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Mechanical integrity of myosin thick filaments of airway smooth muscle in vitro: effects of phosphoryation… Ip, Kelvin 2008

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MECHANICAL INTEGRITY OF MYOSIN THICK FILAMENTS OF AIRWAY SMOOTH MUSCLE in vitro: EFFECTS OF PHOSPHORYATION OF THE REGULATORY LIGHT CHAIN by KELViN IP B.Sc., The University of British Columbia, 2005 A THESIS SUBMIEFED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2008 © Kelvin Ip, 2008 Abstract Background and aims: It is known that smooth muscle possesses substantial mechanical plasticity in that it is able to adapt to large changes in length without compromising its ability to generate force. It is believed that structural malleability of the contractile apparatus underlies this plasticity. There is strong evidence suggesting that myosin thick filaments of the muscle are relatively labile and their length in vivo is determined by the equilibrium between monomeric and filamentous myosin. The equilibrium in turn is governed by the state of phosphorylation of the 20-kD regulatory myosin light chain (MLC2O, or RLC). It is known that phosphorylation of the myosin light chain favors formation of the filaments; it is not known how the light chain phosphorylation affects the lability of the filaments. The major aim of this thesis was to measure the mechanical integrity of the filaments formed from purified myosin molecules from bovine airway smooth muscle, and to determine whether the integrity was influenced by phosphorylation of the myosin light chain. Methods: Myosin was purified from bovine trachealis to form filaments, in ATP containing zero-calcium solution during a slow dialysis that gradually reduced the ionic strength. Sufficient myosin light chain kinase and phosphatase, as well as calmodulin, were retained after the myosin purification and this enabled phosphorylation of RLC within 20-40 s after addition of calcium to the filament suspension. The phosphorylated and non-phosphorylated filaments were then partially disassembled by ultrasonification. The extent of filament disintegration was visualized and quantified by atomic force microscopy. 11 Results: RLC phosphorylation reduced the diameter of the filaments and rendered the filaments more resistant to ultrasonic agitation. Electron microscopy revealed a similar reduction in filament diameter in intact smooth muscle when the cells were activated. Conclusion: Our results suggest that RLC phosphorylation is a key regulatory step in modifying the structural properties of myosin filaments in smooth muscle, where formation and dissolution of the filaments are required in the cells’ adaptation to different cell length. 111 Table of Contents Abstract.ii Table of Contents iv List of Figures vi List of Abbreviations vii Acknowledgements ix CHAPTER 1, Introduction 1 1.1 General Introduction of Airways Smooth Muscle 2 1.2 Structure of Airway Smooth Muscle 4 1.3 The Contractile Apparatus in Airway Smooth Muscle 7 1.4 Mechanism of Muscle Contraction 10 1.5 TheRoleofCalcium 11 1.6 Lability of Smooth Muscle Thick Filament 14 1.7 Regulatory Light Chain Phosphorylation and Thick Filament Formation 17 1.8 Effect of Regulatory Light Chain Phosphorylation in Intact Smooth Muscle 19 1.9 Airway Smooth Muscle and Related Diseases 21 CHAPTER 2, Hypothesis and Specific Aims 23 iv CHAPTER 3, Materials and Methods 26 3.1 Experiment Procedure 26 3.2 Myosin Extraction 29 3.3 SDS-PAGE 31 3.4 Formation of Thick Filaments 33 3.5 Determination of Regulatory Light Chain Phosphorylation 37 3.6 Atomic Force Microscopy 38 3.7 Statistical Analysis 40 CHAPTER 4, Results 41 4.1 Purification of Myosin and Filament Assembly 41 4.2 Testing the Physical Integrity of Myosin Filaments 47 4.3 Measurement of the Number and Diameter of Phosphorylated and Non-Phosphorylated Filaments 50 ChAPTER 5, Discussion 54 5.1 Summary of Results 54 5.2 Purified Myosin and Filament Formation 56 5.3 Structural Integrity and Morphology of Myosin Filaments 57 5.4 Myosin Filament Lability and its Physiological Significance 60 5.5 Myosin Filament Lability and Its Disease Relevance 62 References 64 V List of Fi2ures Figure 1-1 Electron micrograph of a transverse section of ovine tracheal smooth muscle 6 Figure 1-2 Crystal structure of the head and neck region of myosin 9 Figure 1-3 Signal transduction pathway of Ca2 dependent activation through regulatory light chain phosphorylation 13 Figure 3-lA Signal transduction pathway of relaxed condition 35 Figure 3-lB Signal transduction pathway of activated condition 36 Figure 4-1 SDS-PAGE analysis of the myosin purification process 43 Figure 4-2 Myosin molecules from bovine tracheal smooth muscle 44 Figure 4-3 Myosin filaments from bovine tracheal smooth muscle imaged by AFM in scanning mode 45 Figure 4-4 Time course of myosin phosphorylation and dephosphorylation . . .46 Figure 4-5 Effect of brief ultrasonification on myosin filament stability 48 Figure 4-6 Distribution of filament length before and after ultrasonification..49 Figure 4-7 Difference in filament diameter in the relaxed and activated states 52 Figure 4-8 Measurement of myosin filament diameter 53 vi List of Abbreviations ACh - Acetyicholine ANOVA - Analysis of variance ADP - Adenosine diphosphate AFM - Atomic force microscopy ATP - Adenosine triphosphate ASM - Airway smooth muscle BW - Bis Wash CAM - Crude actomyosin CM - Calmodulin COPD - Chronic obstructive pulmonary disease DAG - Diacyiglycerol DI - Deep inspiration EDTA - Ethylenediamine tetraacetic acid EGTA - Ethyleneglycol tetraacetic acid EM - Electronic microscopy HIS - High ionic strength 1P3 - 1 ,4,5-triphosphate LAMES - Low-ionic strength acto-myosin extraction solution MLC - Myosin light chain MLCK - Myosin light chain kinase MLCP - Myosin light chain phosphatase p - Pvalue vii P1 - Inorganic phosphate PiPES - 1, 4-Piperazinebis ethanesulfonic acid PT-PLC - Phosphatidylinositol-specific phospholipase P55 - Physiological saline solution RLC - Regulatory light chain SDS-PAGE - Sodium dodecyl sulphate polyacrylamide gel eleetrophoresis SR - Sarcoplasmic reticulum V - Volts W - Watts viii Acknow1edements I cannot begin to describe how much I appreciate the help my supervisor, Dr. Chun Seow, has given me throughout this project. He has been extremely patient with me in the past 3 years. Personal circumstances have led me to stall progress during the final year of my thesis but Dr. Seow did not give me any pressure at all. I am truly sorry if my slow progress caused any inconvenience in the lab. I would also like to extend my thanks to my committee members, Drs. Peter Pare and Bob Schellenberg, for their valuable inputs on my project. Special thanks go to Dennis Solomon and Yuekan Jiao for their assistance in my project. The experiments would not have been so smooth without their knowledge and experience. In addition, I am grateful for the insights that Dr. Apolinary Sobieszek had given me during his visit and I learned a lot from him. ix CHAPTER 1, Introduction The contractile mechanism and its structural basis in smooth muscle are not well understood. For decades, the theories developed for striated muscle contraction have often been used to explain smooth muscle contraction. It is only in the last decade that we started to realize that the contraction model for striated muscle is not adequate, and in some cases, incorrect for smooth muscle. The best example is the inability of the striated muscle model to describe the subcellular structural plasticity found in smooth muscle (Kuo eta?., 2001; Qi et a?., 2002; Herrera eta?., 2002; Kuo eta?., 2003; Herrera eta?., 2005; Smolensky et a?., 2007). Structural lability of myosin filament is likely an important component of cellular plasticity in smooth muscle (Seow, 2005). To explore further how mechanical properties of the filaments can be regulated through chemical modification, I have focused, in this thesis research, on the effect of phosphorylation of myosin light chain in airway smooth muscle on the physical integrity of the filament structure. Results of my thesis research have been published recently (Ip et a?., 2007). 