Fig. 3: The microtubule–contractility axis regulates T-cell mechanics and migration in 3D environments.

a STED micrograph of a human T cell migrating through 3D collagen matrix. Arrowheads, distinct bleb-to-pseudopodia amoeboid protrusions. Colors: fire, F-actin; white, collagen fibers; blue, nuclei. b Model for how MT-associated signaling integrates amoeboid motility through MT stability and contractility via GEF-H1 cytoplasm⇄MT reversible transitioning. c In our model, T-cell amoeboid motility in 3D environments is linked to actomyosin contractility where cortex tension-induced hydrostatic pressure forces plasma membrane off-cortex peeling (blebbing) that results in pseudopodia formation. Continuous pseudopodial formation facilitates steric interactions between T cells and the 3D microenvironment, i.e., persistent amoeboid motility. d Top, 3D reconstruction of the probed T cells in AFM testing settings. T cells non-specifically adhere to the poly-lysine-coated plasma-treated glass surface (bottom), whereas the AFM cantilever mechanically tests cells from atop. Bottom left, mechanical testing of T cells with atomic force microscopy (AFM) to measure the global compression through a flat cantilever, to measure the overall cortical tension that is counterbalanced with cytoplasm hydrostatic pressure and additional intracellular cytoskeletal structural resistance (microtubules, nucleus, etc). Colors: fire, F-actin; blue, nuclei. Bottom right, AFM measurement of global T-cell surface tension, a measure of cortical contractility. The control group and each of the two MT-targeting treatments (i.e., +DMSO, +Nocodazole, and +Taxol, solid colors) are paired with blebbistatin co-treatment (pale semi-transparent colors), to verify the key role of actomyosin contractility as a regulator of changes in cell rigidity during MT targeting. Alternatively, direct RhoA activation or inhibition is compared to MT perturbation results. Both MT disassembly (+Nocodazole) and direct RhoA activation (+RhoA activator) induce mechanical rigidification of human CD4+ T cells via increased actomyosin tension, as demonstrated by AFM findings after blebbistatin co-treatment. It is noteworthy that MT destabilization or direct RhoA activation induce a rise of hydrostatic pressure. Also, Taxol-induced MT stabilization increases passive (i.e., actomyosin-independent) T-cell rigidity via a direct mechanical contribution from stabilized MT scaffolds (+Taxol vs. +Taxol + Blebbistatin treatments; arrowheads). Individual dots correspond to individual cells. 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. e 3D migration speed and overall 3D cell motility of T cells in 3D collagen-FN matrix: 1, human T cells (hCD4+) in the presence of vehicle (+DMSO), Nocodazole (+Nocodazole), or Taxol (+Taxol); 2, mouse cytotoxic T cells (mCD8+) in the presence of vehicle (+DMSO), Nocodazole (+Nocodazole), or Taxol (+Taxol); and 3, human T cells in the presence of RhoA activator (+RhoA activator) or inhibitor (+RhoA inhibitor). 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, 75th percentile, and whiskers depict the 95% confidence intervals. Statistical tests are one-way ANOVA, post hoc Tukey’s 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.