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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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Related in: MedlinePlus

Depletion of rmd1 mRNA led to cytokinesis and cortical tension defects. (A) Micrographs of 4′,6-diamidino-2-phenylindole (DAPI)-stained cells show that rmd1hp cells have more multinucleated cells than WT control cells. Scale bar, 50 μm. (B) Frequency histogram reveals an increase in the fraction of multinucleated cells in rmd1hp cells. Inset demonstrates the 91% depletion of rmd1 mRNA in rmd1hp cells. (C) The rmd1hp cells had a 20% reduction in cortical tension. (D) Semilog plot of the furrow ingression dynamics of WT vs. rmd1hp cells, showing that the rmd1hp cells had a faster, more linear furrow ingression dynamic than WT control (n = 8–10 cells/genotype).
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Figure 5: Depletion of rmd1 mRNA led to cytokinesis and cortical tension defects. (A) Micrographs of 4′,6-diamidino-2-phenylindole (DAPI)-stained cells show that rmd1hp cells have more multinucleated cells than WT control cells. Scale bar, 50 μm. (B) Frequency histogram reveals an increase in the fraction of multinucleated cells in rmd1hp cells. Inset demonstrates the 91% depletion of rmd1 mRNA in rmd1hp cells. (C) The rmd1hp cells had a 20% reduction in cortical tension. (D) Semilog plot of the furrow ingression dynamics of WT vs. rmd1hp cells, showing that the rmd1hp cells had a faster, more linear furrow ingression dynamic than WT control (n = 8–10 cells/genotype).

Mentions: To test the role of RMD1 using loss-of-function analysis, we depleted rmd1 via small interference RNA (siRNA) using a hairpin construct (rmd1hp), which yielded ∼90–95% reduction of mRNA levels (Figure 5B, inset). Silencing of rmd1 in WT cells induced more binucleated and multinucleated cells (Figure 5, A and B) and reduced the cortical tension of cells (Figure 5C). Commensurate with the mild cytokinesis defect and reduction in cortical tension, rmd1hp cells also showed faster furrow ingression dynamics than the WT control, which had the stereotypical, near-exponential furrow ingression trajectory (Figure 5D). Of note, changes in the furrow ingression trajectory of rmd1hp cells are similar to what we observed previously for 14-3-3hp, myoII-, and several other cell mechanics mutants (e.g., Zhang and Robinson, 2005; Reichl et al., 2008; Zhou et al., 2010).


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)

Depletion of rmd1 mRNA led to cytokinesis and cortical tension defects. (A) Micrographs of 4′,6-diamidino-2-phenylindole (DAPI)-stained cells show that rmd1hp cells have more multinucleated cells than WT control cells. Scale bar, 50 μm. (B) Frequency histogram reveals an increase in the fraction of multinucleated cells in rmd1hp cells. Inset demonstrates the 91% depletion of rmd1 mRNA in rmd1hp cells. (C) The rmd1hp cells had a 20% reduction in cortical tension. (D) Semilog plot of the furrow ingression dynamics of WT vs. rmd1hp cells, showing that the rmd1hp cells had a faster, more linear furrow ingression dynamic than WT control (n = 8–10 cells/genotype).
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Related In: Results  -  Collection

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Figure 5: Depletion of rmd1 mRNA led to cytokinesis and cortical tension defects. (A) Micrographs of 4′,6-diamidino-2-phenylindole (DAPI)-stained cells show that rmd1hp cells have more multinucleated cells than WT control cells. Scale bar, 50 μm. (B) Frequency histogram reveals an increase in the fraction of multinucleated cells in rmd1hp cells. Inset demonstrates the 91% depletion of rmd1 mRNA in rmd1hp cells. (C) The rmd1hp cells had a 20% reduction in cortical tension. (D) Semilog plot of the furrow ingression dynamics of WT vs. rmd1hp cells, showing that the rmd1hp cells had a faster, more linear furrow ingression dynamic than WT control (n = 8–10 cells/genotype).
Mentions: To test the role of RMD1 using loss-of-function analysis, we depleted rmd1 via small interference RNA (siRNA) using a hairpin construct (rmd1hp), which yielded ∼90–95% reduction of mRNA levels (Figure 5B, inset). Silencing of rmd1 in WT cells induced more binucleated and multinucleated cells (Figure 5, A and B) and reduced the cortical tension of cells (Figure 5C). Commensurate with the mild cytokinesis defect and reduction in cortical tension, rmd1hp cells also showed faster furrow ingression dynamics than the WT control, which had the stereotypical, near-exponential furrow ingression trajectory (Figure 5D). Of note, changes in the furrow ingression trajectory of rmd1hp cells are similar to what we observed previously for 14-3-3hp, myoII-, and several other cell mechanics mutants (e.g., Zhang and Robinson, 2005; Reichl et al., 2008; Zhou et al., 2010).

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