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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

Localization of RMD1, mmsdh, and rcdBB proteins in cells. (A) Bar graph shows the cleavage furrow intensity ratio of mCherry-3xAsp myosin II in myoII::mCh-3xAsp cells when GFP-RMD1 and GFP-mmsdh were expressed. These data confirm that GFP-RMD1 and GFP-mmsdh are functional GFP-fusion proteins. Sample sizes and p values are displayed on the graph. (B) Epifluorescence images demonstrate subcellular localization of RMD1, mmsdh, and rcdBB. RMD1 is largely cytoplasmic, with some enrichment around the centrosome (RFP-tubulin is shown for comparison). mmsdh is only cytosolic, and rcdBB was found enriched in the endoplasmic reticulum (RFP-calnexin is shown for comparison). (C) Confocal imaging confirms the largely cytosolic distribution of RMD1, with weak enrichment around the centrosome and the cytosolic distribution of mmsdh. (D) During cytokinesis, RMD1 and mmsdh remain cytosolic, with RMD1 showing weak enrichment around the centrosome. (E) In interphase cells compressed by agarose overlay, which introduces mechanical stress to the cortex, RMD1 remains largely cytosolic, with weak enrichment around the centrosome (RFP-tubulin is shown for comparison). mmsdh also remains cytosolic. (F) In dividing cells compressed by agar overlay, RMD1 and mmsdh remain cytosolic, with RMD1 showing weak enrichment around the centrosome (RFP-tubulin is shown for comparison).
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Figure 4: Localization of RMD1, mmsdh, and rcdBB proteins in cells. (A) Bar graph shows the cleavage furrow intensity ratio of mCherry-3xAsp myosin II in myoII::mCh-3xAsp cells when GFP-RMD1 and GFP-mmsdh were expressed. These data confirm that GFP-RMD1 and GFP-mmsdh are functional GFP-fusion proteins. Sample sizes and p values are displayed on the graph. (B) Epifluorescence images demonstrate subcellular localization of RMD1, mmsdh, and rcdBB. RMD1 is largely cytoplasmic, with some enrichment around the centrosome (RFP-tubulin is shown for comparison). mmsdh is only cytosolic, and rcdBB was found enriched in the endoplasmic reticulum (RFP-calnexin is shown for comparison). (C) Confocal imaging confirms the largely cytosolic distribution of RMD1, with weak enrichment around the centrosome and the cytosolic distribution of mmsdh. (D) During cytokinesis, RMD1 and mmsdh remain cytosolic, with RMD1 showing weak enrichment around the centrosome. (E) In interphase cells compressed by agarose overlay, which introduces mechanical stress to the cortex, RMD1 remains largely cytosolic, with weak enrichment around the centrosome (RFP-tubulin is shown for comparison). mmsdh also remains cytosolic. (F) In dividing cells compressed by agar overlay, RMD1 and mmsdh remain cytosolic, with RMD1 showing weak enrichment around the centrosome (RFP-tubulin is shown for comparison).

Mentions: The genes that rescued 3xAsp myosin II furrow accumulation in the myoII- background might play an important role in myosin II assembly dynamics and/or in pathways involved in myosin II localization and may include “hypothetical receptors” that might be involved in recruiting myosin II to the cleavage furrow during cytokinesis. We focused on three proteins—RMD1, rcdBB, and mmsdh—which are among the top hits in the 3xAsp furrow localization rescue and suspension growth assays. We analyzed the subcellular localizations of these proteins by live-cell imaging of WT cells expressing N-terminal GFP- or mCherry-tagged fusion proteins. Suspension growth and 3xAsp furrow localization assays were repeated to confirm that these fusions did not impair protein function. Both GFP-RMD1 and GFP-mmsdh increased suspension growth of WT::3xAsp cells and rescued the 3xAsp furrow accumulation in myoII- cells (Figure 4A). These data are similar to those acquired for the non–fluorophore-tagged versions, confirming that the N-terminal GFP-fusion proteins were functional. We note that the C-terminal–labeled RMD1 protein lost its spindle localization, indicating that this fusion protein did not function normally.


