Non-uniform impact of extracellular osmotic variations at subcellular level.

Osmotic perturbations, towards understanding basic cellular architectures and to alter cellular mechanics for various purposes, are widely utilized in cell biology. While osmotic perturbations are known to alter whole-cell morphology, their subcellular-level impacts remain poorly characterized. Here, we employ a novel quantitative imaging workflow to demonstrate that extracellular osmolarity induces organelle-specific redistribution patterns in adherent RAW264.7 macrophages, independent of whole-cell morphological changes. At the whole-cell level, we report a decrease in cellular pleomorphism (pixel-intensity-distribution-based heterogeneity) under non-isotonic conditions, with cell membrane and lysosomal pleomorphism decreasing as osmolarity decreases. Remarkably, osmolarity-induced variations observed at whole-cell level are translated to actin and tubulin variations only while nucleus, mitochondria, and endoplasmic reticulum are independent of the whole cell morphology alterations. However, there appears to be 'counterbalancing' of lateral polarity in the distributions of nucleus and endoplasmic reticulum in hypo-osmotic conditions. This work promises to be a key contribution towards understanding cellular architectures.