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Genetic suppression of a phosphomimic myosin II identifies system-level factors that promote myosin II cleavage furrow accumulation.

Ren Y, West-Foyle H, Surcel A, Miller C, Robinson DN - Mol. Biol. Cell (2014)

Bottom Line: How myosin II localizes to the cleavage furrow in Dictyostelium and metazoan cells remains largely unknown despite significant advances in understanding its regulation.Finally, an engineered myosin II with a longer lever arm (2xELC), producing a highly mechanosensitive motor, could also partially suppress the intragenic 3xAsp.Overall, myosin II accumulation is the result of multiple parallel and partially redundant pathways that comprise a cellular contractility control system.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205.

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Two thresholds of myosin II activity determine daughter cell symmetry and furrow ingression kinetics. (A) Furrow-thinning dynamics of WT, WT::rmd1hp, myoII-, myoII::3xAsp, myoII::3xAsp, RMD1, and myoII::2xELC-3xAsp show that only WT cells have the characteristic exponential thinning dynamics. All other cell strains show a transition to more rapid thinning at a relative diameter of 1 (furrow diameter was rescaled by Dx, which is the point at which furrow diameter and length are equal; Zhang and Robinson, 2005). (B) Dot plot shows the distributions of area ratios of the large-/small-daughter cells, and the bars represent the medians. The WT class of cells (WT::EV [EV, empty vector], WT::rmd1hp, myoII::3xAsp,RMD1, and myoII::2xELC-3xAsp) produce symmetrically sized daughter cells (p = 0.12–1). The myoII- class (myoII , myoII::3xAsp, and myoII::3xAsp; mCh) produces highly asymmetrically sized daughter cells (p = 0.69–0.92). The two classes were significantly different (p = 0.0001–0.039). Asterisks denote significance relative to WT with empty vector. The p values were determined by Kruskal–Wallis test (p < 0.0001 for the entire data set), followed by pairwise Wilcoxon test. (C) Time series of a dividing cell expressing GFP-2xELC-3xAsp. Scale bar, 5 μm. (D) TIRF images of myoII- cells expressing GFP-3xAsp, GFP-2xELC-3xAsp, and GFP-WT myosin II. Scale bar, 5 μm. (E) Cartoon depicts two thresholds of myosin II activity. Daughter cell symmetry requires intermediate level of function. WT furrow ingression and daughter cell symmetry require highest level of function. The myoII- scenario has altered ingression dynamics and produces highly asymmetrically sized daughter cells.
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Figure 7: Two thresholds of myosin II activity determine daughter cell symmetry and furrow ingression kinetics. (A) Furrow-thinning dynamics of WT, WT::rmd1hp, myoII-, myoII::3xAsp, myoII::3xAsp, RMD1, and myoII::2xELC-3xAsp show that only WT cells have the characteristic exponential thinning dynamics. All other cell strains show a transition to more rapid thinning at a relative diameter of 1 (furrow diameter was rescaled by Dx, which is the point at which furrow diameter and length are equal; Zhang and Robinson, 2005). (B) Dot plot shows the distributions of area ratios of the large-/small-daughter cells, and the bars represent the medians. The WT class of cells (WT::EV [EV, empty vector], WT::rmd1hp, myoII::3xAsp,RMD1, and myoII::2xELC-3xAsp) produce symmetrically sized daughter cells (p = 0.12–1). The myoII- class (myoII , myoII::3xAsp, and myoII::3xAsp; mCh) produces highly asymmetrically sized daughter cells (p = 0.69–0.92). The two classes were significantly different (p = 0.0001–0.039). Asterisks denote significance relative to WT with empty vector. The p values were determined by Kruskal–Wallis test (p < 0.0001 for the entire data set), followed by pairwise Wilcoxon test. (C) Time series of a dividing cell expressing GFP-2xELC-3xAsp. Scale bar, 5 μm. (D) TIRF images of myoII- cells expressing GFP-3xAsp, GFP-2xELC-3xAsp, and GFP-WT myosin II. Scale bar, 5 μm. (E) Cartoon depicts two thresholds of myosin II activity. Daughter cell symmetry requires intermediate level of function. WT furrow ingression and daughter cell symmetry require highest level of function. The myoII- scenario has altered ingression dynamics and produces highly asymmetrically sized daughter cells.

Mentions: To ascertain the functional state of myosin II in the rmd1hp cells as well as in myoII::3xAsp cells rescued by RMD1, we measured the furrow ingression dynamics and symmetry of daughter cell sizes for these cells. None of the mutants was able to recover the near-exponential WT furrow ingression dynamics (Figure 7A). The products of cell division are two daughter cells, which for WT cells are highly symmetrical in size. To quantify this, we measured the two-dimensional cross-sectional area of the daughter cells and calculated the ratio of the larger cell to the smaller cell (Figure 7B). For WT, this ratio was 1.10 ± 0.021 (mean ± SEM), whereas for myoII- cells, the ratio increased to 1.34 ± 0.084 and was more broadly distributed. As compared with WT cells, depletion of rmd1 did not alter the daughter cell symmetry despite altering furrow ingression kinetics (Figures 5D and 7, A and B). Of interest, RMD1 did not improve the furrow ingression kinetics of myoII::3xAsp, which was identical to myoII- kinetics (Figure 7A), but it did increase the symmetry of the resulting daughter cells (Figure 7B). Thus daughter cell symmetry and furrow ingression kinetics appear to be established by different thresholds of myosin II activity: depletion of rmd1 from WT cells or expression of RMD1 in myoII::3xAsp cells causes the phenotype to converge to an intermediate level of myosin II function, where daughter cell symmetry is normal but furrow ingression kinetics is not (Figure 7E).


