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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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Overexpression of 3xAsp in WT cells reduced growth rate. (A) Diagram of WT myosin II and 3xAsp myosin II. (B) GFP-WT myosin II accumulates at the furrow. Scale bar, 10 μm. (C) Myosin II distribution in myoII::GFP-3xAsp;mCh-WT myosin II was altered by expression of 3xAsp myosin II. GFP-3xAsp and mCherry-WT myosin II colocalized at the cleavage furrow in dividing cells but with an aberrant distribution. Scale bar, 10 μm. (D) WT cells expressing GFP-3xAsp (WT::3xAsp) did not show mechanosensitive myosin II accumulation in response to micropipette aspiration (white arrow). The background-corrected fluorescence intensity ratio of the cortex inside the micropipette (Ip) and the opposite cortex (Io) was measured and used to determine whether the myosin II undergoes mechanosensitive accumulation (Effler et al., 2006). Scale bar, 10 μm. (E) Suspension culture growth for WT control and WT::3xAsp cells. Average growth rate is 0.067 ± 0.008 h−1 for WT cells (n = 11) and 0.032 ± 0.009 h−1 for WT::3xAsp cells (n = 11; p value is on the graph). (F) Western analysis of WT and WT::GFP-3xAsp (where 3xAsp is integrated randomly in the genome) showed that 3xAsp is 40% and WT endogenous myosin II is 60% of the total myosin II in these cells.
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Figure 1: Overexpression of 3xAsp in WT cells reduced growth rate. (A) Diagram of WT myosin II and 3xAsp myosin II. (B) GFP-WT myosin II accumulates at the furrow. Scale bar, 10 μm. (C) Myosin II distribution in myoII::GFP-3xAsp;mCh-WT myosin II was altered by expression of 3xAsp myosin II. GFP-3xAsp and mCherry-WT myosin II colocalized at the cleavage furrow in dividing cells but with an aberrant distribution. Scale bar, 10 μm. (D) WT cells expressing GFP-3xAsp (WT::3xAsp) did not show mechanosensitive myosin II accumulation in response to micropipette aspiration (white arrow). The background-corrected fluorescence intensity ratio of the cortex inside the micropipette (Ip) and the opposite cortex (Io) was measured and used to determine whether the myosin II undergoes mechanosensitive accumulation (Effler et al., 2006). Scale bar, 10 μm. (E) Suspension culture growth for WT control and WT::3xAsp cells. Average growth rate is 0.067 ± 0.008 h−1 for WT cells (n = 11) and 0.032 ± 0.009 h−1 for WT::3xAsp cells (n = 11; p value is on the graph). (F) Western analysis of WT and WT::GFP-3xAsp (where 3xAsp is integrated randomly in the genome) showed that 3xAsp is 40% and WT endogenous myosin II is 60% of the total myosin II in these cells.

Mentions: To establish a system for identifying proteins involved in novel pathways for myosin II cleavage furrow accumulation, we characterized the effects of 3xAsp myosin II on WT myosin II activity (Figure 1A). The 3xAsp mutant myosin II has the threonine residues at positions 1823, 1833, and 2029 mutated to aspartic acid residues, mimicking the phosphorylated WT myosin II and disrupting the ability of this myosin II to assemble into BTFs (Egelhoff et al., 1993). Compared to the uniform cleavage furrow distribution of WT myosin II (Figure 1B), when GFP-3xAsp was expressed along with mCh-WT myosin II, the two proteins colocalized at the cleavage furrow cortex, but the overall myosin II distribution was disrupted, with irregular, asymmetric accumulation often observed (Figure 1C). We also tested whether 3xAsp could accumulate in response to applied stress in the presence of WT myosin II. We found that the mechanosensory responsiveness of 3xAsp myosin II remained disrupted, indicating a severe impairment of myosin II dynamics (Figure 1D; Ren et al., 2009). Finally, although it is well characterized that myoII- cells expressing 3xAsp are completely unable to grow in suspension culture (Egelhoff et al., 1993), WT cells expressing 3xAsp from an episomal plasmid were able to survive in shaking culture, albeit with greatly reduced growth rates as compared with WT cells (unpublished data).


