i c t y o s o m e s d i d not appear t o be l a b e l l e d f o l l o w i n g lanthanum t r e a t m e n t ( F i g . 16). L a b e l l e d v e s i c l e s c o u l d be seen i n the v i c i n i t y of the G o l g i , but not i n d i r e c t c o n n e c t i o n t o the c i s t e r n a e . No s t r u c t u r e c o r r e s p o n d i n g t o the p a r t i a l l y c o a t e d r e t i c u l u m ( P e s a c r e t a and Lucas 1985) c o u l d be seen, a l t h o u g h some v e s i c l e s were s l i g h t l y e l o n g a t e and may r e p r e s e n t a s e c t i o n t h r o u g h a s i m i l a r s t r u c t u r e . The o c c u r r e n c e of d e p o s i t s i n the endoplasmic r e t i c u l u m must be no t e d , however the f r e q u e n c y of t h e i r o c c u r r e n c e was e x t r e m e l y low. A p p r o x i m a t e l y 5% of the c e l l s d i s p l a y e d some d e p o s i t s i n the endoplasmic r e t i c u l u m . 23 F i g s . 1-3: D i f f e r e n t i a t i o n of p r i m a r y r o o t c e l l s from meristem r e g i o n t o mature, v a c u o l a t e c e l l s . F i g 1 : m e r i s t e m a t i c c e l l s a r e c h a r a c t e r i z e d by dense c y t o p l a s m , prominent c e n t r a l n u c l e i , and p r o v a c u o l e s . F i g . 2 : e l o n g a t i n g c e l l s have a r e c t a n g u l a r shape, and p r o v a c u o l e s c o a l e s c i n g . F i g 3 : v a c u o l a t e , mature c e l l s d i s p l a y l a r g e c e n t r a l v a c u o l e , w i t h a p e r i p h e r a l c y t o p l a s m . 25 F i g s . 4-8: comparison of d i f f e r e n t heavy m e t a l t r e a t m e n t s ( L a , Pb) v s . c o n t r o l ( d i s t i l l e d w a t e r ) . F i g . 4: lanthanum forms heavy d e p o s i t s i n r a d i a l c r o s s w a l l s , but does not appear i n ground c y t o p l a s m . F i g . 5: normal u l t r a s t r u c t u r e of L o b e l i a s e e d l i n g r o o t t r e a t e d w i t h water. F i g . 6: l e a d d e p o s i t s can be seen i n the c e l l w a l l as w e l l as ground c y t o p l a s m and o r g a n e l l e s . F i g . 7:x-ray spectrum g e n e r a t e d by r e g i o n scan of heavy d e p o s i t i n c e l l w a l l found i n lanthanum t r e a t e d r o o t , x - r a y s used f o r mapping a r e i n d i c a t e d . F i g . 8:x-ray map shows l o c a t i o n of x - r a y s w i t h c h a r a c t e r i s t i c energy l e v e l f o r lanthanum. 27 F i g s . 9-14: Coated and smooth v e s i c l e s found i n r o o t c e l l s of L o b e l i a e r i n u s . F i g . 9: lanthanum t r e a t e d sample; a p p a r e n t l y u n l a b e l l e d c o a t e d v e s i c l e a s s o c i a t e d w i t h m i c r o t u b u l e . F i g . 10: d i s t i l l e d water t r e a t e d ; c e l l w a l l does not have e l e c t r o n dense d e p o s i t s . F i g s . 11 -14:lanthanum t r e a t e d . arrow i n d i c a t e s lanthanum l a b e l , arrowhead i n d i c a t e s c o a t e d membrane, pm=plasma membrane, mt=microtubule, c w = c e l l w a l l . A l l bars=l00nm. 29 F i g s . 15-19: I n t r a c e l l u l a r s t r u c t u r e s l a b e l l e d w i t h lanthanum. a l l lanthanum t r e a t e d . F i g . l5:smooth e n d o c y t o t i c v e s i c l e s a s s o c i a t e d w i t h m i c r o t u b u l e F i g . 16: e l o n g a t i n g e p i d e r m a l c e l l w i t h a p p a r e n t l y u n l a b e l l e d c e l l w a l l , G o l g i , and p r o v a c u o l e s ; arrow i n d i c a t e s lanthanum d e p o s i t . F i g . 17: V e s i c l e - m i c r o t u b u l e c o n n e c t i o n ( arrowhead). F i g . 18: membrane i n f o l d i n g s form l o n g - n e c k e d s t r u c t u r e s . F i g . 19: m u l t i v e s i c u l a r body. A l l bars=l00 nm. 31 F i g . 20: D e n s i t y of e n d o c y t o t i c v e s i c l e s a l o n g a x i s of d i f f e r e n t i a t i o n i n p r i m a r y r o o t c e l l . M e r i s t e m a t i c c e l l s have r e l a t i v e l y h i g h amount of e n d o c y t o s i s ; e l o n g a t i n g c e l l s have h i g h e s t . The mature, v a c u o l a t e c e l l s have l o w e s t amount of e n d o c y t o s i s . Sample s i z e f o r each s t a g e was 45 c e l l s . Meristematic Cells mean—0.06+0.04 ° oN # <J> o*> £ ( o o- o o o o o o o 0 ofe <$> •$> * «V O ^  4P vesicles/um /cell Elongating Cells |mean=0.08±0.04 vesicles/um /cell Vacuolate Cells mean«=0.04+0.02 i ^ ^ W W W W i . • , • W i , r-° & & O* ^  * ^ •?> ^ . . . 9 . vesicles/um /cell 3 3 DISCUSSION In p r e v i o u s s t u d i e s of e n d o c y t o s i s i n h i g h e r p l a n t c e l l s , a h i g h degree of v a r i a b i l i t y i n the amount of e n d o c y t o s i s has been r e p o r t e d (Joachim and Robinson 1984, Tanchak and Fowke 1987). For t h i s r e a s o n , q u a n t i f i c a t i o n of e n d o c y t o s i s was n e c e s s a r y t o compare d i f f e r e n t c e l l t y p e s . One e x p l a n a t i o n f o r the h i g h v a r i a n c e i s the e f f e c t of p l a n e of s e c t i o n on the o c c u r r e n c e of l a b e l l e d v e s i c l e s . A s e c t i o n a d j a c e n t t o the plasma membrane c o u l d have more v e s i c l e s than a s e c t i o n t h r o u g h the p e r i n u c l e a r r e g i o n , where the v e s i c l e s c o u l d have a l r e a d y f u s e d w i t h m u l t i v e s i c u l a r b o d i e s . In c o n t r a s t t o the r e s u l t s f o r maize r o o t cap (Hubner et a l . 1985) lanthanum, not l e a d , seems t o be the s u p e r i o r marker f o r e n d o c y t o s i s i n p r i m a r y r o o t c e l l s of L o b e l i a e r i n u s . Lanthanum d i d not appear t o c r o s s the plasma membrane; t h i s i s c o n s i s t e n t w i t h e a r l i e r f i n d i n g s ( R e v e l and Karnovsky 1967, T a y l o r and H a l l 1979). In p r e v i o u s s t u d i e s where lanthanum was r e p o r t e d i n the c y t o p l a s m , i n c u b a t i o n s of up t o 15 hours i n h i g h c o n c e n t r a t i o n s of lanthanum were used (Van S t e v e n i n c k e t a_l. 1976). In the p r e s e n t s t u d y , s h o r t time exposure (1 hour) and r e l a t i v e l y low 34 c o n c e n t r a t i o n s (5 mM La) were u t i l i z e d . I t must be noted t h a t lanthanum may d i s p l a c e c a l c i u m i o n s ( M a r t i n and R i c h a r d s o n 1979), which c o u l d b r i n g about changes i n p h o s p h o l i p i d domains, p e c t i c b r i d g e s i n the c e l l w a l l , and the c a l c i u m m e t a bolism of the c e l l . C a l m o d u l i n f u n c t i o n d i d not seem t o be i m p a i r e d because c y t o p l a s m i c systems c o n t r o l l e d by t h i s p r o t e i n appear normal, e.g. m i c r o t u b u l e s ( K e i t h e t a l . 1983). The lanthanum l a b e l l i n g of c e l l u l a r s t r u c t u r e s found here agrees w e l l w i t h the r e s u l t s from ^ s t u d i e s of e n d o c y t o s i s i n p l a n t p r o t o p l a s t s and an i m a l c e l l s . The pr e s e n c e of t u r g o r p r e s s u r e may account f o r some c h a r a c t e r i s t i c d i f f e r e n c e s i n d i f f e r e n t i a t i n g r o o t c e l l s . The smooth membrane i n t e r m e d i a t e s t r u c t u r e s f i r s t d e s c r i b e d by S t a e h e l i n and Chapman (1987) f o r s e c r e t i o n under t u r g o r and observed i n t h i s s t u d y , may r e p r e s e n t a mechanism f o r e n d o c y t o t i c i n v a g i n a t i o n . I n a d d i t i o n , the basket of the c o a t e d membrane forms a g e o d e s i c dome s t r u c t u r e t h a t c o u l d support the membrane a g a i n s t t u r g o r ( P e s a c r e t a and Lucas 1985). In a n i m a l c e l l s , c o a t e d v e s i c l e s have been shown t o p l a y a r o l e i n r e c e p t o r mediated e n d o c y t o s i s ; r e l a t i v e l y l i t t l e i s known about the 35 f u n c t i o n of c o a t e d v e s i c l e s from p l a n t c e l l s (Robinson and Depta 1988). In a n i m a l c e l l s , c o a t e d v e s i c l e s a r e a c t i v e i n s e l e c t i v e l y c o n c e n t r a t i n g i n t e g r a l membrane p r o t e i n s d u r i n g r e c e p t o r mediated e n d o c y t o s i s (Brown e t a l . 1983). The p r o c e s s e s of d i f f e r e n t i a t i o n c o u l d r e q u i r e a changing complement of enzymes, s e c r e t o r y p r o d u c t s , membrane pumps or pores over t i m e . Coated v e s i c l e s c o u l d be c a r r y i n g some of these p r o t e i n s t o and from the endomembrane system of the p l a n t i n a manner analogous t o r e c e p t o r mediated e n d o c y t o s i s i n a n i m a l s . The l o c a l i z a t i o n of p e r o x i d a s e ( G r i f f i n g and Fowke 1985) and g l u c a n s y n t h a s e s ( G r i f f i n g e t a l . 1986; Depta e t a l . 1987) i n c o a t e d v e s i c l e s i s c o n s i s t e n t w i t h t h i s h y p o t h e s i s . On t h e o r e t i c a l c o n s i d e r a t i o n s , i t i s p o s s i b l e t h a t r e c e p t o r mediated e n d o c y t o s i s o c c u r s i n t he t u r g i d h i g h e r p l a n t c e l l (Saxton and B r e i d e n b a c h 1988). The i n t e r n a l i z a t i o n of c o a t e d v e s i c l e s o b s e r v e d i n t h e s e e x p e r i m e n t s may r e p r e s e n t s p e c i f i c uptake of i n t e g r a l membrane p r o t e i n s but f u r t h e r work i s r e q u i r e d i n t h i s a r e a . T h i s i s the f i r s t r e p o r t of c y t o s k e l e t a l elements a s s o c i a t e d w i t h e n d o c y t o s i s i n p l a n t s . I t i s p o s s i b l e t h a t i n d i f f e r e n t i a t i n g p l a n t c e l l s 36 m i c r o t u b u l e s appear c l o s e t o the endosomes as a f o r t u i t o u s consequence of the c l o s e p a c k i n g of m i c r o t u b u l e s as they form a c o i l r e s t r i c t i n g l a t e r a l e x p a n s i o n ( L l o y d and S e a g u l l 1985). However, the p r e s e n c e of f i b r i l l a r c o n n e c t i o n s between v e s i c l e s and m i c r o t u b u l e s can be i n t e r p r e t e d as e v i d e n c e of a f u n c t i o n a l r o l e f o r m i c r o t u b u l e s i n endosome t r a n s p o r t . The r e l a t i o n s h i p between endosomes and m i c r o t u b u l e s i n L o b e l i a r o o t c e l l s a g r e e s w i t h the r e p o r t e d t r a n s p o r t of endosomes i n a n i m a l c e l l s ( K o l s e t e t a l . 1979, V a l e 1987). I t i s p o s s i b l e t o s p e c u l a t e t h a t k i n e s i n or an analogous " m i c r o t u b u l e motor" p r o t e i n may be a c t i n g i n v e s i c u l a r t r a n s p o r t i n e l o n g a t i n g r o o t c e l l s as i t does i n amoeba, neurons, and f i b r o b l a s t s ( V a l e 1987). The a s s o c i a t i o n between m i c r o t u b u l e s and c o a t e d v e s i c l e s has been noted many tim e s i n h i g h e r p l a n t c e l l s (Doohan and P a l e v i t z 1980, Ryser 1979, Emons and T r a a s 1986). The i n c r e a s e d uptake of e x t r a c e l l u l a r medium i n e l o n g a t i n g s e c r e t o r y c e l l s compared t o v a c u o l a t e d n o n - s e c r e t o r y c e l l s , as d e s c r i b e d i n t h i s work, s u p p o r t s the concept t h a t e n d o c y t o s i s i s a t l e a s t one of the mechanisms f o r b a l a n c i n g 37 e x o c y t o s i s . The q u a n t i t a t i v e d a t a i n t h i s s tudy i s c o n s i s t e n t w i t h a r e p o r t t h a t t h e r e were more c o a t e d v e s i c l e s i n "growing c e l l s " than non-growing c e l l s (Emons and Traas 1986). However, i n the absence of e n d o c y t o t i c m a r kers, i t i s i m p o s s i b l e t o t e l l i f those c o a t e d v e s i c l e s were i n v o l v e d i n e n d o c y t o s i s or e x o c y t o s i s . V e s i c u l a r membrane r e c y c l i n g d u r i n g wounding has been demonstrated i n an a l g a , B o e r q e s e n i a ( O ' N e i l and L a C l a i r e 1988). Based on the r e s u l t s of t h i s s tudy and s i m i l a r r e s u l t s i n p r o t o p l a s t s , a model of membrane c y c l i n g i n e l o n g a t i n g r o o t c e l l s can be proposed ( F i g . 2 1 ) . Coated and smooth v e s i c l e s c o u l d i n v a g i n a t e from the plasma membrane, p u l l e d a g a i n s t the f o r c e s of t u r g o r by the c y t o s k e l e t o n . U n c o a t i n g of the e n d o c y t o t i c v e s i c l e would then o c c u r f a i r l y r a p i d l y , w hich c o u l d account f o r the g r e a t e r number of smooth v e s i c l e s than c o a t e d v e s i c l e s . Smooth v e s i c l e s c o u l d a l s o bud o f f the plasma membrane, perhaps v i a the f o r m a t i o n of i n t e r m e d i a t e s t r u c t u r e s d e s c r i b e d by Chapman and S t a e h e l i n (1987) f o r s e c r e t i o n . In t h i s s t u d y , s i m i l a r s t r u c t u r e s were obse r v e d l a b e l l e d w i t h lanthanum, s u g g e s t i n g e n d o c y t o s i s under t u r g o r does o c c u r . 38 After formation of endocytotic v e s i c l e s , the microtubule system may transport vesi c l e s to the multivesicular bodies. Lanthanum accumulates in the multivesicular body. This i s consistent with reports on animal c e l l s where horseradish peroxidase was used as a f l u i d phase marker; i t appeared only in the endosomal and lysosomal compartments but not in the Golgi (Farquhar 1981, de C h a s t e l l i e r et a l . 1987, Storrie et a l . 1984). For membrane recycling to occur, the internalized membrane must make i t s way back to the Golgi, to package the c e l l wall material being secreted. Because vesicles associated with the Golgi were not la b e l l e d in t h i s study, a di r e c t fusion between endocytotic vesi c l e s and Golgi does not appear l i k e l y . Vesicular membrane recycling could s t i l l occur i f small v e s i c l e s , (e.g. with diameter of 50 nm, and with a high surface area to volume ratio) pinch off the multivesicular body or endosome. The formation of tubules or small v e s i c l e s has been proposed as a mechanism of extruding the aqueous contents of the endosome, allowing concentration of membrane components (Rome 1985, Geuze et a l . 1984). Thus membrane recycling could be a two step process, f i r s t the internalized 39 membrane reaches the m u l t i v e s i c u l a r body, then membrane m a t e r i a l i s s h u t t l e d back t o the G o l g i f o r the next round of s e c r e t i o n . I t s h o u l d be n o t e d , however, t h a t p h o s p h o l i p i d t r a n s f e r p r o t e i n s or m i c e l l e s c o u l d a l s o p l a y a r o l e i n membrane c y c l i n g (Robinson 1984, S t e e r 1985, S t a e h e l i n and Chapman 1987). There are many unanswered q u e s t i o n s i n the a r e a of e n d o c y t o s i s i n p l a n t s r e g a r d i n g p o s s i b l e r e c e p t o r s and l i g a n d s , c y t o s k e l e t a l c o n n e c t i o n s , and the r o l e of the l y s o s o m e / c e n t r a l v a c u o l e i n the u l t i m a t e f a t e of the m a t e r i a l i n t e r n a l i z e d . The p r e s e n t study i s p a r t of a growing body of l i t e r a t u r e t h a t s u p p o r t s the view t h a t p l a n t c e l l s have the u l t r a s t r u c t u r a l a p p a r a t u s t o undergo e n d o c y t o s i s and t h a t e n d o c y t o s i s i s an i n t e g r a l p a r t of the membrane ho m e o s t a s i s of the endomembrane system. 4 0 Fig. 21: Model of membrane recycling during cell wall secretion. cv= coated vesicle, sv= smooth vesicle, mt= microtubules stipple= lanthanum, squares= secretory product, triangles* clathrin coat protein. 41 Chapter 2 SECRETION OF CELL WALL COMPONENTS IN ELONGATING ROOT CELLS OF LOBELIA ERINUS INTRODUCTION 42 In a study of e n d o c y t o s i s i t i s a p p r o p r i a t e t o examine the environment i n which e n d o c y t o s i s o c c u r s . The o b j e c t i v e of t h i s study was t o examine the i n t r a c e l l u l a r c o n d i t i o n s and f a c t o r s d i r e c t l y r e l a t e d t o e n d o c y t o s i s , w i t h emphasis on the i n t e r r e l a t i o n s h i p between t h i s p r o c e s s and s e c r e t i o n . G r a n u l o c r i n e s e c r e t i o n can be c o n s i d e r e d the o p p o s i t e of e n d o c y t o s i s , as i t r e p r e s e n t s the v e s i c l e mediated a d d i t i o n of membrane m a t e r i a l t o the plasma membrane t h r o u g h membrane f u s i o n . The aqueous c o n t e n t s of the v e s i c l e a re r e l e a s e d , r e s u l t i n g i n the e x t r u s i o n of s e c r e t o r y p r o d u c t i n t o the e x t e r n a l e nvironment. The membrane i n the s e c r e t o r y c e l l f l o w s from endoplasmic r e t i c u l u m (ER) t o d i c t y o s o m e s , c o l l e c t i v e l y c a l l e d the G o l g i a p p a r a t u s , t o plasma membrane (PM) ( t h e endomembrane h y p o t h e s i s of Morre and M o l l e n h a u e r 1974, H a r r i s 1986). The endomembrane system can be r o u g h l y d i v i d e d i n t o E R -1ike membranes ( n u c l e a r e n v e l o p e , ER, t r a n s i t i o n v e s i c l e s , c i s t e r n a e of the c i s G o l g i ) and P M - l i k e membranes ( c i s t e r n a e of t h e t r a n s G o l g i , t r a n s G o l g i network, s e c r e t o r y v e s i c l e s , endosomes,PM). New membrane m a t e r i a l would mature from the ER 43 type c o m p o s i t i o n t o the PM type c o m p o s i t i o n as i t moves a c r o s s the di c t y o s o m e s t a c k (Morre and M o l l e n h a u e r 1983). The c h a r a c t e r i s t i c endoplasmic r e t i c u l u m l i p i d s a r e p h o s p h a t i d y l c h o l i n e ( P C ) , p h o s p h a t i d y l e t h a n o l a m i n e (PE) w i t h p h o s p h a t i d y l i n o s i t o l ( PI) and p h o s p h a t i d y l g l y c e r o l as minor components ( C h r i s p e e l s 1980). F r e e s t e r o l s , g l u c o c e r e b r o s i d e , PC and PE a r e the p r i n c i p l e l i p i d components i n the plasma membrane ( R o c h e s t e r e t a l . 1987). Both p l a n t and a n i m a l c e l l s e x h i b i t t h i s h i g h s t e r o l t p h o s p h o l i p i d r a t i o a t the plasma membrane (Leonard and Hodges 1980). The, o r g a n e l l e s of the endomembrane system a l s o show p o l a r i t y i n terms of heterogenous p r o t e i n c o m p o s i t i o n s (Morre 1975). C o i n c i d i n g w i t h b i o c h e m i c a l d e t e r m i n a t i o n s i s the e v i d e n c e from f r e e z e f r a c t u r e s t u d i e s , which show "membrane d i f f e r e n t i a t i o n " w i t h r e s p e c t t o the p r o t e i n c o m p o s i t i o n , i . e . changes i n i n t r a membrane p a r t i c l e s (IMP) a c r o s s the G o l g i ( S t a e h e l i n and Kier m a y e r 1970, N o r t h c o t e and Lewis 1968). The endoplasmic r e t i c u l u m i s the major s i t e of p h o s p h o l i p i d s y n t h e s i s (Montague and Ray 1977, Morre 1970) and c o t r a n s l a t i o n a l i n s e r t i o n of i n t e g r a l membrane p r o t e i n s ( W a l t e r and B l o b e l 1982, 44 W a l t e r e t a l . 1984, Wickner and L o d i s h 1985, Von H e i j n e 1985, Boston e_t a l . 1982). T h i s p r o d u c t i o n of membrane components i n the ER s u p p l i e s the o r g a n e l l e s of the c e l l w i t h membrane e i t h e r by v e s i c u l a r t r a n s p o r t or p h o s p h o l i p i d t r a n s f e r p r o t e i n s (Robinson 1985, Morre and M o l l e n h a u e r 1980). The ER must be c o n s i d e r e d a dynamic s t r u c t u r e : r e s i d e n t p r o t e i n s as w e l l as t r a n s i t o r y p r o t e i n s a r e found t h e r e . I n c u l t u r e d a n i m a l c e l l s , t h e r e t e n t i o n of r e s i d e n t p r o t e i n s i n the ER i s h i g h l y s e l e c t i v e ; p r o t e i n s l a c k i n g r e t e n t i o n s i g n a l a r e e x p o r t e d t h r o u g h the G o l g i t o the PM (Wieland e t a l . 1987). In h i g h e r p l a n t c e l l s , the ER i s o f t e n o b s e r v e d l y i n g p a r a l l e l t o the plasma membrane, s e p a r a t e d from i t by the c o r t i c a l c y t o p l a s m ( C h r i s p e e l s 1976, C r a i g and S t a e h e l i n 1988). T h i s c o n f i g u r a t i o n may be r e l a t e d t o the importance of the ER i n m a i n t a i n i n g low i n t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n s (Buckhout 1984, Moore 1986). There a r e many s t r u c t u r a l elements i n c o r t i c a l c y t o p l a s m 2 + t h a t a r e c o n t r o l l e d by Ca c o n c e n t r a t i o n changes, eg. v e s i c l e f u s i o n , p o l y m e r i z a t i o n of c y t o s k e l e t a l e l e m e n t s , and membrane f l u i d i t y ( P i c t o n and S t e e r 1985). 45 In higher plants, the morphological and functional relationships observed between the ER and Golgi have some special features (Chrispeels 1976, Mollenhauer and Morre 1976, Robinson 1980). In contrast to protein secreting animal c e l l s , there are few t r a n s i t i o n v e s i c l e s between the ER and Golgi in plant c e l l s (Morre and Mollenhauer 1976, Robinson 1980, Dauwalder and Whaley 1982). It has been suggested that fewer t r a n s i t i o n v e s i c l e s are required between the ER and Golgi because less protein i s being produced in the plant c e l l (Robinson and Kristen 1982). However, the polysaccharides produced in the plant c e l l w i l l require membrane packaging; the amount of membrane required on the maturing face of the Golgi should be independent of the chemical composition of the ve s i c l e contents. Less membrane addition to the c i s face of the Golgi could be interpreted as support for an e f f i c i e n t membrane recycling mechanism on the trans face. Direct connections between the ER and the trans Golgi have been interpreted as a GERL (Golgi ER Lysosome) network, similar to that described by Novikoff in animals (Harris and Oparka 1983, Harris 1986). Ul t r a r a p i d l y frozen root c e l l s did not show 46 c o n n e c t i o n s between the ER and G o l g i ( C r a i g and S t a e h e l i n 1988). The f u n c t i o n s of the G o l g i i n p l a n t s , a n i m a l s , and f u n g i i n c l u d e g l y c o s y l a t i o n , s o r t i n g , and p a c k a g i n g of s e c r e t o r y p r o d u c t s . The dictyosome u s u a l l y c o n s i s t s of 4-8 s t a c k e d c i s t e r n a e . Each c i s t e r n a i s c h a r a c t e r i z e d by a c o m p a r a t i v e l y s t a b l e c e n t r a l p l a t e c o n t a i n i n g r e s i d e n t p r o t e i n s and a p e r i p h e r a l network from which v e s i c l e s bud. There a r e s e v e r a l models of c i s t e r n a f o r m a t i o n , maintenance, and m a t u r a t i o n (Farquhar 1985, Shannon et a l . 1982, Robinson 1985). The c i s t e r n a l p r o g r e s s i o n model suggests the e n t i r e c i s t e r n a moves t h r o u g h the dictyosome s t a c k , m a t u r i n g as i t goes from c i s t o t r a n s f a c e s (Farquhar 1985). T h i s model i s s u p p o r t e d by the work of Brown and coworkers who s t u d i e d s c a l e s e c r e t i o n i n a Chrysophycean a l g a . One s c a l e per c i s t e r n a i s produced and the e n t i r e c i s t e r n a i s shed from the dictyosome as the s c a l e matures (Brown 1969). An a l t e r n a t e h y p o t h e s i s i s a f i x e d c i s t e r n a model, where each c i s t e r n a e i s s p e c i a l i z e d f o r a subset of g l y c o s y l a t i o n or s o r t i n g f u n c t i o n s . In t h i s model, r e s i d e n t enzymes would be s t a t i o n a r y i n 47 the c i s t e r n a w i t h membrane and s u b s t r a t e b e i n g t r a n s f e r r e d by v e s i c l e s from c i s t e r n a t o c i s t e r n a . T h i s model has r e c e i v e d s u p p o r t from a v a r i e t y of s o u r c e s . S p e c i f i c c y t o c h e m i c a l r e a c t i o n s and g l y c o s y l t r a n s f e r a s e s have been l o c a l i z e d i n c i s , m e d i a l , or t r a n s c i s t e r n a e ( P a v e l k a 1987). V e s i c u l a r t r a n s p o r t has been s i m u l a t e d j_n v i t r o , d e m o n s t r a t i n g t h a t i n t e r c i s t e r n a l t r a n s p o r t i s u n i d i r e c t i o n a l and s e q u e n t i a l ( B a l c h e t a l . 1984). R e c e n t l y the view of the G o l g i as a r e l a t i v e l y f i x e d s t r u c t u r e w i t h dynamic f l o w of membrane and p r o d u c t s has emerged (Farquhar 1985, O r c i e t a l . 1989). The n a t u r e of the i n t e r c i s t e r n a l t r a n s p o r t v e s i c l e s i s c u r r e n t l y the s u b j e c t of i n v e s t i g a t i o n ; the v e s i c l e s a r e c o a t e d but the coa t does not appear t o c o n s i s t of c l a t h r i n ( G r i f f i t h s e_t a l . 1985, O r c i et a l . 1986). The p r o d u c t s of the G o l g i i n c l u d e e x t r a c e l l u l a r m a t r i c e s , membrane p r o t e i n s , g l y c o l i p i d s , and l y s o s o m a l p r o t e i n s . These p r o d u c t s must be s o r t e d t o the c o r r e c t i n t r a c e l l u l a r d e s t i n a t i o n . I n a n i m a l c e l l s , t he e x t e n s i v e network of v e s i c l e s and t u b u l e s a t the t r a n s f a c e of the G o l g i i s b e l i e v e d t o be the r e g i o n where s o r t i n g o c c u r s (Van Deurs e t a l . 1988). 48 The r e t i c u l a r n a t u r e of t h i s r e g i o n was o r i g i n a l l y i n t e r p r e t e d as a s p e c i a l i z e d domain of ER, and based on i t s a l k a l i n e phosphatase a c t i v i t y i t was named GERL, f o r G o l g i - E R p r o d u c i n g lysosomes ( N o v i k o f f 1964). R e c o g n i t i o n of the v a r i e t y of G o l g i p r o d u c t s which t r a v e r s e t h i s t r a n s G o l g i r e g i o n , and t h e l a c k of e v i d e n c e f o r ER c o n n e c t i o n s , have l e d t o i t s r e d e f i n i t i o n as the t r a n s G o l g i network (TGN) ( G r i f f i t h s and Simon 1986). The TGN has been c h a r a c t e r i z e d u s i n g the t r a n s p o r t of lysosome enzymes v i a the mannose-6-phosphate r e c e p t o r and the t r a n s p o r t of the v e s i c u l a r s t o m a t i t u s v i r u s G p r o t e i n as models ( K o r n f e l d and K o r n f e l d 1985, G r i f f i t h s e t a l . 1985). Analogous models have not been d e v e l o p e d f o r p l a n t c e l l s , and l i t t l e i s known about the mechanism of s o r t i n g of G o l g i p r o d u c t s . The s i t u a t i o n i s f u r t h e r c o m p l i c a t e d by an a d d i t i o n a l s o r t i n g d e s t i n a t i o n , the v a c u o l e ( D o r e l e t a l . 1989). W h i l e the b a s i c f u n c t i o n s of the G o l g i can be c o n s i d e r e d u n i v e r s a l (Farquhar and P a l a d e 1981), d i f f e r e n c e s between p h y l a can be seen. I n h i g h e r p l a n t s , the G o l g i t e n d t o occur as i n d i v i d u a l 49 d i c y t o s o m e s , w i t h seemingly random o r i e n t a t i o n ; a n i m a l d i c t y o s o m e s t e n d t o o c c u r as a p e r i n u c l e a r complex, p e r i p h e r a l l y a s s o c i a t e d and w i t h s i m i l a r p o l a r i t y . The c i s t o t r a n s p o l a r i t y i s more pronounced i n p l a n t d i c t y o s o m e s ; the lumen of the c i s t e r n a e g r a d u a l l y narrows i n the t r a n s d i r e c t i o n (Morre and M o l l e n h a u e r 1983, Robinson 1985). The complex membrane arrangements a t the t r a n s f a c e of the p l a n t dictyosome have been i n t e r p r e t e d as the f r a g m e n t a t i o n or s l o u g h i n g o f f of the t r a n s c i s t e r n a ( M o l l e n h a u e r 1971, Robinson 1985) . A p a r t i a l l y c o a t e d r e t i c u l u m (PCR), l o c a t e d near t h e d i c t y o s o m e , has been d e s c r i b e d i n h i g h e r p l a n t c e l l s ( P e s a c r e t a and Lucas 1985). The PCR i s an anastomosing network of v e s i c l e s and t u b u l e s . There i s now a " d e v e l o p i n g c o n t r o v e r s y " as t o the n a t u r e of the PCR (Tanchak e t §_1. 1988). I t has been i n t e r p r e t e d as an endosome, d i s t i n c t from the d i c t y o s o m e ( i b i d . ) ; i t has been c a l l e d an e x t e n s i o n of t h e t r a n s G o l g i c i s t e r n a e ( H i l l m e r ejb a l . 1988, Robinson and Depta 1988). Robinson and coworkers suggest the PCR would become s e p a r a t e d from the d i c t y o s o m e by the s l o u g h i n g o f f of the t r a n s c i s t e r n a e ( H i l l m e r e t a l . 1988). In view of the 50 c o n f u s i o n s u r r o u n d i n g the PCR and t r a n s G o l g i membranes i n h i g h e r p l a n t s , c y t o c h e m i c a l and u l t r a r a p i d f r e e z i n g s t u d i e s were undertaken t o examine t h e s e P M - r e l a t e d membranes. The major component of s e c r e t i o n i n w a l l s e c r e t i n g c e l l s i s p o l y s a c c h a r i d e s . To observe p o l y s a c c h a r i d e s , c y t o c h e m i c a l s t a i n s s p e c i f i c f o r p o l y h y d r o x y compounds can be used. There a r e s e v e r a l p r o t o c o l s f o r s t a i n i n g p o l y s a c c h a r i d e s (Roland 1978) such as m o d i f i e d p e r i o d i c a c i d - S c h i f f s ( T h i e r y 1967, Ryser 1979), a l k a l i n e bismuth (Park e t a l . 1987, S h i n j i e t a l . 1976), ruthenium r e d ( V i a n and R e i s 1972) or a l c i a n b l u e ( T r a c h t e n b e r g and Fahn 1981). The use of t h e s e s t a i n s has p r o v i d e d m o r p h o l o g i c a l e v i d e n c e t h a t complements b i o c h e m i c a l and a u t o r a d i o g r a p h i c e v i d e n c e t h a t p o l y s a c c h a r i d e s a r e assembled i n the G o l g i (Dixon and N o r t h c o t e 1985, Dauwalder and Whaley 1982). In t h i s s t u d y , the G o l g i was o n l y a p a r t of t h e o v e r a l l membrane systems examined f o r p o l y s a c c h a r i d e c o n t e n t . The p a r t i a l l y c o a t e d r e t i c u l u m and m u l t i v e s i c u l a r body, as elements of the P M - l i k e endomembrane system were a l s o examined. C o n v e n t i o n a l t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y sample p r e p a r a t i o n was compared w i t h u l t r a r a p i d 51 f r e e z i n g on a p o l i s h e d copper b l o c k h e l d a t l i q u i d n i t r o g e n t e m p e r a t u r e , f o l l o w e d by f r e e z e s u b s t i t u t i o n . In summary, the o b j e c t i v e s of t h i s s tudy were: 1. In view of the c o n f u s i o n s u r r o u n d i n g the PCR and t r a n s G o l g i membranes i n h i g h e r p l a n t s , c y t o c h e m i c a l and u l t r a r a p i d f r e e z i n g s t u d i e s were undertaken t o examine the s e P M - r e l a t e d membranes. 2. The G o l g i and a s s o c i a t e d membrane systems were examined f o r p o l y s a c c h a r i d e c o n t e n t , i n d i c a t i n g c e l l w a l l m a t r i x s y n t h e s i s . 3. U l t r a r a p i d f r e e z i n g and s t a i n i n g f o r p o l y s a c c h a r i d e s s h o u l d i d e n t i f y which compartments a r e p a r t of the pathway of e x o c y t o s i s . T h i s t e c h n i q u e i m m o b i l i z e s membranes a t the i n s t a n t of f r e e z i n g and s h o u l d p r o v i d e an a c c u r a t e p i c t u r e of the endomembrane system 4. The pathway of e x o c y t o s i s can be compared w i t h the pathway of e n d o c y t o s i s shown i n c h a p t e r 1. METHODS AND MATERIALS 52 C o n v e n t i o n a l TEM F i x a t i o n L o b e l i a e r i n u s s e e d l i n g s were g e r m i n a t e d on f i l t e r paper m o i s t e n e d w i t h d i s t i l l e d water f o r 7 days. The p l a n t s were m a n i p u l a t e d by the c o t y l e d o n s , under a d i s s e c t i n g m i c r o s c o p e . The s e e d l i n g s were f i x e d f o r 1 hour i n a d i l u t e K a r n o v s k y ' s f i x a t i v e c o n s i s t i n g of 1 % g l u t a r a l d e h y d e and 1.5 % formaldehyde, f r e s h l y p r e p a r e d from paraformaldehyde powder i n 50 mM PIPES pH 7.4. A f t e r 2 r i n s e s of 10 minutes each i n b u f f e r , the samples were p o s t f i x e d i n 1 % OsO^, b u f f e r e d as above, o v e r n i g h t a t 4° C. A f t e r a r i n s e i n b u f f e r , the samples were d e h y d r a t e d through a s e r i e s of i n c r e a s i n g c o n c e n t r a t i o n of methanol. P r o p y l e n e o x i d e was used as a t r a n s i t i o n s o l v e n t between methanol and r e s i n . The s e e d l i n g s were i n f i l t r a t e d and embedded i n epon. S i l v e r s e c t i o n s were o b t a i n e d u s i n g a diamond k n i f e on a R e i c h e r t OMU3 or a R e i c h e r t U l t r a c u t E and mounted on uncoated 200 mesh copper g r i d s . The g r i d s were s t a i n e d i n s a t u r a t e d u r a n y l a c e t a t e f o r 25 m i n u t e s , f o l l o w e d by r i n s i n g i n d i s t i l l e d water. S a t o ' s l e a d c i t r a t e was used f o r 5-7 minutes i n a low ca r b o n d i o x i d e environment. 53 P e r o x i d a s e C y t o c h e m i s t r y S e e d l i n g s were t r e a t e d t o l o c a l i z e endogenous p e r o x i d a s e f o l l o w i n g the methods of G r i f f i n g and Fowke (1985). The c e l l s were f i x e d i n 1 % g l u t u a r a l d e h y d e i n 60 mM sodium phosphate b u f f e r , pH 7.4, f o l l o w e d by 5 washes i n the b u f f e r over 1 hour, w i t h the pH d e c r e a s i n g s t e p w i s e from 7.4 t o 5.4. The p e r o x i d a s e s u b s t r a t e and c y t o c h e m i c a l r e a g e n t s were 5 mM hydrogen p e r o x i d e and 4 mg/ml d i a m i n o b e n z i d i n e (DAB). The s e e d l i n g s were i n c u b a t e d w i t h the s u b s t r a t e / r e a g e n t mix f o r 10 m i n u t e s , 30 m i n u t e s , or 60 m i n u t e s . For c o n t r o l t r e a t m e n t , samples were i n c u b a t e d i n DAB a l o n e or b u f f e r a l o n e . At the end of the i n c u b a t i o n p e r i o d , the samples were put on i c e and a g a i n r i n s e d t h o r o u g h l y over 1 hour. P o s t f i x a t i o n u s i n g 1 % OsC»4 was c a r r i e d out o v e r n i g h t a t 4° C. A f t e r s e v e r a l washes i n b u f f e r , m e t h a n o l i c d e h y d r a t i o n and epon embedding was performed as d e s c r i b e d above f o r TEM. S e c t i o n s were o b s e r v e d under the Z e i s s 10C TEM w i t h o u t p o s t s t a i n t o improve the r e l a t i v e c o n t r a s t of the p e r o x i d a s e r e a c t i o n p r o d u c t . 5 4 U l t r a r a p i d F r e e z i n g / F r e e z e S u b s t i t u t i o n For u l t r a r a p i d f r e e z i n g , seeds were g e r m i n a t e d as d e s c r i b e d above and f r o z e n a t 7 days p o s t i m b i b i t i o n . S e e d l i n g s were p l a c e d on a l a y e r of f i l t e r paper, on a l a y e r of p a r a f i l m which was st u c k on a s o f t foam pad w i t h a g l u e s t i c k . The s o f t foam pad was backed w i t h a me t a l p l a n c h e t t e t h a t m a g n e t i c a l l y a t t a c h e s t o the MM80 attachment of the R e i c h e r t - J u n g KF80 f r e e z i n g a p p a r a t u s . The samples were mounted and kept i n a humid environment w h i l e the p o l i s h e d copper m i r r o r ("metal m i r r o r " ) e q u i l i b r i a t e d t o l i q u i d n i t r o g e n t e m p e r a t u r e (-196° C ) . The p l a n c h e t t e , w i t h a t t a c h e d sample, was then r a p i d l y t r a n s f e r r e d t o the MM80 arm and slammed a g a i n s t the me t a l m i r r o r . The samples on the f i l t e r paper were then s e p a r a t e d from the s o f t foam by removing the now b r i t t l e p a r a f i l m . The samples were t r a n s f e r r e d , under l i q u i d n i t r o g e n , t o the R e i c h e r t - J u n g CSAuto f o r f r e e z e s u b s t i t u t i o n . The s u b s t i t u t i o n was c a r r i e d out i n 1 % OsO^ a t -80° C f o r 55 h o u r s . D u r i n g t h i s t i m e , t he water i n the sample was r e p l a c e d w i t h a c e t o n e . The temperature was i n c r e a s e d a t a r a t e of 10° C per hour u n t i l i t reached 0° C. At t h i s t i m e , the 55 osmium acetone mix was r e p l a c e d and r i n s e d w i t h i c e c o l d a b s o l u t e a c e t o n e . A f t e r s e v e r a l r i n s e s , the samples were a l l o w e d t o come t o room te m p e r a t u r e and i n f i l t r a t e d i n S p u r r ' s r e s i n . A f t e r embedding and c u r i n g the r e s i n , s i l v e r s e c t i o n s were c u t on the U l t r a c u t E. P o l y s a c c h a r i d e c y t o c h e m i s t r y S e v e r a l c y t o c h e m i c a l t e c h n i q u e s f o r p o l y s a c c h a r i d e s were t e s t e d i n p r e l i m i n a r y e x p e r i m e n t s (see r e f e r e n c e s Appendix 2 f o r d e t a i l s of methods). Both c o n v e n t i o n a l l y f i x e d and u l t r a r a p i d l y f r o z e n m a t e r i a l was s t a i n e d i n a l k a l i n e b i s m u t h , f o l l o w i n g the p r o c e d u r e of S h i n j i et a l . 1976). A s t o c k s o l u t i o n of 1 g bismuth s u b n i t r a t e , 2 g sodium t a r t r a t e , 5 g sodium h y d r o x i d e and 50 ml d e i o n i z e d d i s t i l l e d water was p r e p a r e d . T h i s was d i l u t e d 1 ml i n 40 ml d e i o n i z e d d i s t i l l e d water t o g i v e the working s o l u t i o n . The g r i d s were s t a i n e d a t 40° C f o r 40 minutes i n a humid environment, then r i n s e d by g e n t l y a g i t a t i n g i n be a k e r s of d e i o n i z e d d i s t i l l e d w ater. The s e c t i o n s were examined and photographed on the Z e i s s 10C o p e r a t e d a t 60 kV. RESULTS 56 The endogenous p e r o x i d a s e s of the r o o t t i p of L. e r i n u s were l o c a l i z e d w i t h the c l a s s i c a l c y t o c h e m i c a l t e c h n i q u e of Graham and Karnovsky (1966). The p r o d u c t i o n of t h i s enzyme can be c o n s i d e r e d an example of c o n s t i t u t i v e p r o t e i n s e c r e t i o n . The r e a c t i o n p r o d u c t f o r p e r o x i d a s e was found i n the c e l l w a l l , v e s i c l e s a s s o c i a t e d w i t h the c o r t i c a l c y t o p l a s m , and i n t r a l u m i n a l v e s i c l e s of the v a c u o l e s ( F i g s . 22-25). In the c e l l w a l l , the p e r o x i d a s e r e a c t i o n p r o d u c t i s s t r a t i f i e d i n t o s e v e r a l w a l l l a y e r s ( F i g s . 22-24). The e p i d e r m a l m u c i l a g e l a y e r i s more d a r k l y s t a i n e d than the c e l l u l o s i c l a y e r , ( F i g . 2 4 ) . The plasma membrane and nearby v e s i c l e s a l s o c o n t a i n r e a c t i o n p r o d u c t ( F i g s . 23, 2 4 ) . The d i ctyosome v e s i c l e s were amorphous i n terms of membrane and c o n t e n t s ( F i g . 25). In some a r e a s the c y t o c h e m i c a l r e a g e n t s d i d not p e n e t r a t e i n t o the t i s s u e making i t i m p o s s i b l e t o draw any c o n c l u s i o n about p e r o x i d a s e l e v e l s between t i s s u e s . To examine o r g a n e l l e s r e l a t e d t o c e l l w a l l s y n t h e s i s , c y t o c h e m i c a l s t a i n s f o r p o l y s a c c h a r i d e were used. Of the s e v e r a l s t a i n s t e s t e d ( T a ble 4) a l k a l i n e bismuth s t a i n i n g gave most c o n s i s t e n t r e s u l t s on both c o n v e n t i o n a l l y f i x e d ( F i g s . 26-36) 57 T a b l e 4: Comparison of c y t o c h e m i c a l s t a i n s l a b e l l i n g p a t t e r n on e r i n u s r o o t t i p s . A l l s t a i n s were v i s u a l i z e d by TEM. S t a i n S t r u c t u r e s l a b e l l e d p e r i o d a t e -s i l v e r m e t h e n a m i n e p o s t s t a i n c e l l w a l l ; c e l l s e a s i l y d i g e s t e d w i t h p e r i o d a t e , r u t h e n i u m red en b l o c outermost c e l l w a l l l a y e r ; r o o t h a i r s ; r o o t cap. a l c i a n b l u e en b l o c p e c t i n a s e - c o l l o i d a l g o l d on Epon s e c t i o n s a l k a l i n e bismuth p o s t s t a i n r o o t cap m u c i l a g e ; e p i d e r m a l o u t e r w a l l l a y e r . no s i g n a l above n o i s e . d i c t y o s o m e s ; v e s i c l e s ; p a r t i a l l y c o a t e d r e t i c u l u m ; plasma membrane; c e l l w a l l 58 and freeze substituted tissue (Figs. 36-38). This method has been used to stain Golgi v e s i c l e s , c e l l wall and plasma membrane of pear leaves (Park et a l . 1987). In Lobelia erinus root c e l l s , well characterized carbohydrate containing structures were stained with alkaline bismuth. Examples of these are the starch granules (Fig. 22, compare control F i g . 23) and the c e l l wall (Figs. 26, 28, 32-34; compare controls Figs. 27, 29, 35). The starch granules are more prominent in the pl a s t i d s of the root cap than in the plas t i d s of epidermal or c o r t i c a l c e l l s . The po s i t i v e reaction of starch granules to al k a l i n e bismuth confirms the s p e c i f i c i t y of t h i s stain for polysaccharide. The c e l l wall appeared s t r a t i f i e d into several layers that varied depending on the c e l l type and sectioning effects (Fig. 26 vs. 28). In general the pattern of c e l l wall staining observed was (from the c e l l surface): a darkly staining e x t r a c e l l u l a r surface of the plasma membrane, a zone of decreased density into which f i b r i l s from the c e l l surface projected, a dense c e l l u l o s i c layer, and a darkly stained middle lamella zone. 59 In c o n v e n t i o n a l l y f i x e d m a t e r i a l , the r e g i o n between the plasma membrane and c e l l w a l l c o n t a i n e d a l k a l i n e bismuth r e a c t i v e v e s i c l e s which can be i d e n t i f i e d as lomasomes (Fowke and S e t t e r f i e l d 1969 F i g s . 28, 29, 34, 3 5 ) . No such v e s i c l e s were i d e n t i f i e d i n u l t r a r a p i d l y f r o z e n m a t e r i a l ( F i g s . 36, 3 7 ) . F i b r i l l a r c o n n e c t i o n s between the plasma membrane and c e l l w a l l c o u l d be v i s u a l i z e d w i t h a l k a l i n e bismuth ( F i g . 28, 33, 3 4 ) . The plasma membrane o f t e n had c o r t i c a l m i c r o t u b u l e s l i n i n g the c y t o p l a s m i c s u r f a c e ; the c e l l w a l l s t a i n i n t h e s e r e g i o n s had no ob v i o u s r e l a t i o n s h i p t o the m i c r o t u b u l e s ( F i g . 3 2 ) . I t i s common t o observe v e s i c l e s i n the c o r t i c a l c y t o p l a s m i n both the c o n v e n t i o n a l l y f i x e d and the u l t r a r a p i d l y f r o z e n ; some of t h e s e s t a i n e d p o s i t i v e l y w i t h a l k a l i n e b i smuth ( F i g s . 28, 30, 32-34, 36-38). I t i s i n t e r e s t i n g t o note however t h a t t h e r e i s a wide v a r i e t y of s t a i n i n g i n t e n s i t i e s between v e s i c l e s ( F i g . 30, 34, 3 6 ) . I t was o f t e n d i f f i c u l t t o d i s t i n g u i s h c o a t e d membranes i n t h e ground c y t o p l a s m on s e c t i o n s t h a t were s t a i n e d f o r p o l y s a c c h a r i d e s o n l y ( F i g . 3 3 ) . T h i s c o u l d be because the p r o t e i n c o a t on the membrane does not r e a c t w i t h the s t a i n f o r p o l y s a c c h a r i d e . 60 The c o n v e n t i o n a l TEM f i x a t i o n d i d not p r e s e r v e the v e s i c l e s of the di c t y o s o m e s ( F i g s . 29, 30) . F i x a t i o n c o n d i t i o n s were v a r i e d i n an attempt t o improve the G o l g i p r e s e r v a t i o n , but thes e s t r u c t u r e s were e a s i l y d i s r u p t e d . D i f f e r e n t c o m b i n a t i o n s of b u f f e r s , d i v a l e n t c a t i o n s , or formaldehyde : g l u t a r a l d e h y d e r a t i o s were t e s t e d (data not shown). Under t h e s e c o n d i t i o n s of poor p r e s e r v a t i o n i t was d i f f i c u l t t o a s s e s s t h e number of t r a n s i t i o n v e s i c l e s between t h e ER and dic t y o s o m e , or the s t a i n i n g p r o p e r t i e s of the dictyosome c i s t e r n a ( F i g . 30 ) . T h i s can be compared w i t h the m a t e r i a l t h a t was u l t r a r a p i d l y f r o z e n where the d i c t y o s o m e s and a d j a c e n t v e s i c l e s a r e c l e a r l y d e l i n e a t e d ( F i g s . 36-38) . V e s i c l e s were observed i n between the ER and d i c t y o s o m e s ; these v e s i c l e s have s m a l l d i a m e t e r s (about 50 nm). A f t e r u l t r a r a p i d f r e e z i n g , d i c t y o s o m e s d i s p l a y e d c i s t o t r a n s p o l a r i t y s i m i l a r t o t h a t d e s c r i b e d by Robinson and K r i s t e n (1982) where the c i s t e r n a w i d t h d e c r e a s e s i n the c i s t o t r a n s d i r e c t i o n ( F i g . 36 ) . A l k a l i n e b i s m u t h s t a i n i n g i n t e n s i t y a l s o d i s p l a y e d a d e f i n i t e p o l a r i t y , w i t h h i g h e s t d e n s i t y i n the t r a n s c i s t e r n a e ( F i g s . 