1 1.1 General Introduction of Airways Smooth Muscle There are three general types of muscles in the human body: Skeletal, smooth, and cardiac muscles. Skeletal and cardiac muscles are striated while smooth muscles are non-striated. Skeletal and cardiac muscles are considered striated because their contractile filaments display regular patterns of striated structure under the microscope. Smooth muscle, on the other hand, does not exhibit such a recurring pattern. The lack of structural information in smooth muscle is one of the reasons research in smooth muscle lagged behind that of striated muscle. Smooth muscles are found embedded in walls of hollow organs such as the bladder, blood vessel, and gastrointestinal and respiratory tracts. The major function of smooth muscle is to modulate the dimensions of these organs. The current project focuses on smooth muscles found in mammalian respiratory tracts - the airways. Airway smooth muscle (ASM) is found in both the trachea and the bronchi. In the trachea, ASM bundles bridge the dorsal side of the cartilage ring. In the bronchi, ASM bundles completely encircle the airways. ASM contraction and relaxation is critical in 2 regulating airflow in the lung. Excessive ASM shortening can lead to obstruction in airflow and respiratory syndromes such as asthma. 3 1.2 Structure of Airway Smooth Muscle The most noticeable difference between smooth and skeletal muscle is the lack of visible striation. Despite the seemingly random intracellular organization, a number of characteristic structural features can still be clearly defined in ASM cells, as shown in Figure 1-1. Like other types of smooth muscle, ASM cells are spindle-shaped with the nucleus located in the centroidal center of the cell. ASM cell length and diameter vary with different amounts of stretch. On average, they are roughly 250 im long and 3-5 pm wide at the center point (Stephens, 2001). Golgi apparatus, mitochondria, and sarcoplasmic reticulum (SR) are often located closely around the two poles of the cigar-shaped nucleus. Under electron microscope, one can see electron-dense patches spread out around the cytosol and the inner cell membrane. These dark patches are termed dense bodies if they are in the cytosol and dense plaques if they are attached to the cell membrane, or sarcolemma. Similar to Z disks in skeletal muscles, dense bodies are thought to function as anchoring points for actin thin filaments since they are mainly composed of ct-actinin, an actin cross linking protein (Fay et al., 1984). Dense plaques serve as both mediators for force transmission between contractile units and extracellular matrix and adhesion points between adjacent cells (Kuo et al., 2004). Invaginated 4 vesicles called caveolae are found throughout the sarcolemma and they alternate with dense plaques along the longitudinal axis of the ASM cell (Stephens, 2001). The function of these caveolae is not well understood but due to the fact that they increase surface area of the ASM cell, it is thought that they can increase the number of receptors and channels embedded in the membrane (Stephens, 2001). 5 Figure 1-1. Electron micrograph of a transverse section of ovine tracheal smooth muscle. 6 1.3 The Contractile Apparatus in Airway Smooth Muscle Although the structure of contractile units in smooth muscle remains a mystery, it is believed that they are similar to that of skeletal muscle and involve interactions between thick and thin filaments (Guilford eta!., 1998). Thick filaments are composed of myosin class II monomers polymerized into filamentous form. They have an average diameter of 15-20 urn (Kuo et al., 2003). A myosin monomer is around 520 kDa and is made up of 6 polypeptides: two 205 kDa heavy chains, two 20 kDa regulatory light chains, and two 17 kDa essential light chains (Adeistein eta!., 1996), as shown in Figure 1-2. Each myosin heavy chain has a globular head region and an alpha helical tail region. The head has two important sites: an actin binding site and an ATP binding site. The actin binding site is the location at which myosin crossbridges connect to the thin filaments. ATP is hydrolyzed in the ATP binding site in order to provide energy for contraction. The alpha helical tail joins with other tails from other myosin monomers to form the backbone of thick filaments and is responsible for transmission of force generated by the myosin heads. Thin filaments are composed of polymers of 42 kDa G-actin monomers and they are around 6-7 urn in diameter. Thin filaments are double stranded helixes and have complete turns every 36 urn (Small et al., 1983). JnASM cells, actin exists in four isoforms: 7 contractile cx. and y and cytoskeletal and 6 isoforms (Kabsch et al., 1992). It is believed that since actins of different isoforms have different affinities to actin binding molecules, they are localized in different regions of the cell and serve different purposes (North et al., 1994). 8 Motor Domain ATP Binding Site Neck Domain Reg u atory Light Chain Actin Binding Region Essential Light Chain Figure 1-2. Crystal structure of the head and neck region of myosin. 9 1.4 Mechanism of Muscle Contraction The sliding filament, crossbridge mechanism similar to that of skeletal muscle is believed to be operative in smooth muscle (Hanson et al., 1953). The most common model for crossbridge cycling involves a four-step mechanism. The very first step occurs when thick and thin filaments are separated. The myosin heads of the thick filament, with ionically bonded ADP and P1 from hydrolysis ofATP, have high affinity for actin. The next step involves myosin heads binding to thin filaments. In the third step, the release of ADP and P1 from the ATP binding site facilitates conformational changes in the myosin head that cause sliding of the thin filament relative to the thick filament. In the final step, ATP binds to the myosin head and releases it from the thin filament. The ATP is subsequently hydrolyzed and shifts the conformation of the myosin head back to that of step one. The myosin can now repeat the cycle for as long as the muscle cell is activated and ATP is available. 10 1.5 The Role of Calcium Like skeletal muscles, calcium plays a major role in initiating smooth muscle contraction. However, smooth muscle handles calcium differently than skeletal muscle. In skeletal muscle, crossbridge cycling is activated via the troponin system. Ca2 binds to troponin, which is a component of the thin filament, and exposes the myosin binding sites on thin filaments. Myosin heads can then bind to these sites and undergo crossbridge cycling. However, there is no troponin in smooth muscle cells. Figure 1-3 illustrates how smooth muscle utilizes calmodulin to regulate itsCa2-dependent activity. During activation, intracellular Ca2 concentration rises and four calcium ions bind to one calmodulin. TheCa2-ca1modulin complex then binds to the myosin light chain kinase (MLCK) and renders it active. The activated MLCK then phosphorylates the regulatory light chain (RLC) of the thick filament to initiate actin-induced myosin ATPase activity. As a result, crossbridge cycling occurs and the muscle contracts. When intracellular Ca2 concentration decreases, myosin light chain phosphatase (MLCP) activity supercedes MLCK activity and dephosphorylation of RLC occurs. The muscle becomes relaxed and is ready for the next round of activation. 