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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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Swelling induces localization of MLCK to foci at the base of the cell, where focal transient tyrosine phosphorylation events are detected. (A) Selected stills from a swollen cell expressing GFP-MLCK show the appearance and disappearance of rings and patches. Representative patches are marked with larger arrows; representative rings are marked with smaller arrows. Insets at higher magnification are marked with the same arrows. (B) Stills taken at the base of a cell overexpressing YFP-dSH2 before and after hypotonic exposure. Arrows mark new dSH2 flashes, sites of tyrosine phosphorylation, at each time point. Time (in min) indicates duration of hypotonic exposure. (C) Graphs of the number of YFP-dSH2 flashes per cell (left) and the number of GFP-MLCK patches (middle) and rings (right) per cell as a function of time. The kinetics of the dSH2 flashes and MLCK patches follow a similar time course. Scale bars are 15 μm.
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Figure 2: Swelling induces localization of MLCK to foci at the base of the cell, where focal transient tyrosine phosphorylation events are detected. (A) Selected stills from a swollen cell expressing GFP-MLCK show the appearance and disappearance of rings and patches. Representative patches are marked with larger arrows; representative rings are marked with smaller arrows. Insets at higher magnification are marked with the same arrows. (B) Stills taken at the base of a cell overexpressing YFP-dSH2 before and after hypotonic exposure. Arrows mark new dSH2 flashes, sites of tyrosine phosphorylation, at each time point. Time (in min) indicates duration of hypotonic exposure. (C) Graphs of the number of YFP-dSH2 flashes per cell (left) and the number of GFP-MLCK patches (middle) and rings (right) per cell as a function of time. The kinetics of the dSH2 flashes and MLCK patches follow a similar time course. Scale bars are 15 μm.

Mentions: MLCK-regulated bleb retraction appears to be one of the responses to cell swelling. However, it cannot fully account for the dependence of cell volume recovery on MLCK activity per se because not all swollen cells bleb (Supplemental Video 4). We therefore hypothesized that MLCK also regulates retrieval of membranes outside of blebs during osmotic stress and looked for localization of MLCK to other dynamic membrane structures. On swelling of GFP-MLCK–expressing cells, we observed two other motile MLCK-positive structures at the base: rings and patches that were ∼0.5 μm in diameter (Figure 2A, Supplemental Video 7). Although patches could be occasionally and transiently visualized under isotonic conditions, they appeared more prominently after hypotonic exposure (Figure 2C).


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)

Swelling induces localization of MLCK to foci at the base of the cell, where focal transient tyrosine phosphorylation events are detected. (A) Selected stills from a swollen cell expressing GFP-MLCK show the appearance and disappearance of rings and patches. Representative patches are marked with larger arrows; representative rings are marked with smaller arrows. Insets at higher magnification are marked with the same arrows. (B) Stills taken at the base of a cell overexpressing YFP-dSH2 before and after hypotonic exposure. Arrows mark new dSH2 flashes, sites of tyrosine phosphorylation, at each time point. Time (in min) indicates duration of hypotonic exposure. (C) Graphs of the number of YFP-dSH2 flashes per cell (left) and the number of GFP-MLCK patches (middle) and rings (right) per cell as a function of time. The kinetics of the dSH2 flashes and MLCK patches follow a similar time course. Scale bars are 15 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Swelling induces localization of MLCK to foci at the base of the cell, where focal transient tyrosine phosphorylation events are detected. (A) Selected stills from a swollen cell expressing GFP-MLCK show the appearance and disappearance of rings and patches. Representative patches are marked with larger arrows; representative rings are marked with smaller arrows. Insets at higher magnification are marked with the same arrows. (B) Stills taken at the base of a cell overexpressing YFP-dSH2 before and after hypotonic exposure. Arrows mark new dSH2 flashes, sites of tyrosine phosphorylation, at each time point. Time (in min) indicates duration of hypotonic exposure. (C) Graphs of the number of YFP-dSH2 flashes per cell (left) and the number of GFP-MLCK patches (middle) and rings (right) per cell as a function of time. The kinetics of the dSH2 flashes and MLCK patches follow a similar time course. Scale bars are 15 μm.
Mentions: MLCK-regulated bleb retraction appears to be one of the responses to cell swelling. However, it cannot fully account for the dependence of cell volume recovery on MLCK activity per se because not all swollen cells bleb (Supplemental Video 4). We therefore hypothesized that MLCK also regulates retrieval of membranes outside of blebs during osmotic stress and looked for localization of MLCK to other dynamic membrane structures. On swelling of GFP-MLCK–expressing cells, we observed two other motile MLCK-positive structures at the base: rings and patches that were ∼0.5 μm in diameter (Figure 2A, Supplemental Video 7). Although patches could be occasionally and transiently visualized under isotonic conditions, they appeared more prominently after hypotonic exposure (Figure 2C).

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