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Cortical Dynamics of Actin and Myosin II during Early Cytokinesis Detected with TIRF Optics

Zhou and Wang., Mol. Biol. Cell. 19:318-326 (2008)

Equatorial concentration of actin and myosin II takes place during early cytokinesis and is believed to represent an important step of the process. Observations of these cortical events have been difficult due to the round cell shape and the high background signals from the cytoplasm. To overcome this limitation, well-spread Normal Rat Kidney epithelial cells were transfected with GFP-tagged myosin IIA, GFP-tagged actin, or mCherry-tagged actin, and observed with total internal reflection fluorescence optics that detect structures within 100-200 nm from the glass surface. The observations were limited to early cytokinesis since the increasing distance between the glass and cell cortex during cytokinesis causes the signal to disappear.

Dynamics of Myosin II during the Assembly of Equatorial Cortex

No directed flow of cortical myosin is detectable. Some myosin dots in the equatorial region gain intensity, while other dots form de novo. Enlarged view of the equatorial region is shown to the right. Recording time, 33 sec.

Domains of Myosin II Assembly in the Equatorial Region

The normalized rate of intensity change is reflective of reaction kinetics, and may be visualized with temporal differential microscopy (TDM, right; regions of assembly are shown in orange and regions of disassembly in blue).  Dynamic domains of myosin assembly/association appear throughout the cortex and some appear to travel across the surface.  Disassembly is suppressed along the equator during cytokinesis, which accounts for the net intensity increase.   Recording time, 87 sec.

Flux of Actin Filaments toward the Equator

Unlike myosin II, actin shows a striking flux toward the equator during early cytokinesis. The juxtaposition of stationary and moving structures argues against a global cortical flow process. The left panel was recorded with a cell expression GFP-actin. The right panel, recorded with cells expressing m-Cherry actin, shows highly active ruffling in a neighboring cell in addition very active flux. Unlike myosin, actin shows no dynamic domains of assembly. Recording time, 134 sec (left), and 70 sec (right).

Dependence of Actin Flux on Myosin II

The cell to the left was treated with a negative control of blebbistatin. Actin flux takes place as in untreated cells. The cell to the right was treated with active blebbistatin, an inhibitor of the myosin II ATPase.  The treatment abolishes actin flux, however equatorial actin concentration remains unaffected at least qualitatively, suggesting that there is a de novo assembly mechanism in addition to the flux. Recording time, 74 sec.