11 The Ca2 needed for activation is generally obtained from the SR. Stimuli such as acetylcholine (ACh) and angiotensin II bind to surface M3 muscarinic receptors, which in turn couples to Gq and phosphatidylinositol-specific phospholipase C (P1-PLC) to produce inositol 1 ,4,5-triphosphate (1P3) and diacylglycerol (DAG) (Biancani et a?., 1994). 1P3 can then stimulate SR to release its calcium content. Since Ca2 is such a critical second messenger to contraction, it is equally important to remove it from the cell cytosol in order to maintain proper homeostasis inside the cell. Years of research have led to a number of proposed mechanisms: Plasma membrane Ca2 ATPases, which use ATP to pump Ca2 out of the cell; Sarcoplasmic/Endoplasmic Reticulum Ca2 ATPases, which 2+ • + 2+ + +transport Ca back mto the SR, Na -Ca -exchangers coupled to the Na -K ATPases, which has an overall function of transporting three Ca2 ions out of the cell and two K ions into the cell, and the mitochondria (Casteels eta?., 1986; Somlyo et a?., 1989; Karaki eta?., 1997). 12 INACTIVE Ac tin Tropomyosin Caldesmon (Cald) courtesy of King, M.W, 1996 Figure 1-3. Signal transduction pathway of Ca2 dependent activation through RLC phosphorylation A TP M PKA ADP Calmodulin + 4Ca’ 4Ca’ Calmodulin (C aCM) 1 CaCM-Cal d ACTIVE Actin Tropomyosin A TP ADP ph osp ho-MLCK low CaCM affinity INACTIVE Myos in p-light chain /MLCPACTIVE Myos in phosp ho-p-light chain 13 1.6 Lability of Smooth Muscle Thick Filament Many studies have shown that the assembly of myosin molecules in smooth muscle is different from skeletal muscle (Craig et al., 1977). Electron Microscopy (EM) studies have shown that thick filaments obtained from smooth muscle have no “central bare zone”. The “central bare zone” observed in skeletal muscles is due to myosin molecules reversing their polarities at the center of the thick filaments. The result is an area where there are only myosin tails but no myosin heads (Onishi et a!., 1978) and this filament structure is termed “bi-polar”, as apposed to the “side-polar” structure of smooth muscle myosin filaments. In addition, smooth muscle thick filaments have been reported to be less stable than those of skeletal muscle, as early microscopists (in the 1950’s and 1960’s) often had difficulties seeing them in smooth muscle cells fixed for EM (Kelly et a!., 1969). To further understand such phenomenon, researchers have attempted to study artificially grown filaments under physiological conditions. These studies have discovered that, in accordance with the early EM observations, thick filaments grown from skeletal muscle myosin monomers are more stable than those formed from smooth muscle myosin monomers (Onishi et a!., 1978). Another interesting observation is that skeletal muscle myosins readily assemble into bi-polar filaments while smooth muscle 14 myosins form side-polar filaments (Onishi eta!., 1978). The concentration of ATP has a major bearing on the stability of these synthetic thick filaments. MgATP concentration in tM range is observed to disassemble smooth muscle thick filaments (Onishi et a?., 1978) while a thousand-fold increase is necessary to disassemble skeletal muscle thick filaments (Harrington et a?., 1972). To examine whether this difference in structural integrity is related to the way myosin monomers are assembled into thick filaments, Suzuki et a?. (1978) grew both hi-polar and side-polar thick filaments from purified smooth muscle myosin monomers through special dilution techniques and assessed their stabilities at various MgATP concentrations. They noticed that these filaments, regardless of their configurations, are readily dissembled by stoichiometric amount of MgATP. Therefore, they concluded that it was the innate differences between smooth muscle and skeletal muscle myosin molecules, not their configuration in filamentous form, was responsible for the difference in integrity. Another characteristic display of smooth muscle myosins is their unusual shape when disassembled into monomeric form by high concentration of MgATP. Myosin monomers in skeletal muscle have a sedimentation coefficient of 6S (Suzuki eta?., 1978). 15 However, disassembled smooth muscle myosins have a sedimentation coefficient of lOS. At first glance, one would predict that these are actually dimers instead of monomers. Yet, EM studies have revealed that these lOS species are indeed monomers but in a different conformation. 6S monomers are those that have extended tail regions while los monomers appear to have tails that are folded into equal thirds, which is a unique property present only in smooth muscle myosin (Onishi et al., 1982; Craig et a!., 1983). The cause of the folding tails is not well understood but it has been speculated that as ATP binds to the myosin head, it shifts its orientation, thus leading to the lOS state and facilitates filament disassembly (Onishi et a!., 1982). 16 1.7 Regulatory Light Chain Phosphorylation and Thick Filament Formation Although smooth muscle thick filaments are not as stable as those of skeletal muscle, phosphorylation at a serine residue in just one of the two RLC’s can tremendously increase their structural integrity (Trybus et at’., 1985). In fact, phosphorylated smooth muscle thick filaments can withstand the same MgATP concentration as skeletal muscles without disassembling (Onishi eta!., 1978). In addition, disassembled lOS monomers can be induced to re-form into thick filaments by phosphorylating RLC with MLCK. This suggests that RLC phosphorylation not only initiates crossbridge cycling, but is also a critical step in thick filament formation (Onishi eta!., 1978). To further understand smooth muscle thick filament assembly, Trybus et al. (1987) attempted to form thick filaments by dialyzing smooth muscle myosin against low ionic strength buffer and evaluating their stability versus that of skeletal muscle. The results showed that skeletal muscle myosin preferentially forms 32S filaments under a wide range of conditions but smooth muscle exhibits no such preference and forms filaments that range from 1 5-30S (Reisler et a!., 1986). It has been observed that antiparallel dimers with one-third overlap at the tail regions are especially prominent (Trybus et a!., 1987). 17 This phenomenon has great implication in both skeletal and smooth muscle thick filament stabilities. Since skeletal muscle thick filaments are bi-polar in structure, the most stable part of the entire filament is the central bare zone, where myosin tails participate in antiparallel interactions. At the same time, such configuration puts a size limit on these filaments at 0.5 im because longer filaments will involve having myosin tails in parallel, which is relatively less stable (Craig et al., 1977). In contrast, smooth muscle thick filaments have no such restraint. Gradual dilution of the buffer solution leads to steady increase in filament length. This implies that smooth muscle thick filaments are antiparallel and are lengthened by step-wise addition of other antiparallel dimers. Moreover, this confirms that electrostatic interaction between myosin tails is the major factor in determining the size of smooth muscle thick filaments (Josephs et al., 1968). Apart from providing stability, the RLC has yet another function which is to amplify the 0.5 nm conformational change of the myosin head during crossbridge cycling into a 10 nm power stroke (Stephens et al., 1998). Unfortunately, even though it is well known how the RLC affects the properties of smooth muscle myosin, there is still no structural data in the submolecular level to confirm these findings. 