Genetic suppression of a phosphomimic myosin II identifies system-level factors that promote myosin II cleavage furrow accumulation.
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.Analysis of several mutant strains revealed that different thresholds of myosin II activity are required for daughter cell symmetry than for furrow ingression dynamics.Finally, an engineered myosin II with a longer lever arm (2xELC), producing a highly mechanosensitive motor, could also partially suppress the intragenic 3xAsp.
Affiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205.Show MeSH
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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).
Affiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205.