Fig. 2

Fig. 2

Osmotic Shock of HeLa Cells Triggers cPla₂ Translocation, AA Release, and Nuclear Swelling Counteracted by F-Actin

(A) Confocal imaging of cPla₂-mK2 in the tail fin of larval zebrafish and HeLa cells, before and after hypotonic exposure (see also Figures S1 and S3). Note the INM-accumulation of cPla₂ (white arrow) upon osmotic shock triggered by epithelial wounding of zebrafish in hypotonic bathing solution, or by diluting the cell culture medium of HeLa cells to 150 mOSM. Scale bars, 10 µm.

(B) [³H] arachidonic acid release triggered by 10 min hypotonic shock, measured in cPLA₂ knock out (k.o.) HeLa cells in the absence (left bars) and presence of heterologous expression of cPla₂-mK2. Error bars, SEM; results from eight to ten preparations. ***p < 0.0005, t test.

(C) Western blot of wild-type, cPLA₂ knock out, and cPla₂-mK2-expressing HeLa cells.

(D) Parallel, single cell measurements of cPla₂-mK2 INM-translocation and cytoplasmic Ca²⁺ signals, using the calcium indicator dye Fluo4. INM-translocation and Fluo4 signals were scored by automated image analysis. Fluo4 signals were normalized to baseline values and ionomycin-induced maximum signals (see the Experimental Procedures for details). Upper panels, plot of cPla₂-mk2 translocation (x axis) as a function [Ca²⁺] (y axis) in the same cell. Cellular responses were triggered by the purinergic agonist ATP (100 µM) or hypotonicity (150 mOSM) in the absence/presence of latrunculin A (LA) pretreatment as indicated. Gray dots, cells with Ca²⁺ signals below the critical threshold required for cPla₂-mK2 translocation. Black dots, cells above the critical Ca²⁺-threshold (i.e., 0.3) that do not show cPla₂-mK2 translocation. Red dots, cells above the critical Ca²⁺-threshold showing cPla₂-mK2 translocation. The percentage of translocating (red) versus non-translocating cells (black) above the critical Ca²⁺-threshold is depicted as bar diagram above each plot. This ratio indicates the efficiency of a suprathreshold Ca²⁺ signal to induce cPla₂-mK2 translocation under the respective experimental condition. Lower panels, average [Ca²⁺]-traces of cells that exhibit cPla₂-mK2 translocation during the stimulation. Error bars, SEM; n, number of cells.

(E) Average nuclear volume evolution after hypotonic shock in untreated and LA-pretreated HeLa cells, measured by confocal imaging of nuclear targeted EGFP. Error bars, SEM; n, number of cells.