36-38) 61 The t r a n s f a c e of the G o l g i shows a r e t i c u l u m of t u b u l e s and v e s i c l e s ; one subset of t h e s e v e s i c l e s a r e l a r g e r ( d i a meter about 100-150 nm) than t h e p e r i p h e r a l v e s i c l e s of the dictyosome ( d i a m e t e r about 50-100 nm). These l a r g e r v e s i c l e s r esembled some of the d a r k l y s t a i n i n g c o r t i c a l v e s i c l e s , thus t h e y may r e p r e s e n t s e c r e t o r y v e s i c l e s ( F i g . 36) . The r e t i c u l u m a t the t r a n s G o l g i f a c e can be d i s t i n g u i s h e d by the pr e s e n c e of t h e s e l a r g e r v e s i c l e s . I n a d d i t i o n t o the t r a n s G o l g i a s s o c i a t e d r e t i c u l u m , a s e p a r a t e complex w i t h s m a l l v e s i c l e s and s t a i n i n g c h a r a c t e r i s t i c s s i m i l a r t o the plasma membrane were obse r v e d ( F i g s . 36-38). T h i s system of anastamosing t u b u l e s and v e s i c l e s can be i n t e r p r e t e d as the p a r t i a l l y c o a t e d r e t i c u l u m ( P e s a c r e t a and Lucas 1985). T h i s s t r u c t u r e was commonly observed i n the u l t r a r a p i d l y f r o z e n c e l l s , but r a r e l y i n the c o n v e n t i o n a l l y f i x e d c e l l s . M u l t i v e s i c u l a r b o d i e s were l i g h t l y s t a i n e d w i t h a l k a l i n e b i smuth ( F i g s . 30, 34, compare c o n t r o l F i g . 3 1 ) . A f t e r c o n v e n t i o n a l f i x a t i o n , a p o p u l a t i o n of s m a l l v e s i c l e s i n the c y t o p l a s m s u r r o u n d i n g the m u l t i v e s i c u l a r body a r e s t a i n e d w i t h a l k a l i n e b i s m u t h . A f t e r u l t r a r a p i d f r e e z i n g , 62 s i m i l a r v e s i c l e s can be seen budding from or f u s i n g w i t h the m u l t i v e s i c u l a r body ( F i g . 38). The most d i s t i n c t i v e f e a t u r e s seen i n the u l t r a r a p i d l y f r o z e n m a t e r i a l compared t o c o n v e n t i o n a l l y f i x e d t i s s u e a r e the p r e s e n c e of an e l a b o r a t e network on the t r a n s G o l g i f a c e , absence of lomasomes, and the p r e s e n c e of many s m a l l s h u t t l e or t r a n s i t i o n v e s i c l e s between o r g a n e l l e s of t h e endomembrane system. The p r o v a c u o l e s and c e n t r a l v a c u o l e showed the same r e l a t i v e a l k a l i n e bismuth r e a c t i v i t y ( F i g s . 28, compare c o n t r o l F i g . 29). I t was d i f f i c u l t t o i n t e r p r e t the n a t u r e of the m a t e r i a l a s s o c i a t e d w i t h the i n n e r s u r f a c e of the v a c u o l e ; the t o n o p l a s t was s t a i n e d by l e a d and u r a n y l a c e t a t e as w e l l . I t i s i n t e r e s t i n g t o compare the p o s t s t a i n e d m a t e r i a l ( F i g s . 26-38) w i t h the p e r o x i d a s e m a t e r i a l which was u n s t a i n e d ( F i g s . 22-25). 63 F i g s . 22-25: Endogenous p e r o x i d a s e a c t i v i t y i n e l o n g a t i n g L o b e l i a r o o t c e l l s . F i g . 22: survey of f i r s t c e l l l a y e r showing p e n e t r a t i o n of DAB p r o d u c t i n c e l l w a l l . Arrow shows i n t r a l u m i n a l v e s i c l e i n c e n t r a l v a c u o l e . F i g . 23: e p i d e r m a l c e l l s l a b e l l e d a l o n g c e l l w a l l and i n c o r t i c a l v e s i c l e s ( a r r o w ) . F i g . 24: c e l l w a l l l a y e r s show d i f f e r e n t i a l s t a i n i n g ; c o r t i c a l v e s i c l e s s t a i n e d . F i g . 25: poor dictyosome p e r s e r v a t i o n . V=vacuole, c w = c e l l w a l l , pm=plasma membrane, pv=provacuole, c = c e l l u l o s i c l a y e r , m=mucilage, d=dictyosome. 65 F i g s . 26-31: A l k a l i n e b ismuth s t a i n i n g of c o n v e n t i o n a l TEM p r e p a r a t i o n s . F i g . 26: s t a r c h g r a n u l e s t a i n e d w i t h a l k a l i n e b i s m u t h . F i g . 27: c o n t r o l s t a r c h g r a n u l e s t a i n e d w i t h u r a n y l a c e t a t e / l e a d c i t r a t e . F i g . 28: s e c r e t o r y g r a n u l e (arrow) and c e l l w a l l s t a i n e d w i t h a l k a l i n e b i s m u t h . F i g . 29: c o n t r o l s t a i n e d w i t h u r a n y l a c e t a t e / l e a d c i t r a t e . F i g . 30: d i c t y o s o m e , m u l t i v e s i c u l a r body s t a i n e d w i t h a l k a l i n e b i s m u t h . F i g . 31: c o n t r o l m u l t i v e s i c u l a r body s t a i n e d w i t h u r a n y l a c e t a t e / l e a d c i t r a t e . Note: plaque on MVB. c w = c e l l w a l l , s = s t a r c h g r a n u l e , pv=provacuole, d=dictyosome, m v b = m u l t i v e s i c u l a r body. 67 F i g s . 32-35: A l k a l i n e bismuth s t a i n i n g of c o n v e n t i o n a l TEM p r e p a r a t i o n s . F i g . 32: t a n g e n t i a l s e c t i o n t h r o u g h c o r t i c a l c y t o p l a s m showing s e c r e t o r y v e s i c l e ( a r r o w ) , and c e l l w a l l s t a i n p o s i t i v e f o r p o l y s a c c h a r i d e s . F i g . 33: c o r t i c a l v e s i c l e , plasma membrane, and c e l l w a l l i n e l o n g a t i n g c e l l . F i g . 34: d i f f e r e n t i a l s t a i n i n g of c o r t i c a l v e s i c l e s ; m u l t i v e s i c u l a r body does not s t a i n f o r p o l y s a c c h a r i d e s . F i g . 35: c o n t r o l s e c t i o n s t a i n e d w i t h u r a n y l a c e t a t e / l e a d c i t r a t e . arrow i n d i c a t e s v e s i c l e , a s t e r i s k i n d i c a t e s u n u s u a l p o l y g o n a l s t r u c t u r e t h a t resembles c o a t e d membranes. mt= m i c r o t u b u l e s , c w = c e l l w a l l , l=lomasome, m v b = m u l t i v e s i c u l a r body. 69 Figs. 36-38: Alkaline bismuth staining of u l t r a r a p i d l y frozen, freeze substituted material. F i g . 36: example of epidermal c e l l u l t r a s t r u c t u r e . ER not stained, dictyosome exhibits p o l a r i t y from c i s to trans, secretory v e s i c l e s at trans Golgi and in c o r t i c a l cytoplasm (arrows). F i g . 37: dictyosome between plasma membrane and developing central vacuole. F i g . 38: dictyosome with extensive trans Golgi reticulum, separate from p a r t i a l l y coated reticulum, multivesicular body. pcr=partially coated reticulum, c=cis Golgi face, t=trans Golgi face, mvb=multivesicular body, d=dictyosome, er=endoplasmic reticulum, t=tonoplast. DISCUSSION 71 The P M - r e l a t e d membranes of the endomembrane system may be c o n s i d e r e d the environment f o r the o v e r l a p p i n g pathways of e x o c y t o s i s and e n d o c y t o s i s . The e x o c y t o t i c pathways shown i n t h i s study were p e r o x i d a s e s e c r e t i o n and c e l l w a l l m a t r i x s e c r e t i o n . The r e s u l t s of the c y t o c h e m i c a l s t u d i e s of c o n v e n t i o n a l l y p r e p a r e d TEM samples agree w e l l w i t h r e s u l t s r e p o r t e d i n o t h e r h i g h e r p l a n t ( V i a n and R e i s 1972, Ryser 1979) and a n i m a l s p e c i e s ( T h i e r y 1967). The c e l l u l a r l o c a t i o n of p e r o x i d a s e i n c o r t i c a l v e s i c l e s , c e l l w a l l , and v a c u o l e i s c o n s i s t e n t w i t h the f u n c t i o n s p o s t u l a t e d f o r t h i s enzyme. P e r o x i d a s e i n the c e l l w a l l may p l a y a r o l e i n c r o s s l i n k i n g t y r o s i n e u n i t s of e x t e n s i n d u r i n g c e l l w a l l s y n t h e s i s , and i n c r o s s l i n k i n g p h e n o l i c u n i t s d u r i n g l i g n i f i c a t i o n ( F r y 1986). S i n c e l i g n i f i c a t i o n does not be g i n u n t i l the c e l l i s f u l l y e l o n g a t e d , the l a t t e r f u n c t i o n i s p r o b a b l y not c r i t i c a l i n the e l o n g a t i n g c e l l . I n a s i m i l a r c y t o c h e m i c a l s t u d y of soybean s u s p e n s i o n c u l t u r e d c e l l s , p e r o x i d a s e was found i n the c e l l w a l l and v a c u o l e s ( G r i f f i n g and Fowke 1985). In c u l t u r e d c e l l s , p e r o x i d a s e r e a c t i o n p r o d u c t was f a i n t i n the t r a n s d i c t y o s o m e , but d i d 72 not appear on the s u r f a c e of t h e plasma membrane. The L o b e l i a r o o t c e l l s ' d i c t y o s o m e s were not l a b e l l e d , whereas the plasma membrane and some a s s o c i a t e d v e s i c l e s were h e a v i l y l a b e l l e d . The c o r t i c a l v e s i c l e s which show p e r o x i d a s e a c t i v i t y may r e p r e s e n t s e c r e t o r y v e s i c l e s d e l i v e r i n g p e r o x i d a s e t o the c e l l w a l l . The l a b e l on t h e c e l l s u r f a c e would then be r e c e n t l y e x t r u d e d s e c r e t o r y p r o d u c t , b e f o r e i t s i n c o r p o r a t i o n i n t o the w a l l . I t i s unexpected t h a t the endoplasmic r e t i c u l u m and dictyosome a r e not l a b e l l e d by t h i s technique", as t h e s e o r g a n e l l e s a re the s i t e s of p r o t e i n s y n t h e s i s and m o d i f i c a t i o n . The pH a t which the c y t o c h e m i c a l t e s t s a r e c a r r i e d out and the f i x a t i o n p r o t o c o l which precedes the c y t o c h e m i c a l t e s t may have c r e a t e d u n f a v o r a b l e c o n d i t i o n s f o r enzyme a c t i v i t y ; the parameters employed had been used t o l o c a l i z e p e r o x i d a s e i n c u l t u r e d c e l l s ( G r i f f i n g and Fowke 1985). I t was d i f f i c u l t t o judge the dictyosome p e r o x i d a s e c o n t e n t due t o poor p r e s e r v a t i o n of these o r g a n e l l e s i n c o n v e n t i o n a l p r e p a r a t i o n s . T h i s i s an a r e a t h a t c r y o t e c h n i q u e s c o u l d be a p p l i e d t o i n the f u t u r e . 7 3 The h i g h l e v e l of endogenous p e r o x i d a s e found i n the e l o n g a t i n g r o o t c e l l s , and i n o t h e r h i g h e r p l a n t s p e c i e s , e l i m i n a t e p e r o x i d a s e as a u s e f u l marker f o r e n d o c y t o s i s i n h i g h e r p l a n t s . H o r s e r a d i s h p e r o x i d a s e i s w i d e l y used i n a n i m a l c e l l b i o l o g y t o s t u d y the endomembranes and k i n e t i c s of e n d o c y t o s i s (Steinman et a l . 1983). The c y t o p l a s m i c s y n t h e s i s and e v e n t u a l d e p o s i t i o n of p e r o x i d a s e can be used as an example of p r o t e i n s e c r e t i o n . However, p r o t e i n s o n l y make up about 10 % of the c e l l w a l l ; 90 % of the w a l l i s p o l y s a c c h a r i d e ( M c N e i l e t a l . 1984). To get a complete p i c t u r e of the e x p c y t o t i c pathway i n the e l o n g a t i n g c e l l s , i t was n e c e s s a r y t o use s t a i n s f o r p o l y s a c c h a r i d e s . A l t h o u g h h i s t o r i c a l l y o t h e r methods, such as the m o d i f i e d PAS r e a c t i o n , have been used t o d e t e c t p o l y s a c c h a r i d e s , a l k a l i n e b i s muth s t a i n i n g was the most r e p r o d u c i b l e and d i s t i n c t i v e s t a i n f o r t h i s t i s s u e . In c o n v e n t i o n a l l y f i x e d samples, the c e l l w a l l , e x t r a c e l l u l a r f a c e of the plasma membrane, and v e s i c l e s r e a c t e d s t r o n g l y w i t h a l k a l i n e b i s m u t h . The i n t e n s i t y of s t a i n i n g of v e s i c l e s was h i g h l y v a r i a b l e . The h e a v i l y s t a i n e d v e s i c l e s c o u l d be s e c r e t o r y v e s i c l e s c a r r y i n g c e l l w a l l 74 p r e c u r s o r s s i n c e the d e n s i t y of s t a i n i n these v e s i c l e s resembles the d e n s i t y of s t a i n i n the c e l l w a l l . The l i g h t l y s t a i n e d v e s i c l e s c o u l d r e p r e s e n t e n d o c y t o t i c v e s i c l e s , but i n t h e absence of markers f o r e n d o c y t o s i s t h i s i n t e r p r e t a t i o n i s s p e c u l a t i v e . Due t o the p r o c e s s of membrane r e c y c l i n g , some endosomes may s t a i n p o s i t i v e l y f o r s e c r e t o r y p r o d u c t . In r a t a c i n a r c e l l , i t was shown t h a t the a p o r t i o n of the p r o t e i n s e c r e t o r y p r o d u c t was r e i n t e r n a l i z e d by e n d o c y t o s i s (Romagnoli and Herzog 1987). T h i s apparent paradox i l l u s t r a t e s the d i f f i c u l t y of i n t e r p r e t i n g t h i s type of e x p e r i m e n t . The c e l l w a l l has been c o n s i d e r e d as l i t t l e more than a t e c h n i c a l impediment t o the stu d y of the plasma membrane and the i n t i m a t e r e l a t i o n s h i p between t h e s e s t r u c t u r e s i s i g n o r e d . The w a l l may i n f l u e n c e the plasma membrane i n a manner analogous t o the r e s t r a i n t s imposed on the plasma membrane by the c y t o s k e l e t o n . In t h i s s t u d y , s t r u c t u r a l c o n t i n u i t y was ob s e r v e d between the e x t r a c e l l u l a r f a c e of the plasma membrane and the c e l l w a l l . The f i b r i l s p r o j e c t i n g from the c e l l s u r f a c e may be c e l l w a l l components t h a t have been s e c r e t e d and are c o a l e s c i n g w i t h the e x i s t i n g c e l l w a l l . A l t e r n a t i v e l y , the f i b r i l s seen p r o j e c t i n g from the 75 c e l l w a l l c o u l d be components of the g l y c o c a l y x , the c a r b o h y d r a t e c o a t on the c e l l s u r f a c e . I f t h i s c o a t i s l i n k e d t o the c e l l w a l l m a t r i x , the e x t r a c e l l u l a r c o n n e c t i o n s c o u l d r e s t r i c t t he m o b i l i t y of the g l y c o l i p i d s and g l y c o p r o t e i n s i n the membrane. The v e s i c l e s found i n the p e r i p l a s m i c space between the c e l l w a l l and plasma membrane, c a l l e d lomasomes, are o n l y o b s e r v e d i n c o n v e n t i o n a l l y f i x e d m a t e r i a l , not i n u l t r a r a p i d l y f r o z e n m a t e r i a l . I n o t h e r s t u d i e s of u l t r a r a p i d l y f r o z e n p l a n t c e l l s , the plasma membrane was r e p o r t e d as smooth and u n p e r t u r b e d (Fernandez and S t a e h e l i n 1985). T h i s s u g g e s t s lomasomes a r e an a r t i f a c t of c h e m i c a l f i x a t i o n , as su g g e s t e d many y e a r s ago by Fowke and S e t t e r f i e l d (1969). One p o s s i b l e mechanism of g e n e r a t i o n of lomasomes c o u l d be the s t a b i l i z a t i o n of plasma membrane-cell w a l l c o n n e c t i o n s by g l u t a r a l d e h y d e d u r i n g p r i m a r y f i x a t i o n . D u r i n g a l d e h y d e f i x a t i o n , l i p i d s a r e not s t a b i l i z e d so the membrane may be c o n t r a c t i n g and expa n d i n g over the f i x a t i o n and r i n s e p e r i o d . I f some domains of t h e membrane a r e h e l d i n p l a c e by a s s o c i a t i o n w i t h the e x t r a c e l l u l a r m a t r i x , t h e s e domains c o u l d be t o r n out of the membrane. 76 Membrane fragments would then vesiculate to form lomasomes. In the conventional TEM micrographs, plant c e l l s often show a " t y p i c a l undulating appearance of plasma membrane" (Leonard and Hodges 1980). In contrast, one of the c r i t e r i a for judging the success of u l t r a r a p i d freezing i s the smooth, unperturbed appearance of the membranes (Gilkey and Staehelin 1986). The endomembranes seen in zones of well frozen material in this study have d i s t i n c t i v e appearances. There are many more vesi c l e s present and the dictysome associated network i s extensive. When stained with a l k a l i n e bismuth, these sections provide information on the location of polysaccharide in the unperturbed c e l l . In u l t r a r a p i d l y frozen material, there are small v e s i c l e s in the zone of cytoplasm between ER and the Golgi. This i s predicted by the endomembrane concept (Morre and Mollenhauer 1974), but has not been commonly observed in conventional preparations (Steer 1985). The presence of these ve s i c l e s suggests that one mechanism for transport of membrane subunits between the ER and the Golgi i s by v e s i c l e s , another possible mechanism i s phospholipid transfer proteins (Robinson 1985, Fernandez and Staehelin 1985). 77 The ER elements do not c o n t a i n enough p o l y s a c c h a r i d e t o g i v e a p o s i t i v e a l k a l i n e b i s m u t h r e a c t i o n . A l t h o u g h f r a c t i o n a t i o n s t u d i e s have su g g e s t e d t h a t some c e l l w a l l m a t r i x components a r e assembled i n the ER (Bowles and N o r t h c o t e 1972, 1974), the ER i s a minor component when compared t o the G o l g i , the predominant s i t e of c e l l w a l l m a t r i x s y n t h e s i s ( F i n c h e r and Stone 1981). In u l t r a r a p i d l y f r o z e n p r e p a r a t i o n s , the G o l g i c i s t e r n a e showed p o l a r i t y a c r o s s the s t a c k of the dicty o s o m e w i t h r e s p e c t t o c i s t e r n a e w i d t h and a l k a l i n e bismuth s t a i n i n g p r o p e r t i e s . The p o l a r i t y has been noted f o r a v a r i e t y of c h a r a c t e r i s t i c s , i . e . p h o s p h o t u n g s t i c a c i d - c h r o m i c a c i d s t a i n i n g , i n t e r c i s t e r n a l e l e m e n t s , and osmium s t a i n i n g (Robinson and K r i s t e n 1982, Shannon e t §_1. 1982). A model f o r m u l a t e d t o e x p l a i n s e n s i t i v i t y of the G o l g i to. i o n o p h o r e s s u g g e s t s a mechanism f o r m a i n t a i n i n g p o l a r i t y ( G r i f f i n g and Ray 1985). In t h i s model, p r o t o n t r a n s p o r t i n g ATPases i n the c i s t e r n a membrane would c r e a t e a pH g r a d i e n t ; the p r o t o n s would a s s o c i a t e w i t h i o n i z e d c a r b o x y l groups of the p e c t i n s i n the c i s t e r n a lumen. Osmotic e f f e c t s would then cause water t o l e a v e the lumen of the c i s t e r n a and f o r c e s e c r e t o r y p r o d u c t 78 out t o the p e r i p h e r a l v e s i c l e s . I t has been suggested t h a t t h e r e a r e two pathways where s o r t i n g of membrane components and s e c r e t o r y p r o d u c t s o c c u r s : the endosome pathway and the t r a n s G o l g i network (Van Deurs et a l . 1988). In p l a n t s and a n i m a l s m u l t i v e s i c u l a r b o d i e s a r e r e c o g n i z e d as endosomes (Tanchak and Fowke 1987, F r i e n d 1969). The m u l t i v e s i c u l a r b o d i e s were not s t a i n e d f o r p o l y s a c c h a r i d e s i n both c o n v e n t i o n a l l y f i x e d and u l t r a r a p i d l y f r o z e n L o b e l i a r o o t s . T h i s s u g g e s t s the e n d o c y t i c pathway and e x o c y t i c pathway a r e s e p a r a t e a t t h i s p o i n t . The endosome i n a n i m a l c e l l s has been d i v i d e d i n t o two m o r p h o l o g i c a l and f u n c t i o n a l compartments: the p e r i p h e r a l t u b u l a r network and the p e r i n u c l e a r m u l t i v e s i c u l a r b o d i e s ( M i l l e r e t a l . 1986). F u n c t i o n a l l y these c o r r e s p o n d t o e a r l y and l a t e endosomes. In the e a r l y s t a g e , endosomes s e r v e as compartments f o r u n c o u p l i n g r e c e p t o r s and l i g a n d s (CURL) (Geuze et a l . 1984, Schmid et a l . 1988). The l a t e endosome i s b e l i e v e d t o be i n v o l v e d i n t r a n s i t t o t h e lysosome ( i b i d . , G r i f f i t h s e t a l . 1988). The p a r t i a l l y c o a t e d r e t i c u l u m o b s e r v e d i n the u l t r a r a p i d l y f r o z e n L o b e l i a r o o t c e l l s i s m o r p h o l o g i c a l l y s i m i l a r t o the e a r l y endosome i n 79 a n i m a l s . F u r t h e r support t o the t h e o r y t h a t PCR may be an endosome, analogous t o t h o s e of a n i m a l s , comes from enzyme c y t o c h e m i s t r y . A c i d phosphatase, the marker enzyme f o r lysosomes, was not found i n the PCR i n h i g h e r p l a n t p r o t o p l a s t s , s u g g e s t i n g they a r e p r e l y s o s o m a l compartments (Record and G r i f f i n g 1988). Thus i n L o b e l i a r o o t t i p c e l l s the b i o s y n t h e t i c pathway f o r p o l y s a c c h a r i d e s i s v i a G o l g i , t r a n s G o l g i network, s e c r e t o r y v e s i c l e , and plasma membrane. O r g a n e l l e s t h a t d i d not s t a i n s t r o n g l y f o r p o l y s a c c h a r i d e s c o i n c i d e w i t h the pathway f o r e n d o c y t o s i s as o u t l i n e d i n Chapter 1. I f the p a r t i a l l y c o a t e d r e t i c u l u m does r e p r e s e n t an e a r l y endosome, then m i c r o t u b u l e s may p l a y a r o l e i n t r a n s p o r t i n g the endosome t h r o u g h the c y t o p l a s m as i t matures i n t o a m u l t i v e s i c u l a r body, as they do i n a n i m a l c e l l s ( M i l l e r e t a l . 1986). The f o l l o w i n g c h a p t e r examines the r o l e s m i c r o t u b u l e s p l a y i n e l o n g a t i n g c e l l s of L o b e l i a . 80 Chapter 3 MICROTUBULES IN ELONGATING ROOT CELLS OF LOBELIA ERINUS: EFFECT OF COLCHICINE ON ENDOCYTOSIS 81 INTRODUCTION The o b j e c t i v e of t h i s s t u d y was t o examine the m i c r o t u b u l e s i n e l o n g a t i n g r o o t c e l l s of L o b e l i a  e r i n u s . Immunofluorescence and TEM were used t o stu d y the c y t o s k e l e t o n , w i t h emphasis on the r o l e of the m i c r o t u b u l e s i n e n d o c y t o s i s d u r i n g c e l l w a l l s y n t h e s i s . The study of m i c r o t u b u l e s i n the d e p o s i t i o n of c e l l u l o s e m i c r o f i b r i l s has overshadowed o t h e r a s p e c t s of the r o l e of the c y t o s k e l e t o n i n c e l l w a l l s e c r e t i o n . There i s l i t t l e i n f o r m a t i o n on how v e s i c l e s a r e moved from the G o l g i t o the plasma membrane d u r i n g s e c r e t i o n , or from the plasma membrane t o i n t e r n a l endomembranes d u r i n g e n d o c y t o s i s . In t he a n i m a l systems i n g e n e r a l , v e s i c l e t r a n s p o r t has been shown t o be p r i m a r i l y m i c r o t u b u l e based ( K o l s e t e t a_l. 1979, Sheetz e_t a l . 1986). V e s i c l e t r a n s p o r t has been d i s s e c t e d i n v i t r o t o i s o l a t e the f o r c e g e n e r a t i n g p r o t e i n s , e.g. k i n e s i n (Schnapp et a l . 