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: 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plasmid were able to survive in shaking culture, albeit with greatly reduced growth rates as compared with WT cells (unpublished data).
Affiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205.