Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells - PubMed (original) (raw)

Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells

Miguel Vicente-Manzanares et al. J Cell Biol. 2007.

Erratum in

J Cell Biol. 2007 Mar 26;176(7):1073

Abstract

We have used isoform-specific RNA interference knockdowns to investigate the roles of myosin IIA (MIIA) and MIIB in the component processes that drive cell migration. Both isoforms reside outside of protrusions and act at a distance to regulate cell protrusion, signaling, and maturation of nascent adhesions. MIIA also controls the dynamics and size of adhesions in central regions of the cell and contributes to retraction and adhesion disassembly at the rear. In contrast, MIIB establishes front-back polarity and centrosome, Golgi, and nuclear orientation. Using ATPase- and contraction-deficient mutants of both MIIA and MIIB, we show a role for MIIB-dependent actin cross-linking in establishing front-back polarity. From these studies, MII emerges as a master regulator and integrator of cell migration. It mediates each of the major component processes that drive migration, e.g., polarization, protrusion, adhesion assembly and turnover, polarity, signaling, and tail retraction, and it integrates spatially separated processes.

PubMed Disclaimer

Figures

Figure 1.

Knockdown of MIIA or MIIB differentially alters cell polarity. (A) CHO.K1 cells were transfected with pSUPER-GFP vector or pSUPER-GFP-RNAi against MIIA or MIIB, and blotted for MIIA or MIIB. GIT1 is a loading control. (B) Representative images of MIIA- (top) and MIIB-depleted cells (bottom) stained for MIIA or MIIB, respectively. Arrows point to transfected cells. (C–E) Time-lapse series of MIIA-depleted (C; Video 1), MIIB-depleted (D; Video 2), or control cells (E; Video 3). In C, arrowheads point to the defect in tail retraction. (D) Arrows point to transfected, unpolarized cells. Videos are representative of >25 cells in 6 independent experiments. (F) Fluorescence images depicting the enlargement of MIIB-deficient cells. Images are representative of >300 cells. (G; top) Effect of MIIB knockdown on cell area. Data are the mean ± the SD of 4 independent experiments comprising >300 cells/experiment. (bottom) Cell areas in control and MIIB-depleted cells. (H) MIIB-deficient cell showing clockwise rotation of the nucleus. Arrowhead points to nucleolus; top-right indicator, clockwise angular displacement (Video 4). (I) Depolarization of the Golgi is observed in MIIB-deficient cells (arrows) and not in nontransfected cells (arrowheads). Bars: (C) 40 μm; (D–F and H) 50 μm. Videos 1–4 are available at

http://www.jcb.org/cgi/content/full/jcb.200612043/DC1

Figure 2.

MIIA and IIB regulate protrusion and differentially control adhesion turnover. (A; top) Kymographs from control (pSUPER), MIIA-depleted (pSUP-IIA), and MIIB-depleted (pSUP-IIB) cells. (bottom) Overlay of periodicity and slope from the kymographs. (B) Protrusion rates from A. At least 12 cells (3–5 protrusions/cell) from four independent experiments were analyzed. Data is presented as the mean ± the SEM. (C) Image sequence of control (top; Video 7) and MIIA- (middle; Video 6) and MIIB-depleted cells (bottom; Video 8) cotransfected with paxillin-GFP. (D) Color-inverted image sequence of MIIA-depleted cell expressing paxillin-GFP (Video 9). Time is shown in seconds. (E) Image sequence of paxillin-GFP–expressing, MIIA- (top) or MIIB-depleted (bottom) cells cotransfected with mChe-MIIA or mChe-MIIB, respectively (not depicted). (F) Image sequence of paxillin-GFP–expressing, MIIA-depleted cell. Arrowheads point to central adhesions; arrows point to impaired disassembly at the trailing edge (Video 10). (G) Image sequence of paxillin-GFP in a MIIB-depleted cell (left) expressing mChe-IIA (not depicted) and a MIIA-depleted cell (right) expressing mChe-IIB (not depicted). Bars: (C) 5 μm; (D) 3 μm; (E) 5 μm; (F) 20 μm; (G) 5 μm. Videos 6–10 are available at

http://www.jcb.org/cgi/content/full/jcb.200612043/DC1

Figure 3.

Contractility-deficient mutants of MIIA and MIIB exhibit differential rescue of actin bundling, protrusion, and adhesion dynamics in MIIA- and MIIB-deficient cells. Inhibited FRAP of GFP-MIIA N93K (A) and GFP-MIIB R709C (B) in actin bundles. Data are the mean ± the SD of >20 individual measurements from four independent experiments. (C) Differential FRAP of GFP-MIIA-WT and GFP-MIIB-WT in actin bundles. (D) A MIIB-depleted cell cotransfected with paxillin-GFP and mChe-MIIB R709C (not depicted). (E) Localization of GFP-MIIB and GFP-MIIB R709C. Arrows show the direction of migration. (F) Polarity index of migrating CHO.K1 cells. Data are the mean ± the SD of >100 cells analyzed/condition. (G) Time-lapse series of a MIIA-depleted cell expressing paxillin-GFP and mChe-MIIA (not depicted). Bars: (D) 3 μm; (G) 15 μm.

Figure 4.

Adhesive signaling near the leading edge of MII-depleted cells. MIIA- or MIIB-depleted or control cells were plated on fibronectin and then fixed and stained for phosphotyrosine (A), phosphoTyr397-FAK (B), and phosphoTyr31-paxillin (C). Bar, 10 μm.

References

1. Adelstein, R.S., and M.A. Conti. 1975. Phosphorylation of platelet myosin increases actin-activated myosin ATPase activity. Nature. 256:597–598. - PubMed
1. Bao, J., S.S. Jana, and R.S. Adelstein. 2005. Vertebrate nonmuscle myosin II isoforms rescue small interfering RNA-induced defects in COS-7 cell cytokinesis. J. Biol. Chem. 280:19594–19599. - PubMed
1. Bear, J.E., T.M. Svitkina, M. Krause, D.A. Schafer, J.J. Loureiro, G.A. Strasser, I.V. Maly, O.Y. Chaga, J.A. Cooper, G.G. Borisy, and F.B. Gertler. 2002. Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility. Cell. 109:509–521. - PubMed
1. Bershadsky, A., M. Kozlov, and B. Geiger. 2006. Adhesion-mediated mechanosensitivity: a time to experiment, and a time to theorize. Curr. Opin. Cell Biol. 18:472–481. - PubMed
1. Bokoch, G.M. 2003. Biology of the p21-activated kinases. Annu. Rev. Biochem. 72:743–781. - PubMed

Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells - PubMed (original) (raw)