Involvement of Both Midzone and Astral Microtubules in Signaling Cytokinesis

J. Cell Biol. 159:45-53 (2002)

While some models assume that astral microtubules are primarily responsible for delivering the ingression signal to the equatorial cortex, other studies strongly implicate midzone microtubules. Aurora B, as a key component for signaling cytokinesis, may serve as a probe for understanding this transport mechanism. In addition to direct imaging, the movement and function of aurora B may be dissected using photobleaching recovery (FRAP) and micromanipulation approaches.

FRAP Analysis of Aurora B-GFP Turnover on the Centromeres of a Prometaphase Cell

Fluorescence of bleached centromeres recovers soon after bleaching, indicating that there is a cytoplasmic pool of aurora B that undergoes exchange with those at centromeres.  Recording time, 220 sec.

Stable Association of Aurora B at the Midbody

The lower half of this midbody was bleached with a laser beam. Contrary to the observations at centromeres, there is little recovery of fluorescence after photobleaching, indicating tight association of aurora B along midzone microtubules. Recording time, 15 min.

Differential Delivery of Aurora B to the Lateral Edges and Interior Regions of the Division Plane

In this key experiment, a large laser beam was used to bleach GFP-tagged aurora B near all separated chromosomes during early anaphase.  Few fluorescent structures emerge in the midzone, consistent with the low mobility of aurora B along midzone microtubules. More importantly, concentrated fluorescence develops along lateral ingressing edges, indicating that there is a separate source and delivery mechanism, which is dependent on astral microtubules as indicated in a separate experiment using low dose nocodazole treatment. Recording time, 8 min.

Requirement of Midzone Signals for Cortical Ingression in Control Cells

As in the previous study (Cao and Wang, 1996), perforation at metaphase or early anaphase near the metaphase plate (phase-light region to the left of chromosomes) causes inhibition of cortical ingression on the side blocked from the mitotic spindle.  The perforation subsequently migrates toward the upper right as a result of biased ingression activities on the right hand side.  Immunostaining shows aurora B concentration only on the ingressing side. Recording time, 10.5 min. 

Overcoming Blockage of Midzone Signals by Overexpressing Aurora B

Similar perforation (phase-light region to the right of chromosomes), when performed on a cell expressing a large amount of aurora B, fails to inhibit cytokinesis. Ingression occurs on both sides of the perforation, causing the perforation to extend into a wide slit.  Immunostaining indicates aurora B concentration on both sides of the perforation. Recording time, 12 min..