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Study of wound healing and the involvement of the cytoskeleton in Vaucheria longicaulis variety macounii Tornbom, Laurie Jean


Vaucheria longicaulis var. macounii is a coenocytic siphonous member of the Division Chrysophyta. This alga grows in the intertidal zone, and it is exposed to grazing damage by slugs and snails, as well as probable fragmentation and sand abrasion due to wave action. V. lonqicaulis exhibits the capacity to heal large transverse wounds, and this wound healing process is the object of this study. The sequence of events leading to wound healing and resumption of growth are studied by light microscopy as well as the use of differential interference contrast optics. Wound healing has been divided into four stages. In Stage 1, a bi-membranous wound healing vesicle is rapidly extruded from the wound site. Loss of turgor pressure, as observed by the appearance of plasmolysis, is observed along the length of the filament. In Stage 2, the wound healing vesicle is sealed off from the filament by the fusion of the plasma membrane and tonoplast at the wound site. Organelles then accumulate at the membranous barrier created by the sealing off procedure. This is followed by chloroplast retraction and return to the wound site, concurrent with restoration of turgor pressure in late Stage 2.In Stage 3 chloroplasts again retract and return to the wound site, in preparation for Stage 4 that finally leads to the resumption of growth on the part of the healed filament. The effect of wound healing on organelle distribution has been studied with the use of selective fluorochromes. The distribution of nuclei, identified by DAPI staining, remains uniform throughout the wound region. CTC fluorescence along the wound site and wound vesicle cytoplasm implies the importance of Ca²⁺-based exocytosis in both wound healing vesicle expansion and its sealing off from the filament. Mitochondria and probable lipid bodies (designated RBIs) are identified by staining with DiOC6 and Rhodamine B, respectively. They are present in the wound healing vesicle cytoplasm and accumulate along the wound site. The cytoskeleton is identified as strand-like cables or tracks by differential contrast microscopy. The cytoskeleton tracks are oriented parallel to the length of the filament, and the organelles are closely associated and appear to move along them. In uncut filaments, the different classes of organelles move independently of one another and travel at different speeds. Chloroplasts, mitochondria, RBIs, and smaller organelles (SSIs) exhibit a bulk pattern of motion characterized by brief reversals in their primary pattern of movement that do not affect their final direction of travel. Nuclei move independently of each other and do not reverse their direction of movement. In cut filaments, cytoskeleton density and relationship to organelles in the wound region during Stage 1 is similar to that in uncut filaments, and extends into the wound healing vesicle. Mitochondria size organelles move in bulk rapidly toward the wound site and into the wound healing vesicle. In Stage 2, an increase in cytoskeleton density is observed in the wound region, and bulk movement of mitochondria and similar size organelles toward the wound occurs. Nuclei also move in bulk unidirectionally toward the wound. Organelle retraction from the wound is restricted to chloroplasts. Both uncut and cut filaments are treated with the cytoskeleton depolymerizers Cytochalasin B and Oryzalin to determine the role(s) of microfilaments and microtubules in organelle movement and the overall role of the cytoskeleton in the wound healing process. In uncut filaments, cytoplasmic streaming is severely reduced by both Cytochalasin B and Oryzalin, with the greatest reduction observed following Cytochalasin B treatment. Work in cut filaments points to a preferential association between microfilaments and chloroplasts, mitochondria, and RBIs, and microtubules with nuclei during the wound healing process. Consequently, bulk movement of chloroplasts and mitochondria (with temporary reversals) in uncut filaments may be microfilament based, and the independent nuclear movement may be microtubule based.

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