Cell cycle-regulated trafficking of Chs2 controls actomyosin ring stability during cytokinesis - PubMed (original) (raw)

Cell cycle-regulated trafficking of Chs2 controls actomyosin ring stability during cytokinesis

Lynn VerPlank et al. Mol Biol Cell. 2005 May.

Abstract

Cytokinesis requires the coordination of many cellular complexes, particularly those involved in the constriction and reconstruction of the plasma membrane in the cleavage furrow. We have investigated the regulation and function of vesicle transport and fusion during cytokinesis in budding yeast. By using time-lapse confocal microscopy, we show that post-Golgi vesicles, as well as the exocyst, a complex required for the tethering and fusion of these vesicles, localize to the bud neck at a precise time just before spindle disassembly and actomyosin ring contraction. Using mutants affecting cyclin degradation and the mitotic exit network, we found that targeted secretion, in contrast to contractile ring activation, requires cyclin degradation but not the mitotic exit network. Analysis of cells in late anaphase bearing exocyst and myosin V mutations show that both vesicle transport and fusion machineries are required for the completion of cytokinesis, but this is not due to a delay in mitotic exit or assembly of the contractile ring. Further investigation of the dynamics of contractile rings in exocyst mutants shows these cells may be able to initiate contraction but often fail to complete the contraction due to premature disassembly during the contraction phase. This phenotype led us to identify Chs2, a transmembrane protein targeted to the bud neck through the exocytic pathway, as necessary for actomyosin ring stability during contraction. Chs2, as the chitin synthase that produces the primary septum, thus couples the assembly of the extracellular matrix with the dynamics of the contractile ring during cytokinesis.

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Figures

Figure 1.

Figure 1.

The exocytic machinery arrives at the bud neck before cytokinesis. (A) Representative images of RLY1994 (Myo1-GFP, Tub1-GFP), RLY1996 (Sec3-GFP, Tub1-GFP), and RLY1997 (Sec4-GFP, Tub1-GFP) captured by time-lapse spinning disk confocal microscopy and shown as 2D projections from the collected z-series. Cells were chosen for imaging by the appearance of an elongated spindle (noted by *), indicating the cell is in anaphase (time point 0). To establish the timing of cytokinesis, actomyosin ring contraction was compared with spindle disassembly (top). Myo1-GFP is indicated by an arrow. Localization of Sec3-GFP and Sec4-GFP in the middle and bottom panels (denoted by the arrowheads) occurs before spindle disassembly, which is highlighted by a box drawn around the aligned images. Images were taken at 30-s intervals in all time-lapse series. Bars, 2 μm. (B) Data compiled from all the time-lapse series show the timing of the localization of the exocytic machinery to the bud neck before contraction of the actomyosin ring and spindle disassembly. (C) RLY2000 and RLY2001 cells imaged 30–45 min after release from nocodazole arrest, at the nonpermissive temperature of 37°C. Bar, 5 μm.

Figure 2.

Figure 2.

Mitotic exit is required for targeted exocytosis at the bud neck. RLY2066, RLY2065 (A) and RLY2003, RLY2005 (B) were arrested with nocodazole in YPGR media to induce expression from GAL-CLB_Δ_DB or an empty GAL plasmid. After release from arrest, samples of the culture were taken every 15 min for viewing of live cells to count for Sec3-GFP or Sec4-GFP localization at the bud neck (▪ and •, left _y_-axis) and morphology (□ and ○, right _y_-axis) to determine cell cycle progression. Cells (200) were counted for each time point, and the averages from three experiments are plotted in A and B. (A) Images of RLY2002 (Sec3-GFP, Tub1-GFP) arrested from overexpression of Clb2ΔDB are shown to the right of the graph. (C) Images of Sec4-GFP in cells with the empty GAL plasmid (RLY2005) and GAL-CLB2_Δ_DB (RLY2003) in YPGR media (a), and WT (RLY2009) and sec3-2 (RLY2008) at 37°C (b), all between 30 and 45 min after release from nocodazole arrest. Bars, 5 μm. RLY1840 (wt) and RLY1838 (sec3-2) were analyzed by EM 30 min after release from nocodazole arrest at 37°C (c). The cells are in anaphase as seen by the nuclei (n). Bars, 0.5 μm. (D) RLY2040, RLY2041, and RLY2042 were arrested with nocodazole in YPD and then elevated to 37°C before release from arrest. After release, samples were taken every 15 min to view Sec3-GFP localization at the bud neck. Cells were counted as in A and B.

Figure 3.

Figure 3.

