Fig. 5: CRISPR ARHGEF2/GEF-H1 knockout influences T-cell migration. | Nature Communications

Fig. 5: CRISPR ARHGEF2/GEF-H1 knockout influences T-cell migration.

From: Engineering T cells to enhance 3D migration through structurally and mechanically complex tumor microenvironments

Fig. 5

Although GEF-H1 knockout (KO) prevents enhanced migration from microtubule destabilization, it enhances migration of untreated T cells. a Combined multi-photon excitation of fluorescence (MPE) and second harmonic generation (SHG) of primary human T cells (green) migrating through 3D matrices. To test the hypothesis that increased contractility from destabilized microtubules is GEF-H1 dependent, we knocked out GEF-H1 using CRISPR and tested migration over time in the presence of destabilized MTs (+Nocodazole). Samples of migrating T cells are shown and tracks of individual cells are highlighted with their tracks (yellow curves: start position, tick; end position, arrowhead). Images are samples from imaging every 1.5 min for >1 h. b Speeds (left) and c motility coefficients (right) for control (Cas9) and GEF-H1 KO T cells migrating within 3D microenvironments under MT-destabilizing conditions (+Nocodazole) showing that loss of GEF-H1 disrupts the contractility-enhanced migration from MT destabilization. Individual dots correspond to the cell motility from time-lapse imaging every 1.5 min for >1 h. Box plots depict the 25th percentile, median, and 75th percentile, and whiskers depict the 95% confidence intervals. Statistical tests are pairwise one-sided t-tests. All n- and p-values are shown on the plots. Number of replicates per condition: 3. Source data are provided as a Source Data file. d Combined MPE/SHG of control and GEF-H1 KO cells without destabilization of MTs. Samples of migrating T cells are shown and tracks of individual cells are highlighted using numerated pins along with their tracks (yellow curves: start position, tick; end position, arrowhead). e Speeds (left) and f motility coefficients (right) for control (Cas9) and GEF-H1 KO T cells migrating within 3D microenvironments with unperturbed MTs showing increased migration through complex 3D environments following genome engineering to KO GEF-H1. Individual dots correspond to the cell motility from time-lapse imaging every 1.5 min for >1 h. Box plots depict the 25th percentile, median, and 75th percentile, and whiskers depict the 95% confidence intervals. Statistical tests are pairwise one-sided t-tests. All n- and p-values are shown on the plots. Number of replicates per condition: 3. Source data are provided as a Source Data file.

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