Protein phosphorylation regulates actomyosin-driven vesicle movement in cell extracts isolated from the green algae, Chara corallina.
In Characean cells endoplasmic streaming stops upon membrane depolarization accompanied by Ca(2+) entry. We investigated the mechanism of this cessation of endoplasmic streaming by reconstituting the vesicle movement in vitro. In a living cell of Chara corallina, there are a number of vesicles moving along actin cables. Vesicles in the endoplasm squeezed out of the cell into a medium containing Mg-ATP showed directional movements under a dark field microscope. When the extracted endoplasm was treated with 20 nM okadaic acid, vesicles showed only movements like the Brownian motion. When it was treated with 50 nM staurosporine, directional movements of vesicles were activated. These movements were analyzed by image processing of videomicroscopic records. Vesicle movements along F-actin filaments were also observed by merging both images of the same field by dark field microscopy and fluorescence microscopy, indicating that myosin on the vesicle surface was responsible for vesicle movements. We also examined the effects of okadaic acid and staurosporine on in vitro sliding of F-actin on Chara myosin. When Chara myosin was treated with 20 nM okadaic acid in the cell extract, the number of sliding F-actin filaments was greatly reduced. In contrast, it increased when Chara myosin was treated with 50 nM staurosporine. In addition, Chara myosin treated with protein kinase C greatly diminished its motility. These results suggest that inactivation of Chara myosin via its phosphorylation is responsible for cessation of endoplasmic streaming.
Morimatsu
Miki
M
Department of Molecular Biology, School of Science Nagoya University, Chikusa-ku Nagoya, Japan.
Hasegawa
Satoshi
S
Higashi-Fujime
Sugie
S
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002457:Cell Extracts; D000460:Chlorophyta / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D003595:Cytoplasmic Streaming / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018715:Microscopy, Video; D009218:Myosins / Q000378:metabolism* / Q000648:ultrastructure; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013347:Subcellular Fractions; D022161:Transport Vesicles / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure
D002457:Cell Extracts; D004791:Enzyme Inhibitors; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D009218:Myosins