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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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Related in: MedlinePlus

cDNA library complementation selection identified 3xAsp suppressors. (A) Strategy of cDNA library suppression. A Dictyostelium cDNA library was transformed into WT::3xAsp cells and subjected to selection using suspension growth. Plasmids were isolated and identified from winner pools based on growth rate. Recovered plasmids were reintroduced into WT::3xAsp cells, which were again subjected to suspension growth to identify strong suppressors. (B) Recapitulation results of “winner” plasmids were sorted according to mean growth rates. All growth rates were normalized over empty vector control (pLD1 control), as shown by the light gray bar. WT control (WT::GFP-myosin II) cells are shown by the dark gray bar. Error bars, SEM. (C) Suspension of growth of the myoII::3xAsp cells transformed with suppressor plasmids and several genes of interest, which included corA, rps2, RMD1, cortexillin I, and 14-3-3 hp. None of these genes was able to rescue the growth of myoII::3xAsp cells in suspension culture, a highly restrictive condition for myosin II–deficient growth.
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Figure 2: cDNA library complementation selection identified 3xAsp suppressors. (A) Strategy of cDNA library suppression. A Dictyostelium cDNA library was transformed into WT::3xAsp cells and subjected to selection using suspension growth. Plasmids were isolated and identified from winner pools based on growth rate. Recovered plasmids were reintroduced into WT::3xAsp cells, which were again subjected to suspension growth to identify strong suppressors. (B) Recapitulation results of “winner” plasmids were sorted according to mean growth rates. All growth rates were normalized over empty vector control (pLD1 control), as shown by the light gray bar. WT control (WT::GFP-myosin II) cells are shown by the dark gray bar. Error bars, SEM. (C) Suspension of growth of the myoII::3xAsp cells transformed with suppressor plasmids and several genes of interest, which included corA, rps2, RMD1, cortexillin I, and 14-3-3 hp. None of these genes was able to rescue the growth of myoII::3xAsp cells in suspension culture, a highly restrictive condition for myosin II–deficient growth.

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.


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)

cDNA library complementation selection identified 3xAsp suppressors. (A) Strategy of cDNA library suppression. A Dictyostelium cDNA library was transformed into WT::3xAsp cells and subjected to selection using suspension growth. Plasmids were isolated and identified from winner pools based on growth rate. Recovered plasmids were reintroduced into WT::3xAsp cells, which were again subjected to suspension growth to identify strong suppressors. (B) Recapitulation results of “winner” plasmids were sorted according to mean growth rates. All growth rates were normalized over empty vector control (pLD1 control), as shown by the light gray bar. WT control (WT::GFP-myosin II) cells are shown by the dark gray bar. Error bars, SEM. (C) Suspension of growth of the myoII::3xAsp cells transformed with suppressor plasmids and several genes of interest, which included corA, rps2, RMD1, cortexillin I, and 14-3-3 hp. None of these genes was able to rescue the growth of myoII::3xAsp cells in suspension culture, a highly restrictive condition for myosin II–deficient growth.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263456&req=5

Figure 2: cDNA library complementation selection identified 3xAsp suppressors. (A) Strategy of cDNA library suppression. A Dictyostelium cDNA library was transformed into WT::3xAsp cells and subjected to selection using suspension growth. Plasmids were isolated and identified from winner pools based on growth rate. Recovered plasmids were reintroduced into WT::3xAsp cells, which were again subjected to suspension growth to identify strong suppressors. (B) Recapitulation results of “winner” plasmids were sorted according to mean growth rates. All growth rates were normalized over empty vector control (pLD1 control), as shown by the light gray bar. WT control (WT::GFP-myosin II) cells are shown by the dark gray bar. Error bars, SEM. (C) Suspension of growth of the myoII::3xAsp cells transformed with suppressor plasmids and several genes of interest, which included corA, rps2, RMD1, cortexillin I, and 14-3-3 hp. None of these genes was able to rescue the growth of myoII::3xAsp cells in suspension culture, a highly restrictive condition for myosin II–deficient growth.
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.

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