1985, V a l e 1987). In e l o n g a t i n g r o o t c e l l s t he m i c r o t u b u l e s a r e a prominent f e a t u r e of the c y t o s k e l e t o n . M i c r o t u b u l e s , f i r s t o b s e r v e d t r a n s v e r s e l y around the l o n g i t u d i n a l a x i s of the c e l l , were i n t e r p r e t e d as hoops of m i c r o t u b u l e s around the c e l l ( L e d b e t t e r 82 and P o r t e r 1963). Immunofluorescence t e c h n i q u e s have a l t e r e d t h i s v i e w : the a r r a y of m i c r o t u b u l e s i n e l o n g a t i n g c e l l s has been d e s c r i b e d as a " h e l i c a l a r r a y " ( L l o y d and S e a g u l l 1985). The t h e o r y t h a t m i c r o t u b u l e s a c t as gu i d e elements f o r the d e p o s i t i o n of c e l l u l o s e m i c r o f i b r i l s has r e c e i v e d s u p p o r t from a v a r i e t y of s o u r c e s (Heath 1974, S e a g u l l 1983, H e p l e r 1985). In g e n e r a l , the o r i e n t a t i o n of newly d e p o s i t e d c e l l u l o s e m i c r o f i b r i l s has been found t o be p a r a l l e l t o the m i c r o t u b u l e s u n d e r l y i n g the c e l l w a l l (Robinson e_t a l . 1 976). Treatment w i t h the drug c o l c h i c i n e d i s r u p t s c o r t i c a l m i c r o t u b u l e s , s u b s e q u e n t l y c e l l u l o s e m i c r o f i b r i l p a t t e r n becomes random ( P i c k e t t - H e a p s 1967). The development of secondary w a l l p a t t e r n s d u r i n g x y l o g e n e s i s has been c i t e d as "one of the be s t examples of the r e l a t i o n s h i p between m i c r o t u b u l e d i s p o s i t i o n and m i c r o f i b r i l o r i e n t a t i o n " ( F a l c o n e r and S e a g u l l 1985). The consequence of d e p o s i t i o n of c e l l u l o s e m i c r o f i b r i l s t r a n s v e r s e t o t h e l o n g a x i s of t h e c e l l i s the r e s t r i c t i o n of l a t e r a l e x p a n s i o n . The meristem c e l l s have s i d e s which a r e r o u g h l y s q u a r e , but which become r e c t a n g u l a r as the c e l l e l o n g a t e s . 83 The role of microtubules in t h i s morphogenic event i s i l l u s t r a t e d by the changes that occur when c e l l s are treated with ethylene (Roberts e_t a l . 1985). Ethylene treatment i n h i b i t s c e l l u l a r elongation and stimulates l a t e r a l expansion. Using immuno-fluorescence, the authors demonstated that the changes in c e l l shape were preceded by changes in microtubule orientation from a transverse to oblique pattern. The prominent microtubule network in the elongating c e l l appears to play important roles in the synthesis of the f i b r i l l a r component of the c e l l wall and in morphogenesis. It i s not known what role, i f any, t h i s network plays in endomembrane turnover during synthesis of the matrix component of the c e l l wall. Elongation i s accompanied by the addition of exocytotic secretory v e s i c l e s (see chapter 2), and the removal of membrane material by endocytotic vesicles (see chapter 1). The microtubules present in elongating c e l l s are good candidates for guiding v e s i c l e movement. However, microtubules are only one part of the complex cytoskeleton found in higher plant c e l l s which consists of microtubules, actin microfilaments (Palevitz 1987, McCurdy et a l . 1988), and intermediate filaments (Powell et a l . 84 1982, Dawson et a l . 1985). In addition, the microtrabecular network interconnects the elements of the cytoskeleton l i s t e d above and the c e l l u l a r organelles (Kobayashi et a l . 1988, Cox et a l . 1986). The microtubules which underlie the plasma membrane were observed amongst lanthanum l a b e l l e d endosomes (see chapter 1). Depending on the plane of sectioning, f i b r i l l a r connections can be observed between the endosome and microtubule. The microtubules are c l o s e l y packed along the cytoplasmic face of the membrane; the endocytotic ve s i c l e s may simply be migrating freely past t h i s region when they are fixed. A l t e r n a t i v e l y the mechanism of endocytotic v e s i c l e transport could be along microtubules. To distinguish between these p o s s i b i l i t i e s , elongating root c e l l s were treated with colchicine to disrupt the microtubules and the pattern of vesicular transport compared with normal c e l l s . Plant c e l l s are considerably less sensitive to co l c h i c i n e than animal c e l l s (Morejohn and Fosket 1986). In animal c e l l s , nanomolar amounts of co l c h i c i n e interfere with mitosis. Plant c e l l s require millimolar amounts to produce the same 85 e f f e c t . In t h i s work, immunofluorescence was used t o d e t e r m i n e the c o n c e n t r a t i o n of c o l c h i c i n e r e q u i r e d t o d i s r u p t the m i c r o t u b u l a r a r r a y i n L o b e l i a e r i n u s . T h i s c o l c h i c i n e l e v e l was then used i n c o n j u n c t i o n w i t h lanthanum as a marker f o r e n d o c y t o s i s . 86 METHODS AND MATERIALS  Immunofluorescence The b u f f e r f o r a l l immunofluorescence e x p e r i m e n t s was m i c r o t u b u l e s t a b i l i z i n g b u f f e r (MSB), c o n s i s t i n g of 50 mM PIPES, 5 mM EGTA, and 5 mM MgSO^ pH 7.2. 7 day o l d L o b e l i a e r i n u s s e e d l i n g s were f i x e d f o r 40 m i n u t e s , 20 m i n u t e s , or 10 minutes i n 4 % b u f f e r e d formaldehyde, f r e s h l y p r e p a r e d from p a r a f o r m a l d e h y d e . A f t e r 3 washes over 15 minutes the c e l l w a l l was p a r t i a l l y d i g e s t e d f o r 20 minutes i n 1 % c e l l u l a s e (Sigma C-7377 from A s p e r g i l l u s n i q e r ) , and 0.5 % p e c t i n a s e (Sigma P-2401 from Rhizopus s p e c i e s ) . P o l y - l - l y s i n e (PLL) c o a t e d c o v e r s l i p s were p r e p a r e d f o l l o w i n g the proc e d u r e of F i s h e r (1982). Square #1 c o v e r s l i p s were s o n i c a t e d i n hot soapy water f o r 5 m i n u t e s , f o l l o w e d by a r i n s e i n d i s t i l l e d w ater. The c o v e r s l i p s were immersed i n hot chromic a c i d - s u l f u r i c a c i d f o r 1-2 hours a t 70° C, then r i n s e d a g a i n i n d i s t i l l e d w a t e r . A drop of one hundred m i c r o l i t r e s of 2.5 mg/ml PLL was l a y e r e d on the c o v e r s l i p and a l l o w e d t o s t a n d f o r 5 m i n u t e s . I f the c o v e r s l i p s were a t a l l d i r t y , t h e r e would be h y d r o p h o b i c r e p u l s i o n , and n e i t h e r the PLL nor the c e l l s would adhere t o the 87 c o v e r s l i p . A f t e r 5 m i n u t e s , the PLL was d r a i n e d o f f the s l i d e and i t was g i v e n a q u i c k wash i n d i s t i l l e d w ater. The c o v e r s l i p s were a i r d r i e d and used w i t h i n 1 hour. The enzyme d i g e s t e d r o o t t i p s were d i s s e c t e d away from the s e e d l i n g s under a d i s s e c t i n g m i c r o s c o p e . About 30 r o o t t i p s would be p l a c e d i n a drop of b u f f e r on the PLL c o a t e d c o v e r s l i p . A c l e a n uncoated c o v e r s l i p was used t o squash the r o o t t i p s and the squashes a l l o w e d t o s e t t l e on the PLL f o r 1-2 h o u r s , i n a humid environment. The c e l l s were made permeable t o a n t i b o d i e s u s i n g e i t h e r 1 % T r i t o n - X - 1 0 0 f o r 1 hour or by p l u n g i n g the c o v e r s l i p s i n t o 50 % methanol a t -10° C f o r 10 mi n u t e s . Non s p e c i f i c p r o t e i n b i n d i n g s i t e s were b l o c k e d w i t h a " p r e i n c u b a t i o n " of 1 % BSA f o r 10-20 m i n u t e s . Aldehydes were reduced w i t h 3 washes of 3 minutes each i n 1 mg/ml sodium or p o t a s s i u m b o r o h y d r i d e . A f t e r 3 washes over 15 m i n u t e s , the c e l l s were ready f o r a n t i b o d y t r e a t m e n t . S t a i n i n g chambers were made by a t t a c h i n g s t r i p s of c o v e r s l i p g l a s s t o a l i g h t m i c r o s c o p e s l i d e w i t h s i l i c o n e s e a l a n t . The c o v e r s l i p w i t h the c e l l s was i n v e r t e d over the s t r i p s , s u s p ending 88 the c e l l i n t o 100 m i c r o l i t r e s of a n t i b o d y s o l u t i o n . The b e n e f i t of t h i s system i s t h a t s m a l l volumes of a n t i b o d y a r e s u f f i c i e n t t o e v e n l y b a t h the c e l l s . The s t a i n i n g chambers were s e t i n a humid environment and m a i n t a i n e d a t 37° C. The p r i m a r y a n t i b o d y was a n t i - t u b u l i n , r a i s e d i n r a b b i t a g a i n s t c h i c k f i b r o b l a s t t u b u l i n ( p o l y c l o n a l , Sigma T3536); the a n t i b o d y was d i l u t e d from i t s s t o c k s o l u t i o n 1:40 or 1:50 i n 50 mM sodium phosphate b u f f e r . The r o o t c e l l s were i n c u b a t e d w i t h the a n t i - t u b u l i n f o r 30 minutes or 1 hour i n the s t a i n i n g chamber as d e s c r i b e d above. Exce s s a n t i b o d y was removed w i t h 3 washes i n b u f f e r of 10 minutes each. The s l i d e s were d r a i n e d of b u f f e r and s t a i n e d w i t h th e secondary a n t i b o d y , a n t i - r a b b i t IgG c o n j u g a t e d t o f l u o r o s c e i n i s o t h i o c y a n a t e (FITC) (Sigma F0382), d i l u t e d 1:50 i n 50 mM sodium phosphate b u f f e r . T h i s was the l e v e l a t which s t a i n i n g w i t h secondary a n t i b o d y a l o n e d i d not produce s i g n i f i c a n t f l u o r e s c e n c e . A n t i - r a b b i t IgG c o n j u g a t e d t o rhodamine was used i n p r e l i m i n a r y e x p e r i m e n t s but the l e v e l of background s t a i n i n g was u n a c c e p t a b l y h i g h . A f t e r a f i n a l s e r i e s of 3 t e r m i n a l washes b u f f e r of 10 m i nutes each, the c o v e r s l i p s were mounted i n 90 % 89 g l y c e r o l and viewed w i t h a L e i t z D i a l u x microscope f i t t e d w i t h e p i f l u o r e s c e n c e i l l u m i n a t i o n . A l l f l u o r e s c e n c e photographs were taken w i t h a s t a n d a r d 2 minute exposure t o a l l o w comparison between t r e a t m e n t ( p r i m a r y and secondary a n t i b o d y ) and c o n t r o l s (no p r i m a r y a n t i b o d y ; secondary a n t i b o d y o n l y ) . Roots were t r e a t e d w i t h 10 mM c o l c h i c i n e f o r 2 hours a t room t e m p e r a t u r e and f i x e d i m m e d i a t e l y i n 4 % formaldehyde. The samples were then p r o c e s s e d f o r immunofluorescence as o u t l i n e d above a l o n g s i d e c o n t r o l (no c o l c h i c i n e ) r o o t s . R e s u l t s from 5 immunofluorescence e x p e r i m e n t s were a n a l y z e d u s i n g the Kontron Image P r o c e s s i n g System ( I P S ) . N e g a t i v e s were i l l u m i n a t e d on a l i g h t t a b l e ; the image of the n e g a t i v e was r e c o r d e d w i t h a v i d e o camera which was o p e r a t e d on l i n e w i t h the IPS. The image was d i g i t i z e d , and the c e l l s were e d i t e d out of the background. The i n t e g r a t e d o p t i c a l d e n s i t y , which d e s c r i b e s the g r e y l e v e l s of the a r e a of the c e l l , was measured f o r c e l l s p hotographed a t t h e same m a g n i f i c a t i o n (315X) and exposure (2 m i n u t e s ) . S t a n d a r d b l a c k and w h i t e i e v e l s were s e t , t a k i n g i n t o account the e f f e c t of s t r a y l i g h t . A b s o l u t e numbers between e x p e r i m e n t s were v a r i a b l e but t h e amount of 90 f l u o r e s c e n c e i n samples t r e a t e d w i t h a n t i t u b u l i n c o u l d be compared t o the amount of f l u o r e s c e n c e i n c o n t r o l samples (no a n t i t u b u l i n ) . When f l u o r e s c e n c e w i t h i n e x p e r i m e n t s was e x p r e s s e d as a p e r c e n t a g e of the c o n t r o l f l u o r e s c e n c e , the between experiment v a r i a t i o n was s m a l l . T r a n s m i s s i o n E l e c t r o n M i c r o s c o p y Seven day o l d s e e d l i n g s were p r e t r e a t e d w i t h 10 mM c o l c h i c i n e , f o r 2 hours at room t e m p e r a t u r e . 5 mM lanthanum n i t r a t e was added t o the 10 mM c o l c h i c i n e s o l u t i o n t o t e s t f o r e n d o c y t o s i s a f t e r m i c r o t u b u l e d i s r u p t i o n . P r e t r e a t e d s e e d l i n g s were i n c u b a t e d i n the l a n t h a n u m / c o l c h i c i n e f o r 1 hour. As c o n t r o l s , s e e d l i n g s were t r e a t e d w i t h d i s t i l l e d d e i o n i z e d w a t e r , lanthanum o n l y , or c o l c h i c i n e o n l y . Samples were f i x e d w i t h o u t r i n s i n g i n d i l u t e K a r n o v s k y ' s f i x a t i v e (1.5 % formaldehyde, 2 % g l u t a r a l d e h y d e i n 50 mM PIPES b u f f e r pH 7.4) f o r 1 hour. A f t e r 3 r i n s e s of 10 minutes each i n b u f f e r , t h e samples were p o s t f i x e d i n 1 % OsO^, o v e r n i g h t a t 4 0 C. D e h y d r a t i o n i n methanol was f o l l o w e d by i n f i l t r a t i o n and embedding i n epon r e s i n . S i l v e r s e c t i o n s were c u t on the R e i c h e r t - J u n g U l t r a c u t E 91 and s t a i n e d w i t h m e t h a n o l i c u r a n y l a c e t a t e f o r 25 m i n u t e s . G r i d s were examined and photographed under the Z e i s s TEM 10C. 92 RESULTS The e l o n g a t i n g L o b e l i a e r i n u s r o o t c e l l s show the c h a r a c t e r i s t i c t r a n s v e r s e m i c r o t u b u l e a r r a y s u n d e r l y i n g the plasma membrane of d i f f e r e n t i a t i n g h i g h e r p l a n t c e l l s ( F i g . 3 9 ) . T h i s arrangement of m i c r o t u b u l e s was obse r v e d i n b o t h lanthanum t r e a t e d ( F i g . 46) and u n t r e a t e d c o n t r o l c e l l s ( F i g s . 39, 40). V e s i c l e s w i t h and w i t h o u t lanthanum l a b e l were o f t e n a s s o c i a t e d w i t h m i c r o t u b u l e s i n the c o r t i c a l c y t o p l a s m ( F i g . 46, see a l s o c h a p t e r 1 ) . The m i c r o t u b u l e s a r e a r r a n g e d i n p a r a l l e l s t r a n d s p e r p e n d i c u l a r t o the a x i s of e l o n g a t i o n . In t h i s c e l l t y p e , groups of 2-3 m i c r o t u b u l e s were more common than s i n g l e m i c r o t u b u l e s ( F i g . 4 0 ) . The b i o c h e m i c a l i d e n t i t y of the s e h o l l o w r o d elements of the c y t o s k e l e t o n as m i c r o t u b u l e s was c o n f i r m e d by immunofluorescence. I n c u b a t i o n i n p r i m a r y a n t i b o d y s p e c i f i c f o r t u b u l i n r e s u l t e d i n b r i g h t c y t o p l a s m i c f l u o r e s c e n c e ( F i g . 4 3 ) . There was low f l u o r e s c e n c e i n the n u c l e u s and c e l l w a l l s . Fragments of c e l l s adhered t o the p o l y - l - l y s i n e c o a t e d c o v e r s l i p s and showed b r i g h t f l u o r e s c e n c e , i n d i c a t i n g the f l u o r e s c e n c e was a s s o c i a t e d w i t h the c o r t i c a l c y t o p l a s m , i . e . f l u o r e s c e n c e was not r e l e a s e d by opening the c e l l . I n d i v i d u a l f i b r i l s 93 u s u a l l y observed i n such immunofluorescence e x p e r i m e n t s , were not c l e a r . The b r i g h t f l u o r e s c e n c e i n c e l l s t r e a t e d w i t h the p r i m a r y a n t i b o d y , a n t i - t u b u l i n , was compared w i t h low f l u o r e s c e n c e i n c o n t r o l c e l l s which were t r e a t e d w i t h secondary a n t i b o d y o n l y ( F i g s . 43, 44). The b r i g h t n e s s of the f l u o r e s c e n c e was measured by the parameter of i n t e g r a t e d o p t i c a l d e n s i t y (IOD) on the Kon t r o n I P S; b r i g h t f l u o r e s c e n c e g i v e s a lower i n t e g r a t e d o p t i c a l d e n s i t y ( F i g . 5 0 ) . For c e l l s t r e a t e d w i t h a n t i - t u b u l i n , the i n t e g r a t e d o p t i c a l d e n s i t y was 33 + 2.3 % (n=3 r e p l i c a t e e x p e r i m e n t s ) of c o n t r o l c e l l s . The a b s o l u t e b r i g h t n e s s of the c e l l s v a r i e d between r e p l i c a t e s , but when b r i g h t n e s s was e x p r e s s e d as a p e r c e n t a g e of the c o n t r o l , t he r e l a t i v e b r i g h t n e s s d i d not v a r y between r e p l i c a t e s . When c e l l s were t r e a t e d w i t h 10 mM or 100 mM c o l c h i c i n e , the p a t t e r n of f l u o r e s c e n c e changed ( F i g . 4 5 ) . The p e r i p h e r a l c y t o p l a s m was no l o n g e r b r i g h t l y f l u o r e s c e n t , and the n u c l e a r f l u o r e s c e n c e i n c r e a s e d . The p e r i n u c l e a r zone appeared as a b r i g h t band around the n u c l e u s . The p e r i p h e r a l c y t o p l a s m had uneven f l u o r e s c e n c e . When the amount of f l u o r e s c e n c e i n th e s e c e l l s was q u a n t i f i e d , the 94 i n t e g r a t e d o p t i c a l d e n s i t y i n c e l l s t r e a t e d w i t h c o l c h i c i n e and a n t i - t u b u l i n was 43 + 17 % of c o n t r o l s . T h i s i n d i c a t e s t h a t the f l u o r e s c e n c e was dimmer i n these c e l l s than i n c e l l s not t r e a t e d w i t h c o l c h i c i n e ; t h e f l u o r e s c e n c e i s more v a r i a b l e as w e l l . S i n c e 10 mM and 100 mM c o n c e n t r a t i o n of c o l c h i c i n e gave s i m i l a r r e s u l t s , t h e lower c o n c e n t r a t i o n was used i n a l l subsequent TEM e x p e r i m e n t s . A f t e r 2 hour i n c u b a t i o n i n 10 mM c o l c h i c i n e , the o r g a n e l l e s of the e l o n g a t i n g r o o t c e l l s showed minor u l t r a s t r u c t u r a l changes (compare c o n t r o l F i g . 41 w i t h F i g . 4 2 ) . The n u c l e i i n c o n t r o l c e l l s were s p h e r i c a l i n shape, whereas i n c o l c h i c i n e t r e a t e d c e l l s t he n u c l e i become i r r e g u l a r i n shape. The plasma membrane of c o l c h i c i n e t r e a t e d c e l l s u n d u l a t e d and showed many more lomasomes and b l e b s ( F i g . 4 2 ) . T a n g e n t i a l s e c t i o n s t h r o u g h t h e plasma membrane and c o r t i c a l c y t o p l a s m d i s p l a y e d the normal t r a n s v e r s e m i c r o t u b u l e a r r a y s ( F i g . 4 6 ) . A f t e r c o l c h i c i n e t r e a t m e n t , t h e s e a r r a y s a r e almost e n t i r e l y absent ( F i g . 4 7 ) . O c c a s i o n a l l y a m i c r o t u b u l e would s u r v i v e t h e c o l c h i c i n e t r e a t m e n t ; t h e s e remnants were always seen i m m e d i a t e l y below the plasma membrane, never deeper i n the c y t o p l a s m 95 than 50 nm from the membrane. The remnants were u s u a l l y d i r e c t l y c o n n e c t e d t o the plasma membrane by a t h i n arm ( d a t a not shown). When c e l l s were p r e t r e a t e d w i t h c o l c h i c i n e , f o l l o w e d by an i n c u b a t i o n i n lanthanum and c o l c h i c i n e , the e f f e c t s of m i c r o t u b u l e d i s r u p t i o n on e n d o c y t o s i s can be seen. There were fewer lanthanum l a b e l l e d v e s i c l e s i n the c e l l s t r e a t e d w i t h c o l c h i c i n e p l u s lanthanum than i n c e l l s t r e a t e d w i t h lanthanum a l o n e . The m u l t i v e s i c u l a r b o d i e s i n c o l c h i c i n e t r e a t e d c e l l s were not l a b e l l e d as o f t e n as were the m u l t i v e s i c u l a r b o d i e s i n c o n t r o l c e l l s (compare c o n t r o l F i g . 48 w i t h F i g . 4 9 ) . The d i c t y o s o m e s appeared normal a f t e r c o l c h i c i n e t r e a t m e n t . The i n t e r c i s t e r n a l s p a c i n g was s i m i l a r t o the d i c t y o s o m e s i n c o n t r o l c e l l s , and a m u l t i v e s i c u l a r body was o f t e n o b s e r v e d near by ( F i g . 4 6 ) . 96 Figs. 39-42: Microtubules in elongating c e l l s . F i g . 39: Plasma membranes of adjacent c e l l s joined by plasmodesmata. Microtubules l i n e cytoplasmic face of plasma membrane (arrow). F i g . 40: Tangential section through the c e l l wall, plasma membrane, and c o r t i c a l cytoplasm. Microtubules appear in groups (arrow). F i g . 41: No colchicine control c e l l s have spherical n u c l e i , smooth membranes. Fi g . 42: Colchicine treated c e l l s have irr e g u l a r nucleus, wavy membranes. cw=cell wall, p=plasmodesmata, pm=plasma membrane, m=mitochondrion, v=vesicle, pv=provacuole, Nu=nucleus. 98 F i g s . 43-45: A n t i - t u b u l i n Immunofluorescence. F i g . 43: C e l l s t r e a t e d w i t h a n t i - t u b u l i n p r i m a r y a n t i b o d y ; a n t i t u b u l i n d e t e c t e d w i t h secondary a n t i b o d y - F I T C . F l u o r e s c e n c e b r i g h t i n c y t o p l a s m , dim i n n u c l e u s and c e l l w a l l . F i g . 44: C e l l s not t r e a t e d w i t h a n t i - t u b u l i n , j u s t secondary a n t i b o d y - F I T C , c o n t r o l f o r n o n s p e c i f i c b i n d i n g . F i g . 45: C o l c h i c i n e i n c u b a t i o n b e f o r e f i x a t i o n , a n t i - t u b u l i n p r i m a r y a n t i b o d y , secondary a n t i b o d y - F I T C . F l u o r e s c e n c e i n n u c l e a r p e r i p h e r y b r i g h t , w h i l e c o r t i c a l c y t o p l a s m f l u o r e s c e n c e p a t c h y . A l l bars=5 urn A l l t r e a t m e n t s photographed f o r s t a n d a r d t i m e s and c o n d i t i o n s . 4 ® 4 4 100 F i g s . 46-49: E f f e c t of c o l c h i c i n e on e n d o c y t o s i s i n e l o n g a t i n g r o o t c e l l s . F i g . 46: C e l l w a l l and v e s i c l e s l a b e l l e d w i t h lanthanum o n l y , no c o l c h i c i n e . T a n g e n t i a l s e c t i o n t h r o u g h c e l l w a l l , plasma membrane, c o r t i c a l c y t o p l a s m shows m i c r o t u b u l e a r r a y c h a r a c t e r i s t i c of e l o n g a t i n g c e l l s . Arrow i n d i c a t e s l a b e l l e d e n d o c y t o t i c v e s i c l e . F i g . 47: Region of c o r t i c a l c y t o p l a s m a f t e r c o l c h i c i n e t r e a t m e n t . Wavy plasma membrane; no m i c r o t u b u l e s p r e s e n t ; p a t t e r n p e r s i s t s i n c e l l u l o s e m i c r o f i b r i l s i n c e l l w a l l (open a r r o w ) . F i g . 48: M u l t i v e s i c u l a r body-dictyosome a s s o c i a t i o n i n c o l c h i c i n e t r e a t e d c e l l s . MVB u n l a b e l l e d . F i g . 49: M u l t i v e s i c u l a r body i n no c o l c h i c i n e , lanthanum o n l y t r e a t e d c e l l . Arrow i n d i c a t e s lanthanum d e p o s i t s . A l l bars=0.1 urn 102 F i g . 