C-terminal alignment of FMNL1 isoforms. S1086 of FMNL1β shows a high degree of homology with PKC-phosphorylated S1072 in FMNL2.

Panel A) amino acid sequences of the C termini of FMNL2, FMNL1α and FMNL1β, as well as FMNL1γ containing a C-terminal intron retention, and sharing the final C-terminal amino acids with FMNL1α (shown underlined). The three FMNL1 isoforms share identical sequence (100% identity) from amino acid residue 1 to T1070, and diverge in the C-terminal region, which includes the DAD autoinhibitory domain (36). The DAD domain sequence, which is responsible for autoinhibition in the murine homolog is highlighted in green (36), and the identical C-terminal amino acids shared by FMNL1α and FMNL1γ are underlined. The arginine-rich, polybasic region common to other formins such as FMNL2 (32), FHOD3 (50) and FHOD1 (49) (see Discussion) is also shown overlined. Panel B), C-terminal amino acid sequences of the two different point mutations at S1086 in FMNL1β used in this study.

Expression of YFP-FMNL1β S1086 variants in FMNL1-silenced cells.

Panel A), C3 control clone was untransfected (Control YFP–) (first row) or transfected with FMNL1-interfering (shFMNL1-HA-YFP) (second row), or FMNL1-interfering expressing interference-resistant YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT) (third row), YFP-FMNL1βS1086A (fourth row) or YFP-FMNL1βS1086D (fifth row) constructs. Subsequently, cells were challenged with CMAC-labelled SEE-pulsed Raji cells (blue) for 1 h, fixed, stained with anti-FMNL1, and imaged by epifluorescence microscopy. Representative MIP images of merged transmittance (TRANS), CMAC (blue), YFP (yellow), and anti-FMNL1 (red) channels are indicated for the different cell groups. Panel B), cell lysates corresponding to the indicated cell groups were analysed by WB developed with anti-FMNL1 antibody (two different expositions). The lower bar graph depicts the WB quantification showing the endogenous FMNL1 expression in the different cell groups relative to control untransfected (YFP–) cells. Results are representative of data from several independent experiments (n=3) with similar results.

S1086 in FMNL1β is phosphorylated upon PKC activation.

C3 control clone was unstransfected (Control YFP–) or transfected with either FMNL1-interfering expressing interference-resistant YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT), YFP-FMNL1βS1086A or YFP-FMNL1βS1086D constructs. Subsequently, cells were stimulated or not (-) with PKCδ activator PMA for 30 min. The different cell groups were lysed and immunoprecipitated with anti-FMNL1. These IPs were analysed by WB, first with anti-Phospho-Ser PKC substrate antibody (two different expositions) and then reprobed with anti-FMNL1 to normalize Phospho-Ser PKC substrate signal for FMNL1 protein levels. The lower graph represents the normalized fold induction of phosphorylation of the different FMNL1 variants. Results are representative of data from several independent experiments (n=3) with similar results.

FMNL1β phosphorylation at S1086 is involved in MTOC/MVB polarization towards the immune synapse.

C3 control clone was untransfected (Control YFP–) (first row) or transfected with FMNL1-interfering (shFMNL1-HA-YFP) (second row), or FMNL1-interfering expressing interference-resistant YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT) (third row), YFP-FMNL1βS1086A (fourth row) or YFP-FMNL1βS1086D (fifth row) constructs. Subsequently, cells were challenged with CMAC-labelled, SEE-pulsed Raji cells (blue) for 1 h, fixed, stained with anti-pericentrin (magenta) to label the MTOC and anti-CD63 (red) to label MVB, and imaged by epifluorescence microscopy. Panel A), representative MIP images with the indicated merged channels for each of the specified cell groups, along with a schematic diagram on the right representing the measured parameters used to calculate the MTOC and MVB PI. This includes the distance in microns between the MTOCC (or MVBC) projection on the vector defined by the CellC–synapse axis and the CellC (“B” or “A” distance, respectively), and the distance between the CellC and the synapse (“C” distance). Panel B), dot plots of MVB and MTOC PI from each of the indicated cell groups, corresponding to the indicated number of synapses from a experiment similar to that described in panel A) are depicted. NS, not significant. ***, p ≤0.05. Results and ANOVA analyses are representative of data from several independent experiments (n=3) with similar results.

