Date

5-26-2009

UMMS Affiliation

Graduate School of Biomedical Sciences, Interdisciplinary Graduate Program

Document Type

Dissertation, Doctoral

Subjects

Cytokinesis; Actins; Myosin Type II; Monomeric GTP-Binding Proteins; Mitosis; Academic Dissertations; Dissertations, UMMS

Disciplines

Cell and Developmental Biology | Life Sciences | Medicine and Health Sciences

Abstract

Myosin II and actin are two major components of the ingressing cortex during cytokinesis. However, their structural dynamics and functions during cytokinesis are still poorly understood. To study the role of myosin II in cortical actin turnover, dividing normal rat kidney (NRK) cells were treated with blebbistatin, a potent inhibitor of the non-muscle myosin II ATPase. Blebbistatin caused a strong inhibition of actin filament turnover and cytokinesis. Local release of blebbistatin at the equator caused inhibition of cytokinesis, while treatment in the polar region also caused a high frequency of abnormal cytokinesis, suggesting that myosin II may play a global role. These observations indicate that myosin II ATPase is essential for actin turnover and remodeling during cytokinesis.

To further study the mechanism of myosin II and actin recruitment to the cytokinetic furrow, equatorial cortex were observed with total internal reflection fluorescence microscope (TIRF-M) coupled with spatial temporal image correlation spectroscopy (STICS) and a new approach termed temporal differential microscopy (TDM). The results indicated at least partially independent mechanisms for the early equatorial recruitment of myosin II and actin filaments. Cortical myosin II showed no detectable directional flow toward the equator. In addition to de novo equatorial assembly, localized inhibition of disassembly appeared to contribute to the formation of the equatorial myosin II band. In contrast, actin filaments underwent a striking, myosin II dependent flux toward the equator. However, myosin II was not required for equatorial actin concentration, suggesting that there was a flux-independent, de novo mechanism.

The study was then extended to retraction fibers found typically on mitotic adherent cells, to address the hypothesis that they may facilitate post-mitotic spreading. Cells with retraction fibers showed increased spreading speed in post-mitotic spreading compared to cells without retraction fibers. In addition, micromanipulation study suggested that retraction fibers may guide the direction of post-mitotic spreading. Focal adhesion proteins were present at the tips of retraction fibers, and may act as small nucleators for focal adhesions reassembly that help cell quickly respread and regrow focal adhesions. These findings may suggest a general mechanism utilized by adherent cells to facilitate post-mitotic spreading and reoccupy their previous territory.

Comments

This dissertation is accompanied by supplemental video files. See Additional Files below.

Video 2.1.mov (904 kB)
The field as shown in Fig. 2.1A recorded over 51 minutes. Circles in the beginning indicate three dividing cells. Even though cytokinesis fails, these cells show striking shape changes throughout the period of division. Both mitosis and post-mitotic spreading appear to take place normally.

Video 2.2.mov (712 kB)
The same sequence as in the upper row of Fig. 2.4A, showing the inhibition of ingression when blebbistatin is applied near the equator.

Video 2.3.mov (598 kB)
The same sequence as in the lower row of Fig. 2.4A, showing the misplacement of ingression when blebbistatin is applied near the pole.

Video 3.1.avi (1218 kB)
TIRF-M of an NRK cell expressing GFP-nonmuscle myosin IIA heavy chain during the assembly of equatorial cortex. No directed flow of cortical myosin is detectable. Enlarged view of the equatorial region is shown to the right.

Video 3.2.avi (1474 kB)
TIRF-M and the corresponding TDM of GFP-myosin in an NRK cell during the assembly of the equatorial cortex. Dynamic domains of myosin assembly/association appear throughout the cortex. Some assembly domains appear to travel across the cell cortex. The total duration of recording is 87 seconds.

Video 3.3.avi (1268 kB)
Recruitment of GFP-myosin to the equator in cell treated with Y-27632. TIRF-M shows that myosin in the equatorial region is very stably associated with cortex, in contrast to dots outside the equator. The total duration of recording is 66 seconds.

Video 3.4.avi (2323 kB)
Equatorial recruitment of GFP-myosin in a cell treated with ML-7. Cytokinesis is delayed. The total duration of recording is 3.75 minutes.

Video 3.5.avi (695 kB)
Equatorial recruitment of GFP-myosin in a cell treated with (- )-blebbistatin. Cortical myosin intensity shows steady increase both in and outside the equatorial region, while cytokinesis is inhibited. The total duration of recording is 34 seconds.

Video 3.6.avi (352 kB)
Equatorial recruitment of GFP-myosin in a cell treated with latrunculin B, despite the inhibition of furrow ingression. Equatorial myosin dots appear to scatter over a wide region than in control cells. The total duration of recording is 4.5 minutes.

Video 3.7.avi (2988 kB)
TIRF-M of an NRK cell expressing GFP-actin during the assembly of equatorial cortex, showing dramatic fluxes of actin filaments toward the equator.

Video 3.8.avi (2550 kB)
TIRF-M of an NRK cell expressing mCherry-actin during the assembly of equatorial cortex, showing dramatic fluxes of actin filaments toward the equator, as well as active ruffles of a neighboring cell. The total duration of recording is 70 seconds.

Video 3.9.avi (3557 kB)
TIRF-M of NRK cells expressing mCherry-actin treated with (+)-blebbistatin (negative control, left) or (-)-blebbistatin (myosin ATPase inhibitor, right). (+)-blebbistatin has no effect on actin flux, while (-)-blebbistatin abolishes actin flux. Despite the inhibition of flux, equatorial actin concentration remains unaffected.

Video 3.10.avi (1548 kB)
TIRF-M and the corresponding TDM of mCherry-actin in an NRK cell treated with (-)-blebbistatin. Equatorial band appears despite the inhibition of actin flux. Bands of de novo assembly activities are visible in the TDM image along the 82 equator during the first half of the recording. The total duration of recording is 75 seconds.

Video 4.1.avi (7680 kB)
Post-mitotic spreading involves rapid expansion and extension of cytoplasm along retraction fibers toward their tips (arrowheads).

Video 4.2.avi (3625 kB)
Retraction fibers guide the direction of post-mitotic spreading.

Video 4.3.avi (7092 kB)
Retraction fibers cannot prevent neighboring cells from moving into the space left by mitotic cell, but help post-mitotic cells regain their space.

Video 4.4.avi (115 kB)
Focal adhesions grow directly from the tips of retraction fibers.