50: Q u a n t i f i c a t i o n of a n t i - t u b u l i n immunofluorescence. I n t e g r a t e d o p t i c a l d e n s i t y (IOD) measures b r i g h t n e s s of f l u o r e s c e n c e , i . e . b r i g h t e r f l u o r e s c e n c e i s l e s s d e n s i t y . "+Ab" i n d i c a t e s t r e a t e d w i t h a n t i - t u b u l i n p r i m a r y a n t i b o d y . "-Ab" i n d i c a t e s no p r i m a r y a n t i b o d y , t r e a t e d w i t h secondary a n t i b o d y - F I T C o n l y . " + c o l " i n d i c a t e s t r e a t e d w i t h 10 mM c o l c h i c i n e . Microtubule Immunofluorescence antitubulin/colchicine 104 DISCUSSION M i c r o t u b u l e s p l a y an i m p o r t a n t r o l e i n the maintenance of normal c e l l s t r u c t u r e i n e l o n g a t i n g L o b e l i a r o o t c e l l s . They can be c o n s i d e r e d t o be a s t r u c t u r a l framework, m a i n t a i n i n g c e l l shape. D i s r u p t i o n of t h i s c y t o s k e l e t a l element w i t h c o l c h i c i n e l e d t o the r e d i s t r i b u t i o n of immunofluorescence p a t t e r n s and changes i n e n d o c y t o s i s , such as fewer lanthanum l a b e l l e d v e s i c l e s and l e s s m u l t i v e s i c u l a r body l a b e l l i n g . Whole L o b e l i a e r i n u s r o o t c e l l s showed b r i g h t f l u o r e s c e n c e when t r e a t e d w i t h p o l y c l o n a l a n t i t u b u l i n a n t i b o d i e s , however, the b r i g h t f l u o r e s c e n t s t r a n d s of m i c r o t u b u l e p a t t e r n were not c l e a r . The s p e c i a l p r o p e r t i e s of the p l a n t c e l l make the o b s e r v a t i o n of i n d i v i d u a l s t r a n d s p r o b l e m a t i c . The t h i c k n e s s of the c e l l , the presence of the c e l l w a l l , and the d e n s i t y of c l o s e l y packed c o r t i c a l m i c r o t u b u l e s can a l l c o n t r i b u t e t o obscure the s t r i a t i o n i n the image (Van der V a l k e t a l . 1980, Wick e t a l . 1981, Simmonds e t a l . 1983). In the f i r s t r e p o r t of m i c r o t u b u l e immunofluorescence i n i n t a c t h i g h e r p l a n t c e l l s , i t was noted t h a t " t h i c k n e s s of an i n t a c t c e l l can make r e s o l u t i o n of i n d i v i d u a l 105 b u n d l e s d i f f i c u l t . . . w h e r e m i c r o t u b u l e d e n s i t y i s low enough t h i n f l u o r e s c e n t f i b r e s , a lmost c e r t a i n l y i n d i v i d u a l m i c r o t u b u l e s , can be t r a c e d , as i n the case of w e l l s p r e a d a n i m a l c e l l s " (Wick e t a l . 1981). The h i g h m i c r o t u b u l e d e n s i t y , s m a l l s i z e , and b o x l i k e shape of the L o b e l i a r o o t c e l l s a r e i m p o r t a n t c o n s i d e r a t i o n s when i n t e r p r e t i n g t h i s t y p e of ex p e r i m e n t . Immunofluorescence of c r y o s e c t i o n s can be a good a l t e r n a t i v e t o whole mounts (Sakaguchi et a l . 1988). The c r y o t e c h n i q u e would have the added b e n e f i t of b y p a s s i n g the p r o c e s s i n g s t e p s of c e l l w a l l d i g e s t i o n , membrane p e r m e a b i l i z i n g by T r i t o n or methanol, and s q u a s h i n g on PLL c o a t e d c o v e r s l i p s . The f l u o r e s c e n c e observed i n the L o b e l i a r o o t c e l l s appears o n l y i n c e l l s t r e a t e d w i t h the s p e c i f i c p r i m a r y a n t i b o d y . C o n t r o l samples i n c u b a t e d i n b u f f e r i n s t e a d of p r i m a r y a n t i b o d y d i d not show s i g n i f i c a n t f l u o r e s c e n c e when i n c u b a t e d w i t h secondary a n t i b o d y - F I T C . I t i s p o s s i b l e t h a t t h e b r i g h t f l u o r e s c e n c e i s the r e s u l t of p r i m a r y a n t i b o d y b i n d i n g n o n s p e c i f i c a l l y t o c y t o p l a s m i c components. However, t h e d r a s t i c changes i n the f l u o r e s c e n c e p a t t e r n s of c e l l s t r e a t e d 106 c o l c h i c i n e s u g gests f l u o r e s c e n c e i s the r e s u l t of s p e c i f i c p r i m a r y a n t i b o d y b i n d i n g t o m i c r o t u b u l e s . In a d d i t i o n , the c e l l s were p r e t r e a t e d w i t h b o v i n e serum albumin b e f o r e a n t i b o d y i n c u b a t i o n t o s a t u r a t e n o n s p e c i f i c p r o t e i n b i n d i n g s i t e s . The change i n m i c r o t u b u l e immunofluorescence p a t t e r n s i n c o l c h i c i n e t r e a t e d c e l l s was s t r i k i n g when compared t o the u l t r a s t r u c t u r e of c o l c h i c i n e t r e a t e d c e l l s viewed under the TEM. The f l u o r e s c e n c e p a t t e r n was dim and p a t c h y i n the c o r t i c a l c y t o p l a s m i n c o l c h i c i n e t r e a t e d compared t o u n t r e a t e d c e l l s . T h i s change i n p a t t e r n seemed t o r e f l e c t t r u e d i f f e r e n c e s i n the m i c r o t u b u l e s because w i t h the e x c e p t i o n of the d i s a s s e m b l y of m i c r o t u b u l e s , the c o r t i c a l c y t o p l a s m i t s e l f was not r a d i c a l l y changed when viewed under t h e TEM. The membrane p r o f i l e s a r e wavy, but t h e r e were no g r o s s changes t h a t c o u l d account f o r the change i n the immunofluorescence p a t t e r n . The endomembranes i n v o l v e d i n e n d o c y t o s i s , as o u t l i n e d i n c h a p t e r 1, showed d e c r e a s e d l a b e l l i n g a f t e r t r e a t m e n t w i t h c o l c h i c i n e . There was no pronounced g a t h e r i n g of e n d o c y t o t i c v e s i c l e s near the plasma membrane, as e x p e c t e d i f the v e s i c l e s were budding o f f t h e membrane but not b e i n g 107 t r a n s p o r t e d . I n s t e a d , the d i s r u p t i o n of m i c r o t u b u l e s l e d t o fewer v e s i c l e s i n the c o r t i c a l c y t o p l a s m and the membrane had a wavy appearance. T h i s c o u l d be the r e s u l t of fewer v e s i c l e s budding o f f the plasma membrane. I t has been noted t h a t e n d o c y t o s i s i n p l a n t s o c c u r s i n a h i g h t u r g o r environment (Cram 1980, Gradmann and Robinson 1989), and t h i s i s e s p e c i a l l y t r u e of e l o n g a t i n g c e l l s . M i c r o t u b u l e s may a c t as s u p p o r t beams upon which m i c r o t u b u l e a s s o c i a t e d p r o t e i n s (MAPs) would b r a c e t o move v e s i c l e s i n t o the c o r t i c a l c y t o p l a s m . A f t e r d i s r u p t i o n of m i c r o t u b u l e s by c o l c h i c i n e , t h i s mechanism f o r budding would no l o n g e r be a c t i v e . There a r e good p r e c e d e n t s f o r a microtubule-MAP based v e s i c l e t r a n s p o r t system. MAPs a c t as m i c r o t u b u l e motors t o t r a n s p o r t membranous o r g a n e l l e s i n s q u i d g i a n t axon, b o v i n e b r a i n , and amoeba (Hirokawa e t a l . 1989). D i f f e r e n t motors move v e s i c l e s i n o p p o s i t e d i r e c t i o n s a l o n g t h e p o l a r m i c r o t u b u l e : k i n e s i n moves v e s i c l e s towards the p l u s end (Brady e t a l . 1982, V a l e e t a l . 1985), w h i l e d y n e i n (MAP 1C) moves v e s i c l e s towards the minus end ( P a s c h e l e t a l . 1987, Gibbons 1988). The t h r e e d i m e n s i o n a l s t r u c t u r e of k i n e s i n 108 has been described in quick freeze, deep etch studies of reconstituted microtubules, kinesin, and latex beads (Hirokawa et §_1. 1989). The kinesin head, which has ATPase a c t i v i t y , attaches to the microtubule; the kinesin foot attaches to the latex bead or v e s i c l e (Yang et a_l. 1989). The low l e v e l of multivesicular body l a b e l l i n g in c o l c h i c i n e treated c e l l s i s consistent with reports from the animal l i t e r a t u r e , where colchic i n e inhibited uptake of ligand and transfer of ligand to the lysosome (Kolset et a_l. 1979). In the root c e l l s , the decrease in multivesicular body label may be considered morphological evidence of in h i b i t e d transfer of membranes between internal organelles. The lack of vesi c l e s in the c o r t i c a l cytoplasm, together with the diminished multivesicular body l a b e l , support the theory that microtubules are important in endocytosis during elongation in root c e l l s . It i s d i f f i c u l t to chemically dissect one component of the cytoskeleton without e f f e c t i n g the other interacting components. The a c t i n microfilament system i s found in the c o r t i c a l cytoplasm with microtubules (Parthasarathy 1985, Seagull et a l . 1987, McCurdy et a l . 1988); general 109 d i s r u p t i o n of t h i s m i c r o e n v i r o n m e n t w i t h c o l c h i c i n e may be p r o d u c i n g i n d i r e c t e f f e c t s on the m i c r o f i l a m e n t s . To d i s r u p t m i c r o f i l a m e n t s , c e l l s can be t r e a t e d w i t h l e a d , c y t o c h a l a s i n B, or c y t o c h a l a s i n D. There a r e a v a r i e t y of changes seen i n the t r e a t e d c e l l s : c y t o p l a s m i c s t r e a m i n g i s h a l t e d i n mature v a c u o l a t e c e l l s , v e s i c l e s a r e a c c u mulated around the d i c t y o s o m e , and G o l g i c i s t e r n a e d i l a t e (Yamagoshi and Nagai 1981, Shannon e t a l . 1984, Vaughn and Vaughn 1987). None of t h e s e o r g a n e l l e changes were p r e s e n t i n c o l c h i c i n e t r e a t e d L o b e l i a r o o t c e l l s , which s u g g e s t s the i n t e g r i t y of the m i c r o f i l a m e n t system was not s i g n i f i c a n t l y d i s r u p t e d . There were few v e s i c l e s around the G o l g i , u n l i k e the r e p o r t e d d i l a t e d or fragmented G o l g i a r e a i n c y t o c h a l a s i n t r e a t e d c e l l s ( M o l l e n h a u e r and Morre 1976). Dictyosome s t r u c t u r e was not a l t e r e d by c o l c h i c i n e t r e a t m e n t i n L o b e l i a , or i n the maize r o o t cap ( i b i d ) . M i c r o f i l a m e n t s have been shown t o be i m p o r t a n t i n s e c r e t i o n of s l i m e i n r o o t cap c e l l s (Morre and M o l l e n h a u e r 1976, Shannon e t a l . 1984) and s e c r e t i o n of c e l l w a l l i n p o l l e n tubes of T r a d e s c a n t i a ( P i c t o n and S t e e r 1981). I t i s 110 d i f f i c u l t t o v i s u a l i z e m i c r o f i l a m e n t s i n t h i n s e c t i o n s because they a r e o n l y 6 nm i n d i a m e t e r ( i n c o m p a r i s o n , m i c r o t u b u l e s a r e 25 nm i n d i a m e t e r ) . In s e c t i o n s of L o b e l i a r o o t c e l l s , m i c r o f i l a m e n t s were r a r e l y o b s e r v e d u n l i k e m i c r o t u b u l e s which were abundant. In c o n c l u s i o n , t h e d i s r u p t i o n of m i c r o t u b u l e s w i t h c o l c h i c i n e produced changes i n lanthanum l a b e l p a t t e r n s i n d i c a t i n g e n d o c y t o s i s was p e r t u r b e d . I t i s not c l e a r from t h i s p r e l i m i n a r y s t u d y i f t h i s i s a d i r e c t e f f e c t on a microtubule-MAP v e s i c l e t r a n s l o c a t i o n mechanism or an i n d i r e c t e f f e c t of d i s r u p t i n g t h i s i m p o r t a n t c y t o s k e l e t a l element of the e l o n g a t i n g c e l l . 111 Chapter 4: PRIMARY CELL WALL POROSITY IN ROOT TIPS OF LOBELIA ERINUS 112 INTRODUCTION The composition of the e x t r a c e l l u l a r medium bathing the higher plant c e l l i s determined by the physical and chemical barrier of i t s c e l l wall. During endocytosis, solutes and solution from the e x t r a c e l l u l a r medium are i n t e r n a l i z e d . This interaction between the c e l l and i t s environment i s limited to molecules and p a r t i c l e s that can cross the c e l l wall. The environment of the apoplast determines what can be taken up by endocytosis. The objective of t h i s study was to determine the size of the p a r t i c l e s capable of d i f f u s i n g through the pores of the c e l l wall, and to explore what components of the c e l l wall define the pore s i z e . Morphologically the c e l l wall of the epidermal and c o r t i c a l c e l l s has an outer pectic layer and an inner c e l l u l o s i c layer ( S e t t e r f i e l d and Bayley 1957, Foster 1982). There are several l i n e s of evidence for t h i s layered structure: l i g h t microscope cytochemistry, fluorescence (Miki et a l . 1980), TEM cytochemistry, (Moore and Staehelin 1988) , and TEM apoplast tracer studies (Owen et a l . 1988, Crowdy and Tanton 1970). The dicot primary c e l l wall, l i k e many e x t r a c e l l u l a r matrices, i s composed of a fibrous network in an 113 embedding m a t r i x . In h i g h e r p l a n t s i n g e n e r a l , the f i b r o u s network i s made up of the c r y s t a l l i n e c e l l u l o s e m i c r o f i b r i l s cemented t o g e t h e r w i t h a m a t r i x of p e c t i n s , h e m i c e l l u l o s e s , and p r o t e i n s ( M c N e i l e t a l . 1984). In d i c o t s , t h e predominant p e c t i n s a r e homogalacturons, rhamnogalacturon I and I I ; the main h e m i c e l l u l o s e i s x y l o g l u c a n (Varner and L i n 1989). A l t h o u g h the c o v a l e n t and i o n i c i n t e r a c t i o n s between the components of the c e l l w a l l a r e not w e l l u n d e r s t o o d , a w o r k i n g model of a network of c e l l u l o s e m i c r o f i b r i l s c o a t e d w i t h x y l o g l u c a n i n a m a t r i x of p e c t i n s and p r o t e i n s ( K e e g s t r a e_t a l . 1973, P r e s t o n 1974) w i l l be assumed h e r e . The p r o t e i n s of the c e l l w a l l a r e enzymes as w e l l as s t r u c t u r a l p r o t e i n s (Cassab and V a r n e r 1988). I t has been s u g g e s t e d t h a t bonds between s t r u c t u r a l p r o t e i n s and p o l y s a c c h a r i d e s "might d e t e r m i n e the p o t e n t i a l pore s i z e of an i n t e r m o l e c u l a r l y c r o s s l i n k e d e x t e n s i n network" (Cooper e t a l . 1987). The pore s i z e s of h i g h e r p l a n t c e l l w a l l s have been d e t e r m i n e d u s i n g s e v e r a l d i f f e r e n t m e t h o d o l o g i e s . The a b i l i t y of a compound t o cause p l a s m o l y s i s or c y t o r r h y s i s was used as an a s s a y of the compounds a b i l i t y t o c r o s s the c e l l w a l l of 1 14 c u l t u r e d Acer p s e u d o p l a t a n u s ; t h e l i m i t i n g d i a m e t e r of p o r e s was r e p o r t e d t o be 3-4 nm ( C a r p i t a e t a l . 1979). C e l l w a l l s of P h a s e o l u s  v u l g a r i s L. s e e d l i n g s were ground, f r o z e n thawed, and packed i n t o a g e l f i l t r a t i o n chromatography column; the e x c l u s i o n of p r o t e i n s of m o l e c u l a r weight g r e a t e r than 60 000 d a l t o n s was t a k e n as e v i d e n c e t h a t the pore s i z e i s about 3 nm ( T e p f e r and T a y l o r 1981a). The passage of f l u o r e s c e i n a t e d d e x t r a n s and p r o t e i n s i n t o the c e l l w a l l and p e r i p l a s m of c u l t u r e d , p l a s m o l y z e d G l y c i n e max c e l l s showed u n h i n d e r e d p o r o s i t y of 3.3 nm and a r e s t r i c t e d p o r o s i t y of 6.6-8.6 nm ( B a r o n - E p e l e t a l . 1988). In t h i s s t u d y , e l e c t r o n dense markers of a range of s i z e s were used t o d e t e r m i n e t h e pore s i z e i n the i n t a c t p r i m a r y r o o t of L o b e l i a e r i n u s . 115 METHODS AND MATERIALS L o b e l i a e r i n u s s e e d l i n g s were g e r m i n a t e d on f i l t e r paper w e t t e d w i t h d i s t i l l e d d e i o n i z e d w a t e r . A f t e r 7 days, the s e e d l i n g s were immersed i n the a p o p l a s t t r a c e r s o l u t i o n s f o r 2 or 12 h o u r s . The t r a c e r s were c o l l o i d a l g o l d o r 5 mM lanthanum n i t r a t e s o l u t i o n s . For d e t a i l s of lanthanum t e c h n i q u e , see c h a p t e r 1. C o l l o i d a l g o l d was p r e p a r e d by the method of S l o t and Geuze (1985), u s i n g 5 ml of 1 % t a n n i c a c i d , 5 ml 25 mM r^CO^, 4 ml 1 % sodium c i t r a t e , 6 ml d e i o n i z e d d i s t i l l e d water t o reduce the c h l o r o a u r i c a c i d s o l u t i o n c o n s i s t i n g of 1 ml 1 % c h l o r o a u r i c a c i d and 89 ml d e i o n i z e d d i s t i l l e d w a t e r . The s o l u t i o n was b o i l e d under r e f l u x f o r 1 hour and a l l o w e d t o c o o l t o room t e m p e r a t u r e . C o l l o i d a l g o l d p a r t i c l e s i z e was measured u s i n g the K o n t r o n Image P r o c e s s i n g System (IPS) and the Z e i s s 1OC t r a n s m i s s i o n e l e c t r o n m i c r o s c o p e . The m i c r o s c o p e was c a l i b r a t e d by measuring the c r y s t a l l a t t i c e s p a c i n g of n e g a t i v e l y s t a i n e d c a t a l a s e c r y s t a l s . An a l i q u o t of g o l d s o l u t i o n was l a y e r e d on formvar c o a t e d g r i d s , e x c e s s was b l o t t e d o f f , and t h e g r i d s were a i r d r i e d . The c o l l o i d a l g o l d p a r t i c l e s i z e was measured as the maximum di a m e t e r of each p a r t i c l e . 116 EGTA t r e a t m e n t c o n s i s t e d of immersing s e e d l i n g s i n m i c r o t u b u l e s t a b i l i z i n g b u f f e r (50 mM PIPES pH 6.9, 5 mM M g 2 ( S 0 4 ) , and 5 mM EGTA) f o r 1 hour, f o l l o w e d by 12 hours i n 3-5 nm c o l l o i d a l g o l d s o l u t i o n . P a r t i a l c e l l w a l l d i g e s t i o n s were c a r r i e d out on 7 day s e e d l i n g s f o r 15 minutes a t room temp e r a t u r e i n the f o l l o w i n g enzyme s o l u t i o n s . ( i ) P e c t i n a s e (Sigma P-2401, E.C. 3.2.1.15, from R h i z o p u s s p e c i e s ) was d i s s o l v e d i n 50 mM PIPES b u f f e r pH 7.4 t o make a s t o c k s o l u t i o n of 10 mg/ml. T h i s was f u r t h e r d i l u t e d t o 1 mg/ml, 0.1 mg/ml, 0.025, 0.05 mg/ml, and 0.01 mg/ml. ( i i ) P r o t e a s e (Sigma P-5005, type V, from Streptomyces q r i s e u s ) , and ( i i i ) c e l l u l a s e (Sigma C-7377, E.C. 3.2.1.4, from A s p e r g i l l u s n i q e r ) were d i s s o l v e d i n PIPES a t s t o c k c o n c e n t r a t i o n s of 10 mg/ml, and f u r t h e r d i l u t e d t o 1 mg/ml. A f t e r 15 m i n u t e s , the s e e d l i n g s were r i n s e d i n b u f f e r 3 t i m e s 5 minutes and then i n c u b a t e d i n c o l l o i d a l g o l d s o l u t i o n f o r 12 h o u r s . At the end of the g o l d i n c u b a t i o n , samples were f i x e d i n d i l u t e K a r n o v s k y ' s f i x a t i v e (1.5 % formaldehyde, 2 % g l u t a r a l d e h y d e i n 50 mM PIPES pH 7.4) f o r 1 hour. A f t e r 3 r i n s e s of 5 minutes each, 1 17 p o s t f i x a t i o n i n 1 % OsO^ was c a r r i e d out o v e r n i g h t a t 4 0 C. S e e d l i n g s were d e h y d r a t e d i n methanol and embedded i n epon. S i l v e r s e c t i o n s were c u t on t h e R e i c h e r t - J u n g OMU3 or U l t r a c u t E. Three r o o t s were trimmed and s e c t i o n e d f o r each p e c t i n a s e c o n c e n t r a t i o n f o r 2 r e p l i c a t e f i x a t i o n s . The s e c t i o n s were p i c k e d up on uncoated copper g r i d s and examined w i t h o u t p o s t s t a i n i n g . F o r each r o o t , the d i a m e t e r of the c o l l o i d a l g o l d p a r t i c l e s i n the e p i d e r m a l e l o n g a t i n g c e l l s was sampled i n a t l e a s t 5 d i f f e r e n t c e l l s . The d i a m e t e r of t h e g o l d p a r t i c l e s i n r o o t s e c t i o n s was measured on the Ko n t r o n I PS, as o u t l i n e d above, f o r t h e d e t e r m i n a t i o n of g o l d p a r t i c l e s i z e . The mean maximum d i a m e t e r of g o l d p a r t i c l e p e n e t r a t i n g the c e l l w a l l f o r each p e c t i n a s e c o n c e n t r a t i o n was p l o t t e d . L i n e a r r e g r e s s i o n a n a l y s i s was used t o t e s t the n u l l h y p o t h e s i s t h a t probe s i z e was independent of p e c t i n a s e c o n c e n t r a t i o n . F r o z e n h y d r a t e d samples were p r e p a r e d and viewed i n the B i o l o g i c a l S c i e n c e s E l e c t r o n M i c r o s c o p y L a b o r a t o r y a t the U n i v e r s i t y of C a l i f o r n i a a t B e r k e l e y . Seven day L o b e l i a e r i n u s s e e d l i n g s were mounted on the SEM s t u b and f r o z e n i n l i q u i d n i t r o g e n s l u s h . The s t u b was 118 t r a n s f e r r e d a t l i q u i d n i t r o g e n t e m p e r a t u r e t o the c o l d s tage of t h e SEM, where a c o n t r o l l e d r i s e i n temperature t o -110 ° C was used t o s u b l i m e the o u t e r l a y e r s of i c e c o v e r i n g the sample. The s t u b was t r a n s f e r r e d t o a c o l d s tage i n the c o a t i n g chamber of the SEM and s p u t t e r c o a t e d w i t h g o l d . The r o o t s were r e t u r n e d t o t h e c o l d s t a g e of the SEM, viewed and photographed a t 20 kv a c c e l e r a t i n g v o l t a g e . 119 RESULTS The pore s i z e of the p r i m a r y c e l l w a l l of L o b e l i a e r i n u s was d e t e r m i n e d u s i n g e l e c t r o n dense a p o p l a s t markers. Lanthanum f r e e l y d i f f u s e s a c r o s s the c e l l w a l l (see c h a p t e r s 1 and 5 ) , s u g g e s t i n g the p o r o s i t y of the c e l l w a l l i s a t l e a s t 0.8 nm. C o l l o i d a l g o l d p a r t i c l e s of s i z e range 2 nm-5 nm d i d not c r o s s the i n t a c t c e l l w a l l . The o n l y a r e a where c o l l o i d a l g o l d was found w i t h i n the r o o t t i p was a s c a t t e r i n g of p a r t i c l e s i n t h e m i d d l e l a m e l l a e of the e p i d e r m a l c e l l s . T h i s appears t o be a s i z e r e s t r i c t i o n and not the consequence of c h a r g e , because when c o l l o i d a l g o l d p a r t i c l e s were c o a t e d w i t h amino a c i d s of d i f f e r e n t c h a r g e s ( l y s i n e , p o s i t i v e c h a r g e ; g l y c i n e , n e u t r a l ; a s p a r t i c a c i d , n e g a t i v e c h a r g e ) , the c o l l o i d a l g o l d was s t i l l e x c l u d e d ( d a t a not shown). The e x t e r i o r c u t i c l e l a y e r of the i n t a c t r o o t a c t s as a b a r r i e r t o m a t e r i a l e n t e r i n g t h e c e l l w a l l . In a r e a s where t h i s l a y e r was d i s r u p t e d , a s m a l l amount of c o l l o i d a l g o l d were o b s e r v e d w i t h i n the p e c t i c l a y e r of the c e l l w a l l . T h i s e x t e r i o r l a y e r was not s t a i n e d f o r p o l y s a c c h a r i d e s w i t h a l k a l i n e b i smuth ( F i g . 