Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2007 Sep 24;178(7):1207–1221. doi: 10.1083/jcb.200706012

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

Copyright © 2007, The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

Figure 5. — Motility initiation requires Rho kinase–dependent myosin activity. (A, top) Keratocyte fixed and stained for tubulin to visualize microtubules. (bottom) Live stationary keratocyte expressing YFP-myosin regulatory light chain. (B) Motility initiation frequency of stationary keratocytes within 30 min of a temperature shift from 20 to 30°C. Successful motility initiation was defined as persistent polarized morphology and cell movement over at least four cell lengths. Shape changers were cells that had irregular morphologies. Depolymerization of microtubules with 1 μg/ml nocodazole, inhibition of PI-3 kinase with 50 μM LY294002, and inhibition of MLCK with 10 or 25 μM ML-7 had no effect on the frequency of motility initiation (P = 0.7498, P = 0.0173, and P = 0.5562, respectively). Myosin II inhibition with 40 or 100 μM blebbistatin and Rho kinase inhibition with 10 or 25 μM Y-27632 decreased the frequency of motility initiation (P < 0.0001). In contrast, myosin phosphatase inhibition with 10 or 25 nM calyculin A increased the frequency of motility initiation (P < 0.0001). When two drug concentrations were used, there was no significant difference between the results (P > 0.2); results from the two concentrations were pooled. (C) Stationary keratocytes were treated with the indicated drugs for 10–20 min. F-actin was visualized by fixing the cells and staining with phalloidin. Treatment with ML-7 and calyculin A retained the circular bands of F-actin in the perinuclear region. Treatment with blebbistatin or Y-27632 reduced F-actin in the perinuclear region, and circular bands were no longer visible (yellow brackets). (D and E) Changes in perinuclear and peripheral F-actin radial velocity and directional coherence (see Materials and methods) before and after treatment with the indicated drugs. Error bars indicate the SD of the mean over time; gray lines indicate correspondence between data points representing the same cell before and after treatment. In some cases, the SD is smaller than the size of the data point. (D) Blebbistatin treatment decreased the perinuclear and peripheral radial velocity; conversely, calyculin A treatment increased the perinuclear and peripheral radial velocity. Y-27632 or ML-7 treatment had no effect. (E) Blebbistatin and Y-27632 treatment decreased the directional coherence of the actin flow in the perinuclear and peripheral zones. The effect was greater in the perinuclear zone than the peripheral zone. ML-7 and calyculin A had no effect.