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Hypotonic Stress Induces Fast, Reversible Degradation of the Vimentin Cytoskeleton via Intracellular Calcium Release.

Advanced science (Weinheim, Baden-Wurttemberg, Germany) (2019-09-29)
Leiting Pan, Ping Zhang, Fen Hu, Rui Yan, Manni He, Wan Li, Jingjun Xu, Ke Xu
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, protease free, essentially globulin free, pH 7, ≥98%
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Glucose Oxidase from Aspergillus niger, Type VII, lyophilized powder, ≥100,000 units/g solid (without added oxygen)
Sigma-Aldrich
MES monohydrate, BioXtra, ≥99.0% (T)
Sigma-Aldrich
2-Aminoethyl diphenylborinate, 97%
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Cysteamine, ≥98.0% (RT)
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Fura 2-AM, ≥95% (HPLC)
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Anti-Vimentin Antibody, serum, Chemicon®
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MDL 28170, ≥90% (TLC)
Sigma-Aldrich
LDN-57444, ≥98% (HPLC)