YFP-FMNL1βS1086D expression does not rescue deficient MTOC polarization in PKCδ-interfered cells.

PKCδ-interfered P5 clone was untransfected (Control YFP–) (first row) or transfected with FMNL1-interfering (shFMNL1-HA-YFP) (second row), FMNL1-interfering expressing interference-resistant YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT) (third row), YFP-FMNL1βS1086A (fourth row) or YFP-FMNL1βS1086D (fifth row) constructs. In parallel, C3 control clone was untransfected (Control YFP–) or transfected with FMNL1-interfering (shFMNL1-HA-YFP) construct. Subsequently, cells were challenged with CMAC-labelled, SEE-pulsed Raji cells (blue) for 1 h, fixed, stained with anti-pericentrin (magenta) to label the MTOC and anti-CD63 (red) to label MVB, and imaged by epifluorescence microscopy. Panel A), representative MIP images with the indicated merged channels for each of the specified cell groups along with their corresponding diagrams as in Fig. 4, representing the measured parameters used for calculating the MTOC and MVB PI are shown. This includes the distance in microns between the MTOCC (or MVBC) projection on the vector defined by the CellC-synapse axis and the CellC (“B” or “A” distance, respectively), and the distance between the CellC and the synapse (“C” distance). Panel B), dot plots of MTOC PI of each cell group. Untransfected (Control YFP–) and FMNL1-interfering (shFMNL1-HA-YFP) C3 clone synapses corresponding to the data shown in Suppl.Fig. S3B were included in the far-right columns, as a reference. NS, not significant; ***, p ≤0.05. Results and ANOVA analyses are representative of data from several independent experiments (n=3) with similar results.

FMNL1β recruitment to the immune synapse is PKCδ-independent.

C3 control and P5 PKCδ-interfered cells were transfected with FMNL1-interfering expressing interference-resistant YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT) plasmid. Subsequently, both transfected clones were simultaneously challenged with CMAC-labelled, SEE-pulsed Raji cells (blue) attached to slides and time-lapse acquisition of emerging synapses was performed as indicated in Materials and Methods. The videos (7 fps) (Suppl. Video 3) were captured and in panel A), left, representative frames from videos of each clone are shown. White arrows indicate accumulations of YFP-FMNL1βWT at the IS. WB analysis of cell lysates from both clones (top inset) shows PKCδ silencing in P5 clone. In the right side, YFP-FMNL1βWT MFI within the cell ROI (grey line) and the IS ROI (red line) are represented. The inserts in the graphs include the cell ROI (white) and the IS ROI (red) used for the time-lapse measurements on representative frames for both clones. Results are representative of the data from several videos (n=6 for each clone) with similar results. Panel B), control C3 (upper rows) and P5 PKCδ-interfered cells (lower rows) were simultaneously challenged with CMAC-labelled SEE-pulsed Raji cells (blue) attached to slides. After 1 h, synapses were fixed and immunofluorescence developed with anti-FMNL1 (red) to label endogenous FMNL1 and phalloidin (magenta) to label F-actin. Synapses were imaged with confocal fluorescence microscopy and colocalization pixels between FMNL1 (red) and F-actin (acquired in magenta, changed to blue in the fourth column) are represented in white. Representative optical sections of synapses formed by both clones are shown in the left columns. The colocation coefficients were: First row, Pearson= 0.50, Manders-= 0.877; Second row, Pearson = 0.431, Manders= 0.864; Third row: Pearson= 0.434, Manders= 0.838; Fourth row, Pearson= 0. 484, Manders= 0.794. MIP images of the same synapses are shown in the two far right columns. Results and ANOVA analyses are representative of data from several independent experiments (n=3) with similar results.

