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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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Src inhibition leads to the accumulation of internalized membrane marker within MLCK rings. MLCK rings may represent MLCK-coated vesicular intermediates. (A) Cells transfected with GFP-MLCK (green) were treated with exogenous FM4-64X (red) and fluorescently labeled CTXB (blue) during hypotonic exposure in the presence of PP2 (10 μM) for 1 min before fixation. Arrows mark distinct MLCK and FM4-64X membrane dye rings. (B) Stills from live imaging of cells transfected with GFP-MLCK (green) taken at the base and treated with PP2 (10 μM) during hypotonic treatment in the presence of exogenous FM4-64X. An arrow marks an MLCK ring that appears to encircle internalized membrane marker. (C) Cells transfected with GFP-MLCK (green) were treated with exogenous FM4-64X (red) and fluorescently labeled CTXB (blue) during hypotonic exposure in the presence of PP2 (10 μM) for 5 min before fixation. An arrow marks an MLCK ring that appears to encircle a lumen of FM4-64X–bound membrane dye. Single channel signals for MLCK, FM4-64X, and CTXB (left) show that FM4-64X signal is present in the ring, whereas CTXB signal is not. A three-dimensional reconstruction of the cell (right) with magnified panels adjacent shows that the MLCK ring surrounds FM4-64X dye. (D) Model for the visualization of rings. Side views of potential involuting membrane structures labeled with FM4-64X dye and recruited MLCK are marked with a plane of focus. The corresponding predicted two-dimensional patterns of FM4-64X and MLCK signal, as seen from above, are adjacent. Scale bars are 15 μm.
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Figure 7: Src inhibition leads to the accumulation of internalized membrane marker within MLCK rings. MLCK rings may represent MLCK-coated vesicular intermediates. (A) Cells transfected with GFP-MLCK (green) were treated with exogenous FM4-64X (red) and fluorescently labeled CTXB (blue) during hypotonic exposure in the presence of PP2 (10 μM) for 1 min before fixation. Arrows mark distinct MLCK and FM4-64X membrane dye rings. (B) Stills from live imaging of cells transfected with GFP-MLCK (green) taken at the base and treated with PP2 (10 μM) during hypotonic treatment in the presence of exogenous FM4-64X. An arrow marks an MLCK ring that appears to encircle internalized membrane marker. (C) Cells transfected with GFP-MLCK (green) were treated with exogenous FM4-64X (red) and fluorescently labeled CTXB (blue) during hypotonic exposure in the presence of PP2 (10 μM) for 5 min before fixation. An arrow marks an MLCK ring that appears to encircle a lumen of FM4-64X–bound membrane dye. Single channel signals for MLCK, FM4-64X, and CTXB (left) show that FM4-64X signal is present in the ring, whereas CTXB signal is not. A three-dimensional reconstruction of the cell (right) with magnified panels adjacent shows that the MLCK ring surrounds FM4-64X dye. (D) Model for the visualization of rings. Side views of potential involuting membrane structures labeled with FM4-64X dye and recruited MLCK are marked with a plane of focus. The corresponding predicted two-dimensional patterns of FM4-64X and MLCK signal, as seen from above, are adjacent. Scale bars are 15 μm.

Mentions: Some forms of endocytosis are regulated by lipid raft formation (Parton and Richards, 2003), which we have found to be important in cell volume control (Barfod et al., 2007). In particular, Src activation has been suggested to control endocytosis mediated by GM1 ganglioside–containing lipid rafts (Shajahan et al., 2004). To determine whether the sites of volume-sensitive membrane turnover we have observed were also enriched in such lipid raft domains, we exposed cells expressing GFP-MLCK to fluorescently labeled GM1 ganglioside marker cholera toxin B (CTXB) in addition to FM4-64X. When these cells were swollen in the presence of PP2 before fixation, distinct rings of MLCK and FM4-64X appeared within 1 min (Figure 7A), but no rings of CTXB were seen, although this marker was clearly internalized to separate compartments. However, the CTXB and FM4-64X signals did colocalize in intracellular vesicles at longer time points (10–15 min) after swelling (unpublished data), suggesting that the initial uptake mechanisms were distinct but that membrane mixing could occur subsequently in endosomes.


