Fig. 2: Stress vesicles induce rapid intracellular changes.

a Representative live imaging frames illustrating the formation of stress vesicles during the application of positive pressure. Similar results were obtained from at least three independent experiments. Scale bar: 2 µm. See also Movie S1. b Three-dimensional renderings of basal epidermal cells before and after the application of positive pressure. The nucleus is shown in green, the plasma membrane in magenta, and stress vesicles in red. Scale bar: 2 µm. c High-resolution electron micrograph of basal epidermal cells in the mouse epidermis. A large cytoplasmic vesicle is visible in the stressed cell. The red dashed line marks the boundary between the epidermis and dermis. Similar results were obtained from two independent experiments. Scale bar: 1 µm. d Schematic of the experimental strategy used to track the internalization of extracellular fluid into epidermal cells following the application of negative pressure. e Quantification of epidermal cells with stress vesicles labeled with the extracellular fluid tracer Dextran Alexa-488. Data are from N = 4 mice, n = 16 areas measured. Statistical analysis: unpaired t-test. f Representative images of Dextran internalization in normal skin and skin subjected to 10 min of negative pressure. Scale bar: 10 µm. g Representative images of epidermal cells labeled with mGFP (membrane-bound) and cytTom (cytoplasmic) following negative force application. Stress vesicles are composed of plasma membrane, with lumens devoid of red signal, indicating the absence of cytoplasmic content. Scale bar: 5 µm. h Quantification of stress vesicle growth. Data are from N = 3 mice, n = 20 vesicles. i Representative images of stress vesicles filled containing Dextran-FITC dye, previously injected intradermally in a R26-mTom reporter mouse; Dextran-FITC labeled stress vesicles were processed through 3D rendering in Imaris. j Quantification of stress vesicle size measured by 3D volumetric analysis. Data are from N = 4 mice, n = 120 vesicles. k Representative images of stress vesicle disappearance after the removal of force. Scale bar: 5 µm. l Quantification of the decrease in vesicle numbers 1 h and 2 h after the removal of force loading. Data are from N = 5 mice for each group. Statistical analysis: two-tailed unpaired t-test. Data are presented as mean ± SEM Scale bars: 2 µm (c) and 5 µm (e).