FMNL1β phosphorylation at S1086 regulates the F-actin architecture of the immune synapse.

C3 control clone was untransfected (Control YFP–) (first column) or transfected with FMNL1-interfering (shFMNL1-HA-YFP) (second column), FMNL1-interfering expressing interference-resistant YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT) (third column), YFP-FMNL1βS1086A (fourth column) or YFP-FMNL1βS1086D (fifth column) constructs. Subsequently, cells were challenged with CMAC-labelled, SEE-pulsed Raji cells (blue) for 1 h, fixed, stained with phalloidin AF647 (magenta) to label F-actin, and imaged by confocal microscopy. Panel A), the upper rows display the top, yx views corresponding to the MIP images of the specified, merged channels of a representative example from each of the indicated cell groups. White arrows indicate the direction to visualize the en face views of the IS (IS interface) enclosed by the ROIs (white rectangles), as shown in Suppl. Video 5. In the lower panels, the enlarged ROIs (2x zoom) used to generate the IS interface, zx images of each cell group are shown. The areas of the F-actin-low region at the cIS (Fact-low cIS area) (yellow line) and the synapse (IS area) (white line) were defined and measured as indicated in Materials and Methods, and the relative area of the F-actin-low region at the cIS (Fact-low cIS area / IS area) was calculated and represented. Panel B), relative area (Fact-low cIS area / IS area) dot plot distributions and average area ratios (red horizontal lines) for the indicated number of IS conjugates developed by each cell group are shown. This figure is related to Suppl. Video 5. NS, not significant; ***, p≤0.05. Results are representative of data from several independent experiments (n=3) with similar results.

Three-dimensional distribution and colocalization of FMNL1 and F-actin at the immune synapse interface.

C3 control clone was untransfected (Control YFP–) (first column) or transfected with FMNL1-interfering (shFMNL1-HA-YFP) (second column), FMNL1-interfering expressing interference-resistant YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT) (third and fourth column), YFP-FMNL1βS1086A (fifth column) or YFP-FMNL1βS1086D (sixth column) constructs. Subsequently, cells were challenged with CMAC-labelled SEE-pulsed Raji cells (blue) for 1 h, fixed, and stained with phalloidin (magenta) and anti-FMNL1 (red). The corresponding shFMNL1 construction is in yellow, and synaptic conjugates were imaged by confocal fluorescence microscopy. Please realize that for the IS interface and since interface colocalization in NIS-AR only works with red, green and blue channels, F-actin (acquired in magenta) was changed to blue color, and YFP (acquired in yellow) was changed to green in the second row of each panel. Panel A), the upper row includes top, yx views corresponding to the MIP images of representative examples of each cell group. Vertical white arrows indicate the direction to visualize the en face views of the IS (IS interface) enclosed by the ROIs (white rectangles), as shown in Suppl. Video 6. In the second row, the enlarged ROIs (2x zoom) used to generate the IS interface, zx images of each cell group are shown. Subsequently, interface colocalization pixels (white) were generated by merging the indicated channels in the second row of each panel (F-actin in blue merged to anti-FMNL1 in red), at the IS interfaces of the synaptic areas (generated as shown in Suppl. Video 6). The last frame of these videos corresponds to the en face view (interface) (second row in both panels). MFI profiles along the indicated line (horizontal white arrow) of each separate channel (Factin in blue, anti-FMNL1 in red) and the colocalization pixels (grey) are shown below the IS interfaces. Panel B), same as panel A, but the top views show YFP-expressing constructs signal instead of the anti-FMNL1 signal. The IS interfaces and the MFI profiles show F-actin (magenta changed to blue) and YFP (yellow changed to green). This figure is related to Suppl. Video 6. At least 6 synapses of each cell group were analysed. Results are representative of data from several independent experiments (n=3) with similar results.

