Quantitation of the distribution and flux of myosin-II during cytokinesis.
BACKGROUND
During cytokinesis, the cell's equator contracts against the cell's global stiffness. Identifying the biochemical basis for these mechanical parameters is essential for understanding how cells divide. To achieve this goal, the distribution and flux of the cell division machinery must be quantified. Here we report the first quantitative analysis of the distribution and flux of myosin-II, an essential element of the contractile ring.


RESULTS
The fluxes of myosin-II in the furrow cortex, the polar cortex, and the cytoplasm were examined using ratio imaging of GFP fusion proteins expressed in Dictyostelium. The peak concentration of GFP-myosin-II in the furrow cortex is 1.8-fold higher than in the polar cortex and 2.0-fold higher than in the cytoplasm. The myosin-II in the furrow cortex, however, represents only 10% of the total cellular myosin-II. An estimate of the minimal amount of this motor needed to produce the required force for cell cleavage fits well with this 10% value. The cell may, therefore, regulate the amount of myosin-II sent to the furrow cortex in accordance with the amount needed there. Quantitation of the distribution and flux of a mutant myosin-II that is defective in phosphorylation-dependent thick filament disassembly confirms that heavy chain phosphorylation regulates normal recruitment to the furrow cortex.


CONCLUSIONS
The analysis indicates that myosin-II flux through the cleavage furrow cortex is regulated by thick filament phosphorylation. Further, the amount of myosin-II observed in the furrow cortex is in close agreement with the amount predicted to be required from a simple theoretical analysis.
Robinson
Douglas N
DN
Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307, USA. Douglas.Robinson@jhu.edu
Cavet
Guy
G
Warrick
Hans M
HM
Spudich
James A
JA
D000818:Animals; D002455:Cell Division / Q000235:genetics / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D003593:Cytoplasm / Q000737:chemistry; D003599:Cytoskeleton / Q000737:chemistry; D004023:Dictyostelium / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008938:Mitosis / Q000235:genetics / Q000502:physiology; D024462:Myosin Type II / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000737:chemistry; D014162:Transfection
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D024462:Myosin Type II