18 1.8 Effect of Regulatory Light Chain Phosphorylation in Intact Smooth Muscle It has been well established in in vitro experiments that RLC has stabilizing effects on smooth muscle thick filaments. It is still crucial to determine whether the effects are preserved in intact smooth muscle cells. EM images of intact ASM cells have indicated that thick filaments exist even in the relaxed muscles, where RLC phosphorylation is supposed to be minimal (Somlyo et al., 1981). It is not clear whether RLC phosphorylation is required for initiating filament formation in intact muscle. However, it is certain that RLC phosphorylation can facilitate filament assembly. Manipulating MgATP concentration in intact smooth muscle cells to assess thick filament stability is not feasible with the currently available technologies. Fortunately, mechanical perturbation applied to intact muscle cells has been shown to be a viable alternative for disassembling the thick filaments. By applying length oscillation to tracheal smooth muscle in the relaxed state, Kuo et al. (2001) were able to show that the thick filament mass in the muscle cells decreased immediately after the oscillation, matching a similar decrease in isometric force, and both the force and filament mass returned to the pre-oscillation level after a period (25 mm) of recovery during which the 19 muscle was stimulated briefly (12 seconds) at 5-mm intervals. In a separate study, Qi et al. (2002) have demonstrated that RLC phosphorylation by MLCK is necessary for both muscle contraction and filament formation. By using wortmannin, a potent MLCK inhibitor, they observed partial dissolution of thick filaments, similar to the experiment of Kuo et al. (2001). In the presence of wortmannin, both thick filament mass and muscle force do not recover after length oscillation, unlike that observed by Kuo et a!. (2001). But once the wortmannin is removed, thick filament mass recovers to the pre-oscillation level, and muscle force recovers almost totally. The results suggest that the facilitation of MLC phosphorylation in thick filament formation shown in vitro is preserved in vivo. An important note is that although wortmannin is used extensively as a MLCK inhibitor, it is not specific and can also inhibit phosphatidylinositol 3-kinase and mitogen-activated protein kinase. Therefore, the results observed can be due to pathways downstream of these two enzymes. All in all, our understanding of smooth muscle remains limited and more research is needed in order to unmask the mysteries behind it. 20 1.9 ASM and Related Diseases One of the most well known syndromes associated with ASM is asthma. Asthma is characterized by excessive airways shortening in response to agonists (Woolcock et al., 1984). However, the mechanisms that lead to the syndrome remain unknown. Some suggests that ASM increase in number and collectively result in greater tendency for the airways to narrow (Lambert eta!., 1993). Others have speculated that a shift ofASM phenotype into one that has greater shortening capacity is responsible for excessive airway narrowing during an asthma attack (Halayko et al., 1994). Whichever is the case, given the intimate relationship between RLC phosphorylation, force generation, and thick filament formation, it is logical to assume RLC phosphorylation plays a key role in the pathogenesis of asthma. Furthermore, deep inspiration has been shown to have bronchodilative effect in normal ASM due to stretch-induced break down of crossbridge attachment andior thick filaments themselves (Wang et a!., 2000). In asthmatics, such protection by deep inspiration may be compromised and results in excessive shortening. It is not known whether asthamatics’myosin filaments are more stable, and therefore more resistant to length perturbation, compared with myosin filaments from normal ASM. 21 However, it is know that the extent of RLC phosphorylation appears to be greater in ASM from asthmatic airways due to an increased content of MLCK (Ma et aL, 2002; Ammit et al., 2000). The results reported in this thesis research regarding the effect of RLC phosphorylation on the structural stability of myosin filaments therefore have direct relevance to asthma. 22 CHAPTER 2 Hypothesis and Specific Aims As discussed in the Introduction, one of the key features of smooth muscle is myosin filament evanescence, the ability of the filaments to break down and then reassemble. It has been demonstrated that RLC phosphorylation is a key regulator in both crossbridge activation and thick filament formation. However, the actual physical and morphological differences between thick filaments formed with and without RLC phosphorylation remains to be explored. The goal of this thesis project is to investigate the mechanical and structural changes that RLC phosphorylation has on myosin thick filaments. We attempt to answer the following questions: 1. Since RLC phosphorylation facilitates thick filament formation and prevents disassembly by MgATP, do phosphorylated filaments possess greater physical integrity than their non-phosphorylated counterparts? 2. Are phosphorylated thick filaments structurally different than non-phosphorylated ones? 23 3. How quickly does RLC phosphorylation occur upon activation of myosin filaments in vitro? 4. What implications do our findings have on ASM related diseases/syndromes such as asthma? From the literature review (described in Introduction) it can be concluded that the thick filaments in ASM is labile and the lability may be important in allowing the muscle to be malleable and therefore able to adapt to different lengths. What regulates the thick filament lability is not entirely clear, although RLC phosphorylation may be involved. However, how RLC phosphorylation affects thick filament stability has never been directly tested. The guiding hypothesis of my thesis research is that RLC phosphorylation enhances the physical integrity of the thick filaments. The following specific aims are designed to test the hypothesis: 1. RLC phosphorylation has a stabilizing effect on myosin thick filaments, so phosphorylated thick filaments are more resistant to mechanical perturbation than their non-phosphorylated counterparts. 2. Since phosphorylated thick filaments are more stable, their monomers are 24 organized more tightly than non-phosphorylated thick filaments. 3. Previous studies have shown the ability of smooth muscles to adapt to length changes in a matter of minutes, so RLC phosphorylation must happen quicker than that in order to prepare the cell for structural reorganization and contraction. 25 CHAPTER 3, Materials and Methods 3.1 Experiment Procedure The overall experimental procedure is briefly described in this section; detailed procedures for specific methods are described under separate subtitles. Fresh bovine tracheas were obtained from a local abattoir immediately after the animals were sacrificed. The smooth muscle tissue was carefully dissected and stored in ice-cold physiological saline solution (PSS) with the following composition (in mM): 118 NaCl, 4.5 KC1, 1.2 NaHPO4,22.5 NaHCO3,2 MgSO4,2 CaC1, and 2g/1 dextrose. The tissue pieces were pulverized in liquid nitrogen and, after thawing, extensively washed to prepare myofibrils from which actomyosin was extracted according to the methods of Sobieszek and Bremel (Sobieszek and Bremel, 1975). This actomyosin was immediately subjected to ammonium sulphate fractionation to separate myosin and actin filament fractions as described previously by Sobieszek (Sobieszek, 1994). Myosin filaments were formed from purified myosin monomers in solution during slow dialysis that gradually decreased the ionic strength of the solution. Phosphorylated 26 and non-phosphorylated myosin filaments were grown in solutions with or without calcium. To assess filament stability, suspensions of phosphorylated and non-phosphorylated myosin filaments were subjected to brief ultrasonification and the extent of filament breakdown was visualized and quantified with atomic force microscopy (AFM). The filament length was measured using ImageJ 1 .36b software (National Institutes of Health, Bethesda, USA). The time course of RLC phosphorylation was determined using urea/glycerol gel (Sobieszek and Jertschin, 1986). For visualization of myosin filaments in intact ovine trachealis using electron microscopy, smooth muscle cell bundles were dissected from the tracheas. The muscle strips were then preconditioned (equilibrated) in PSS (37°C, aerated with 95% 02, 5% CO2 to maintain a pH of 7.4) for one hour during which the muscles were stimulated electrically once every 5 minutes for 12 seconds (bipolar sinusoidal wave, 60 Hz, 20 V). For muscle strips fixed in the activated state, ACh (1 0 M) was used for activation (after the muscles had been preconditioned) and the muscles were fixed at the plateau of contraction 2 minutes after introduction ofACh into the muscle bath. The reason that ovine (instead of bovine) tissue was used for the EM examination was because the ovine 27 tracheal smooth muscle layer possesses the ideal thickness for chemical fixation. The bovine tracheal muscle tissue was chosen for myosin purification because large amount of smooth muscle tissue could be obtained per trachea. 28 3.2 Myosin Extraction The myosin extraction process must be done the day the animals were sacrificed as myosins could readily degrade over time. The tracheas were cut lengthwise along the cartilage side. Afterwards, the tracheas were opened up with four corners pinned down in 4°C physiological saline (Krebs) solution. To expose the smooth muscle layer, the inner epithelial layer was peeled off. The smooth muscle layer was then carefully dissected out and placed on ice. We strived to remove as much connective tissue as possible both during and after the dissection. When the muscle was clean, we quickly froze it with liquid nitrogen. The tissue pieces were then pulverized with mortar and pestle into a fine powder. The frozen tissue powder was placed in a beaker submerged in warm water to thaw. Myosin has a tendency to degrade if the surrounding temperature is changed slowly, so putting the powder in warm water can rapidly increase the temperature and limit degradation. The tissue paste was then extensively washed to prepare myofibril as described by Sobieszek and Bremel (1975). The tissue paste was subject to 3 washes in order to remove all the contaminants. In the first wash, it was re-suspended using a Polytron (a type of tissue grinder from Kinematica, Newark, USA) in Bis Wash (BW) Buffer (composition in mM: 40 KC1, 2 MgCl, 10 imidazole, 0.5 DTT, and 10 Tris base 29 with pH adjusted to 6.6 at 4°C) with 0.5% Triton-100 to break open the cells and remove the cellular contents. The suspension was then ultracentrifuged at 39,100 x g for 30 minutes at 4°C. The resulting pellet was re-suspended again in BW buffer with 0.3% Triton-X 100 and spun down as before. The resulting pellet was re-suspended the third time in BW buffer and spun down. Actomyosin was then separated from the myofibrils with Low-ionic strength Acto-Myosin Extraction Solution (LAMES) (composition in mM: 90 KC1, 2 EDTA, 2 EGTA, 7.5 Na2ATP, 1 DTT, and 0.16 imidazole with pH adjusted to 7.2 at 4°C). To facilitate separation of actin from myosin, an excess amount of ATP was added to the actomyosin along with MgC12.Actin and other contaminants were precipitated with ammonium sulphate at 40% saturation. The myosin that remained in the supernatant was precipitated with amnionium sulphate at 60% saturation. The myosin pellet was re-suspended in minimal amount of BW buffer and dialyzed overnight with 50% BW buffer and an additional 3 hours with BW buffer the following day. To further purify the myosin, it was re-suspended and spun down at 30,000 x g for 30 minutes at 4°C twice. The purified myosin was frozen with liquid nitrogen in 1 ml aliquots and stored at -80°C. 30 3.3 SDS PAGE SDS PolyAcrylamide Gel Electrophoresis was used to determine the purity and the relative concentration of the purified myosin. In order to visualize both myosin heavy and light chains, a step gradient separating gel of 8% and 12%, with a 4% stacking gel, was used. For liquid samples, 20 1tL of SDS mix (0.45M Tris base, 17.5% SDS, and 7% 3-mercaptoethano1 with pH adjusted to 6.8) and 20 jil of SDS Dye (75% glycerol, 0.075% bromophenol blue) were added to every 100 p1 of sample. For solid samples, they were first dissolved in 8 M urea and then SDS mix and dye were added at the same proportion as liquid samples. After that, the samples were boiled for approximately 1 minute before applying to the gel. Standard amounts of samples were applied to the gel because the samples had varying concentrations. The amounts were as follows (in gil): 5 supematant of first wash, 10 supematant of second wash, 15 supernatant of third wash, 10 crude actomyosin (CAM), 5 myofibrils, 5 40% ammonium sulphate pellet, 20 60% ammonium sulphate supematant, and 5 purified myosin. The gel was run in SDS buffer (25 mM Tris base, 250 mM glycine, 0.1% SDS) in a Bio-Rad Mini-Protean apparatus. For the first 15 minutes, the gels were run at constant 31 current of 30 mA at room temperature. After that, it was switched to 50 mA for approximately 45 minutes or until the dye was just about to leave the gel. When electrophoresis was finished, the gels were removed from the plates and washed with deionized water for 2 x 10 minutes to remove SDS. They were then stained with GelCode Blue Stain Reagent for at least 1 hour and then washed again with deionized water for at least another additional hour. Finally, the gels were analyzed with Odyssey Infrared Imaging System. 32 3.4 Formation of Thick Filaments Approximately 1.3 mg of frozen myosin was re-suspended using a positive displacement pipette in imi of High Ionic Strength (HIS) solution (composition in mM: 5 EGTA, 1 MgC12,5 MgATP, 10 PIPES, sufficient KCL to make 500mM ionic strength, with pH adjusted to 7.0 at room temperature). The myosin solution was further diluted 4x and separated into “relaxed” and “activated” conditions. Figure 3-lA and 1 B illustrate how theCa2-dependent ASM activation pathway is manipulated. In the relaxed condition, 5 mM of staurosporine and 5 mM wortmannin, which were both MLCK inhibitors, were added in order to prevent RLC phosphorylation. In the activated condition, nothing was added. The two solutions were dialyzed separately over 24 hours. The initial external buffer was HIS solution, which had ionic strength of 500 mM. It was decrease to 88 mM over 17 hours with addition of 2 mM MgC12.The myosin solutions were then allowed to further dialyze for an additional 7 hours. After dialysis, the myosin solutions were harvested and kept at 4°C. 5 mM ofATP was added to the relaxing solution. At the same time, 5 mM ATP, 2 mM CaC12,and 100 nM microcystin, a potent MLCP inhibitor, were added to the activating solution to facilitate RLC phosphorylation. Both solutions were divided into two portions and incubated for 2 minutes at 4°C. After 2 33 minutes, one portion from the two conditions was used as control while the other was subjected to ultrasonication. The duration of ultrasonication was 10 seconds at 5.1W using Vibra-Cell ultrasonicator at 4°C. At the end of the experiment, we ended up with four samples: relaxed, relaxed sonicated, activated, and activated sonicated. All four samples were plated on mica surface and visualized under the Atomic Force Microscope (AFM). 