Journal: eLife

Article Title: Formin-like 1 mediates effector T cell trafficking to inflammatory sites to enable T cell-mediated autoimmunity

doi: 10.7554/eLife.58046

Figure Lengend Snippet: Ex vivo activated control LifeAct-GFP and FMNL1 KO LifeAct-GFP T cells were stained with Hoechst, added to PDMS microchannels and imaged by spinning-disk confocal time-lapse microscopy. ( A ) Schematic of the PDMS microchannels with constrictions used in the experiments. ( B ) FMNL1 deficiency impairs the ability of T cells to migrate through 3 µm constrictions. Quantification of the percentage of T cell passage through 3 µm constrictions within microchannel devices. ( C ) Example images of WT LifeAct-GFP (Left panels and ) and FMNL1 KO/LifeAct-GFP (Right panels and ) T cells engaging microchannel constrictions. Top images, LifeAct-GFP (green) and Hoechst (DNA, blue) overlaid on the brightfield channel (gray). Bottom images, pseudocolor rendition of the LifeAct-GFP channel. White dashed arrows indicate cell direction, red arrows point to areas of F-actin accumulation at the back of the cell, black arrows indicate F-actin accumulation at the front of the cell. Time is min:sec, white scale bar = 10 µm. ( D ) FMNL1 promotes actin polymerization at the back of the nucleus during migration under confinement. Top, example of the image masking process to quantify F-actin distribution relative to the front and back of the nucleus. Bottom, quantification of the back-to-front ratio of F-actin distribution during unconfined and confined migration. ( E ) Paired analysis of individual T cells undergoing unconfined and confined migration. Data for cells that increase their F-actin back-to-front ratio under confinement are shown in black, data for cells that decrease the back-to-front ratio are in red. Data in B are the mean ± SEM from 3 independent experiments with a total of 83 control and 68 KO cells analyzed. Data in D are the mean ± SEM and data in E are pooled from 3 independent experiments with a total of 47 control and 35 KO cells analyzed. Statistics in B and E calculated using a two-tailed paired t-test; statistics in D calculated using One-way ANOVA with Sidak’s multiple comparisons. n.s. = not significant. Figure 7—source data 1. Data points for the graphs in are provided as an Excel spreadsheet.

Article Snippet: PDMS (polydimethylsiloxane) microchannel fluidic devices with constrictions were purchased from 4DCell (Montreuil, France).

Techniques: Ex Vivo, Control, Staining, Time-lapse Microscopy, Migration, Two Tailed Test

Journal: eLife

Article Title: Formin-like 1 mediates effector T cell trafficking to inflammatory sites to enable T cell-mediated autoimmunity

doi: 10.7554/eLife.58046

Figure Lengend Snippet:

Article Snippet: PDMS (polydimethylsiloxane) microchannel fluidic devices with constrictions were purchased from 4DCell (Montreuil, France).

Techniques: Expressing, Construct, Recombinant, Plasmid Preparation, Control