5 4 ) . To s t u d y which components of the c e l l w a l l a r e 120 important in c e l l wall porosity, enzyme digestions were performed. Treatment of roots with 10 mg/ml ce l l u l a s e or protease had no eff e c t on the porosity of the c e l l wall (Fig. 51, 53). In contrast, treatment of roots with pectinase concentrations of 10 mg/ml and less produced extensive changes in c e l l wall ultrastructure and porosity (Fig. 53). The pectic c e l l . w a l l layer became less coherent and less dense. C o l l o i d a l gold probes were found throughout the c e l l wall. The root c e l l s were completely disrupted by pectinase treatment. This could be observed by diminished fluoroscein diacetate staining, increased Evan's Blue staining (data not shown), and by the dr a s t i c a l t e r a t i o n in ult r a s t r u c t u r e . This disruption occurred whether or not the roots were pretreated with plasmolyzing medium. A range of concentrations of pectinase were tested for i t s a b i l i t y to change porosity, from 10 ug/ml to 10 000 ug/ml (10 mg/ml), see Table 5. The eff e c t of the enzyme varied between c e l l types. For example, in roots treated with 50 ug/ml pectinase, in the elongation zone there was gold p a r t i c l e s in the c e l l wall; in mature c e l l s there was no gold in the c e l l wall. Even within one c e l l 121 type, the response to pectinase treatment was variable. After treatment with 100 ug/ml pectinase, the gold was always seen in the pectic layer of elongating c e l l s but the gold did not always penetrate the c e l l u l o s i c layer (Fig. 55). The size of the c o l l o i d a l gold p a r t i c l e s in the c e l l wall after pectinase treatment was measured using the Kontron IPS. The gold p a r t i c l e s that had penetrated furthest into the wall were measured for each treatment. According to linear regression analysis, a s i g n i f i c a n t increase in the pore size i s observed with increasing concentrations of pectinase (Fig. 59). The e f f e c t of the calcium chelator EGTA was similar to the e f f e c t of low concentrations of pectinase treatment: the pectic layer became di f f u s e , the porosity of the wall in t h i s layer increased, and the gold did not penetrate the c e l l u l o s i c layer (Fig. 56) . The root c e l l s had distended endoplasmic reticulum a f t e r EGTA treatment, but in general were less disrupted than the pectinase treated. The external surface of the root in the conventional preparations was delineated by the non-polysaccharide c u t i c l e layer and some fine 122 wisps of polysaccharide (Fig. 55). In samples prepared using cryotechniques, the mucilage on the surface of the root looks very d i f f e r e n t . Whole frozen hydrated roots were viewed under the SEM with a cold stage (Fig. 57); the r e l a t i o n s h i p between the surface of the root hairs and i t s environment was observed (Fig. 58). The extensive network of f i b r i l s radiating from the c e l l wall (Fig. 58) was also present in freeze substituted material (see chapter 5). In summary, the c e l l wall of Lobelia erinus consists of several layers of varying composition and porosity. The exterior of the c e l l wall i s connected to the surrounding medium by a network of f i b r i l s , as observed on frozen samples under the SEM. The most exterior layer of the c e l l wall proper i s a non-polysaccharide layer which r e s t r i c t s access of gold p a r t i c l e s but not lanthanum. This exterior layer covers the pectic layer which i s e a s i l y disrupted by low concentrations of pectinase enzyme or EGTA, but not c e l l u l a s e or protease. Beneath the pectic layer i s the c e l l u l o s e layer which forms another barrier layer, stopping c o l l o i d a l gold p a r t i c l e s from reaching the c e l l i f they penetrate the pectic 123 l a y e r . T h i s c e l l u l o s i c l a y e r i s o n l y d i s r u p t e d by h i g h p e c t i n a s e c o n c e n t r a t i o n s . The most i n t e r n a l l a y e r i s the plasma membrane s u r f a c e c o a t (see c h a p t e r 2 ) . 124 T a b l e 5 E f f e c t of p e c t i n a s e t r e a t m e n t on c e l l w a l l p o r o s i t y [ p e c t i n a s e ] ug/ml 10 c o l l o i d a l g o l d c e l l w a l l p o r o s i t y probe g o l d does not e n t e r w a l l e x c e p t where b r e a k s i n c u t i c l e l a y e r o c c u r 25 g o l d e n t e r s p e c t i c l a y e r c o m p l e t e l y , e x c l u d e d from c e l l u l o s i c l a y e r i n some c a s e s 50 g o l d e n t e r s p e c t i c l a y e r c o m p l e t e l y , e x c l u d e d from c e l l u l o s i c l a y e r i n some c a s e s 100 g o l d c r o s s e s p e c t i c l a y e r , e n t e r s c e l l u l o s i c l a y e r i n most c a s e s 1000 g o l d c r o s s e s p e c t i c l a y e r , c r o s s e s c e l l u l o s i c l a y e r c o m p l e t e l y 125 F i g s . 51-53: I n f l u e n c e of w a l l components on c e l l w a l l p o r o s i t y . A l l s e c t i o n s , no p o s t s t a i n . F i g . 51: 10 mg/ml c e l l u l a s e t r e a t m e n t , f o l l o w e d by 12 hours i n 3-5 nm c o l l o i d a l g o l d s o l u t i o n . G o l d p e n e t r a t i o n of w a l l e x t r e m e l y l i m i t e d . Note some p e n e t r a t i o n a t break i n c u t i c l e l a y e r ( a r r o w ) . 52: 0*1 mg/ml p e c t i n a s e t r e a t m e n t , f o l l o w e d by 12 hours i n 3-5 nm c o l l o i d a l g o l d s o l u t i o n . G o l d c r o s s e s p e c t i c l a y e r of c e l l w a l l , and t o l e s s e r e x t e n t c r o s s e s c e l l u l o s i c l a y e r t o p e n e t r a t e t o c e l l lumen. F i g . 53: 10 mg/ml p r o t e a s e t r e a t m e n t f o l l o w e d by 12 hours i n 3-5 nm c o l l o i d a l g o l d s o l u t i o n . R e s u l t s s i m i l a r . t o no enzyme c o n t r o l s . p l = p e c t i c l a y e r , c l = c e l l u l o s i c l a y e r , c y=cytoplasm, arrowhead i n d i c a t e s c o l l o i d a l g o l d p a r t i c l e s , arrow i n d i c a t e s e x t e r i o r c u t i c l e l a y e r . 127 F i g s . 54-56: C e l l w a l l u l t r a s t r u c t u r e . F i g . 54: c e l l w a l l s t a i n e d w i t h a l k a l i n e b ismuth f o r p o l y s a c c h a r i d e s . C u t i c l e l a y e r does not r e a c t . Root t r e a t e d w i t h 10 ug/ml p e c t i n a s e , f o l l o w e d by 12 hours i n 3-5 nm c o l l o i d a l g o l d s o l u t i o n . G o l d does not e n t e r w a l l where e x t e r i o r l a y e r i n t a c t . F i g . 55: c e l l w a l l l a y e r s a f t e r d i g e s t i o n w i t h 1.0 mg/ml p e c t i n a s e , f o l l o w e d by 12 hours i n 3-5 nm c o l l o i d a l g o l d s o l u t i o n . G o l d i s seen i n p e c t i c l a y e r but not i n c e l l u l o s i c l a y e r . C y t o p l a s m of c e l l s d i s r u p t e d . P o s t s t a i n e d w i t h u r a n y l a c e t a t e / l e a d c i t r a t e . F i g . 56: 5 mM EGTA t r e a t m e n t f o l l o w e d by 12 hours i n 3-5 nm c o l l o i d a l g o l d . G o l d e n t e r s p e c t i c , but not c e l l u l o s i c l a y e r s of c e l l w a l l . ER lumen d i s t e n d e d . P o s t s t a i n e d w i t h u r a n y l a c e t a t e / l e a d c i t r a t e . p l = p e c t i c l a y e r , c l = c e l l u l o s i c l a y e r , er=endoplasmic r e t i c u l u m , arrowhead i n d i c a t e s c o l l o i d a l g o l d p a r t i c l e s , arrow i n d i c a t e s c u t i c l e c e l l w a l l l a y e r . 129 F i g s . 57-58: f r o z e n h y d r a t e d samples of L o b e l i a  e r i n u s , viewed on c o l d s t a g e of SEM. F i g . 57: Whole f r o z e n h y d r a t e d r o o t , showing i c e s u r r o u n d i n g r o o t h a i r s ; .root t i p e p i d e r m a l s u r f a c e s l i g h t l y f r e e z e - d r i e d . Mature e p i d e r m a l c e l l s can be seen where i c e has s u b l i m e d away. F i g . 58: Root h a i r s embedded i n i c e . F i b r i l s r a d i a t i n g out from s u r f a c e of r o o t . r t = r o o t t i p , rh=root h a i r , me=mature e p i d e r m a l c e l l s . 131 Fig. 59: Effect of pectinase on cell wall porosity. Treatment with varying concentrations of pectinase allowed different sizes of colloidal gold probe to enter cell wall. effect of pectinase on wall porosity DISCUSSION 133 The e x t r a c e l l u l a r medium f o r a h i g h e r p l a n t c e l l c o n s i s t s of the c e l l w a l l components and the m o l e c u l e s c a p a b l e of d i f f u s i n g a c r o s s the c e l l w a l l . Based on the pore s i z e of 1-2 nm i n i n t a c t c e l l s and 2-3 nm i n p e c t i n a s e t r e a t e d c e l l s , p r e d i c a t i o n s can be made about what t y p e s of m o l e c u l e s may be e n c o u n t e r e d by the e l o n g a t i n g L o b e l i a e r i n u s r o o t c e l l s . M o l e c u l e s such as i o n s and s i m p l e s u g a r s would d i f f u s e f r e e l y t h r o u g h t h e w a l l , p e n e t r a t i n g t h r o u g h o u t the a p o p l a s t as lanthanum does. In c o n t r a s t , l a r g e p r o t e i n s and p a r t i c l e s , e x c l u d e d from t h e c e l l w a l l , would not come i n d i r e c t c o n t a c t w i t h the p l a n t c e l l s u r f a c e . T h i s i s an i m p o r t a n t c o n s i d e r a t i o n f o r a s s a y s of e n d o c y t o s i s and a n t i b o d y s t u d i e s on i n t a c t h i g h e r p l a n t t i s s u e s . I t i s d i f f i c u l t t o r e c o n c i l e a homogenous w a l l w i t h p o r o s i t y of 1-3 nm w i t h p r o c e s s e s l i k e A g r o b a c t e r i u m i n f e c t i o n , movement of o l i g o s a c c h a r i d e f r a g m e n t s , and changes i n shape and s i z e . The heterogenous c o m p o s i t i o n of the c e l l w a l l p r o v i d e s a range of s u b s t r a t e s f o r m i c r o b i a l enzymes such as p e c t i n a s e , p r o t e a s e and c e l l u l a s e ( C o l l m e r and Keen 1986). I t seems l i k e l y t h a t i n the s o i l t he c o n d i t i o n , p o r o s i t y , and 134 u l t r a s t r u c t u r e of the e p i d e r m a l c e l l w a l l s would be i n f l u e n c e d by the nearby d i f f e r e n t i a t e d c e l l s , s o i l b a c t e r i a , and m y c o r h i z z a e . In a d d i t i o n , the a b r a s i o n of the r o o t s u r f a c e by s o i l p a r t i c l e s d u r i n g growth c o u l d a l t e r p o r o s i t y i n m i c r o e n v i r o n m e n t s around the r o o t t i p . I t i s u s e f u l t o r e l a t e c o n d i t i o n s t h a t o c c u r i n s o i l w i t h t h e i_n v i t r o e x p e r i m e n t s on c e l l w a l l p o r o s i t y . The new approach of t h i s s t u d y was t o r e l a t e p o r o s i t y t o the c o n d i t i o n s which may o c c u r i n s o i l such as the presence of m i c r o b i a l enzymes. T h i s approach i n c l u d e d t h e use of ( i ) i n t a c t t i s s u e s , i . e . not c u l t u r e d c e l l s ( B a r o n - E p e l e t a l . 1988) and ( i i ) i n t a c t c e l l w a l l , i . e . not c e l l w a l l fragments ( T e p f e r and T a y l o r 1981a). The v a r i e d c e l l w a l l p o r o s i t i e s of d i f f e r e n t i a t e d c e l l t y p e s ( e l o n g a t i n g and v a c u o l a t e ; e l o n g a t i n g and r o o t cap) obse r v e d e a r l i e r i n t h i s s t u d y i s analogous t o the d i f f e r e n c e s i n w a l l p o r o s i t y o b s e r v e d e a r l i e r by C a r p i t a e_t a l . ( 1979) i n e l o n g a t i n g c e l l s and r o o t h a i r s of Raphanus s a t i v u s . S i m i l a r l y , d i s t i n c t l y d i f f e r e n t w a l l p o r o s i t i e s have been r e p o r t e d i n the b r o m e l i a d B r o c c h i n i a r e d u c t a , where lanthanum p e n e t r a t e d the c e l l w a l l of the t r i c h o m e and 135 surrounding mesophyll c e l l s but not the wall of the epidermal c e l l s (Owen e_t ajL. 1988). These differences between c e l l types further r e f l e c t the heterogenous nature of the c e l l wall. The mucilage layer covering the c e l l wall proper of the epidermal c e l l s and root hairs does not present a porosity b a r r i e r to the probes tested here. There are two d i s t i n c t i v e types of root mucilage: root cap mucilage and epidermal mucilage (Miki et a l . 1980, Ray et a l . 1988). It i s generally very d i f f i c u l t to preserve t h i s c e l l wall layer in TEM preparations (Chaboud and Rougier 1986, Foster 1982). Using cryotechniques the mucilage can be seen radiating from the root surface. The functions of t h i s type of mucilage are believed to be ion exchange and conditioning of the s o i l to allow root to push through the s o i l during growth (Ray et a l . 1988). The d i r e c t contact between these "rhizoplane f i b r i l s " and s o i l p a r t i c l e s may allow d i r e c t transfer of cations from the s o i l p a r t i c l e to the root (Leppard and Ramamoorthy 1975). In the elongating Lobelia erinus c e l l s , the entire c e l l wall i s covered by a thin, nonpolysaccharide layer of c u t i c l e . A similar 136 s t r u c t u r e , "a d e f i n i t e c u t i c l e - l i k e membrane...near the s u r f a c e of the g e l " was r e p o r t e d i n f i e l d grown p l a n t s such as c l o v e r and wheat ( F o s t e r 1982). The pre s e n c e of a c u t i c l e i n r o o t s has been noted i n s e v e r a l c e l l t y p e s ( S c o t t e t a_l. 1958, Chaboud and R o u g i e r 1986). The c u t i c l e may a c t as b a r r i e r t o e n t r y t o the r o o t c e l l w a l l : i n many c a s e s , c o l l o i d a l g o l d o n l y p e n e t r a t e d the c e l l w a l l a t br e a k s i n the c u t i c l e . B r e a k s i n the c u t i c l e may occ u r where the r o o t i s i n c o n t a c t w i t h s o i l p a r t i c l e s ( F o s t e r 1982). The c u t i c l e l i e s over the g e l of the o u t e r p e c t i c c e l l w a l l l a y e r . The p o r o s i t y of the p e c t i c l a y e r was a l t e r e d by t r e a t m e n t w i t h p e c t i n a s e and EGTA, but not c e l l u l a s e and p r o t e a s e . A l t e r a t i o n i n c e l l w a l l p o r o s i t y by p e c t i n a s e t r e a t m e n t was a l s o r e p o r t e d r e c e n t l y i n s u s p e n s i o n c u l t u r e d G l y c i n e max c e l l s , u s i n g a f l u o r e s c e n c e m i c r o s c o p y a s s a y ( B a r o n - E p e l e t a l . 1988). However p r o t e a s e , c e l l u l a s e , and EGTA d i d not change p o r o s i t y i n thos e c e l l s . The EGTA d i s r u p t i o n i n t h i s s t u d y was l i m i t e d t o the p e c t i c c e l l w a l l l a y e r ; the p o r o s i t y of the c e l l u l o s i c l a y e r d i d not change. The p e c t i c l a y e r i s r i c h i n c a r b o x y l g roups, which can be c r o s s l i n k e d by c a l c i u m b r i d g e s ( T e p f e r 137 and T a y l o r 1981b). T h i s i o n i c bonding seems t o c o n t r i b u t e t o m a i n t a i n i n g a t i g h t l y c r o s s l i n k e d m a t r i x , because d i s r u p t i o n of the b r i d g e s w i t h EGTA l e d t o i n c r e a s e d pore d i a m e t e r . Based on the f l u o r e s c e n c e s t u d i e s of B a r o n - E p e l et a_l. ( 1988) and the p e c t i n a s e c o l l o i d a l g o l d a s s a y s p r e s e n t e d h e r e , i t appears t h a t p e c t i n i s i m p o r t a n t i n m a i n t a i n i n g c e l l w a l l p o r o s i t y . The g e l f o r m i n g p r o p e r t i e s of p e c t i n s c o u l d be e s s e n t i a l t o c r e a t i n g t h i s m o l e c u l a r network. An aqueous g e l can be c o n s i d e r e d the sum of t h r e e p a r t s : 1. j u n c t i o n zones where polymers j o i n 2. i n t e r j u n c t i o n segments where the polymers a r e m o b i l e , a l t h o u g h l e s s m o b i l e than i n f r e e s o l u t i o n 3. water e n c l o s e d by t h i s network ( J a r v i s 1984). In t h e c e l l w a l l the j u n c t i o n would be formed by b l o c k s of unbranched g a l a c t u r o n a n s , c r o s s l i n k e d by c a l c i u m ; the i n t e r j u n c t i o n would be b l o c k s of e s t e r i f i e d g a l a c t u r o n a n s which a r e u n a b le t o b i n d c a l c i u m ( i b i d ) . I f the i n t e r j u n c t i o n zones a r e c o n s i d e r e d p o r e s , t h e i r w a l l s would be l e s s h i g h l y c h a r g e d than the j u n c t i o n zones. The pore s i z e c o u l d v a r y by removal of c a l c i u m from the j u n c t i o n 138 zones by EGTA. The p o s i t i o n of the p e c t i c l a y e r on the o u t e r c e l l w a l l makes i t the f i r s t l i n e of d e f e n s e a g a i n s t pathogens ( C o l l m e r and Keen 1986). Fragments of the c e l l w a l l , o l i g o s a c c h a r i n s , a c t as s i g n a l l i n g m o l e c u l e s when p l a n t s a r e a t t a c k e d by some pathogens, and p e c t i c f r a c t i o n s a r e amongst the most a c t i v e o l i g o s a c c h a r i n s ( D a r v i l l e t a l . 1985, Ryan 1987). I t was s t r i k i n g i n t h i s s t u d y how even low c o n c e n t r a t i o n of p e c t i n a s e l e d t o c e l l d e a t h ; s i m i l a r p h y t o t o x i c e f f e c t s f o l l o w i n g p e c t i n a s e t r e a t m e n t have been r e p o r t e d (Basham and Bateman 1975, Hahne and L o r z 1988). I t has been s u g g e s t e d on t h e o r e t i c a l grounds t h a t a p r o t e i n network of e x t e n s i n may mediate c e l l w a l l p o r o s i t y (Cooper e t a l _ . 1987). Treatment of r o o t s w i t h p r o t e a s e d i d not change the p o r o s i t y i n t h e s e e x p e r i m e n t s , which s u g g e s t s p r o t e i n s a r e not an i m p o r t a n t f a c t o r i n p o r o s i t y . E x t e n s i n s have been l o c a l i z e d t h r o u g h o u t the c e l l w a l l w i t h the e x c e p t i o n of the m i d d l e l a m e l l a ( S t a f s t r o m and S t a e h e l i n 1988). The m i d d l e l a m e l l a between c e l l s i s c o n t i n u o u s w i t h the o u t e r p e c t i c l a y e r of the e p i d e r m a l c e l l s . The l e v e l of s u b s t r a t e may have been t o o low i n the o u t e r e p i d e r m a l c e l l w a l l l a y e r 139 t o change the p o r o s i t y s i g n i f i c a n t l y . The c e l l u l o s e r i c h r e g i o n of the c e l l w a l l i s i n t e r i o r t o t h e p e c t i c l a y e r as w e l l , which may be why the c e l l u l a s e t r e a t m e n t does not i n f l u e n c e p o r o s i t y . The c e l l u l o s i c l a y e r s u r r o u n d i n g the p r o t o p l a s t a c t e d as a b a r r i e r t o the passage of c o l l o i d a l g o l d p r o b e s , a l t h o u g h i t was d i s r u p t e d by h i g h e r c o n c e n t r a t i o n of p e c t i n a s e . T h i s s u g g e s t s t h a t p e c t i n s form p a r t of t h e network s u r r o u n d i n g the c e l l u l o s e m i c r o f i b r i l s , i n a d d i t i o n t o the x y l o g l u c a n s (Hayashi et a_l. 1987, Moore and S t a e h e l i n 1988). In c o n c l u s i o n , c u t i n and p e c t i n a r e the most i m p o r t a n t components i n c e l l w a l l p o r o s i t y i n i n t a c t L o b e l i a e r i n u s r o o t s , when compared t o c e l l u l o s e and p r o t e i n s . The pore s i z e of the c e l l w a l l would s t r i c t l y l i m i t t h e m o l e c u l e s t h a t may i n t e r a c t w i t h t h e r o o t c e l l , but t h i s p o r o s i t y i s p l a s t i c depending on exposure t o f a c t o r s which m o d i f y p e c t i n . 140 Chapter 5 : A RE-EXAMINATION OF ENDOCYTOSIS USING ULTRA RAPID FREEZING AND FREEZE SUBSTITUTION 141 INTRODUCTION E n d o c y t o s i s o c c u r s on a t i m e s c a l e of m i l l i s e c o n d s over d i s t a n c e s of m i c r o m e t e r s . T r a n s m i s s i o n e l e c t r o n m i c r o s c o p y (TEM) must be used t o s t u d y t h e s e s u b c e l l u l a r s t r u c t u r e s , but the c o n v e n t i o n a l p r e p a r a t i v e t e c h n i q u e s used i n TEM a r e p o o r l y s u i t e d f o r dynamic e v e n t s . The s i z e s c a l e of e n d o c y t o s i s r e q u i r e s TEM, but the time s c a l e of e n d o c y t o s i s makes c o n v e n t i o n a l f i x a t i o n t e c h n i q u e s i n a p p r o p r i a t e . An a l t e r n a t i v e t o c o n v e n t i o n a l t e c h n i q u e s i s f i x a t i o n by u l t r a r a p i d f r e e z i n g and f r e e z e s u b s t i t u t i o n . . There a r e l i m i t a t i o n s a s s o c i a t e d w i t h c o n v e n t i o n a l TEM f i x a t i o n t e c h n i q u e s ( G i l k e y and S t a e h e l i n 1986, Menco 1986, S i t t e e t a l . 1987, K n o l l e t a l . 1987). The most s e r i o u s of t h e s e l i m i t a t i o n s f o r e n d o c y t o s i s i s the slowness of the f i x a t i v e p e n e t r a t i o n and c r o s s r e a c t i o n which may l e a d t o v e s i c l e and o r g a n e l l e rearrangements d u r i n g f i x a t i o n . The c h e m i c a l s used i n f i x a t i o n must d i f f u s e i n t o t h e c e l l s b e f o r e c r o s s l i n k i n g r e a c t i o n s may b e g i n ; t h i s time p e r i o d may be s e v e r a l seconds t o minutes (Mersey and M c C u l l y 1978, B a j e r and M o l e - B a j e r 1969). In a d d i t i o n , not a l l m o l e c u l e s a r e c r o s s l i n k e d by any one f i x a t i v e , 142 e.g. l i p i d s a r e p o o r l y f i x e d by a l d e h y d e s but r e a c t w e l l w i t h osmium t e t r a o x i d e (Hayat 1981). T h i s i s r e l e v a n t i n the study of e n d o c y t o s i s because the time l a g between f i x a t i o n of the membrane p r o t e i n s w i t h a l d e h y d e and the f i x a t i o n of membrane l i p i d s w i t h osmium may be as l o n g as 1 t o 2 h o u r s . D u r i n g t h i s t i me p e r i o d , l i p a s e s may s t i l l be a c t i v e and membrane b l e b b i n g can o c c u r (Wetzel and Scow 1980, Hasty and Hays 1978). Aldehyde f i x a t i o n i s accompanied by r e l e a s e and r e d i s t r i b u t i o n of i o n s + 2 + such as H and Ca which may produce a r t i f a c t s ( H a l l and Gupta 1984, Z i e r o l d and S t e i n b r e c h t 1987). These problems can be a v o i d e d by u l t r a r a p i d f r e e z i n g , which i m m o b i l i z e s m o l e c u l e s and o r g a n e l l e s by s o l i d i f y i n g the s o l v e n t s u r r o u n d i n g them. The time f o r f i x a t i o n changes from seconds t o m i l l i s e c o n d s : f o r example, a t i s s u e i s f r o z e n t o a depth of 10 urn w i t h i n 0.5 m i l l i s e c o n d s (Jones 1984). U l t r a r a p i d f r e e z i n g t e c h n i q u e s a r e based on the p r o p e r t i e s of w a ter, one of the most n e g l e c t e d of the c e l l c o n s t i t u e n t s . Water p l a y s i n t e g r a l s t r u c t u r a l ( h y d r a t i o n s h e l l s ) and f u n c t i o n a l ( s o l v e n t and r e a c t a n t ) r o l e s i n c e l l s . 