34 A TP Calmodulin + Calmodulin aC M) INACTIVE Actin Tropomyosin Caldesmon (Cald) CaCM-Cald ACTIVE lActin [rropomyosin A OP phospho-MLCK low CaCM affinity ADP INACTIVE Myosin p-light chain] /MLCPACTIVE Myos in phospho-p-light chain courtesy of King, M. W, 1996 Figure 3-lA. Signal transduction pathways under relaxed condition. Ca2 is not added to the solution and MLCK is inhibited by staurosporine and wortmannin. This allows MLCP to dephosphorylate all phosphorylated MLC. 35 Calmodulin INACTIVE Myosin p-light chaj ________ ACTIVE courtesy of King, M.W, 1996 Figure 3-lB. Signal transduction pathways under activated condition. Ca2 and ATP are added to facilitate MLCK activation. MLCP activity is inhibited by microcystin to prevent dephosphorylation of MLC. INAC11VE Actin Tropomyosin Caldesmon (Cald? Al? MLCK( ADP phospho-MLCK low CaCM affinity CaCM-Cald ADP ACTIVE Actin Tro porn yes in Myosin phospho-p-light chain 36 3.5 Determination of Regulatory Light Chain Phosphorylation RLC phosphorylation was determined by polyacrylamide urea-glycerol gel electrophoresis. To visualize RLC, 10% polyacrylamide separating gels and 40% glycerol with 4% stacking gels were used. The samples were given 20 il of Urea Dye (8.5M urea, 1% f3-mercaptoethanol, and 1% bromophenol blue) for every 100 il of sample. 20 tl of samples were applied to each well and the gels were run at constant current of 10 mA for 6.5 hours. Due to the voltage limitation of the power source (Biorad) at 300 V, current would drop as voltage reached its maximum. After electrophoresis was finished, the gels were washed with deionized water for 5 minutes and then stained with GelCode Blue Stain Reagent for at least 1 hour. After staining, they were washed again with deionized water for at least 1 hour before being analyzed with Odyssey Infrared Imaging System. 37 3.6 Atomic Force Microscopy (AFM) Visualization of myosin monomer and filaments was done under AFM on mica slides. The mica was taped to normal light microscope slides using double sided tape. To prepare fresh mica, a piece of masking tape was taped on the mica surface and then quickly peeled off. The top layer of the mica would be removed along with the tape, exposing a fresh mica surface. The mica surface was first washed with 5 x 1 ml of deionized water and then blown dry with nitrogen gas. It was then treated with 50 jil of filtered 5 mM MgC12 and incubated for 5 minutes. MgCl2 gave the mica a positively charged surface for myosin filaments, which are negatively charged, to adhere. Care was taken to prevent dust from settling on the mica surface as it would cause problems when scanning with AFM. After incubation, the mica was rinsed with 5 x 1 ml of deionized water and blown dry with nitrogen gas. 50 il of sample was pipetted on the MgCl2 treated mica and incubated for 5 minutes. At the same time, two 1% gluteraldehyde solutions, one made from the final external buffer used in forming relaxed filaments and the other from activated filaments, were prepared. 10 tl of this solution was added to each of their respective samples after the 5 minute incubation. The slides were tilted lightly back and forth for an even mix of the two solutions and let stand for an additional 38 3 minutes. The slides were rinsed with 5 x 90 mM ammonium acetate and then blew dry with nitrogen gas. They were now ready for imaging with AFM in contact mode. For each sample group, 10 images, dimension 10 pm x 10 IIm, were taken at random spots on the slide. The images were analyzed using ImageJ and the myosin filament lengths were manually measured. 39 3.7 Statistical Analysis For analysis of distributions of filament lengths, Kruskall-Wallace test (JMP1N; SAS Institute Inc., Cary, USA) was used to evaluate the significance of differences among the distributions. Comparison of means was done by ANOVA. Values reported in this study were means ± standard errors of the means. 40 CHAPTER 4, Results 4.1 Purification of Myosin and Filament Assembly As described in Methods, a purified native-like myosin preparation was obtained from bovine tracheal smooth muscle. To check the quality of the myosin molecules after the purification process, the molecules were analyzed using SDS-PAGE and imaged under AFM. Figure 4-1 is the SDS-PAGE analysis of the products obtained from each step of the purification process. The dense band on lane 8 is myosin heavy chain and it indicates the high purity of the isolated myosin. Figure 4-2 shows individual non-phosphorylated myosin molecules in solution of high ionic strength (500 mM). The head and tail regions of the molecules can be easily identified, suggesting that the molecules remained intact in terms of their secondary and tertiary structures. The purified myosin molecules were able to form filaments when the ionic strength of the solution was lowered, especially when the RLC was phosphorylated, as shown in Figure 4-3. The purified protein also contained calmodulin (CM), MLCK, MLCP, and the myosin filaments could be phosphorylated by addition of calcium, and dephosphorylation 41 occurred when MgATP became depleted over time due to its hydrolysis by myosin. The time course of phosphorylation is shown in Figure 4-4a and 4b, which indicates that in our preparation, RLC phosphorylation reaches a maximal plateau between 0.5-10 mm. The experiments described immediately below were performed within this time period. 42 •9 I L i- 4 •1 3 4 56 Figure 4-1. SDS analysis of the myosin purification process. Lane 1: first wash; lane 2: second wash; lane 3: third wash; lane 4: CAM; lane 5: myofibrils dissolved in 8 M urea; lane 6: 40%ammonium sulphate pellet dissolved in 8 M urea; lane 7: 60% ammonium sulphate supernatant; lane 8: lx washed myosin. 43 -. Myosn Heavy Chain Figure 4-2: Myosin molecules from bovine tracheal smooth muscle. Purified myosin at 1 ig/m1 were diluted in relaxing solution at high ionic strength (500 mM) and settled onto a mica surface before they were fixed with 0.1% glutaraldehyde. The image was obtained using AFM in tapping mode. The myosin heavy chains (grey curvy lines, —470 nm) are seen with globular myosin heads (bright spots) attached to one end. Other spots without tails are contaminant or partially degraded proteins in the sample. Grey scale: 0-15 nm. Image size: 2 x 2 im. 44 Figure 4-3: Myosin filaments from bovine tracheal smooth muscle imaged by AFM in contact mode. The filaments were formed in solution from purified myosin molecules in activating solution at low ionic strength (80 mM) at a concentration of 2 ig/ml. a) Height image, grey scale: 0-20 nm. b) Same image as in a, but produced from displacement signal of scanning tip of the AFM. This type of image eliminates the effect of unevenness of mica surface and produces a cleaner picture. Image size: 6.7 x 6.7 tm. 45 70 - __________ __________ C) - E 50- 0 - o30- 20- C\J 10- C) 0- Time (mm) Figure 4-4: Time course of myosin phosphorylation and dephosphorylation determined by a) urea-glycerol PAGE and b) y32P incorporation in radioactive counts per thousand per minute (cpm3).MLC2Oa and MLC2Ob are two forms of non-phosphorylated MLC2O (the 20 kD myosin light chain, or RLC). P-MLC2Oa and P-MLC2Ob are two forms of phosphorylated MLC2O. MLC17a and MLC17b are two forms of essential light chain of the myosin. a) .1 • . Time: 0 7.5” 15” 25” 40” 1’ 2255.25’ 12’ 18’ 27’ 40’ b) — MLC2Oa — +— MLC2Ob/P-MLC2Oa 4— P-MLC2Ob - MLC17a .