Yamashiro S et al. (2018), Myosin-dependent actin stabilization as reveale...

XB-ART-54967

Mol Biol Cell 2018 Aug 08;2916:1941-1947. doi: 10.1091/mbc.E18-01-0061.

Show Gene links Show Anatomy links

Myosin-dependent actin stabilization as revealed by single-molecule imaging of actin turnover.

Yamashiro S , Tanaka S , McMillen LM , Taniguchi D , Vavylonis D , Watanabe N .

???displayArticle.abstract???
How mechanical stress applied to the actin network modifies actin turnover has attracted considerable attention. Actomyosin exerts the major force on the actin network, which has been implicated in actin stability regulation. However, direct monitoring of immediate changes in F-actin stability on alteration of actomyosin contraction has not been achieved. Here we reexamine myosin regulation of actin stability by using single-molecule speckle analysis of actin. To avoid possible errors attributable to actin-binding probes, we employed DyLight-labeled actin that distributes identical to F-actin in lamellipodia. We performed time-resolved analysis of the effect of blebbistatin on actin turnover. Blebbistatin enhanced actin disassembly in lamellipodia of fish keratocytes and lamellar of Xenopus XTC cells at an early stage of the inhibition, indicating that actomyosin contraction stabilizes cellular F-actin. In addition, our data show a previously unrecognized relationship between the actin network-driving force and the actin turnover rates in lamellipodia. These findings point to the power of direct viewing of molecular behavior in elucidating force regulation of actin filament turnover.

???displayArticle.pubmedLink??? 29847209
???displayArticle.pmcLink??? PMC6232968
???displayArticle.link??? Mol Biol Cell
???displayArticle.grants??? [+]

GO keywords: actin binding

???attribute.lit??? ???displayArticles.show???

	FIGURE 1:. (A) Live image of DyLight550-actin (DL-actin) speckles in a keratocyte with epifluorescence microscopy. (B) Positions of appearance of DL-actin speckles in a 34-s time window (left) and disappearance of these speckles (right) are indicated by pink dots over an image of the keratocyte. Bars, 10 μm. (C) Lifetime distribution of DL-actin speckles in lamellipodia of keratocytes.
	FIGURE 2:. Blebbistatin enhanced actin disassembly in lamellipodia of keratocytes. (A) Time-lapse images of a keratocyte loaded with DL-actin at 60-s intervals. Blebbistatin (50 μM) was added at 0 s. (B) The graph shows variations in the normalized cell width (%) of blebbistatin-treated keratocytes (red lines) and control cells (blue lines). Blebbistatin induced lateral expansion of keratocytes within 2 min. (C) Trajectories of speckles before and 90 s after the blebbistatin treatment. (D) Regression measurements of DL-actin speckles in lamellipodia of keratocytes before (blue dots) and after (red dots) treatment with 50 μM blebbistatin (left). Images were acquired at 2-s intervals. The data were normalized for photobleaching (Watanabe and Mitchison, 2002; Yamashiro et al., 2014). For the regression analysis of DL-actin speckles after the treatment, the first frame of the analysis is within 90–180 s after adding blebbistatin. Right, control cells. The single exponential curve (black lines) fitted to the data gave half-life of speckles (t1/2). The dissociation rate of actin speckles was significantly higher in the cells after the blebbistatin treatment than that in the cells before the treatment (p < 0.05, two-tailed paired t test).
	FIGURE 3:. Blebbistatin enhanced actin disassembly in lamella of XTC cells. (A) Live images of Lifeact-EGFP (left) and DL-actin speckles (right) in a XTC cell. DL-actin speckles in the lamellar region between two white lines were analyzed. Blue arrows indicate fluorescent organelle-like structures, which is apparently an artifact of electroporation. Bar, 5 μm. (B) The retrograde flow speeds in lamella before and 70–132 s after the 50 μM blebbistatin treatment. Each colored line represents data from an individual cell (n = 6 cells). The retrograde flow speeds at each point show the average speeds of 10 actin speckles in lamella. (C) Regression measurements of long-lived population of DL-actin speckles (Yamashiro et al., 2014) in lamella of XTC cells before (blue dots) and 70–132 s after (red dots) the blebbistatin treatment. To evaluate long-lived population of DL-actin speckles, speckles that exist at least 40 s were measured. The dissociation rate of long-lived actin speckles was significantly higher in the cells after the blebbistatin treatment than that in the cells before the treatment (p < 0.05, two-tailed paired t test).
	FIGURE 4:. (A) Relationship between the half-life of lamellipodial actin filaments and cell migration speed (blue dots, Pearson correlation coefficient, r = -0.623, p = 0.0172) or actin flow speed with respect to substratum (red dots, r = -0.402, p = 0.154) in individual keratocytes. (B) Kymograph of actin speckles at the cell periphery showing retrograde actin flow toward the cell center with respect to the substratum. Bar, 2 μm. The line in C indicates the region for the kymograph. Bar, 10 μm.

References [+] :

Anderson, Coordination of protrusion and translocation of the keratocyte involves rolling of the cell body. 1996, Pubmed