143 When water f r e e z e s , i t i s a two s t e p p r o c e s s : f i r s t t he n u c l e a t i o n of embryo i c e c r y s t a l s o c c u r s , then t h e i c e c r y s t a l s expand and grow. In b i o l o g i c a l m a t e r i a l , n u c l e a t i o n of i c e c r y s t a l s i s "homogenous n u c l e a t i o n " because n u c l e i a r e c l u s t e r s of pure water; i n "heterogenous n u c l e a t i o n " , f o r e i g n p a r t i c l e s or s u r f a c e s a c t as n u c l e i around which c r y s t a l s grow (Mazur 1970, Robards and S l e y t r 1985). The s i z e of the i c e c r y s t a l s formed depends p r i m a r i l y on the r a t e of c o o l i n g , but sample geometry and s o l u t e c o n c e n t r a t i o n a r e a l s o i m p o r t a n t ( P l a t t n e r and Bachmann 1982). The q u a l i t y of u l t r a r a p i d f r e e z i n g i s de t e r m i n e d by thes e " c r y s t a l l i z a t i o n k i n e t i c s . . . n u c l e a t i o n f r e q u e n c y and c r y s t a l growth r a t e " (Bachmann and Mayer 1987). I f the r a t e of c o o l i n g i s too slow, l a r g e i c e c r y s t a l s ( s e v e r a l m i c r o n s i n d i a m e t e r ) ar e formed. These c r y s t a l s have a c h a r a c t e r i s t i c l a t t i c e s t r u c t u r e of hexagonal u n i t s , thus t h i s common form of f r o z e n water i s c a l l e d hexagonal i c e (Robards and S l e y t r 1985). Hexagonal i c e s i g n i f i c a n t l y d i s r u p t s u l t r a s t r u c t u r e due t o the l a r g e s i z e of i t s c r y s t a l s . Slow c o o l i n g can a l s o r e s u l t i n the s e g r e g a t i o n of s o l u t i o n or c y t o p l a s m 144 c r y s t a l s of pure water t h a t e x c l u d e s o l u t e s . T h i s s e g r e g a t i o n of s o l u t e s i n t o an " e u t e c t i c " phase w i t h d i f f e r e n t f r e e z i n g p r o p e r t i e s has been a c o n s i d e r a t i o n i n f r e e z e f r a c t u r e e x p e r i m e n t s f o r many y e a r s (Hudson e t a l . 1979). The u l t i m a t e g o a l of c r y o f i x a t i o n i s the s o l i d i f i c a t i o n of water w i t h o u t the f o r m a t i o n of i c e c r y s t a l s : the amorphous s o l i d c a l l e d v i t r e o u s i c e . True v i t r i f i c a t i o n o n l y o c c u r s under r i g o r o u s c o n d i t i o n s of r a p i d f r e e z i n g ( c o o l i n g r a t e >10^ °K/ sec) and v e r y s m a l l sample volume ( S i t t e et. a l . 1987, Dubochet and McDowell 1981, Dubochet et a l . 1982). V i t r i f i e d i c e i s found o n l y i n t h e t o p few m i c r o n s of u l t r a r a p i d l y f r o z e n t i s s u e b l o c k s ; t r u e v i t r i f i c a t i o n t h r o u g h the d e p t h of u n c r y o p r o t e c t e d t i s s u e i s not p o s s i b l e (Dubochet et a l . 1987). The p r a c t i c a l g o a l of c r y o f i x a t i o n i s t o a c h i e v e a f a s t r a t e of c o o l i n g t o m i n i m i z e the s i z e of t h e r e s u l t i n g i c e c r y s t a l s ( P l a t t n e r and Bachmann 1982, G i l k e y and S t a e h e l i n 1986). " I d e a l f i x a t i o n and specimen p r e p a r a t i o n r e q u i r e t h a t c o n s t i t u e n t s of the system keep t h e i r p o s i t i o n w i t h i n a range t h a t i s s m a l l e r than the r e s o l u t i o n of the o b s e r v a t i o n method" (Bachmann and Mayer 1987). The f r e e z i n g r a t e must be i n the range of 1 45 >10^ °K/sec t o have c r y s t a l s no b i g g e r than the l i m i t of r e s o l u t i o n of the TEM ( S i t t e e t a l . 1987). For t i s s u e s , t h i s f a s t r a t e of c o o l i n g can be a c h i e v e d by b r i n g i n g the sample i n t o c o n t a c t w i t h a h i g h l y p o l i s h e d m e t a l s u r f a c e (copper or s i l v e r ) which i s h e l d a t l i q u i d n i t r o g e n ( o r l i q u i d h e l i u m ) t e m p e r a t u r e . Only the s u r f a c e c o n t a c t i n g the copper b l o c k and the u n d e r l y i n g 10-40 um a r e u l t r a r a p i d l y c o o l e d . Beyond t h i s depth t h e e f f e c t of t h e low t h e r m a l c o n d u c t i v i t y of i c e overcomes the e f f e c t of the h i g h t h e r m a l c o n d u c t i v i t y of the copper b l o c k ( P l a t t n e r and Bachmann 1982). T h i s t e c h n i q u e was p i o n e e r e d by Van H a r r e v e l d and coworkers (Van H a r r e v e l d and C r o w e l l 1964, Van H a r r e v e l d e t a l . 1974). I t has been i m p o r t a n t , when used w i t h deep e t c h p l a t i n u m carbon r e p l i c a s , i n s t u d i e s of n e u r o t r a n s m i t t e r e x o c y t o s i s and subsequent e n d o c y t o s i s a t neuromuscular j u n c t i o n s (Heuser e t a l . 1979, M i l l e r and Heuser 1984), and c o a t e d p i t budding i n f i b r o b l a s t s (Heuser and Evans 1980, Heuser 1989).. Copper b l o c k f r e e z i n g has been used w i t h f r e e z e s u b s t i t u t i o n t o p r e s e r v e d e l i c a t e s t r u c t u r e s , f o r example s i l k moth antennae ( S t e i n b r e c h t 1980) and c i l i a r y p a t t e r n s on Paramecium (Barlow and S l e i g h 1979). 146 P r e v i o u s l y most p l a n t t i s s u e s were f r o z e n by p l u n g i n g i n t o l i q u i d c r yogen such as F r e o n , or propane (Hereward and N o r t h c o t e 1972, F i s h e r 1975, Browning and Gunning 1977, M c C u l l y and Canny 1985, L a n c e l l e e t a l . 1986). Other methods of u l t r a r a p i d f r e e z i n g such as propane j e t - f r e e z i n g and h i g h p r e s s u r e f r e e z i n g have been a p p l i e d t o c u l t u r e d h i g h e r p l a n t c e l l s u s p e n s i o n s and i n t a c t t i s s u e s (Fernandez and S t a e h e l i n 1985, S t a e h e l i n and Chapman 1987, C r a i g and S t a e h e l i n 1988). There have been few a p p l i c a t i o n s of the copper b l o c k method of u l t r a r a p i d f r e e z i n g f o r p l a n t m a t e r i a l , except f o r s u s p e n s i o n s of u n i c e l l u l a r a l g a e or c h l o r o p l a s t s (Kugrens and Lee 1987, N i s h i z a w a and M o r i 1989). U l t r a r a p i d l y f r o z e n m a t e r i a l can be s l o w l y d e h y d r a t e d and f i x e d by f r e e z e s u b s t i t u t i o n . The samples a r e immersed i n s u b s t i t u t i o n medium such as acetone or methanol w i t h osmium t e t r a o x i d e f o r 2 days and m a i n t a i n e d a t -80 ° C. D u r i n g t h i s t i m e , the s o l v e n t d i s s o l v e s the i c e , i . e . t h e water i n the sample i s removed ( S t e i n b r e c h t and M u l l e r 1987). The temperature i s s l o w l y a l l o w e d t o r i s e , and osmium f i x a t i o n s t a b i l i z e s the c e l l u l a r s t r u c t u r e s . 147 The copper b l o c k u l t r a r a p i d f r e e z i n g method was used i n the p r e s e n t s t u d y because i t i s s p a t i a l l y w e l l s u i t e d f o r e p i d e r m a l c e l l s , and t e m p o r a l l y w e l l s u i t e d f o r e n d o c y t o s i s . W i t h t h e s e new t o o l s of u l t r a r a p i d f r e e z i n g and f r e e z e s u b s t i t u t i o n , the p r o c e s s of e n d o c y t o s i s i n e l o n g a t i n g r o o t c e l l s of L o b e l i a e r i n u s can be r e e v a l u a t e d . When the e x t r a c e l l u l a r medium i s l a b e l l e d w i t h lanthanum, e n d o c y t o s i s can be a c c u r a t e l y t r a c e d as l a b e l i s i n t e r n a l i z e d and t r a n s p o r t e d t h r o u g h i n t r a c e l l u l a r compartments. The u l t r a r a p i d f r e e z i n g / f r e e z e s u b s t i t u t i o n s t u d y of s e c r e t i o n (see c h a p t e r 2 ) showed e x c e l l e n t p r e s e r v a t i o n of o r g a n e l l e s such as G o l g i and PCR. The o b j e c t i v e of t h i s s tudy i s t o re-examine e n d o c y t o s i s i r i s i t u , w i t h i n the new c o n t e x t of the u l t r a r a p i d l y f r o z e n endomembrane system, u n p e r t u r b e d by c h e m i c a l f i x a t i o n . 148 METHODS AND MATERIALS L o b e l i a e r i n u s s e e d l i n g s were ge r m i n a t e d on f i l t e r paper m o i s t e n e d w i t h d i s t i l l e d d e i o n i z e d water f o r 7 days. S e e d l i n g s were immersed i n e i t h e r 5 mM lanthanum n i t r a t e or i n d i s t i l l e d d e i o n i z e d water f o r 1 hour a t room t e m p e r a t u r e , as d e s c r i b e d i n c h a p t e r 1. In the l a s t 5 minutes of the i n c u b a t i o n p e r i o d , samples were removed from the i n c u b a t i n g s o l u t i o n , mounted on s o f t foam pads f o r copper b l o c k f r e e z i n g , and m a i n t a i n e d i n a humid environment. Two c o m m e r c i a l l y a v a i l a b l e u l t r a r a p i d f r e e z i n g d e v i c e s were used i n t h i s s t u d y : R e i c h e r t - Jung MM-80 a t the E l e c t r o n M i c r o s c o p y F a c i l i t y a t UBC or RMC MF7000 a t the RMC c r y o t e c h n i q u e s c o u r s e i n the E l e c t r o n M i c r o s c o p y L a b o r a t o r y a t UC B e r k e l e y . U s i n g the RMC s e t u p , s e e d l i n g s were mounted on an aluminum p l a n c h e t t e which was backed w i t h s o f t foam a t t a c h e d t o a s t e e l d i s k . When the copper b l o c k reached l i q u i d n i t r o g e n t e m p e r a t u r e , the d i s k was mounted on a magnet a t t a c h e d t o the p l u n g i n g arm of the a p p a r a t u s . Immediately the sample was r e l e a s e d t o slam i n t o the copper b l o c k ; an e l e c t r o m a g n e t l o c k e d the sample a g a i n s t the b l o c k upon impact t o p r e v e n t rebounce a r t e f a c t . The 149 seedlings were removed from the aluminum planchette under l i q u i d nitrogen by scraping the planchette with a razor blade. As the well frozen surface could be damaged, the samples were mounted on f i l t e r paper in subsequent experiments. After release from the planchettes, the samples were transferred to small p l a s t i c containers and processed for freeze substitution. The protocol using the Reichert-Jung KF-80 varied s l i g h t l y : the seedlings were mounted on f i l t e r paper, stuck to parafilm, glued to the soft foam pad which i s backed by a steel planchette. When the copper block reached l i q u i d nitrogen temperature, excess water was blotted off the f i l t e r paper and the planchette was stuck on the magnet of the MM80 arm. The sample was slammed against the polished copper surface, then kept at l i q u i d nitrogen temperature. The parafilm next to the f i l t e r paper becomes b r i t t l e at t h i s temperature, allowing easy separation of the samples from the foam. The f i l t e r paper containing the sample was then processed for freeze substitution in a Reichert-Jung CS Auto. Freeze substitution was ca r r i e d out at -80° C for 48-56 hours, followed by a gradual r i s e in 150 temp e r a t u r e of 10° C/hour t o a f i n a l t e m p e r a t u r e of 0° C. The s u b s t i t u t i o n medium was 1 % O s 0 4 i n a b s o l u t e a c e t o n e . When the RMC equipment was used, the t e m p e r a t u r e s d u r i n g s u b s t i t u t i o n were a p p r o x i m a t e . When the R e i c h e r t - J u n g equipment was used, t h e s e v a l u e s were c o n t r o l l e d p r e c i s e l y . The r e s u l t s f o r the two s e t u p s were i d e n t i c a l . At the end of the f r e e z e s u b s t i t u t i o n p r o c e d u r e , the s u b s t i t u t i o n medium was removed and r e p l a c e d s e v e r a l t i m e s w i t h a b s o l u t e a c e t o n e . The acetone then a c t e d as the s o l v e n t f o r i n f i l t r a t i o n i n S p u r r ' s r e s i n . S i l v e r s e c t i o n s were c u t on a R e i c h e r t - J u n g U l t r a c u t E u s i n g a diamond k n i f e . S e c t i o n were p i c k e d up on formvar c o a t e d g r i d s and viewed w i t h o u t p o s t s t a i n i n g or a f t e r 25 minutes i n a s a t u r a t e d s o l u t i o n of u r a n y l a c e t a t e i n 70 % methanol. A l l g r i d s were viewed and photographed a t 60 kV on a Z e i s s 10C TEM. 151 RESULTS When L o b e l i a e r i n u s r o o t t i p s a r e u l t r a r a p i d l y f r o z e n by c o n t a c t i n g a copper b l o c k a t -196 0 C, a g r a d i e n t of i c e c r y s t a l s i z e s was formed ( F i g . 60) as e x p e c t e d i n an u n c r y o p r o t e c t e d sample. Near the e p i d e r m a l c e l l s u r f a c e , m a t e r i a l may be v i t r i f i e d or the i c e c r y s t a l s formed were e x t r e m e l y s m a l l due t o the r a p i d r a t e of c o o l i n g . The p r e s e r v a t i o n of u l t r a s t r u c t u r e i n the s e r e g i o n s was e x c e l l e n t . In c o n t r a s t , a t the b a s a l p o r t i o n of the e p i d e r m a l c e l l s , c l e a r h o l e s r e p r e s e n t i n g c y t o p l a s m i c damages by hexagonal i c e c r y s t a l s were i n e v i d e n c e . Root cap c e l l s c o v e r e d the e x t e r i o r s u r f a c e of the r o o t i n the zone of e l o n g a t i o n . In some c a s e s , the r o o t cap c e l l s had c o l l a p s e d due t o e x c e s s i v e impact b e f o r e f r e e z i n g . The two ty p e s of c e l l s , e p i d e r m a l and r o o t cap, had d i f f e r e n t f r e e z i n g p r o p e r t i e s . The e p i d e r m a l c e l l s o f t e n demonstrated w e l l p r e s e r v e d a p i c a l c y t o p l a s m a d j a c e n t t o the l e s s w e l l f r o z e n b a s a l p o r t i o n of the r o o t cap. In the w e l l p r e s e r v e d p o r t i o n s of the e l o n g a t i n g e p i d e r m a l c e l l s , the pathways of e n d o c y t o s i s were l a b e l l e d u s i n g lanthanum as an marker. Roots t h a t had been i n c u b a t e d i n lanthanum 152 n i t r a t e b e f o r e f r e e z i n g showed e l e c t r o n dense d e p o s i t s i n the a p o p l a s t and w i t h i n some membrane bound o r g a n e l l e s . These d e p o s i t s were i d e n t i c a l i n e l e c t r o n d e n s i t y as those found i n e x p e r i m e n t s u s i n g c o n v e n t i o n a l f i x a t i o n t e c h n i q u e s . The a p o p l a s t i c l a b e l l i n g i n the u l t r a r a p i d l y f r o z e n m a t e r i a l was s p a r s e . T h i s may be due t o l o s s of a p o p l a s t i c lanthanum d u r i n g epoxy r e s i n i n f i l t r a t i o n ; the r e s i n took l o n g e r t o i n f i l t r a t e i n t o u l t r a r a p i d l y f r o z e n m a t e r i a l than c o n v e n t i o n a l l y p r e p a r e d m a t e r i a l . W i t h i n the u l t r a r a p i d l y f r o z e n c e l l s , t he lanthanum l a b e l l i n g p a t t e r n was c o n s i s t e n t w i t h e a r l i e r e x p e r i m e n t s , i . e . v e s i c l e s and m u l t i v e s i c u l a r b o d i e s c o n t a i n e d lanthanum d e p o s i t s . The s u p e r i o r p r e s e r v a t i o n of o t h e r o r g a n e l l e s , such as t h e p a r t i a l l y c o a t e d r e t i c u l u m and d i c t y o s o m e s , r e v e a l e d t h a t t h e s e endomembrane components a r e a l s o l a b e l l e d w i t h lanthanum. Lanthanum l a b e l l e d v e s i c l e s were obse r v e d i n the c o r t i c a l c y t o p l a s m ( F i g s . 61-63). The v e s i c l e d i a m e t e r s were s i m i l a r t o tho s e o b s e r v e d i n c o n v e n t i o n a l t e c h n i q u e s , i . e . about 100 nm. There were few c o a t e d v e s i c l e s near the plasma membrane of the a p i c a l s u r f a c e of w e l l f r o z e n e p i d e r m a l 153 c e l l s . C o r t i c a l m i c r o t u b u l e s were found i n the v i c i n i t y of the l a b e l l e d v e s i c l e s ( F i g . 6 3 ) . The c o r t i c a l c y t o p l a s m of u l t r a r a p i d l y f r o z e n m a t e r i a l o f t e n c o n t a i n e d p a r t i a l l y c o a t e d r e t i c u l u m (PCR) ( F i g . 6 7 ) , the network of c o a t e d and smooth t u b u l e s and v e s i c l e s d e s c r i b e d by P e s a c r e t a and Lucas (1985). T h i s s t r u c t u r e was d i f f i c u l t t o d i s t i n g u i s h i n c o n v e n t i o n a l p r e p a r a t i o n s . A f t e r u l t r a r a p i d f r e e z i n g and f r e e z e s u b s t i t u t i o n , the PCR was d i s t i n c t , s e p a r a t e from the t r a n s G o l g i network (see c h a p t e r 2) and c o n s i s t e d of s m a l l t u b u l a r or v e s i c u l a r p o r t i o n s as w e l l as d i l a t e d v a c u o l e s (not shown) about the same s i z e as m u l t i v e s i c u l a r b o d i e s . The PCR was l a b e l l e d w i t h lanthanum, s u g g e s t i n g t h i s o r g a n e l l e r e c e i v e s membrane m a t e r i a l and v e s i c l e c o n t e n t s from the plasma membrane f o l l o w i n g e n d o c y t o s i s . Another endomembrane component t h a t was c l e a r l y l a b e l l e d a f t e r lanthanum t r e a t m e n t and u l t r a r a p i d f r e e z i n g was the m u l t i v e s i c u l a r body ( F i g s . 64-67). The lanthanum was c l u s t e r e d over the i n t r a l u m i n a l v e s i c l e s of the m u l t i v e s i c u l a r body. Lanthanum l a b e l l e d v e s i c l e s were a d j a c e n t t o the m u l t i v e s i c u l a r body: t h e s e appear t o be budding from or f u s i n g w i t h t h i s o r g a n e l l e ( F i g s . 64-65). 154 In many c a s e s , the m u l t i v e s i c u l a r body was p o s i t i o n e d a d j a c e n t t o a dictyosome ( F i g s . 65, 6 8 ) . T h i s a s s o c i a t i o n was much more common i n u l t r a r a p i d l y f r o z e n m a t e r i a l than i n c o n v e n t i o n a l l y f i x e d m a t e r i a l . The G o l g i of the e l o n g a t i n g e p i d e r m a l c e l l s as r e v e a l e d by u l t r a r a p i d f r e e z i n g and f r e e z e s u b s t i t u t i o n c o n t a i n s w e l l p r e s e r v e d c e n t r a l p o l a r d i c t y o s o m e s t a c k , an e x t e n s i v e p e r i p h e r a l network of s m a l l , r e g u l a r v e s i c l e s and the t r a n s G o l g i network c o n t a i n i n g l a r g e r v e s i c l e s ( F i g s . 68-70, see a l s o c h a p t e r 2 ) . Lanthanum l a b e l appeared i n dicty o s o m e a s s o c i a t e d v e s i c l e s but not i n the c i s t e r n a e of the d i c t y o s o m e . I t i s i n t e r e s t i n g t h a t , i n g e n e r a l , the subset of v e s i c l e s l a b e l l e d w i t h lanthanum appear t o c o n s t i t u t e a d i f f e r e n t p o p u l a t i o n from the v e s i c l e s which were s t a i n e d f o r p o l y s a c c h a r i d e s u s i n g a l k a l i n e b i s m u t h . Lanthanum l a b e l l e d v e s i c l e s were s m a l l e r (60-100 nm) and l o c a t e d around the p e r i p h e r y of the d i c t y o s o m e , w h i l e the p o l y s a c c h a r i d e c o n t a i n i n g v e s i c l e s were l a r g e r (100-150 nm) and l o c a t e d i n the t r a n s G o l g i network (see c h a p t e r 2 ) . In some c a s e s , l a r g e r v e s i c l e s had lanthanum d e p o s i t s ( F i g . 70) but t h e s e were r e l a t i v e l y i n f r e q u e n t . L a b e l l e d v e s i c l e s were 155 a s s o c i a t e d w i t h the dic t y o s o m e i n about 50 % of the di c t y o s o m e s i n u l t r a r a p i d l y f r o z e n m a t e r i a l , as opposed t o about 10 % of the d i c t y o s o m e s i n c o n v e n t i o n a l l y p r e p a r e d m a t e r i a l . In summary, lanthanum was used as an as s a y f o r e n d o c y t o s i s , f o l l o w e d by u l t r a r a p i d f r e e z i n g and f r e e z e s u b s t i t u t i o n t o c o n s e r v e the v e s i c l e and o r g a n e l l e d i s t r i b u t i o n s i n the e l o n g a t i n g e p i d e r m a l r o o t c e l l s . The r e s u l t s c o n f i r m e d e a r l i e r , c o n v e n t i o n a l TEM e x p e r i m e n t s (see c h a p t e r 1) showing lanthanum l a b e l s c o r t i c a l v e s i c l e s and m u l t i v e s i c u l a r b o d i e s . In a d d i t i o n , s u p e r i o r p r e s e r v a t i o n of endomembranes u s i n g c r y o t e c h n i q u e s , showed lanthanum l a b e l l e d the p a r t i a l l y c o a t e d r e t i c u l u m and dictyosome a s s o c i a t e d v e s i c l e s . 156 F i g . 60: G r a d i e n t of f r e e z i n g . C r o s s s e c t i o n of e l o n g a t i n g e p i d e r m a l r o o t c e l l s , p e r p e n d i c u l a r t o s u r f a c e t h a t c o n t a c t e d l i q u i d n i t r o g e n c o o l e d copper m i r r o r ( s u r f a c e i n d i c a t e d by open arrow h e a d s ) . The c y t o p l a s m of the e p i d e r m a l c e l l near the r o o t s u r f a c e shows l i t t l e or no i c e c r y s t a l damage ( a s t e r i x ) . In the b a s a l p o r t i o n of the c e l l , i c e c r y s t a l s have formed, d i s p l a c i n g c y t o p l a s m and c h r o m a t i n . A f t e r f r e e z e s u b s t i t u t i o n c l e a r spaces i n d i c a t e where i c e c r y s t a l s had formed. F i g s . 61-63: lanthanum l a b e l l e d v e s i c l e s . F i g . 61: High m a g n i f i c a t i o n of c o r t i c a l c y t o p l a s m of c e l l shown i n F i g . 60; lanthanum l a b e l l e d v e s i c l e i n c o r t i c a l c y t o p l a s m . F i g . 62: lanthanum l a b e l l e d v e s i c l e deeper i n the c y t o p l a s m F i g . 63: Plasma membrane a s s o c i a t e d lanthanum l a b e l l e d v e s i c l e s , near c o r t i c a l m i c r o t u b u l e s a l l c l o s e d arrows i n d i c a t e s lanthanum d e p o s i t s . pm=plasma membrane, r c = r o o t cap c e l l , c w = c e l l w a l l , er=endoplasmic r e t i c u l u m , m=mitochondion, Nu=nucleus, mt=microtubule 157 p m rc • cw er 61 O.lLim m Pv Nu .-•4 62 f; 0.1 |jm O.ILI m 60 1.0pm 63 m t 1 58 F i g s . 64-66: M u l t i v e s i c u l a r b o d i e s l a b e l l e d w i t h lanthanum. F i g . 64: m u l t i v e s i c u l a r body w i t h v e s i c l e s f u s i n g or b u d ding. I n t r a l u m i n a l v e s i c l e s l a b e l l e d w i t h lanthanum. S e c t i o n s not p o s t s t a i n e d . F i g . 65: dictyosome a d j a c e n t t o m u l t i v e s i c u l a r body; t h e s e o r g a n e l l e s a r e o f t e n a s s o c i a t e d . S e c t i o n s s t a i n e d w i t h u r a n y l a c e t a t e / l e a d c i t r a t e . F i g . 