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)

Src inhibition leads to the accumulation of internalized membrane marker within MLCK rings. MLCK rings may represent MLCK-coated vesicular intermediates. (A) Cells transfected with GFP-MLCK (green) were treated with exogenous FM4-64X (red) and fluorescently labeled CTXB (blue) during hypotonic exposure in the presence of PP2 (10 μM) for 1 min before fixation. Arrows mark distinct MLCK and FM4-64X membrane dye rings. (B) Stills from live imaging of cells transfected with GFP-MLCK (green) taken at the base and treated with PP2 (10 μM) during hypotonic treatment in the presence of exogenous FM4-64X. An arrow marks an MLCK ring that appears to encircle internalized membrane marker. (C) Cells transfected with GFP-MLCK (green) were treated with exogenous FM4-64X (red) and fluorescently labeled CTXB (blue) during hypotonic exposure in the presence of PP2 (10 μM) for 5 min before fixation. An arrow marks an MLCK ring that appears to encircle a lumen of FM4-64X–bound membrane dye. Single channel signals for MLCK, FM4-64X, and CTXB (left) show that FM4-64X signal is present in the ring, whereas CTXB signal is not. A three-dimensional reconstruction of the cell (right) with magnified panels adjacent shows that the MLCK ring surrounds FM4-64X dye. (D) Model for the visualization of rings. Side views of potential involuting membrane structures labeled with FM4-64X dye and recruited MLCK are marked with a plane of focus. The corresponding predicted two-dimensional patterns of FM4-64X and MLCK signal, as seen from above, are adjacent. Scale bars are 15 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 7: Src inhibition leads to the accumulation of internalized membrane marker within MLCK rings. MLCK rings may represent MLCK-coated vesicular intermediates. (A) Cells transfected with GFP-MLCK (green) were treated with exogenous FM4-64X (red) and fluorescently labeled CTXB (blue) during hypotonic exposure in the presence of PP2 (10 μM) for 1 min before fixation. Arrows mark distinct MLCK and FM4-64X membrane dye rings. (B) Stills from live imaging of cells transfected with GFP-MLCK (green) taken at the base and treated with PP2 (10 μM) during hypotonic treatment in the presence of exogenous FM4-64X. An arrow marks an MLCK ring that appears to encircle internalized membrane marker. (C) Cells transfected with GFP-MLCK (green) were treated with exogenous FM4-64X (red) and fluorescently labeled CTXB (blue) during hypotonic exposure in the presence of PP2 (10 μM) for 5 min before fixation. An arrow marks an MLCK ring that appears to encircle a lumen of FM4-64X–bound membrane dye. Single channel signals for MLCK, FM4-64X, and CTXB (left) show that FM4-64X signal is present in the ring, whereas CTXB signal is not. A three-dimensional reconstruction of the cell (right) with magnified panels adjacent shows that the MLCK ring surrounds FM4-64X dye. (D) Model for the visualization of rings. Side views of potential involuting membrane structures labeled with FM4-64X dye and recruited MLCK are marked with a plane of focus. The corresponding predicted two-dimensional patterns of FM4-64X and MLCK signal, as seen from above, are adjacent. Scale bars are 15 μm.
Mentions: Some forms of endocytosis are regulated by lipid raft formation (Parton and Richards, 2003), which we have found to be important in cell volume control (Barfod et al., 2007). In particular, Src activation has been suggested to control endocytosis mediated by GM1 ganglioside–containing lipid rafts (Shajahan et al., 2004). To determine whether the sites of volume-sensitive membrane turnover we have observed were also enriched in such lipid raft domains, we exposed cells expressing GFP-MLCK to fluorescently labeled GM1 ganglioside marker cholera toxin B (CTXB) in addition to FM4-64X. When these cells were swollen in the presence of PP2 before fixation, distinct rings of MLCK and FM4-64X appeared within 1 min (Figure 7A), but no rings of CTXB were seen, although this marker was clearly internalized to separate compartments. However, the CTXB and FM4-64X signals did colocalize in intracellular vesicles at longer time points (10–15 min) after swelling (unpublished data), suggesting that the initial uptake mechanisms were distinct but that membrane mixing could occur subsequently in endosomes.

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