—MLC17b 0 Ca2 I I I I I 0 10 20 30 40 46 4.2 Testing the Physical Integrity of Myosin Filaments Filaments with phosphorylated and non-phosphorylated RLC were subjected to a brief period (10 seconds) of ultrasound agitation (ultrasonification) in solution, and the extent of filament disassembly was examined under AFM. Greater extent of filament breakdown in the non-phosphorylated group compared with the phosphorylated group is shown in Figure 4-5. The breakdown was quantified by measuring the distribution of filament length before and after ultrasonification in both groups, and the results are summarized in Figure 4-6. The total number of filaments measured for each group was 6269 (non-phosphorylated), 10105 (non-phosphorylated and sonicated), 4555 (phosphorylated), and 7532 (phosphorylated and sonicated). Because the experiments were repeated 3 times using tracheal smooth muscle from 3 animals, the measurements from each animal were first averaged before the means (n=3) were averaged. 47 Figure 4-5: Effect of brief ultrasonification on myosin filament stability, a) Filaments formed in relaxing solution with a protein concentration of 2 igIml. b) Same filaments as in Panel a, after 10 s of ultrasonification. C) Filaments formed in activating solution with a protein concentration of 0.5 pg/mI. d) Same filaments as in Panel C, after 10 s of ultrasonification. Image size: 10 x 10 pm. Grey scale: 0-25 nm. 48 0.35 0.30 Cl) C 0) 0.25 E 0.20 0.15 0.10 0.05 0.00 4 Filament Length (jim) Figure 4-6: Distribution of filament length before and after ultrasonification in terms of fractions of the total number of myosin filaments measured versus the ifiament length under the 4 conditions described in Figure 4-5. 0 1 2 3 49 4.3 Measurement of the Number and Diameter of Phosphorylated and Non-Phosphorylated Filaments As shown in Figures 4-7a and 7b, myosin filaments formed in the relaxing solution appear to have larger diameters than those formed in the activating solution. Also, the number of filaments in the activating solution appears to be greater compared with that in the relaxing solution. Counting the number of filaments in 10 x 10 .tm areas (images) randomly sampled by AFM, we found a significant increase in filament number in the activating solution (169±4) compared with that in the relaxing solution (50±2). (20 images were used for each group that included data from 2 separate experiments using different animals, p<0.O5). Measurements of the width of the filaments confirmed that the difference was statistically significant (p<0.05). The results are summarized in Figure 4-8. We have noticed in our electron micrographs of cross-sections of airway smooth muscle that the diameters of myosin filaments in cells fixed in the relaxed state appear larger compared with those fixed in the activated state (Kuo et al., 2003); an example is shown in Figure 4-7c, and 7d. We however have never quantified this difference. In this study we 50 measured the diameter of 120 myosin filaments in EM cross-sections in 3 cells (40 per cell) from tracheal smooth muscle fixed in the relaxed state and equal number of filaments in cells fixed in the activated state, and showed that the difference was statistically significant (p<O.O5) The results are summarized in Figure 4-8. The absolute diameters of the two groups are different. Over estimation of diameter for filaments formed in solution by AFM is an artifact due to the relatively large scanning tip of the AFM compared with the real filament diameter. The diameter ratios (activated/relaxed) are 0.66 and 0.63 for the filaments in solution and in intact cells, respectively. The height measurements by AFM indicated that the average diameter of the filaments laying flat on mica was slight less than 20 nm. 51 Figure 4-7: Myosin filaments imaged by AFM and EM showing the difference in filament diameter in the relaxed and activated state. a) Filaments formed in relaxing solution at low ionic strength (88 mM, 0.5 gIml). Image size: 5 x 5 m. b) Filaments formed in activating solution under the same conditions as in Panel a. Image size: 5 x 5 pm. c) Electron micrograph showing a cross-section of an ovine tracheal smooth muscle cell fixed in the relaxed state. Image size: 0.5 x 0.5 pm. d) Electron micrograph showing a cross-section of an ovine tracheal smooth muscle cell fixed in the activated state. Arrows indicate some selected myosin filaments. Image size: 0.5 x 0.5 pm. 52 70 - 60E _____ L 50 __ _ __ ci) ci) E4° 30 Cu LL 10 0 Filaments in Intact Cells Figure 4-8: Measurement of myosin filament diameter in the four groups described in Figure 4-7. Note that the diameter is reduced in the activated state in both measurements using EM and AFM. See text for more details. ?t*? denotes statistically significant difference (p<O.O5) from respective controls (relaxed filaments). * I I Relaxed ØY% Activated I I Relaxed Activated * Filaments in Solution 53 CHAPTER 5 Discussion 5.1 Summary of Results The role of phosphorylation of RLC in activating the actomyosin interaction in smooth muscle is well known. For myosin in solution, its phosphorylation promotes formation of myosin filaments (Trybus et al., 1987; Kendrick-Jones et al., 1987). The in vivo significance of this in vitro effect of RLC phosphorylation has not been fully explored. The demonstration that the number of myosin filaments increases during contractile activation in some smooth muscle cells (Herrera et al., 2002; Kuo et a!., 2003) suggests that RLC phosphorylation may have a second important in vivo function, and that is, to facilitate formation of myosin filaments. The observation in intact airway smooth muscle that inhibition of RLC phosphorylation abolishes myosin filament reassembly (Qi et a!., 2002) also supports the notion that the in vitro dependence of filament formation on RLC phosphorylation (Trybus et al., 1987; Kendrick-Jones et al., 1987) is likely taking place in vivo. 54 Structural changes and structural integrity of the phosphorylated versus non-phosphorylated myosin filaments are little known. Results from the present study demonstrated that these filaments are different, in both morphology and physical properties. Upon ultrasonic agitation, a 54% decrease in average length is observed in non-phosphorylated thick filaments while a 25% decrease occurs in phosphorylated filaments. Furthermore, the number of filaments that are formed after RLC phosphorylation has an astounding 3.4 fold increase compared to the non-phosphorylated samples. Therefore, such changes in thick filament morphology can be directly linked to the state of RLC phosphorylation. 