66: m u l t i v e s i c u l a r body i n c o r t i c a l c y t o p l a s m . S e c t i o n s not p o s t s t a i n e d . F i g . 67: P a r t i a l l y c o a t e d r e t i c u l u m l a b e l l e d w i t h lanthanum. Note p r o v a c u o l e f u s i o n . pm=plasma membrane, m v b = m u l t i v e s i c u l a r body, v = v e s i c l e , d=dictyosome, m=mitochondrion, p c r = p a r t i a l l y c o a t e d r e t i c u l u m , pv=provacuole. a l l arrowheads i n d i c a t e lanthanum d e p o s i t s mv 1S~9 -? *: * v y * i .-i c i ^ * - • • i i • • «• v. . " - • mvb - , b i r " • ^ V ; . :-• . + ' . • v • i * N -^K. S V- " * d * « * ±fc 65 2s pm per pv 4 I 0.1pm F i g s . 68-70: G o l g i w i t h l a b e l l e d v e s i c l e s . 160 p e r i p h e r a l lanthanum F i g . 68: Dictyosome and m u l t i v e s i c u l a r body l a b e l l e d w i t h lanthanum. V e s i c l e s near d i c t y o s o m e ar e s m a l l e r s i z e c l a s s than t r a n s G o l g i network v e s i c l e s . M u l t i v e s i c u l a r body i n t r a l u m i n a l v e s i c l e s l a b e l l e d . F i g . 68: H i g h e r m a g n i f i c a t i o n view of a d i c t y o s o m e s t a c k w i t h nearby endoplasmic r e t i c u l u m . Note s m a l l lanthanum l a b e l l e d v e s i c l e near d i c t y o s o m e . F i g . 69: L o n g i t u d i n a l s e c t i o n t h r o u g h d i c t y o s o m e p e r i p h e r a l network. Lanthanum l a b e l s s m a l l p e r i p h e r a l v e s i c l e s as w e l l as margin of l a r g e r v e s i c l e t y p e . pm=plasma membrane, m v b = m u l t i v e s i c u l a r body, d=dictyosome, er=endoplasmic r e t i c u l u m , a l l arrowheads i n d i c a t e lanthanum d e p o s i t s 162 DISCUSSION The r e s u l t s of t h i s u l t r a r a p i d f r e e z i n g study agree w e l l w i t h p r e v i o u s s t u d i e s of e n d o c y t o s i s u s i n g c o n v e n t i o n a l t e c h n i q u e s ( c h a p t e r 1, Joachim and Robinson 1984, Tanchak e_t a l . 1984, Hubner e t a l . 1985, Tanchak and Fowke 1987). The lanthanum a s s a y , when combined w i t h u l t r a r a p i d f r e e z i n g and f r e e z e s u b s t i t u t i o n , shows membrane and/ or c o n t e n t s a r e d e l i v e r e d t o the p a r t i a l l y c o a t e d r e t i c u l u m , m u l t i v e s i c u l a r body, and G o l g i . Cryo t e c h n i q u e s have never been used t o study e n d o c y t o s i s i n h i g h e r p l a n t s and a b r a n c h i n g , l e s s v e s i c u l a t e d endomembrane system i s r e v e a l e d . The p r e s e n c e of lanthanum l a b e l l e d v e s i c l e s i n u l t r a r a p i d l y f r o z e n c e l l s s u g g ests e n d o c y t o t i c v e s i c l e s do e x i s t i n h i g h e r p l a n t c e l l s and a r e not an a r t i f a c t of c h e m i c a l f i x a t i o n . C r u c i a l t o the st u d y of e n d o c y t o s i s i s the a b i l i t y t o d e f i n e the n a t u r e of the v e s i c l e s r e l a t e d t o the endomembrane system, i . e . one must be a b l e t o d i s t i n g u i s h between e n d o c y t o t i c v e s i c l e s and o t h e r t r a n s p o r t v e s i c l e s such as s e c r e t o r y v e s i c l e s . The p o p u l a t i o n of e n d o c y t o t i c v e s i c l e s i n t h i s study were d e f i n e d by the presence of lanthanum l a b e l . U l t r a r a p i d f r e e z i n g t e c h n i q u e 163 a l l o w s f o r the f u r t h e r d i s t i n c t i o n between e n d o c y t o t i c and s e c r e t o r y v e s i c l e s t h r o u g h more a c c u r a t e morphology and t h e i r d i s t i n c t a s s o c i a t i o n w i t h d i f f e r e n t p a r t s of the G o l g i . E n d o c y t o t i c v e s i c l e s were a s s o c i a t e d w i t h the p e r i p h e r y of the dictyosome and the s e c r e t o r y v e s i c l e s were a s s o c i a t e d w i t h the t r a n s G o l g i . When the r e s u l t s of t h i s e n d o c y t o s i s study a r e c o n s i d e r e d w i t h the r e s u l t s of the s e c r e t i o n s t u d y on the same c e l l type ( c h a p t e r 2 ) , i t can be suggested t h a t t h e r e a r e heterogenous v e s i c l e p o p u l a t i o n s i n the c y t o p l a s m . V e s i c l e h e t e r o g e n e i t y s h o u l d be c o n s i d e r e d when morphometric s t u d i e s of s e c r e t i o n a r e c a r r i e d o u t . P r e v i o u s morphometric s t u d i e s have assumed t h a t a l l v e s i c l e s i n the c o r t i c a l c y t o p l a s m a r e s e c r e t o r y (Cunningham and H a l l 1986, Q u a i t e et a l . 1983, K r i s t e n and Lockhausen 1983, P h i l l i p s e t a l . 1988). T h i s e n d o c y t o s i s s t u d y shows a p o p u l a t i o n of v e s i c l e s moving from the plasma membrane t o m u l t i v e s i c u l a r body and dict y o s o m e which a r e d i s t i n c t from the s e c r e t o r y v e s i c l e s . The presence of t h i s p o p u l a t i o n of v e s i c l e s can be i n t e r p r e t e d as e v i d e n c e of v e s i c u l a r membrane r e c y c l i n g i n L o b e l i a e r i n u s r o o t s e c r e t o r y 164 c e l l s . The e n d o c y t o t i c v e s i c l e s l a b e l l e d w i t h lanthanum c o u l d be c a r r y i n g away e x c e s s membrane added by e x o c y t o s i s of c e l l w a l l components. A p o p u l a t i o n of v e s i c l e s whose p r i m a r y f u n c t i o n i s moving b u l k membrane m a t e r i a l from the plasma membrane back t o the endomembranes of the g r a n u l o c r i n e s e c r e t o r y c e l l has been p r e d i c t e d on t h e o r e t i c a l grounds (Raven 1987, Robinson 1985, S t e e r 1985). The p r o p e r t i e s of the e n d o c y t o t i c v e s i c l e s o b s e r v e d i n t h i s study a r e c o n s i s t e n t w i t h a r o l e i n membrane r e c y c l i n g . The s m a l l r a d i u s of the v e s i c l e s (100 nm) means a l a r g e s u r f a c e area/volume r a t i o which i s an e f f i c i e n t mechanism f o r p a c k i n g membrane m a t e r i a l (see d i s c u s s i o n c h a p t e r 2 ) . C o n s i d e r a t i o n s of t u r g o r as a r e s t r a i n i n g f o r c e on membrane i n t e r n a l i z a t i o n have s u g g e s t e d the work n e c e s s a r y t o i n t e r n a l i z e a v e s i c l e depends i n p a r t on the volume of the v e s i c l e . " I t f o l l o w s t h a t the uptake of fewer, l a r g e , c o a t e d v e s i c l e s a g a i n s t t u r g o r may be t h e r m o d y n a m i c a l l y i m p o s s i b l e i n c o n t r a s t ' t o the uptake of many s m a l l ones" (Gradmann and Robinson 1989). These a u t h o r s assume a l l r e c y c l i n g v e s i c l e s would be c o a t e d but the argument h o l d s f o r uncoated s m a l l v e s i c l e s as w e l l . In a d d i t i o n t o a s m a l l r a d i u s , a n other i m p o r t a n t p r o p e r t y of the e n d o c y t o t i c v e s i c l e s a r e 165 t h e i r l o c a t i o n i n the c o r t i c a l c y t o p l a s m when c e l l s a r e u l t r a r a p i d l y f r o z e n . Lanthanum l a b e l l e d v e s i c l e s were seen amongst the c o r t i c a l m i c r o t u b u l e s next t o the plasma membrane, as i n c o n v e n t i o n a l TEM p r e p a r a t i o n s of e l o n g a t i n g r o o t s (see c h a p t e r 1 ) . They a r e a l s o found deeper i n the c y t o p l a s m ; t h i s i s s i g n i f i c a n t because v e s i c l e p r o f i l e s near the plasma membrane may j u s t r e p r e s e n t t h e neck of a plasma membrane i n f o l d i n g ( P a s t a n and W i l l i n g h a m 1983). I f lanthanum l a b e l l e d v e s i c l e s a r e more than 1-2 um from the plasma membrane, the' p r o b a b i l i t y t h a t the v e s i c l e i s s e p a r a t e from the plasma membrane i s h i g h e r . In the c o n v e n t i o n a l s t u d y of e n d o c y t o s i s , l o n g necked s t r u c t u r e s on the plasma" membrane were obser v e d ( c h a p t e r 1) which were not o b s e r v e d i n t h i s u l t r a r a p i d f r e e z i n g s t u d y . These s t r u c t u r e s were o r i g i n a l l y d e s c r i b e d i n an u l t r a r a p i d f r e e z i n g s tudy by S t a e h e l i n and Chapman (1987) so t h e s e r e s u l t s a r e unexpected. In some c a s e s v e s i c l e s f u s i n g w i t h the plasma membrane d i d have a f l a t t e n e d ( o v a l ) appearance, but f u r t h e r work i s r e q u i r e d i n t h i s a r e a . A f t e r u l t r a r a p i d f r e e z i n g , the w e l l p r e s e r v e d s e c t i o n s of the e p i d e r m a l c e l l had few c o a t e d 166 v e s i c l e s . The coated membranes may not have been detected because most sampling was done on sections without poststain. The use of unstained sections means the majority of the electron density comes from osmium (membranes) and lanthanum (label for endocytosis), which i s useful for looking at t o t a l endocytosis. Unfortunately, i t i s not conducive to detecting the protein coat on the cytoplasmic surface of 100 nm lanthanum l a b e l l e d coated v e s i c l e s . In t h i s study, u l t r a r a p i d freezing provides a view of the endomembrane system with extensive branching of r e t i c u l a , for example the p a r t i a l l y coated reticulum, endoplasmic reticulum, and trans Golgi network. These branching structures are not as extensive in c e l l s prepared with conventional methods (see chapters 1 and 2). One reason for th i s could be that conventional techniques use glutaraldehyde which has been shown to induce vesiculation of the endoplasmic reticulum (Mersey and McCully 1978). In other higher plant systems prepared using u l t r a rapid freezing techniques, a "continuous extensive network" of endoplasmic reticulum has been reported (Craig and Staehelin 1988, Fernandez and Staehelin 1985). The present 167 study used a d i f f e r e n t method of u l t r a r a p i d f r e e z i n g (copper b l o c k r a t h e r than propane j e t or h i g h p r e s s u r e f r e e z i n g ) but the r e s u l t s o b t a i n e d s u p p o r t the concept of d e l i c a t e , r e t i c u l a t e endo-membrane compartments. A s i m i l a r i n d uced v e s i c u l a t i o n of the p a r t i a l l y c o a t e d r e t i c u l u m by g l u t a r a l d e h y d e c o u l d e x p l a i n the absence of t h i s s t r u c t u r e i n the e a r l i e r s tudy of e n d o c y t o s i s u s i n g c o n v e n t i o n a l TEM t e c h n i q u e s (see c h a p t e r 1 ) . The lanthanum l a b e l l e d p a r t i a l l y c o a t e d r e t i c u l u m was found i n the c o r t i c a l c y t o p l a s m , which i s c o n s i s t e n t w i t h the t h e o r y t h a t the PCR i s an e a r l y endosome ( c h a p t e r 2, P e s a c r e t a and Lucas 1985, Tanchak e t a_l. 1988). The PCR morphology, l i k e t he m u l t i v e s i c u l a r body morphology, has been d e s c r i b e d i n a n i m a l c e l l s as a type of endosome ( M i l l e r et. a_l. 1986). The m u l t i v e s i c u l a r body was one of the c e n t e r s of v e s i c l e budding and f u s i n g o b s e r v e d i n t h i s s t u d y . A c c o r d i n g t o the lanthanum a s s a y f o r e n d o c y t o s i s , t h i s o r g a n e l l e r e c e i v e s membrane and/or c o n t e n t s of v e s i c l e s d e r i v e d from the plasma membrane. In a n i m a l s , endosomes have complex f u n c t i o n s such as u n c o u p l i n g and s o r t i n g of r e c e p t o r s and l i g a n d s , and membrane r e c y c l i n g t o 168 the plasma membrane i n c e l l s w i t h h i g h e n d o c y t o s i s such as macrophages (Schmid e t a l . 1988, de C h a s t e l l i e r e t a l . 1987). The pathways of r e c e p t o r s and l i g a n d s have not been e l u c i d a t e d i n h i g h e r p l a n t s , but the d e l i v e r y of v e s i c l e s t o the m u l t i v e s i c u l a r body seen i n t h i s s tudy i s c o n s i s t e n t w i t h a r o l e i n membrane t u r n o v e r . The m u l t i v e s i c u l a r endosome has been c o n s i d e r e d a " l a t e " endosome, i . e . l o c a t e d i n the p e r i n u c l e a r r e g i o n of the a n i m a l c e l l and d e s t i n e d f o r f u s i o n w i t h a p r i m a r y lysosome ( M i l l e r e t a l . 1986, G r i f f i t h s e t a l . 1988). In t h i s s t u d y , the l o c a t i o n of the m u l t i v e s i c u l a r body i n the c o r t i c a l c y t o p l a s m s u g g e s t s t h i s o r g a n e l l e does not undergo an analogous m i g r a t i o n t o the c e l l i n t e r i o r . T h i s may be a r e f l e c t i o n of the d i f f e r e n c e s i n the o r g a n i z a t i o n of the G o l g i i n p l a n t s and a n i m a l s . I n a n i m a l s the G o l g i complex i s i n t h e p e r i n u c l e a r zone w h i l e i n the h i g h e r p l a n t c e l l s t h e di c t y o s o m e s a r e s c a t t e r e d throughout the c y t o p l a s m . The i n t r a l u m i n a l v e s i c l e s of the m u l t i v e s i c u l a r body were o f t e n l a b e l l e d w i t h lanthanum i n t h i s s t u d y . T h i s may i n d i c a t e t h a t t h e s e s t r u c t u r e s have d i f f e r e n t charge or l i p i d 169 c o m p o s i t i o n than the l i m i t i n g membrane of the m u l t i v e s i c u l a r body. I t i s d i f f i c u l t t o t e l l i f thes e i n t r a l u m i n a l s t r u c t u r e s a r e v e s i c l e s or t u b u l e s i n t h r e e d i m e n s i o n s . The f o r m a t i o n of t u b u l e s was c o n s i d e r e d a mechanism f o r e x t r u d i n g aqueous m a t r i x and f o r m i n g membrane e n r i c h e d domains of the endosome i n a n i m a l c e l l s (Rome 1985, Geuze e t a l . 1984). I t would be i n t e r e s t i n g t o lo o k a t the a c i d i t y of the p l a n t m u l t i v e s i c u l a r body; i n a n i m a l endosomes the s l i g h t l y a c i d pH i s b e l i e v e d t o be i m p o r t a n t i n s e p a r a t i n g the r e c e p t o r s from t h e i r l i g a n d s ( H e l e n i u s e t a l . 1983, Anderson and O r c i 1988). The r a t e of u l t r a r a p i d f r e e z i n g d e t e r m i n e s t h a t the sampl i n g i s a s m a l l s l i c e of t i m e : the o r g a n e l l e s i n the w e l l p r e s e r v e d s u r f a c e zone a r e stopped w i t h i n 1-2 m i l l i s e c o n d s (Jones 1984). A l t h o u g h the TEM r e q u i r e s t h i n s e c t i o n e d m a t e r i a l and t h e r e f o r e s t i l l o n l y p r o v i d e s a s t a t i c image, t h i s i m m o b i l i z a t i o n of the c y t o p l a s m by u l t r a r a p i d f r e e z i n g p r o v i d e s a p r e c i s e s t a t i c image. In c o n c l u s i o n , u l t r a r a p i d f r e e z i n g t e c h n i q u e s have c o n f i r m e d the e x i s t e n c e of e n d o c y t o s i s i n i n t a c t r o o t t i s s u e of L o b e l i a e r i n u s . A d i s t i n c t p o p u l a t i o n of v e s i c l e s was l a b e l l e d w i t h lanthanum. 170 The s i z e and l o c a t i o n of t h e s e v e s i c l e s makes them good c a n d i d a t e s f o r c a r r y i n g e x c e s s plasma membrane added d u r i n g c e l l w a l l s e c r e t i o n . The i n t r a c e l l u l a r l a b e l l i n g of PCR and m u l t i v e s i c u l a r b o d i e s add s u p p o r t t o the t h e o r i e s t h a t t h e s e s t r u c t u r e s can be c o n s i d e r e d endosomes, and t h a t t h e s e s t r u c t u r e s a r e a l s o i m p o r t a n t i n the f l o w of membrane t h r o u g h the p l a n t c e l l . 171 GENERAL CONCLUSIONS The r e s u l t s of the c o n v e n t i o n a l TEM and u l t r a r a p i d f r e e z i n g s t u d i e s suggest e n d o c y t o s i s does occur i n e l o n g a t i n g r o o t s of L o b e l i a e r i n u s . When the number of e n d o c y t o t i c v e s i c l e s per c e l l was measured, the g r e a t e s t amount of e n d o c y t o s i s was found i n e l o n g a t i n g c e l l s . The g r e a t e r amount of e n d o c y t o s i s o b s e r v e d i n c e l l s which a r e a c t i v e l y s e c r e t i n g , compared t o n o n - s e c r e t o r y c e l l s , can be i n t e r p r e t e d as e v i d e n c e of v e s i c u l a r membrane r e c y c l i n g . The v e s i c l e s i n the c y t o p l a s m d i s p l a y e d h e t e r o g e n e i t y w i t h r e s p e c t t o p o l y s a c c h a r i d e c o n t e n t , s i z e , o r g a n e l l e a s s o c i a t i o n s , and a b i l i t y t o t a k e up lanthanum. I t was demonstrated t h a t a p o p u l a t i o n of s m a l l v e s i c l e s i n v o l v e d i n e n d o c y t o s i s a r e d i s t i n c t from the l a r g e r , p o l y s a c c h a r i d e c o n t a i n i n g s e c r e t o r y v e s i c l e s . The membrane i n t e r n a l i z e d by e n d o c y t o s i s becomes i n c o r p o r a t e d i n t o m u l t i v e s i c u l a r b o d i e s and p a r t i a l l y c o a t e d r e t i c u l u m . U s i n g a f u n c t i o n a l d e f i n i t i o n of an endosome as an o r g a n e l l e which r e c e i v e s membrane from the plasma membrane v i a e n d o c y t o s i s , and c o n s i d e r i n g the m o r p h o l o g i c a l s i m i l a r i t i e s t o a n i m a l systems, t h e s e o r g a n e l l e s 172 can be c o n s i d e r e d p l a n t endosomes. Membrane r e c y c l i n g from the plasma membrane t o the G o l g i c o u l d be o c c u r r i n g v i a these i n t e r m e d i a t e o r g a n e l l e s . P r e l i m i n a r y e x p e r i m e n t s suggest m i c r o t u b u l e s p l a y a r o l e i n v e s i c l e movement d u r i n g e n d o c y t o s i s i n e l o n g a t i n g L o b e l i a r o o t c e l l s . A f t e r d i s r u p t i o n of m i c r o t u b u l e s w i t h c o l c h i c i n e , t h e r e were s i g n s t h a t e n d o c y t o s i s had been p e r t u r b e d , e.g. fewer lanthanum l a b e l l e d v e s i c l e s and l e s s l a b e l i n the m u l t i v e s i c u l a r b o d i e s . The c e l l w a l l p o r o s i t y of the e l o n g a t i n g c e l l s of L o b e l i a e r i n u s was dependent on the i n t e g r i t y of the p e c t i n component of the c e l l w a l l . The pore s i z e i n c r e a s e d d r a m a t i c a l l y upon t r e a t m e n t w i t h low c o n c e n t r a t i o n s of p e c t i n a s e . The s e n s i t i v i t y t o d i s r u p t i o n p r o b a b l y means t h a t the 1-2 nm pore s i z e o b s e r v e d i n i n t a c t l a b o r a t o r y grown r o o t s r e p r e s e n t s a minimum t h a t would r a r e l y , i f e v e r , occur i n the s o i l where the r o o t i s exposed t o m i c r o b i a l enzymes and a b r a s i o n . In c o n c l u s i o n , e n d o c y t o s i s can be c o n s i d e r e d an i m p o r t a n t p a r t of the h o m e o s t a t i c mechanism f o r c o n t r o l l i n g plasma membrane s u r f a c e a r e a i n the e l o n g a t i n g p r i m a r y r o o t c e l l s of L o b e l i a e r i n u s . 173 The o r g a n e l l e s of the endomembrane system t h a t were shown t o be i n v o l v e d i n e n d o c y t o s i s a r e o u t l i n e d i n F i g . 71. T h i s r e v i s e d model emphasizes the importance of endosomes i n membrane f l o w , as w e l l as the r e t i c u l a r n a t u r e of the endomembrane system and the heterogenous c o m p o s i t i o n of v e s i c l e s i n the c y t o p l a s m . These e x p e r i m e n t s demonstrated the f i r s t s t e p i n t h i s membrane r e c y c l i n g model, i . e . movement of v e s i c l e s from plasma membrane t o endosome ( F i g . 7 1 ) . The second s t e p i n membrane r e c y c l i n g , i . e . endosome t o G o l g i , i s proposed t o be v e s i c l e mediated as w e l l , a l t h o u g h f u r t h e r work i s r e q u i r e d i n t h i s a r e a . The endomembrane system of h i g h e r p l a n t s has been d e s c r i b e d i n terms of ER, G o l g i , and s e c r e t o r y v e s i c l e s . In the f u t u r e , the presence of endosomes and e n d o c y t o t i c v e s i c l e s s h o u l d a l s o r e c o g n i z e d as an i n t e g r a l p a r t of the endomembrane system. 174 F i g . 71: Revised model of membrane flow i n L o b e l i a  e r i n u s endomembrane system. Endocytosis of v e s i c l e s t o endosomes shown by small arrowheads, based on lanthanum l a b e l l i n g experiments. Proposed pathway of membrane r e c y c l i n g from endosomes to G o l g i shown by c i r c l e s . S e c r e t i o n of c e l l w a l l components shown with l a r g e r arrowheads. stipple=lanthanum, squares=secretory product, G=Golgi, c = c i s , t=trans, er=endoplasmic r e t i c u l u m , sv=secretory v e s i c l e , cv=coated v e s i c l e , mvb=multivesicular body, p c r = p a r t i a l l y coated r e t i c u l u m , mt=microtubules, pm=plasma membrane. 175 APPENDIX 1: CALCULATIONS OF AMOUNT OF PM ADDED TO CELL SURFACE DURING WALL SECRETION; COMPARISON WITH CELL SURFACE OBSERVED SUGGESTS MEMBRANE RECYCLING. If V = volume of the c e l l wall excluding end walls then V= 4 x length x width x wall thickness for meristematic c e l l s V = 4 x 20 x 20 x 0.15 = 240 urn for vacuolate c e l l s V = 4 x 80 x 20 x 0.40 = 2560 um3 Total volume of wall added during elongation = 2560 - 240 = 2320 um3 20% of volume consists of c e l l u l o s e which i s added by plasma membrane bound enzymes rather than secreted, so volume of wall matrix secreted i s 2320 - 0.2 (2320) = 1856 um3 Each c e l l contributes half the wall thickness, so the volume of wall matrix synthesized by each c e l l i s 1856 x 0.5 = 928 um3 Assuming secretion v i a spherical Golgi derived v e s i c l e s of 100 nm diameter (radius =50 nm = 0.05 um) (Quaite et a l . 1983), and assuming the volume of matrix in the secretory v e s i c l e s and the c e l l wall are the same, the volume of one secretory v e s i c l e can be calculated: 176 V= 4/3 x pi x r 3 V= 4/3 x pi x 0.053 = 0.000523 um 3/vesicle The number of ve s i c l e s required to produce the c e l l wall observed i s 928 um3 / 0.000523 um3 = 1,773,361 ve s i c l e s per c e l l The surface area (A) of one v e s i c l e 2 A = 4 x p i x r A = 4 x 3.14 x 0.052 um =0.031 um2 The t o t a l surface area predicted (Ap) to be added by the v e s i c l e s during elongation i s : A p = 1' 7 73'361 x 0.031 um2 = 54,974 um2 This can be compared to the surface area observed in the mature c e l l (A ): o A Q = 4 x 20 x 80 = 6400 um2 A p / A o = 54,974 / 6400 Therefore the surface area of plasma membrane which i s expected to be added by ve s i c l e s during elongation i s about 8.5 times the surface area of the plasma membrane observed in the mature c e l l . 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