Disruption of vesicle transport to or fusion at the plasma membrane blocks completion of cytokinesis, but it does not disrupt the cell cycle. (A) RLY1838, RLY1840, RLY1900, RLY1901, RLY1998, RLY1999, RLY2006, and RLY2007 were analyzed for their effect on cytokinesis. Cells were arrested with nocodazole, switched into nonpermissive conditions, and then released from the arrest. Fixed cells were zymolyase treated and were scored at the time of release from the arrest and 2 h after the release for completion of cytokinesis. (B) Localization of Cdc14-GFP was used to show completion of mitotic exit. RLY2012 and RLY2013 cells were lightly fixed in formaldehyde, stained for DAPI to monitor cell cycle, and Cdc14-GFP release to the cytoplasm from the nucleolus was quantified.

Figure 4.

Figure 4.

Actomyosin rings form in the absence of membrane addition at the bud neck. RLY1838, RLY1840, RLY1998, RLY1999, RLY2006, and RLY2007 were synchronized and subjected to the nonpermissive conditions as described in text. Samples were taken every 15 min after release from arrest and fixed with formaldehyde. Fixed cells were stained with rhodamine-conjugated phalloidin and counted for the number of actin rings seen at the bud neck. Images of RLY1838, RLY1999, and RLY1840 stained with rhodamine phalloidin at the 30-min time point are shown to the right of the graphs. Bar, 5 μm.

Figure 5.

Figure 5.

Abnormal actomyosin ring contraction in exocyst mutants. RLY1898, RLY1899, RLY2010, and RLY2011 were treated with nocodazole at the nonpermissive temperature (37°) as discussed in text. At the time of release, cells were prepared for time-lapse imaging, at 37°C. Imaging began as cells reached anaphase, indicated by the appearance of an elongated spindle (time 0). (A) Still images from examples of three time-lapse series produced from reconstruction of z-images of each time point. Initiation of cytokinesis is noted by contraction of the actomyosin ring (arrow) and disassembly of spindle (*). Contraction proceeds until the Myo1-GFP signal is seen as a point in WT cells (top) or as a smaller band in exocyst mutants (middle and bottom), before disappearing altogether. Bars, 2 μm. (B) Kymographs of the Myo1-GFP signal for WT, sec3-2, and sec10-2 cells. Each horizontal line of the kymograph represents each time point of the image series, derived from a line drawn across the Myo1-GFP signal (shown in drawing).

Figure 6.

Figure 6.

Chs2 is targeted to the plasma membrane of the bud neck before cytokinesis. (A) Relative intensity profiles of three representative time-lapse series of RLY2062 (Tub1-GFP, Chs2-GFP). Ten cells in total were imaged by acquisition with a spinning disk confocal microscope to provide 2D projection movies, where the intensity of Chs2-GFP was measured for each time point. Time 0 defines spindle disassembly. RLY2060, RLY2061 (B), and RLY2062, RLY2063 (C) expressing Chs2-GFP (pLP31) (Tolliday et al., 2003) were arrested with nocodazole at 25°C. Once arrested, cultures were shifted to 37°C for 30 min and then released from arrest. Samples were taken every 15 min, lightly fixed in 1% formaldehyde for 30 min, and then visualized for Chs2-GFP localization at the bud neck. Examples of the 30-min time points are shown below the graphs. Bars, 5 μm. (D) Samples from the 45-min time point from C were imaged with a spinning disk confocal microscope, deconvolved, and reconstructed to produce 3D images. Bars, 2 μm.

Figure 7.

Figure 7.

Chs2 is required for proper actomyosin ring contraction. Time-lapse images of RLY1450, RLY2011, and RLY2064 were captured with a spinning disk confocal microscope at room temperature, with the exception of RLY2011, which was treated and imaged as discussed for Figure 5. Cells were seen in planes that allowed visualization of actomyosin rings from above. Five planes were captured for every 30-s time point, which encompasses the ring. Still images of example cells are excerpts of time-lapse series, including all the time points from before initiation of contraction or disassembly of the actomyosin ring through to completion. The still images were produced from reconstruction of the z-images for each time point. Arrows indicate the direction of the zipping motion for the mutants. Bars, 2 μm.

Figure 8.

Figure 8.

(A) Cell cycle control of cytokinetic events. Contractile ring activation is controlled directly by the MEN, which promotes CDK1 inactivation but also is inhibited by CDK1. Targeted secretion at cell division site requires CDK1 inactivation but not the MEN. (B) Model for the role of exocytic delivery of Chs2 in cytokinesis. At cytokinesis onset, the septin hourglass, which serves as a scaffold for the actomyosin ring, splits into two separate rings sandwiching the contractile ring. Deposition of Chs2 through vesicles fusion at the bud neck has a necessary stabilizing effect on the actomyosin ring during contraction. CW, cell wall; PM, plasma membrane.

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