Fig. 2: Microvilli allow T cells to overcome a glycocalyx barrier.

a Cartoon depiction of experiments testing whether dynamic actin remodeling of microvilli is needed to establish close-contacts in the presence of a glycocalyx barrier. Jasplakinolide (Jasplak) enhances the nucleation and stabilization of actin filaments, resulting in ‘paralysis’ of the actin cytoskeleton and microvillar activity. Latrunculin B (Lat B) and cytochalasin D (Cyto D) prevent the formation of actin filaments by sequestering actin monomers and by blocking their recruitment to pre-existing filaments, respectively, resulting in filament breakdown and subsequent loss of membrane topography. Cells in the cartoon are shown interacting with an SLB2. b Confocal fluorescence images of fixed J8-GECI cells imaged at the midplane after treatment with DMSO or an actin-modifying drug. A digitally-magnified region (white box) is shown below. Images are representative of J8-GECI cells for n = 3 independent experiments. c Examples of contact formation in the presence of DMSO or the actin-modifying drugs, for J8-GECI cells interacting with an SLB2 presenting pMHC^null plus pMHC^9V-lo, i.e., ~1 pMHC^9V molecule/μm². Close contacts are indicated by black holes in the SLB2 glycocalyx fluorescence (with point of initiation indicated by white arrows). See Supplementary Movie 3. Images are representative of drug-treated J8-GECI cells for n = 4 independent experiments. d Fraction of cells that formed detectable close contacts. Data are from the same experiment as in (c). Shown is the mean (±S.D.) of n = 4 independent SLBs with 13–113 cells imaged per SLB. e Time taken for cells to form a close contact versus first appearance of membrane fluorescence. Data are from the same experiment as in (c). Data were pooled from four independent SLBs with n = 93 (DMSO), 88 (Cyto D), 18 (Jasplak), and 64 (Lat B) total contact-forming cells analyzed. The red line indicates the median. In d and e, means were tested using one-way ANOVA with Dunnett correction using DMSO as the control group. f Fraction of DMSO- or actin-modifying drug-treated J8-GECI cells that exhibit calcium release on an SLB2 or SLB2Δglycocalyx (i.e., SLB1 + CD58) presenting pMHC^null plus pMHC^9V-lo. Shown is the mean (±S.D.) of n = 4 independent SLBs with 138-785 cells analyzed per SLB. Two-way ANOVA with Šidák correction was used to compare means between glycocalyx-positive and -negative SLBs for each treatment. Source data are provided in the Source data file.