55 5.2 Purified Myosin and Filament Formation Myosin molecules from tracheal smooth muscle (Figure 4-2) are morphologically similar to those from turkey gizzard (Sheng et at., 2003), except, under our experimental conditions, where the two globular heads (Si) of the molecule were closely apposed (Figure 4-2). The difference could be because of the high ionic strength of our solution; it also could be due to the intrinsic difference in the two isoforms (from two animal species). The close apposition of myosin heads is thought to indicate the conformation of myosin in the inactivated state (Sheng et at., 2003). We did not see the folded tails of non-phosphorylated myosin monomers (Onishi et a!., 1982; Trybus eta!., 1987), instead, we observed extended tails of the molecules (Figure 4-2), as did Sheng et a!. This could be due to the high ionic strength solution we used, although there may be other reasons. Slow dialysis to gradually lower the ionic strength produced long filaments with tapered ends (Figure 4-3). Note that short filaments (—0.5-0.6 jtm) could be formed if the ionic strength of the solution was lowered rapidly (results not shown). The short filaments are likely not the same as the longer filaments found in intact cells (Small, 1977). 56 5.3 Structural Integrity and Morphology of Myosin Filaments Ultrasonification was used to agitate the solution containing myosin filaments. The level of energy for breaking up the filaments was chosen so that some degree of filament disassembly could be observed in both the phosphorylated and the non-phosphorylated groups. The different extents of filament dissolution were quantified by measuring the lengths of the filaments under four conditions (Figure 4-5). We used the fractional length distributions (normalized to the total number of filaments measured) to gauge the extent of filament disassembly, as shown in Figure 4-6. The distribution shifts are all statistically significant (p<O.05) except for the distributions between the non-phosphorylated non-sonicated and phosphorylated non-sonicated groups (p0. 13). The different distributions of the two sonicated groups demonstrated that phosphorylated myosin filaments are more resistant to ultrasonic agitation; we interpret this as due to a tighter packing of myosin molecules in the filament structure that also made the filaments more stable. The notion of thick filaments in tight and flared (loosely packed) conformations originates from Cross et al. (1991). In their observation, two myosin molecules form a 57 dimer (tail-to-tail), and in the tight conformation, these dimers with straight tails are packed into a sheet, whereas in the flared conformation, the curvy tails result in a loosely packed filament with an increased filament diameter. Our hypothesis that phosphorylated myosin filaments are formed with more tightly packed myosin molecules is supported by the measurement of the diameters of myosin filaments in the relaxed and activated states in both synthetic filaments in solution and native filaments in intact muscle cells (Figure 4-7 and 4-8). However, it is not clear whether the relaxed and activated filaments from our experiments are the same as the tightly and loosely packed filaments described by Cross et al. (1991), because the experimental conditions used in the two studies are different, and the status of RLC phosphorylation was not determined in the studies by Cross et at. (1991). A recent study (Smolensky et a!., 2007) provided another explanation for the difference in diameter of myosin filaments. It was suggested that the apparent smaller diameter of the filaments in the activated muscle is due to crossbridges moving away from the myosin filament backbone and attaching to the actin filaments. The resolution of our filament image was not high enough to resolve crossbridges on the filaments; our results therefore cannot offer support to their explanation. We favor the explanation that the increased filament integrity after RLC phosphorylation is due to a 58 change in the mechanical properties of the filament backbone, and is related to the observed change in the filament diameter that reflects a more tightly packed filament structure. We also suggest that the tight and loose conformations of the filaments associated respectively with phosphorylated and non-phosphorylated RLC occur both in solution and in intact cells. Our previous studies (Kuo et al., 2001) showed that large amplitude length oscillation applied to relaxed smooth muscle strips could induce partial dissolution of myosin filaments in intact cells, implying that the filaments in vivo are unstable. Therefore the lability of non-phosphorylated myosin filaments may serve an important physiological role, as discussed below. 59 5.4 Myosin Filament Lability and its Physiological Significance The large volume change of hollow organs such as urinary bladder and stomach suggests that the smooth muscle cells lining the organ wall are able to function over a large length range. By fixing the rabbit urinary bladders at different volumes, Uvelius (1976) has demonstrated a remarkable length range of smooth muscle cells associated with the different volumes. Many studies using different types of smooth muscle have also shown that the muscle is able to generate maximal or near maximal force over a large length range (Harris et a!., 1991; Pratusevich et a!., Wang et a?., 2001; McParland et al., 2005). It is believed that structural malleability of the contractile and cytoskeletal filaments in smooth muscle is what enables the muscle to adapt and function at the extremes of cell lengths (Kuo eta?., 2003; Herrera et a?., 2005, Wang eta?., 2001; Gunst eta?., 1995, Fabry etal., 2001, Bursac eta?., 2005, Deng eta?., 2006). Akey component of the cell malleability may be related to the ability of the myosin filaments to dissolve and reform at different intracellular locations as the cell changes its length (Seow, 2005), in order to maintain optimal overlap between the myosin and actin filaments and thus optimal function of the cell. If evanescence of myosin filaments is part of the mechanism of cell adaptation, then it is important that the stability of the filaments can be regulated. 60 Here we propose that REC phosphorylation is a key step in that regulation. 61 5.5 Myosin Filament Lability and Its Disease Relevance Excessive shortening of ASM is implicated in the pathogenesis of asthma. The mechanisms that lead to the syndrome, however, remain unknown. Some suggest that an increase in ASM cell number results in a greater ability for the airways to narrow (Lambert et al., 1993). Others have speculated that a shift ofASM phenotype to one that has greater shortening capacity is responsible for excessive airway narrowing during an asthma attack (Halayko et al., 1994). Whichever is the case, we believe that the increased ASM shortening in asthmatics may contribute to the observed refractoriness of airways to dilate after a deep inspiration (DI). In normal ASM in vitro, a cycle of stretch and release (mimicking the effect of a DI) caused a temporary decrease in muscle force (Wang et al., 2000). This may explain the transient broncho-dilating effect of DI observed in normal subjects. A possible reason for this normal response could be that DI causes partial dissolution of the filaments. By examining the factors that affect stability of myosin filaments in solution, we discovered that phosphorylated myosin filaments are mechanically more stable than the non-phosphorylated ones, suggesting that if the baseline myosin light chain phosphorylation in asthmatic ASM is higher than normal, the corresponding thick filaments could be more resistant to mechanical disruption and thus 62 contribute to the refractoriness of ASM to DI. 63 Reference Adeistein, R.S., and Sellers, J.R. (1996), Myosin structure and function. In: Barany M, ed. Biochemistry of Smooth Muscle Contraction. 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