| Calmodulin (CaM) is a major cytoplasmic Ca2+ receptor and modulates many basic cellular processes such as cell proliferation, differentiation, cell survival and cell motility. It has been revealed that the specific temporal and spatial localization of CaM correlates with its specific functions. By using GFP-CaM fusion protein, we have observed the detailed dynamic redistribution of CaM in HeLa cells during cytokinesis. In tri-polar cell model, we have studied the relationship between the distribution pattern of CaM and the formation of the cleavage furrow. Furthermore, we have made the study on the regulation mechanism of CaM during the whole process of cytokinesis by inhibiting its activity at different cytokinesis stages. Our main results are listed below:1 . By using the GFP-labeling technique , we have found that GFP-CaM colocalized with all the characterized structures formed during cytokinesis. After the cell entered cytokinesis, in addition of the spindle poles distributed CaM, another fraction of CaM appeared under the equatorial cortex; then large amount of CaM began to aggregate with central spindle along with the progress of cytokinesis; when the midbody formed, CaM colocalized with the two sides of the midbody; at the final stage of cytokinesis, CaM redistributed evenly in the cytoplasm following the midbody dissolving. This dynamic colocolazation pattern of CaM with different cytokinesis apparatuses indicates that CaM involves in the whole process of cytokinesis.2. The tri-polar cells were induced by Cytochalasin B treatment, by using the tri-polar cell model, it's quite convenient to compare the difference of central spindle structure and the difference of CaM distribution pattern between each two poles in the same cell; moreover, it's easy to see whether these differences would affect the furrow formation. We found that either the abnormal central spindle structure or the absence of central spindle distributed CaM could inhibit the furrow formation. It indicates thatthe central spindle distributed CaM might involve in the regulation of furrow formation. Additionally, we found that CaM either appeared simultaneously at central spindle and the equatorial cortex or was absent simultaneously at those two sites. These phenomena further suggest that there might exist a crosstalk between these two fractions of CaM, and it is possible that this crosstalk might be critical to the furrow formation.3. When the cells were treated with W7, a Ca2+/CaM inhibitor, the central spindle assembly was affected, and the abnormal central spindle was formed; besides, when W7 treatment was applied at an early stage of cytokinesis, the division furrow index would decrease substantially. These results show that CaM is really involved in the regulation of furrow formation and this involvement may be fulfilled by regulating the dynamic structure of central spindle.4. We made a living cell observation on the effect of W7 treatment to the ingression of the cleavage furrow. W7 was added when the cells entered anaphase, we found that although some cells had elongated, the distribution patternof CaM at central spindle was normal, and it seemed that a cleavage furrow would possibly form soon, however, these cells stopped cytokinesis at this stage, no furrow ingression followed. This result suggests that central spindle distributed CaM may also involve in the regulation of furrow ingression. Inhibiting the activity of CaM may interrupt the coordination of central spindle and equatorial cortex, thus block the furrow ingression.5.We have found that CaM and y-tubulin colocolized at the two sides of the midbody. When the cells synchronized at a late cytokinesis stage were treated with W7, the dissolving of the midbody was delayed; furthermore, we found that the dissociation of y-tubulin from the midbody was also inhibited. We suggest that y-tubulin is plausibly a kind of stabilizing factor to the midbody microtubules. The fact that inhibiting the activity of CaM would affect the y-tubulin dissociation from the...
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