Fig. 3: Nuclear deformation links stress vesicles to cell differentiation.

a Schematic representation of the genetic alleles and strategy for lineage tracing of epidermal cells via in vivo photo-labeling. Prior to mechanical force application, equivalent groups of basal epidermal cells were scanned to activate the K14H2BPAGFP reporter. The same areas were re-imaged to track population changes over time. b Representative time-lapse images of photo-labeling and cell tracking following mechanical force application. Yellow arrows indicate the direction of cell movement as cells differentiate and move from the basal to the suprabasal layer. Scale bar: 10 µm. c, d Quantification of the relative fractions of cell proliferation and differentiation in tracked epidermal cells 1 day after mechanical force application. N = 3 mice, n = 12 images analyzed per condition. Statistical analysis: two-tailed unpaired t-test. e Experimental strategy for collecting mouse skin tissue immediately after applying negative pressure for panels F-I. f Representative immunofluorescence images showing stress vesicle formation in K10+, Ki67+, and K10-/Ki67- basal epidermal cells. Yellow arrows indicate basal layer cells with stress vesicles. A white dashed line marks the boundary between the epidermis and dermis. Scale bar: 5 µm. g Quantification of stress vesicle formation in three distinct groups of basal epidermal cells following mechanical force application. One-way ANOVA test (P < 0.0001) with Tukey’s mulitiple comparisons test: K10-; Ki67- vs Ki67+, P = 0.0089; K10-; Ki67- vs K10 + , P < 0.0001; Ki67+ vs K10+, P < 0.0001. Data are presented as mean ± SEM, N = 7 mice. h Representative whole-mount immunofluorescence images of stressed epidermis stained with Ki67 (nuclear signals) and Krt10 (membrane signals) antibodies. Scale bar: 10 µm. i Quantification of the ratio of stress vesicle formation in three different groups of basal epidermal cells from whole-mount-stained epidermis. One-way ANOVA test (P < 0.0001) with Tukey’s mulitiple comparisons test: K10-; Ki67- vs Ki67 + , P < 0.0001; K10-; Ki67- vs K10 + , P < 0.0001; Ki67+ vs K10 + , P < 0.0001. Data are presented as mean ± SEM, n = 16 images analyzed from 4 mice for each group. J Representative time-lapse images from live imaging lineage tracing, capturing varying levels of nuclear deformation and the subsequent fates of epidermal cells bearing nuclear deformation. Labeled epidermal cells were categorized into three groups based on their fates: undifferentiated cells (remaining in the basal layer), differentiated cells (delaminating and moving into the suprabasal layer), and proliferating cells (producing two daughter cells in the basal layer). Scale bar: 2 µm. k, l Quantification of nuclear deformation as a function of cell fate. Each data point represents a single nucleus. Statistical comparisons between groups were performed using One-way ANOVA with Tukey’s multiple comparisons test; p-values are indicated on the graph. Circularity: N = 4 mice, n = 59 (undifferentiated cells), n = 59 (differentiated cells), and n = 60 (proliferated cells). Statistical analysis: P < 0.0001. Angle: N = 4 mice, n = 93 (undifferentiated cells), n = 99 (differentiated cells), and n = 89 (proliferated cells). m, n Quantification of nuclear volume in differentiated cells (left graph) or proliferated cells (right graph) compared to undifferentiated cells. Data are from N = 3 mice, n = 40 undifferentiated cells, n = 23 differentiated cells, and n = 22 proliferated cells. Statistical analysis: two-tailed unpaired t-test. Data are presented as mean ± SEM.