Abstract
The dynamic response of the cell to osmotic changes is critical to its physiology and is widely exploited for cell manipulation. Here, using three‐dimensional stochastic optical reconstruction microscopy (3D‐STORM), a super‐resolution technique, the hypotonic stress‐induced ultrastructural changes of the cytoskeleton of a common fibroblast cell type are examined. Unexpectedly, these efforts lead to the discovery of a fast, yet reversible dissolution of the vimentin intermediate filament system that precedes ultrastructural changes of the supposedly more dynamic actin and tubulin cytoskeletal systems as well as changes in cell morphology. In combination with calcium imaging and biochemical analysis, it is shown that the vimentin‐specific fast cytoskeletal degradation under hypotonic stress is due to proteolysis by the calcium‐dependent protease calpain. The process is found to be activated by the hypotonic stress‐induced calcium release from intracellular stores, and is therefore efficiently suppressed by inhibiting any part of the IP3‐Ca2+‐calpain pathway established in this study. Together, these findings highlight an unexpected, fast degradation mechanism for the vimentin cytoskeleton in response to external stimuli, and point to the significant, yet previously overlooked physiological impacts of hypotonic stress‐induced intracellular calcium release on cell ultrastructure and function.
Highlights
The intricate inner machinery of the cell depends critically on water homeostasis to regulate intracellular concentrations
Using 3D-STORM (Huang et al, 2008; Rust et al, 2006) super-resolution microscopy (SRM), here we examine the hypotonic stress-induced ultrastructural changes of the cytoskeleton of a common fibroblast cell type at high spatial resolution
COS-7 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS following standard tissue-culture protocols
Summary
The intricate inner machinery of the cell depends critically on water homeostasis to regulate intracellular concentrations. Recent work further established osmotic volume change as a key method to control intracellular protein concentration for studies on protein crowding and protein-protein interactions (Boersma et al, 2015; Sukenik et al, 2017). It is of both fundamental and practical importance to understand if osmotic effects would lead to significant intracellular structural changes, and if so, how fast do such changes occur, whether they are reversible and/or suppressible, as well as what mechanisms drive such processes. The cytoskeleton plays essential roles in the cell volume regulation under osmotic stress (Pedersen et al, 2001), a natural consequence given the pivotal role of the cytoskeleton in cell structure and function (Fletcher and Mullins, 2010; Lowery et al, 2015; Pollard and Cooper, 2009)
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