FMNL1β phosphorylation at S1086 regulates exosome secretion at the immune synapse.

C3 control clone was transfected with exosome reporter GFP-CD63 alone (Control YFP–) or co-transfected with the exosome reporter GFP-CD63 and 3-fold molar excess of either FMNL1-interfering (shFMNL1-HA-YFP), or FMNL1-interfering expressing interference-resistant YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT), YFP-FMNL1βS1086A or YFP-FMNL1βS1086D constructs. Subsequently, the different cell groups were challenged with unpulsed (-) or SEE-pulsed Raji cells for 4 h and exosomes were collected and purified from cell culture supernatants and analysed by NTA and WB, as indicated in Materials and Methods. Panel A), left, NTA analyses corresponding to nanovesicles isolated from cell culture supernatants of control YFP– Jurkat cells stimulated with unpulsed Raji cells (-) or SEE-pulsed Raji cells (SEE). Right, concentration and SEE-stimulated nanoparticle concentration increase (fold) induction of secreted nanovesicles from several NTA analyses for the indicated cell groups. Panel B), left, WB analyses of exosomal GFP-CD63 reporter isolated from the indicated cell culture supernatants. Right, quantification of normalized, fold induction of exosomal GFP-CD63 secretion from three experiments similar to the one described in the left panel. NS, not significant; ***, p≤0.05. Results and ANOVA analyses are representative of data from several independent experiments (n=3) with similar results.

STED image of CD63+ nanovesicles at the synaptic cleft.

C3 control clone cells untrasfected or expressing shFMNL1-HA-YFP were challenged with CMAC-labelled SEE-pulsed Raji cells (blue) for 1h, fixed, stained with phalloidinAF647 (magenta) and anti-CD63 (red) and imaged simultaneously by confocal and STED microscopy. Two representative control YFP– and FMNL1-interfering (shFMNL1-HA-YFP) Jurkat cells forming IS with Raji cells are shown. Confocal (left) and STED (right) optical sections are shown, and enlarged (2.5 and 3X zoom) views of the IS areas are shown in the right side. The yellow arrows on the enlarged IS images label the edges of the synaptic cleft, which is the narrow, lane-shaped space between the two cells enclosed by the two F-actin-rich (magenta) plasma membrane leaflets. CD63+ MVB from the Jurkat cells are located nearby to the IS and some CD63+ nanovesicles (white arrows) are located at the synaptic cleft in the control YFP– example. The diagrams used to calculate the MVB PI data in both cell groups are represented in the right side of the images. The zoom of the indicated ROIs (white square) is included below the diagrams. The percentage of synaptic conjugates on which was evident the presence of CD63+ nanovesicles at the synaptic cleft was 17% for control YFP–, and 0% for FMNL1-interfering (shFMNL1-HA-YFP), respectively, with at least 35 synapses analysed per condition. Images are representative of the data from several independent experiments (n=3) with similar results.

Expression of YFP-FMNL1β constructs and correlation between YFP-FMNL1β variants expression and the anti-FMNL1 antibody signal.

Panel A), Scatter plots of the MFI values of YFP or YFP-FMNL1 variants and the anti-FMNL1 signal in cells transfected with FMNL1-interfering (shFMNL1-HA-YFP), YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT), YFP-FMNL1βS1086A, or YFP-FMNL1βS1086D constructs are depicted. The trend line (linear regression) is displayed in red color in FMNL1β constructions. Panel B), Box plot representing the different anti-FMNL1 MFI signals in the control and the different transfected cell groups. This figure is related to Fig. 2. ***, p≤0.05. Results and ANOVA analyses are representative of the data from several independent experiments (n=3) with similar results.

SEE-induced polarization of MVB and MTOC to the immune synapse and calculation of their polarization indexes.

