Details

Autor(en) / Beteiligte

• DePina, Ana Santos

Titel

Identification and characterization of a vesicle-associated myosin II in extracts of clam oocytes

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

2000

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• The transport of vesicles and the retention of organelles at specific locations are fundamental processes in cells. Actin filaments and myosin motors have been shown to be required for both of these tasks. The identification of over fifteen classes of myosin motors requires the identification of those members that function as vesicle motors. In order to identify myosins that function as vesicle motors actin-based vesicle transport was investigated in oocyte extracts of the surf clam, Spisula solidissima. Single actin filaments and large actin bundles assembled spontaneously in the extracts. Fast vesicle transport on single actin filaments and actin bundles was observed by AVEC-DIC microscopy. The fast rate of movement suggested that a fast motor such as myosin II was involved in vesicle transport. Immunoblot analysis revealed that myosin II was present in the extracts and associated with isolated vesicles. Movement of isolated vesicles was reconstituted on rabbit muscle actin filaments, suggesting that the vesicle-associated myosin II-like motor was active, and did not require soluble factors from the extract. The vesicle-associated myosin II-like motor was isolated and microsequenced. The peptide sequences obtained showed strong homology to nonmuscle myosin II from various species, confirming that the vesicle-associated motor represented a myosin II motor. Isolated myosin II exhibited low K+-EDTA Chase activity compared to rabbit muscle myosin II. Its activity was inhibited by the general myosin inhibitor BDM, and by ML-7, a myosin light chain kinase (MLCK) inhibitor. Possible roles of myosin II in vesicle transport during embryonic development are discussed.