Myosin IIA drives membrane bleb retraction

Abstract

Membrane blebs are specialized cellular protrusions that play diverse roles in processes such as cell division and cell migration. Blebbing can be divided into three distinct phases: bleb nucleation, bleb growth, and bleb retraction. Following nucleation and bleb growth, the actin cortex, comprising actin, cross-linking proteins, and nonmuscle myosin II (MII), begins to reassemble on the membrane. MII then drives the final phase, bleb retraction, which results in reintegration of the bleb into the cellular cortex. There are three MII paralogues with distinct biophysical properties expressed in mammalian cells: MIIA, MIIB, and MIIC. Here we show that MIIA specifically drives bleb retraction during cytokinesis. The motor domain and regulation of the nonhelical tailpiece of MIIA both contribute to its ability to drive bleb retraction. These experiments have also revealed a relationship between faster turnover of MIIA at the cortex and its ability to drive bleb retraction.

FIGURE 1:

MIIA but not MIIB is necessary for bleb retraction. (A) Laser-induced polar cortex ablation in control, MIIA- or MIIB-depleted HeLa cells. Representative control DIC montage shows the ablation ROI (magenta circle) used to create a membrane bleb (yellow arrow). Dotted yellow line represents ROI used to create kymographs. Representative kymographs for each condition are shown below. White arrows show the measurement method for calculating retraction rates. (B) Tukey plots comparing bleb retraction rates for controlled and spontaneous blebs in control vs. MIIA^lo or MIIB^lo cells. Controlled blebs: n = 25 control, 15 MIIA^lo and 25 MIIB^lo cells from three independent experiments. Spontaneous blebs: n = 18 control blebs from 9 cells, 15 MIIA^lo blebs from 10 cells, 15 MIIB^lo blebs from 10 cells over three independent experiments. (C) Representative time montage of HeLa cell coexpressing MIIA mApple and MIIB mEmerald showing the ablation ROI (magenta circle). Representative kymographs created using the solid white line show MIIA and MIIB recruitment to the bleb. Yellow ROI shows the region of the kymograph compared for recruitment (first 60 s). (D) Comparison of IIA and IIB recruitment to blebs in HeLa and HAP1 fibroblasts. n = 10 cells for each cell line over three independent experiments. Exact p values stated over respective bars. Solid black circles represent outliers. Scale bar: 10 µm.

FIGURE 2:

The motor domain and nonhelical tailpiece of MIIA are sufficient to drive bleb retraction. (A) Representative kymographs from HAP1 parental and myh9 KO cells following cortex ablation. n = 21 parental cells and 12 KO cells over three independent experiments. (B) Representative DIC and fluorescence images showing the localization of MII paralogues and mutants in HAP1 KO cells. (C) Representative kymographs from MIIA, MIIB, and MIIC expressing HAP1 KO cells following cortex ablation, as in Figure 1. Tukey plots comparing retraction rates in HAP1 KO cells expressing MIIA, MIIB, or MIIC, and Cos7 cells expressing MIIA, MIIB, MIIC, or untransfected (UT). For HAP1 KO cells, n = 27 MIIA, 10 MIIB, and 15 MIIC expressing cells over more than three independent experiments. For Cos7 cells, n = 16 untransfected, 16 MIIA, 11 MIIB, and 10 MIIC expressing cells over three independent experiments. (D) Representative kymographs showing MIIA N93K, MIIA/B, MIIB/A, and MIIA/B/A expressing HAP1 KO cells following cortex ablation. (E) Retraction rates comparing mutants shown in D. n = 21 N93K, 18 MIIA/B, 8 MIIB/A, and 21 MIIA/B/A expressing cells over more than three independent experiments. MIIA bar is from the same data set as C and is displayed only for comparison. Exact p values stated over respective bars. Solid circles in Tukey plots represent outliers. Scale bar: 10 µm.

FIGURE 3:

MIIA shows fast turnover compared with MIIB and MIIC at the cortex. (A) Representative time montages from two separate cells showing FRAP of MIIA and MIIB mEGFP expressed in HeLa cells. Inset shows an enlarged view of the yellow box. Dotted white box represents the bleaching region. (B) Averaged FRAP curves for MIIA and MIIB in HeLa cells. n = 15 cells each for MIIA and MIIB over three independent experiments. See Materials and Methods for the curve fitting method. (C, D) Averaged FRAP curves for MIIA and MIB mEGFP (C), and MIIC mEGFP (D) expressed in HAP1 KO cells. (E) Tukey plots showing time for half-maximal recovery for MIIA, MIIB, and MIIC in HAP1 KO cells. n = 11 MIIA, 13 MIIB, and 11 MIIC expressing cells over three independent experiments. (F) Tukey plots showing time for half-maximal recovery for MIIA, MIIB, and MIIC in Cos7 cells. n = 11 MIIA, 9 MIIB, and 10 MIIC expressing cells over three independent experiments. Exact p values stated over respective bars.

FIGURE 4:

The motor domain and nonhelical piece of MIIA both contribute to turnover at the cortex. (A) Averaged FRAP curves for full-length MIIA (from Figure 3C) and MIIA N93K mutant. (B) Tukey plots comparing time for half-maximal recovery for MIIA, MIIA N93K, MIIA/B, MIIB, and MIIA/B/A in HAP1 KO cells. n = 12 N93K, 10 MIIA/B, and 13 MIIA/B/A expressing cells over three independent experiments. The MIIA and MIIB data sets are the same as Figure 3C and are only shown for comparison. (C) Averaged FRAP curves for MIIA (from Figure 3C), MIIA/B, and MIIA/B/A chimera. Exact p values stated over respective bars.

FIGURE 5:

Phosphorylation of the nonhelical tailpiece regulates turnover at the cortex. (A) Representative DIC and fluorescence images showing localization of MII mutants in HAP1 KO cells. (B) Tukey plots comparing time for half-maximal recovery for MIIA tail mutants. n = 10 MIIA Δtailpiece and 12 MIIA S1943A expressing cells over three independent experiments. The MIIA FRAP data set is the same as Figure 3C and is shown only for comparison. (C) Representative kymographs for MIIA Δtailpiece and MIIA S1943A following cortex ablation in HAP1 KO cells. (D) Tukey plots comparing bleb retraction rates in HAP1 KO cells for MIIA tail mutants. n = 17 MIIA Δtailpiece and 15 MIIA S1943A expressing cells over three independent experiments. The MIIA data set is the same as in Figure 2C and is shown only for comparison. (E) DIC image of Scr control M2 cell 5 h postplating. (F) Representative kymographs for bleb retraction in Scr vs. MIIA^lo, MIIB^lo, and MIIC^lo M2 cells during interphase. (G) Tukey plots comparing bleb retraction rates for Scr vs. MIIA^lo, MIIB^lo, and MIIC^lo M2 cells. Scale bars in A and E: 10 and 5 µm, respectively. Exact p values stated over respective bars. Solid circles represent outliers.