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)

Localization of RMD1, mmsdh, and rcdBB proteins in cells. (A) Bar graph shows the cleavage furrow intensity ratio of mCherry-3xAsp myosin II in myoII::mCh-3xAsp cells when GFP-RMD1 and GFP-mmsdh were expressed. These data confirm that GFP-RMD1 and GFP-mmsdh are functional GFP-fusion proteins. Sample sizes and p values are displayed on the graph. (B) Epifluorescence images demonstrate subcellular localization of RMD1, mmsdh, and rcdBB. RMD1 is largely cytoplasmic, with some enrichment around the centrosome (RFP-tubulin is shown for comparison). mmsdh is only cytosolic, and rcdBB was found enriched in the endoplasmic reticulum (RFP-calnexin is shown for comparison). (C) Confocal imaging confirms the largely cytosolic distribution of RMD1, with weak enrichment around the centrosome and the cytosolic distribution of mmsdh. (D) During cytokinesis, RMD1 and mmsdh remain cytosolic, with RMD1 showing weak enrichment around the centrosome. (E) In interphase cells compressed by agarose overlay, which introduces mechanical stress to the cortex, RMD1 remains largely cytosolic, with weak enrichment around the centrosome (RFP-tubulin is shown for comparison). mmsdh also remains cytosolic. (F) In dividing cells compressed by agar overlay, RMD1 and mmsdh remain cytosolic, with RMD1 showing weak enrichment around the centrosome (RFP-tubulin is shown for comparison).
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Related In: Results  -  Collection

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Figure 4: Localization of RMD1, mmsdh, and rcdBB proteins in cells. (A) Bar graph shows the cleavage furrow intensity ratio of mCherry-3xAsp myosin II in myoII::mCh-3xAsp cells when GFP-RMD1 and GFP-mmsdh were expressed. These data confirm that GFP-RMD1 and GFP-mmsdh are functional GFP-fusion proteins. Sample sizes and p values are displayed on the graph. (B) Epifluorescence images demonstrate subcellular localization of RMD1, mmsdh, and rcdBB. RMD1 is largely cytoplasmic, with some enrichment around the centrosome (RFP-tubulin is shown for comparison). mmsdh is only cytosolic, and rcdBB was found enriched in the endoplasmic reticulum (RFP-calnexin is shown for comparison). (C) Confocal imaging confirms the largely cytosolic distribution of RMD1, with weak enrichment around the centrosome and the cytosolic distribution of mmsdh. (D) During cytokinesis, RMD1 and mmsdh remain cytosolic, with RMD1 showing weak enrichment around the centrosome. (E) In interphase cells compressed by agarose overlay, which introduces mechanical stress to the cortex, RMD1 remains largely cytosolic, with weak enrichment around the centrosome (RFP-tubulin is shown for comparison). mmsdh also remains cytosolic. (F) In dividing cells compressed by agar overlay, RMD1 and mmsdh remain cytosolic, with RMD1 showing weak enrichment around the centrosome (RFP-tubulin is shown for comparison).
Mentions: The genes that rescued 3xAsp myosin II furrow accumulation in the myoII- background might play an important role in myosin II assembly dynamics and/or in pathways involved in myosin II localization and may include “hypothetical receptors” that might be involved in recruiting myosin II to the cleavage furrow during cytokinesis. We focused on three proteins—RMD1, rcdBB, and mmsdh—which are among the top hits in the 3xAsp furrow localization rescue and suspension growth assays. We analyzed the subcellular localizations of these proteins by live-cell imaging of WT cells expressing N-terminal GFP- or mCherry-tagged fusion proteins. Suspension growth and 3xAsp furrow localization assays were repeated to confirm that these fusions did not impair protein function. Both GFP-RMD1 and GFP-mmsdh increased suspension growth of WT::3xAsp cells and rescued the 3xAsp furrow accumulation in myoII- cells (Figure 4A). These data are similar to those acquired for the non–fluorophore-tagged versions, confirming that the N-terminal GFP-fusion proteins were functional. We note that the C-terminal–labeled RMD1 protein lost its spindle localization, indicating that this fusion protein did not function normally.

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