Dynamic redistribution of calmodulin in HeLa cells during cell division as revealed by a GFP-calmodulin fusion protein technique.
It has been suggested by many studies that Ca2+ signaling plays an important role in regulating key steps in cell division. In order to study the down stream components of calcium signaling, we have fused the gene of calmodulin (CaM) with that of green fluorescent protein (GFP) and expressed it in HeLa cells. The GFP-CaM protein was found to have similar biochemical properties as the wild-type CaM, and its distribution was also similar to that of the endogenous CaM. Using this GFP-tagged CaM as a probe, we have conducted a detailed examination of the spatial- and temporal-dependent redistribution of calmodulin in living mammalian cells during cell division. Our major findings are: (1) high density of CaM was found to distribute in two sub-cellular locations during mitosis; one fraction was concentrated in the spindle poles, while the other was concentrated in the sub-membrane region around the cell. (2) The sub-membrane fraction of CaM became aggregated at the equatorial region where the cleavage furrow was about to form. The timing of this localized aggregation of CaM was closely associated with the onset of cytokinesis. (3) Using a TA-CaM probe, we found that the sub-membrane fraction of CaM near the cleavage furrow was selectively activated during cell division. (4) When we injected a CaM-specific inhibitory peptide into early anaphase cells, cytokinesis was either blocked or severely delayed. These findings suggest that, in addition to Ca2+ ion, CaM may represent a second signal that can also play an active role in determining the positioning and timing of the cleavage furrow formation.
Li
C J
CJ
Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.
Heim
R
R
Lu
P
P
Pu
Y
Y
Tsien
R Y
RY
Chang
D C
DC
D002147:Calmodulin / Q000378:metabolism*; D002453:Cell Cycle; D002455:Cell Division / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism*; D008938:Mitosis; D008954:Models, Biological; D011993:Recombinant Fusion Proteins; D013997:Time Factors
D002147:Calmodulin; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins