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Myosin light chain kinase and Src control membrane dynamics in volume recovery from cell swelling.

Barfod ET, Moore AL, Van de Graaf BG, Lidofsky SD - Mol. Biol. Cell (2011)

Bottom Line: On hypotonic exposure, we found that there was time-dependent phosphorylation of the MLCK substrate myosin II regulatory light chain.Hypotonic exposure evoked increased biochemical association of the cell volume regulator Src with MLCK and with the endocytosis regulators cortactin and dynamin, which colocalized within these structures.Inhibition of either Src or MLCK led to altered patch and ring lifetimes, consistent with the concept that Src and MLCK form a swelling-induced protein complex that regulates volume recovery through membrane turnover and compensatory endocytosis under osmotic stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Vermont, Burlington, VT 05405 Department of Medicine, University of Vermont, Burlington, VT 05405, USA.

ABSTRACT
The expansion of the plasma membrane, which occurs during osmotic swelling of epithelia, must be retrieved for volume recovery, but the mechanisms are unknown. Here we have identified myosin light chain kinase (MLCK) as a regulator of membrane internalization in response to osmotic swelling in a model liver cell line. On hypotonic exposure, we found that there was time-dependent phosphorylation of the MLCK substrate myosin II regulatory light chain. At the sides of the cell, MLCK and myosin II localized to swelling-induced membrane blebs with actin just before retraction, and MLCK inhibition led to persistent blebbing and attenuated cell volume recovery. At the base of the cell, MLCK also localized to dynamic actin-coated rings and patches upon swelling, which were associated with uptake of the membrane marker FM4-64X, consistent with sites of membrane internalization. Hypotonic exposure evoked increased biochemical association of the cell volume regulator Src with MLCK and with the endocytosis regulators cortactin and dynamin, which colocalized within these structures. Inhibition of either Src or MLCK led to altered patch and ring lifetimes, consistent with the concept that Src and MLCK form a swelling-induced protein complex that regulates volume recovery through membrane turnover and compensatory endocytosis under osmotic stress.

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Actin and cortactin transiently localize with MLCK at the base of the cell in response to swelling. (A) Stills from live imaging of cells transfected with GFP-MLCK and RFP-actin are depicted at different times (in min) after hypotonic exposure and show simultaneous appearance and disappearance of MLCK (green) and actin (red) to patches at the base of the cell. A patch that is transiently induced by cell swelling is marked with an arrow. A kymograph of the patch indicated in the magnified insets is shown below. (B) Stills from live images of cells transfected with GFP-MLCK (green) and RFP-cortactin (red) at different times (in min) after hypotonic treatment. The midsection of each cell base is magnified below. An arrow indicates simultaneous GFP-MLCK and RFP-cortactin accumulation in patches in response to cell swelling. A kymograph including the patch indicated in the magnified insets is shown below; the top patch is the one marked with an arrow above. Scale bars are 15 μm for the top panels of (A) and (B). The scale bars in the kymograph in (A) are 2 μm and in the kymograph in (B) are 1 μm.
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Figure 4: Actin and cortactin transiently localize with MLCK at the base of the cell in response to swelling. (A) Stills from live imaging of cells transfected with GFP-MLCK and RFP-actin are depicted at different times (in min) after hypotonic exposure and show simultaneous appearance and disappearance of MLCK (green) and actin (red) to patches at the base of the cell. A patch that is transiently induced by cell swelling is marked with an arrow. A kymograph of the patch indicated in the magnified insets is shown below. (B) Stills from live images of cells transfected with GFP-MLCK (green) and RFP-cortactin (red) at different times (in min) after hypotonic treatment. The midsection of each cell base is magnified below. An arrow indicates simultaneous GFP-MLCK and RFP-cortactin accumulation in patches in response to cell swelling. A kymograph including the patch indicated in the magnified insets is shown below; the top patch is the one marked with an arrow above. Scale bars are 15 μm for the top panels of (A) and (B). The scale bars in the kymograph in (A) are 2 μm and in the kymograph in (B) are 1 μm.

Mentions: To examine the temporal association of actin, cortactin, and MLCK in swelling-induced patches, we performed live imaging of cells transfected with GFP-MLCK and RFP-actin or RFP-cortactin during hypotonic challenge. Under these conditions, the dynamics of actin recruitment to patches paralleled that of MLCK (Figure 4A, Supplemental Figure S7, Supplemental Video 9). The association between MLCK and cortactin in patches was different (Figure 4B, Supplemental Figure S8, Supplemental Video 10). Detection of cortactin appeared to precede bulk MLCK signal within patches, and cortactin signal remained constant, while MLCK signal rose and fell as it did with actin. This is reminiscent of the dynamic association between regulators of receptor-mediated endocytosis at sites of membrane internalization (Merrifield et al., 2005).


Myosin light chain kinase and Src control membrane dynamics in volume recovery from cell swelling.

Barfod ET, Moore AL, Van de Graaf BG, Lidofsky SD - Mol. Biol. Cell (2011)

Actin and cortactin transiently localize with MLCK at the base of the cell in response to swelling. (A) Stills from live imaging of cells transfected with GFP-MLCK and RFP-actin are depicted at different times (in min) after hypotonic exposure and show simultaneous appearance and disappearance of MLCK (green) and actin (red) to patches at the base of the cell. A patch that is transiently induced by cell swelling is marked with an arrow. A kymograph of the patch indicated in the magnified insets is shown below. (B) Stills from live images of cells transfected with GFP-MLCK (green) and RFP-cortactin (red) at different times (in min) after hypotonic treatment. The midsection of each cell base is magnified below. An arrow indicates simultaneous GFP-MLCK and RFP-cortactin accumulation in patches in response to cell swelling. A kymograph including the patch indicated in the magnified insets is shown below; the top patch is the one marked with an arrow above. Scale bars are 15 μm for the top panels of (A) and (B). The scale bars in the kymograph in (A) are 2 μm and in the kymograph in (B) are 1 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 4: Actin and cortactin transiently localize with MLCK at the base of the cell in response to swelling. (A) Stills from live imaging of cells transfected with GFP-MLCK and RFP-actin are depicted at different times (in min) after hypotonic exposure and show simultaneous appearance and disappearance of MLCK (green) and actin (red) to patches at the base of the cell. A patch that is transiently induced by cell swelling is marked with an arrow. A kymograph of the patch indicated in the magnified insets is shown below. (B) Stills from live images of cells transfected with GFP-MLCK (green) and RFP-cortactin (red) at different times (in min) after hypotonic treatment. The midsection of each cell base is magnified below. An arrow indicates simultaneous GFP-MLCK and RFP-cortactin accumulation in patches in response to cell swelling. A kymograph including the patch indicated in the magnified insets is shown below; the top patch is the one marked with an arrow above. Scale bars are 15 μm for the top panels of (A) and (B). The scale bars in the kymograph in (A) are 2 μm and in the kymograph in (B) are 1 μm.
Mentions: To examine the temporal association of actin, cortactin, and MLCK in swelling-induced patches, we performed live imaging of cells transfected with GFP-MLCK and RFP-actin or RFP-cortactin during hypotonic challenge. Under these conditions, the dynamics of actin recruitment to patches paralleled that of MLCK (Figure 4A, Supplemental Figure S7, Supplemental Video 9). The association between MLCK and cortactin in patches was different (Figure 4B, Supplemental Figure S8, Supplemental Video 10). Detection of cortactin appeared to precede bulk MLCK signal within patches, and cortactin signal remained constant, while MLCK signal rose and fell as it did with actin. This is reminiscent of the dynamic association between regulators of receptor-mediated endocytosis at sites of membrane internalization (Merrifield et al., 2005).

Bottom Line: On hypotonic exposure, we found that there was time-dependent phosphorylation of the MLCK substrate myosin II regulatory light chain.Hypotonic exposure evoked increased biochemical association of the cell volume regulator Src with MLCK and with the endocytosis regulators cortactin and dynamin, which colocalized within these structures.Inhibition of either Src or MLCK led to altered patch and ring lifetimes, consistent with the concept that Src and MLCK form a swelling-induced protein complex that regulates volume recovery through membrane turnover and compensatory endocytosis under osmotic stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Vermont, Burlington, VT 05405 Department of Medicine, University of Vermont, Burlington, VT 05405, USA.

ABSTRACT
The expansion of the plasma membrane, which occurs during osmotic swelling of epithelia, must be retrieved for volume recovery, but the mechanisms are unknown. Here we have identified myosin light chain kinase (MLCK) as a regulator of membrane internalization in response to osmotic swelling in a model liver cell line. On hypotonic exposure, we found that there was time-dependent phosphorylation of the MLCK substrate myosin II regulatory light chain. At the sides of the cell, MLCK and myosin II localized to swelling-induced membrane blebs with actin just before retraction, and MLCK inhibition led to persistent blebbing and attenuated cell volume recovery. At the base of the cell, MLCK also localized to dynamic actin-coated rings and patches upon swelling, which were associated with uptake of the membrane marker FM4-64X, consistent with sites of membrane internalization. Hypotonic exposure evoked increased biochemical association of the cell volume regulator Src with MLCK and with the endocytosis regulators cortactin and dynamin, which colocalized within these structures. Inhibition of either Src or MLCK led to altered patch and ring lifetimes, consistent with the concept that Src and MLCK form a swelling-induced protein complex that regulates volume recovery through membrane turnover and compensatory endocytosis under osmotic stress.

Show MeSH
Related in: MedlinePlus