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)

Overexpression of 3xAsp in WT cells reduced growth rate. (A) Diagram of WT myosin II and 3xAsp myosin II. (B) GFP-WT myosin II accumulates at the furrow. Scale bar, 10 μm. (C) Myosin II distribution in myoII::GFP-3xAsp;mCh-WT myosin II was altered by expression of 3xAsp myosin II. GFP-3xAsp and mCherry-WT myosin II colocalized at the cleavage furrow in dividing cells but with an aberrant distribution. Scale bar, 10 μm. (D) WT cells expressing GFP-3xAsp (WT::3xAsp) did not show mechanosensitive myosin II accumulation in response to micropipette aspiration (white arrow). The background-corrected fluorescence intensity ratio of the cortex inside the micropipette (Ip) and the opposite cortex (Io) was measured and used to determine whether the myosin II undergoes mechanosensitive accumulation (Effler et al., 2006). Scale bar, 10 μm. (E) Suspension culture growth for WT control and WT::3xAsp cells. Average growth rate is 0.067 ± 0.008 h−1 for WT cells (n = 11) and 0.032 ± 0.009 h−1 for WT::3xAsp cells (n = 11; p value is on the graph). (F) Western analysis of WT and WT::GFP-3xAsp (where 3xAsp is integrated randomly in the genome) showed that 3xAsp is 40% and WT endogenous myosin II is 60% of the total myosin II in these cells.
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Figure 1: Overexpression of 3xAsp in WT cells reduced growth rate. (A) Diagram of WT myosin II and 3xAsp myosin II. (B) GFP-WT myosin II accumulates at the furrow. Scale bar, 10 μm. (C) Myosin II distribution in myoII::GFP-3xAsp;mCh-WT myosin II was altered by expression of 3xAsp myosin II. GFP-3xAsp and mCherry-WT myosin II colocalized at the cleavage furrow in dividing cells but with an aberrant distribution. Scale bar, 10 μm. (D) WT cells expressing GFP-3xAsp (WT::3xAsp) did not show mechanosensitive myosin II accumulation in response to micropipette aspiration (white arrow). The background-corrected fluorescence intensity ratio of the cortex inside the micropipette (Ip) and the opposite cortex (Io) was measured and used to determine whether the myosin II undergoes mechanosensitive accumulation (Effler et al., 2006). Scale bar, 10 μm. (E) Suspension culture growth for WT control and WT::3xAsp cells. Average growth rate is 0.067 ± 0.008 h−1 for WT cells (n = 11) and 0.032 ± 0.009 h−1 for WT::3xAsp cells (n = 11; p value is on the graph). (F) Western analysis of WT and WT::GFP-3xAsp (where 3xAsp is integrated randomly in the genome) showed that 3xAsp is 40% and WT endogenous myosin II is 60% of the total myosin II in these cells.
Mentions: To establish a system for identifying proteins involved in novel pathways for myosin II cleavage furrow accumulation, we characterized the effects of 3xAsp myosin II on WT myosin II activity (Figure 1A). The 3xAsp mutant myosin II has the threonine residues at positions 1823, 1833, and 2029 mutated to aspartic acid residues, mimicking the phosphorylated WT myosin II and disrupting the ability of this myosin II to assemble into BTFs (Egelhoff et al., 1993). Compared to the uniform cleavage furrow distribution of WT myosin II (Figure 1B), when GFP-3xAsp was expressed along with mCh-WT myosin II, the two proteins colocalized at the cleavage furrow cortex, but the overall myosin II distribution was disrupted, with irregular, asymmetric accumulation often observed (Figure 1C). We also tested whether 3xAsp could accumulate in response to applied stress in the presence of WT myosin II. We found that the mechanosensory responsiveness of 3xAsp myosin II remained disrupted, indicating a severe impairment of myosin II dynamics (Figure 1D; Ren et al., 2009). Finally, although it is well characterized that myoII- cells expressing 3xAsp are completely unable to grow in suspension culture (Egelhoff et al., 1993), WT cells expressing 3xAsp from an episomal plasmid were able to survive in shaking culture, albeit with greatly reduced growth rates as compared with WT cells (unpublished data).

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