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: These WT::3xAsp cells were subjected to cDNA library suppression to select for genes that could rescue the WT::3xAsp cytokinesis defects in suspension growth (Figure 2A). A total of 77 pools of ∼1800 transformants/pool (140,000 total transformants) were generated and grown in suspension culture for ∼3–4 wk. Once a pool showed a growth rate increase of >30% over the empty vector control pool, the cDNAs were isolated, and individual cDNAs were identified (Robinson and Spudich, 2000; Zhou et al., 2010). After sequence analysis, we recovered 25 independent genes from the selection. One cDNA included the myosin-coding sequence spanning nucleotides 4459 to the poly A tail, which is essentially the coding sequence of the light meromyosin (LMM) region of the myosin heavy chain tail. However, the only in-frame ATG did not occur until nucleotide 5671. Thus LMMTF is predicted to encode only the myosin tail fragment (TF), which spans amino acids 1891–2116. The recovery of LMMTF demonstrated that our cDNA library suppression approach could identify genes involved in myosin II function.
Affiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205.