Polo-like kinase controls vertebrate spindle elongation and cytokinesis

Abstract

During cell division, chromosome segregation must be coordinated with cell cleavage so that cytokinesis occurs after chromosomes have been safely distributed to each spindle pole. Polo-like kinase 1 (Plk1) is an essential kinase that regulates spindle assembly, mitotic entry and chromosome segregation, but because of its many mitotic roles it has been difficult to specifically study its post-anaphase functions. Here we use small molecule inhibitors to block Plk1 activity at anaphase onset, and demonstrate that Plk1 controls both spindle elongation and cytokinesis. Plk1 inhibition did not affect anaphase A chromosome to pole movement, but blocked anaphase B spindle elongation. Plk1-inhibited cells failed to assemble a contractile ring and contract the cleavage furrow due to a defect in Rho and Rho-GEF localization to the division site. Our results demonstrate that Plk1 coordinates chromosome segregation with cytokinesis through its dual control of anaphase B and contractile ring assembly.

Figure 1. Plk1 inhibition prevents cytokinesis.

A) DIC images taken from timelapse recordings of HeLa cells progressing through cytokinesis in the presence or absence of Plk1 inhibitors. Untreated cells are shown in the top row and cells treated with either BTO-1 or BI-2536 are shown in the middle and bottom rows, respectively. Time (minutes) after anaphase onset is shown in the bottom right corner of each image. B) Degree of furrow contraction in the presence or absence of Plk1 inhibitors. The measured width of the furrow is normalized to the pre-anaphase width. The half-time of furrow ingression for the control cells is 15.32±2.77 minutes. No ingression was observed in cells treated with BTO-1 or BI-2536. T_1/2 is shown±SD. Each curve represents the average of multiple timelapse recordings (Control n = 10, BTO-1 n = 10, and BI-2536 n = 10).

Figure 2. Plk1 inhibition blocks anaphase B spindle elongation.

A) Fluorescence images taken from timelapse recordings of GFP-Tubulin expressing PtK2 cells in the presence or absence of Plk1 inhibitor. Time shown in lower right had corner represents minutes after anaphase onset. Untreated cells are shown in the top row and BTO-1 treated cells are shown in the bottom row. B) Degree of anaphase B inhibition in PtK₂ cells treated with BTO-1. Spindle length is normalized to the length of the pre-anaphase spindle. The half-time of spindle elongation for untreated PtK₂ cells is 6.57±0.58 minutes. None of the cells treated with BTO-1 achieved greater than a 10% increase in spindle length. Curves represent an average of Control (n = 22) and BTO-1 (n = 10) recordings. C) Degree of anaphase B inhibition in HeLa cells treated with BTO-1 or BI-2536. Graphs are as described in B (Control n = 10, BTO-1 n = 10, BI-2536 n = 10). The half-time of spindle elongation untreated HeLa cells is 2.31±0.66 minutes. No spindle pole separation was observed in Plk1 inhibited cells. All measurements are shown±SD.

Figure 3. Reversibility of Plk1 inhibition of cytokinesis and anaphase B.

A) DIC images from a timelapse video of HeLa cells treated with BTO-1 for 15 minutes after anaphase onset. Inhibitor was removed at 15 minutes, second panel represents first timelapse image after inhibitor removal. Time in minutes after anaphase is shown in the lower right hand corner. B) Normalized spindle length in cells after inhibitor washout. Control data is the same data shown previously in Figure 2C for reference. BTO-1 washout (closed triangles) represents an average of 5 timelapse recordings. C) Normalized furrow width showing cytokinesis recovery after BTO-1 washout (closed triangles) (n = 5). Dashed lines represent time of washout. The half-time of furrow ingression following BTO-1 washout is 23.46±3.72 minutes post anaphase onset. T_1/2 is shown±SD.

Figure 4. Plk1 inhibition prevents cleavage furrow assembly.

A) Fluorescence images of control or BI-2536 treated HeLa cells. Top row shows localization of anillin, tubulin and DNA in untreated cells, bottom row shows the same in BI-2536 treated cells. B) Same as depicted in A but localization of myosin II is shown. Scale bar represents 5 µm.

Figure 5. Plk1 inhibition blocks the localization of Rho and Rho-GEF but not Rho-GAP or MKLP1.

A) Fluorescence images of control or BI-2536 treated HeLa cells. Top row shows localization of Rho, Tubulin and DNA in untreated cells and bottom row shows localization after BI-2536 treatment. B) Localization of Rho-GEF as described for A. C) Localization of Rho-GAP (MgcRacGAP) as described for A. D) Localization of MKLP1 as described for A. Scale bar represents 5 µm.