Ionic imbalance, in addition to molecular crowding, abates cytoskeletal dynamics and vesicle motility during hypertonic stress

Abstract

Cell volume homeostasis is vital for the maintenance of optimal protein density and cellular function. Numerous mammalian cell types are routinely exposed to acute hypertonic challenge and shrink. Molecular crowding modifies biochemical reaction rates and decreases macromolecule diffusion. Cell volume is restored rapidly by ion influx but at the expense of elevated intracellular sodium and chloride levels that persist long after challenge. Although recent studies have highlighted the role of molecular crowding on the effects of hypertonicity, the effects of ionic imbalance on cellular trafficking dynamics in living cells are largely unexplored. By tracking distinct fluorescently labeled endosome/vesicle populations by live-cell imaging, we show that vesicle motility is reduced dramatically in a variety of cell types at the onset of hypertonic challenge. Live-cell imaging of actin and tubulin revealed similar arrested microfilament motility upon challenge. Vesicle motility recovered long after cell volume, a process that required functional regulatory volume increase and was accelerated by a return of extracellular osmolality to isosmotic levels. This delay suggests that, although volume-induced molecular crowding contributes to trafficking defects, it alone cannot explain the observed effects. Using fluorescent indicators and FRET-based probes, we found that intracellular ATP abundance and mitochondrial potential were reduced by hypertonicity and recovered after longer periods of time. Similar to the effects of osmotic challenge, isovolumetric elevation of intracellular chloride concentration by ionophores transiently decreased ATP production by mitochondria and abated microfilament and vesicle motility. These data illustrate how perturbed ionic balance, in addition to molecular crowding, affects membrane trafficking.electrolyte | cell volume | hypertonicity | membrane trafficking | mitochondria

Regulation of Plasma Membrane Localization of the Na+-Taurocholate Cotransporting Polypeptide (Ntcp) by Hyperosmolarity and Tauroursodeoxycholate

“…Thus, TUDC exerts its choleretic effect by a coordinated insertion of both Ntcp and Bsep into plasma and canalicular membrane, respectively. A recent study showed that vesicle motility and ATP production is transiently reduced in a variety of cell types, including hepatocytes after treatment with hyperosmotic medium (65). Movement of dextran-loaded endosomes was tracked by live-cell imaging.…”

“…Because dextran is distributed to early, late, and recycling endosomes, this approach, however, will not distinguish between differences in motility among these compartments. Whether the motility is restricted also in newly endocytosed vesicles therefore remains unclear (65).…”

Peptide affinity analysis of proteins that bind to an unstructured NH2-terminal region of the osmoprotective transcription factor NFAT5

“…Therefore, we are intrigued that we now find protein-to-protein association of NFAT5, with many proteins involved in effects of hypertonicity, but not known to be transcriptional targets of NFAT5. Thus, hypertonicity interrupts the cell cycle (9), and NFAT5 associates with numerous proteins involved in cell proliferation (see RESULTS); development is aberrant in NFAT5 Ϫ/Ϫ fetuses (37,40), and NFAT5 associates with proteins involved in development (see RE-SULTS); hypertonicity affects activity of numerous enzymes (19), and we find that NFAT5 associates with several enzymes (see RESULTS); hypertonicity affects the cytoskeleton (8,39,45,48), and NFAT5 associates with cytoskeletal proteins and their regulators (see results); hypertonicity affects mitochondria (42,54,56), and NFAT5 associates with mitochondrial ribosomal proteins, transporters and structural proteins. We do not know whether and, if so, how protein-to-protein association of NFAT5 with these other proteins affects them, but the association raises interesting questions.…”

Intracellular Chloride Concentration Changes Modulate IL-1β Expression and Secretion in Human Bronchial Epithelial Cultured Cells

“…In order to test whether or not [Cl − ] i can modulate IL‐1β expression, we used a double ionophore strategy (ionophores nigericin and tributyltin) that equilibrates the intracellular and extracellular chloride and H + concentrations [Krapf et al, ], while keeping constant the pH, cell volume [Nunes et al, ], and membrane potential (since these ionophores are non‐electrogenic) [Antonenko Yu, ; Orlov et al, ]. In these conditions, the extracellular and intracellular Cl − concentrations are equal [Krapf et al, ; Valdivieso et al, , ].…”