Identification and organization of the cytoskeleton in the alga Vaucheria longicaulis var. macounii
Creators: name: Peat, Lucinda Jane
Publisher: name: University of British Columbia
Year: 2009

description: The presence of the cytoskeletal proteins actin and tubulin in the alga Vaucheria lonqicaulis Hoppaugh var. macounii Blum is studied by SDS-PAGE and immunoblotting techniques. These techniques also indicate the presence of the mechanomotor protein myosin. The overall organization of the cytoskeleton in the cytoplasm of intact filaments, their identification and distribution, are investigated by immunofluorescence and epifluorescence microscopy using monoclonal anti-B-tubulin and anti-actin antibodies and FITC-labelled phalloidin. ' Anti-myosin antibodies were also utilized, but proved to be inadequate for my work. Phalloidin labelling of F-actin proved to be more suitable for visualizing microfilaments than anti-actin antibodies. Phalloidin labelling of F-actin reveals a dense array of microfilament cables in the cortical cytoplasm of vegetative filaments, which appear to be sub-divided into two morphologically distinct sets. One set consists of straighter elements, preferentially occupying the cytoplasm adjacent to the plasma membrane and possibly providing tracks for organelle motility. A second set is made up of wavy elements and extends deeper into the cytoplasm where it may be part of the force generating system responsible for organelle motility. Immunofluorescence for tubulin reveals that the microtubule array is much less dense than that made up of microfilaments. Microtubule bundles appear shorter and straighter and are located throughout the width of the cytoplasm. They show no particular relationship to organelles, except nuclei. With respect to nuclei, they seem to be involved in their arrangement within the filament, particularly in the apical region, and this may have implications for the organization of the tip-growth processes. High resolution scanning electron microscopy is also utilized in the study of the organization and distribution of the cytoskeleton. Differential interference contrast microscopy reveals cytoplasmic tracks originating from focal regions in living vegetative filaments of the coenocytic alga Vaucheria longicaulis var. macounii. Fluorescein-labelled phalloidin staining also reveals regions (foci) of similar structure and dimensions among the F-actin array. Immunofluorescence using monoclonal antibodies to B-tubulin shows punctate fluorescence in association with the microtubule array. Cytochalasins are used to breakdown the F-actin array, an effect that is concentration dependent. Cytochalasin D causes a gradual breakdown of the F- actin array, revealing the close association between foci and F- actin fluorescence. Recovery from treatment with this inhibitor confirms this association and suggests that foci act as organizing centers for the F-actin array. Cold temperature, Oryzalin and taxol are used to disrupt the microtubule bundle array. Depolymerization is evident in the appearance of many fluorescent spots (foci), which may be co-localized with nuclei or associated with the ends of microtubule bundles. Recovery from these treatments suggests that spot-like foci act as nucleation centers for microtubule bundles. The existence of microtubule-associated foci is supported by the results of taxol treatment. The different roles played by microfilaments and microtubules in the organization of the polarized structure of the cell are discussed with respect to the function of their respective organizing centers.