Genetic suppression of a phosphomimic myosin II identifies system-level factors that promote myosin II cleavage furrow accumulation.

Ren Y, West-Foyle H, Surcel A, Miller C, Robinson DN - Mol. Biol. Cell (2014)

Two thresholds of myosin II activity determine daughter cell symmetry and furrow ingression kinetics. (A) Furrow-thinning dynamics of WT, WT::rmd1hp, myoII-, myoII::3xAsp, myoII::3xAsp, RMD1, and myoII::2xELC-3xAsp show that only WT cells have the characteristic exponential thinning dynamics. All other cell strains show a transition to more rapid thinning at a relative diameter of 1 (furrow diameter was rescaled by Dx, which is the point at which furrow diameter and length are equal; Zhang and Robinson, 2005). (B) Dot plot shows the distributions of area ratios of the large-/small-daughter cells, and the bars represent the medians. The WT class of cells (WT::EV [EV, empty vector], WT::rmd1hp, myoII::3xAsp,RMD1, and myoII::2xELC-3xAsp) produce symmetrically sized daughter cells (p = 0.12–1). The myoII- class (myoII , myoII::3xAsp, and myoII::3xAsp; mCh) produces highly asymmetrically sized daughter cells (p = 0.69–0.92). The two classes were significantly different (p = 0.0001–0.039). Asterisks denote significance relative to WT with empty vector. The p values were determined by Kruskal–Wallis test (p < 0.0001 for the entire data set), followed by pairwise Wilcoxon test. (C) Time series of a dividing cell expressing GFP-2xELC-3xAsp. Scale bar, 5 μm. (D) TIRF images of myoII- cells expressing GFP-3xAsp, GFP-2xELC-3xAsp, and GFP-WT myosin II. Scale bar, 5 μm. (E) Cartoon depicts two thresholds of myosin II activity. Daughter cell symmetry requires intermediate level of function. WT furrow ingression and daughter cell symmetry require highest level of function. The myoII- scenario has altered ingression dynamics and produces highly asymmetrically sized daughter cells.
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Figure 7: Two thresholds of myosin II activity determine daughter cell symmetry and furrow ingression kinetics. (A) Furrow-thinning dynamics of WT, WT::rmd1hp, myoII-, myoII::3xAsp, myoII::3xAsp, RMD1, and myoII::2xELC-3xAsp show that only WT cells have the characteristic exponential thinning dynamics. All other cell strains show a transition to more rapid thinning at a relative diameter of 1 (furrow diameter was rescaled by Dx, which is the point at which furrow diameter and length are equal; Zhang and Robinson, 2005). (B) Dot plot shows the distributions of area ratios of the large-/small-daughter cells, and the bars represent the medians. The WT class of cells (WT::EV [EV, empty vector], WT::rmd1hp, myoII::3xAsp,RMD1, and myoII::2xELC-3xAsp) produce symmetrically sized daughter cells (p = 0.12–1). The myoII- class (myoII , myoII::3xAsp, and myoII::3xAsp; mCh) produces highly asymmetrically sized daughter cells (p = 0.69–0.92). The two classes were significantly different (p = 0.0001–0.039). Asterisks denote significance relative to WT with empty vector. The p values were determined by Kruskal–Wallis test (p < 0.0001 for the entire data set), followed by pairwise Wilcoxon test. (C) Time series of a dividing cell expressing GFP-2xELC-3xAsp. Scale bar, 5 μm. (D) TIRF images of myoII- cells expressing GFP-3xAsp, GFP-2xELC-3xAsp, and GFP-WT myosin II. Scale bar, 5 μm. (E) Cartoon depicts two thresholds of myosin II activity. Daughter cell symmetry requires intermediate level of function. WT furrow ingression and daughter cell symmetry require highest level of function. The myoII- scenario has altered ingression dynamics and produces highly asymmetrically sized daughter cells.
Mentions: To ascertain the functional state of myosin II in the rmd1hp cells as well as in myoII::3xAsp cells rescued by RMD1, we measured the furrow ingression dynamics and symmetry of daughter cell sizes for these cells. None of the mutants was able to recover the near-exponential WT furrow ingression dynamics (Figure 7A). The products of cell division are two daughter cells, which for WT cells are highly symmetrical in size. To quantify this, we measured the two-dimensional cross-sectional area of the daughter cells and calculated the ratio of the larger cell to the smaller cell (Figure 7B). For WT, this ratio was 1.10 ± 0.021 (mean ± SEM), whereas for myoII- cells, the ratio increased to 1.34 ± 0.084 and was more broadly distributed. As compared with WT cells, depletion of rmd1 did not alter the daughter cell symmetry despite altering furrow ingression kinetics (Figures 5D and 7, A and B). Of interest, RMD1 did not improve the furrow ingression kinetics of myoII::3xAsp, which was identical to myoII- kinetics (Figure 7A), but it did increase the symmetry of the resulting daughter cells (Figure 7B). Thus daughter cell symmetry and furrow ingression kinetics appear to be established by different thresholds of myosin II activity: depletion of rmd1 from WT cells or expression of RMD1 in myoII::3xAsp cells causes the phenotype to converge to an intermediate level of myosin II function, where daughter cell symmetry is normal but furrow ingression kinetics is not (Figure 7E).

Bottom Line: How myosin II localizes to the cleavage furrow in Dictyostelium and metazoan cells remains largely unknown despite significant advances in understanding its regulation.Finally, an engineered myosin II with a longer lever arm (2xELC), producing a highly mechanosensitive motor, could also partially suppress the intragenic 3xAsp.Overall, myosin II accumulation is the result of multiple parallel and partially redundant pathways that comprise a cellular contractility control system.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205.

Show MeSH
Related in: MedlinePlus