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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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3xAsp suppressors restored 3xAsp cleavage furrow accumulation. (A) Expression of 3xAsp suppressors increased furrow accumulation of GFP-3xAsp in myoII- cells. Epifluorescence images of dividing cells at telophase and final bridge stages. Scale bar, 10 μm. Frequency histograms show the 3xAsp fluorescence intensity ratio of furrow/pole (If/Ip). (B) TIRF images show extent of myosin II assembly in the cortex. GFP-3xAsp myosin is imaged for myoII::GFP-3xAsp cells expressing empty vector control, RMD1, and mCherry-RMD1 (mCh-RMD1). mCh-RMD1 was used to confirm that RMD1 was expressed in these cells. GFP-WT myosin II (myoII::GFP-myosin II) was examined as a positive control. Four examples of each are shown. Scale bar, 10 μm.
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Figure 3: 3xAsp suppressors restored 3xAsp cleavage furrow accumulation. (A) Expression of 3xAsp suppressors increased furrow accumulation of GFP-3xAsp in myoII- cells. Epifluorescence images of dividing cells at telophase and final bridge stages. Scale bar, 10 μm. Frequency histograms show the 3xAsp fluorescence intensity ratio of furrow/pole (If/Ip). (B) TIRF images show extent of myosin II assembly in the cortex. GFP-3xAsp myosin is imaged for myoII::GFP-3xAsp cells expressing empty vector control, RMD1, and mCherry-RMD1 (mCh-RMD1). mCh-RMD1 was used to confirm that RMD1 was expressed in these cells. GFP-WT myosin II (myoII::GFP-myosin II) was examined as a positive control. Four examples of each are shown. Scale bar, 10 μm.

Mentions: We then tested whether the suppressors could promote 3xAsp cleavage furrow localization in the myoII::3xAsp cells. When expressed alone, 3xAsp seldom showed accumulation at the cleavage furrow cortex (Figure 3A and Table 2). Based on our experimental design, if a gene plays a pivotal role in modulating myosin II assembly or targeting myosin II to the site of cell division, expression of this suppressor may help drive 3xAsp myosin II toward the cleavage furrow. We expressed each of the recapitulated suppressors in myoII::3xAsp cells, as well as five others that were just above the p < 0.05 threshold. These five included two actin cross-linking proteins (cortexillin I and coronin), RMD1, rps2, and 14-3-3, which we previously showed is involved in the myosin II–RacE pathway that controls myosin II cortical accumulation and dynamics (Zhou et al., 2010). From our conception of how 14-3-3 modulates myosin II assembly, we also tested the 14-3-3hp, which reduces 14-3-3 expression by 70% and also leads to overassembled myosin II in an otherwise WT background (Zhou et al., 2010). We quantified the myosin II levels at the cleavage furrow cortex by measuring the myosin II intensity at the furrow (If) and polar (Ip) cortices and then calculating the intensity ratio (If/Ip) for each cell. Histograms of the distribution of 3xAsp If/Ip ratios were plotted for each cell line and compared with the empty vector control (Figure 3A). Nine of 15 plasmids showed an enhancement of the 3xAsp furrow localization with p < 0.10. These plasmids were 14-3-3hp, rcdBB, mmsdh (methylmalonate-semialdehyde dehydrogenase), rmd1, a novel protein (gene ID: DDB_G0272460), actin, coronin, efbA, and cortexillin II (Table 2). As one example, the mean 3xAsp If/Ip ratio for the pLD1 empty vector control was 1.17 ± 0.034, whereas a strong suppressor plasmid rcdBB had a ratio of 1.64 ± 0.11 (Student's t test, p < 0.0001; Figure 3A and Table 2).


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)

3xAsp suppressors restored 3xAsp cleavage furrow accumulation. (A) Expression of 3xAsp suppressors increased furrow accumulation of GFP-3xAsp in myoII- cells. Epifluorescence images of dividing cells at telophase and final bridge stages. Scale bar, 10 μm. Frequency histograms show the 3xAsp fluorescence intensity ratio of furrow/pole (If/Ip). (B) TIRF images show extent of myosin II assembly in the cortex. GFP-3xAsp myosin is imaged for myoII::GFP-3xAsp cells expressing empty vector control, RMD1, and mCherry-RMD1 (mCh-RMD1). mCh-RMD1 was used to confirm that RMD1 was expressed in these cells. GFP-WT myosin II (myoII::GFP-myosin II) was examined as a positive control. Four examples of each are shown. Scale bar, 10 μm.
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Figure 3: 3xAsp suppressors restored 3xAsp cleavage furrow accumulation. (A) Expression of 3xAsp suppressors increased furrow accumulation of GFP-3xAsp in myoII- cells. Epifluorescence images of dividing cells at telophase and final bridge stages. Scale bar, 10 μm. Frequency histograms show the 3xAsp fluorescence intensity ratio of furrow/pole (If/Ip). (B) TIRF images show extent of myosin II assembly in the cortex. GFP-3xAsp myosin is imaged for myoII::GFP-3xAsp cells expressing empty vector control, RMD1, and mCherry-RMD1 (mCh-RMD1). mCh-RMD1 was used to confirm that RMD1 was expressed in these cells. GFP-WT myosin II (myoII::GFP-myosin II) was examined as a positive control. Four examples of each are shown. Scale bar, 10 μm.
Mentions: We then tested whether the suppressors could promote 3xAsp cleavage furrow localization in the myoII::3xAsp cells. When expressed alone, 3xAsp seldom showed accumulation at the cleavage furrow cortex (Figure 3A and Table 2). Based on our experimental design, if a gene plays a pivotal role in modulating myosin II assembly or targeting myosin II to the site of cell division, expression of this suppressor may help drive 3xAsp myosin II toward the cleavage furrow. We expressed each of the recapitulated suppressors in myoII::3xAsp cells, as well as five others that were just above the p < 0.05 threshold. These five included two actin cross-linking proteins (cortexillin I and coronin), RMD1, rps2, and 14-3-3, which we previously showed is involved in the myosin II–RacE pathway that controls myosin II cortical accumulation and dynamics (Zhou et al., 2010). From our conception of how 14-3-3 modulates myosin II assembly, we also tested the 14-3-3hp, which reduces 14-3-3 expression by 70% and also leads to overassembled myosin II in an otherwise WT background (Zhou et al., 2010). We quantified the myosin II levels at the cleavage furrow cortex by measuring the myosin II intensity at the furrow (If) and polar (Ip) cortices and then calculating the intensity ratio (If/Ip) for each cell. Histograms of the distribution of 3xAsp If/Ip ratios were plotted for each cell line and compared with the empty vector control (Figure 3A). Nine of 15 plasmids showed an enhancement of the 3xAsp furrow localization with p < 0.10. These plasmids were 14-3-3hp, rcdBB, mmsdh (methylmalonate-semialdehyde dehydrogenase), rmd1, a novel protein (gene ID: DDB_G0272460), actin, coronin, efbA, and cortexillin II (Table 2). As one example, the mean 3xAsp If/Ip ratio for the pLD1 empty vector control was 1.17 ± 0.034, whereas a strong suppressor plasmid rcdBB had a ratio of 1.64 ± 0.11 (Student's t test, p < 0.0001; Figure 3A and Table 2).

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