Panel A), In the left panels, synaptic cell conjugates made by Jurkat cells with CMAC-labeled, SEE-pulsed Raji cells (blue) were fixed, permeabilized and stained with anti-CD63 (red) and anti-pericentrin (magenta) antibodies to label MVB and the MTOC, respectively. The white arrow indicates the IS area. In the right-side scheme, the distances in color (“A”, blue; “B”, cyan and “C”, black) used for the calculation of both MVB and MTOC PI (A/C and B/C, respectively) are indicated. The dark-red dot represents the cell geometric center (Cellc), whereas the yellow and green dots indicates the MTOC and MVB center of mass (MTOCc and MVBc), respectively. The line “C” represents the distance between the Cellc and the synapse, and the projections of both MTOCc and MVBc on the “C” line are labelled with red crosses (MTOC/MVBproj). Since the CellC position was taken as the origin to measure distances, those “A” or “B” values in the opposite direction to the synapse were taken as negative. Thus, PI ranged from +1 to -1. The Raji cells and the Jurkat clones are labelled with discontinuous and continuous white lines, respectively. Panel B), C3 control and PKCδ-interfered P5 clone were untransfected (Control YFP–) or transfected with FMNL1-interfering (shFMNL1-HA-YFP). Subsequently, cells were challenged with CMAC-labelled, unpulsed (SEE-) or SEE-pulsed (SEE+) Raji cells for 1 h, fixed, stained with anti-pericentrin (magenta) to label the MTOC and imaged by epifluorescence microscopy to measure MTOC PI as indicated in panel A. The dot plot shows the MTOC PI of each indicated cell group are shown. NS, not significant; ***, p ≤0.05. Results and ANOVA analyses are representative of the data from several independent experiments (n=3) with similar results.

FMNL1β phosphorylation at S1086 is involved in MTOC / MVB polarization towards the immune synapse. Confocal analysis.

Untransfected, C3 clone (Control YFP–) (first row) and C3 clone transfected (yellow) with either FMNL1-interfering (shFMNL1-HA-YFP) (second row) or FMNL1-interfering and expressing interference-resistant YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT) (third row), or YFP-FMNL1βS1086A (fourth row), or YFP-FMNL1βS1086D (fifth row) constructs, were challenged with CMAC-labelled, SEEpulsed Raji cells (blue) for 1 h to form IS. Then, cell conjugates were fixed, stained with anti-pericentrin to label the MTOC (magenta) and anti-CD63 to label MVB (red), and imaged by confocal fluorescence microscopy. Panel A), representative MIP images with merged channels of each cell group specified in the left side, and the diagrams on the right represent the measurements of the parameters used to calculate the MTOC and MVB PI, including the distance between the IS and the center of the cell (CellC) and the IS (line C), the MTOCC (line B) and the MVBC (line A). Jurkat cell outlines are labelled with a continuous line, Raji cells are labelled with a dashed line, whereas ROI containing MVB are labelled with a continuous red line. Panel B), MTOC and MVB PI dot plot for the different cell groups corresponding to the indicated number of synapses from a similar experiment to that described in panel A) are represented. This figure is related to Fig. 4. NS, not significant. ***, p ≤0.05. Results and ANOVA analyses are representative of the data from several independent experiments (n=3) with similar results.

T cell en face analysis of F-actin and MTOC distribution at the immune synapse interface.

C3 control clone was untransfected (Control YFP–) (first column) or transfected with FMNL1-interfering (shFMNL1-HA-YFP) (second column), or FMNL1-interfering expressing interference-resistant YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT) (third column), YFP-FMNL1βS1086A (fourth column), or YFP-FMNL1βS1086D (fifth column) constructs. Subsequently, cells were challenged with CMAC-labelled SEE-pulsed Raji cells (blue) for 1 h, fixed, stained with phalloidin (magenta) and anti-γ-tubulin (red). The corresponding shFMNL1 constructions are in yellow, and cells were imaged by confocal fluorescence microscopy. Please realize that F-actin (acquired in magenta) was changed to blue color. The upper row includes the top, yx views corresponding to the MIP images of representative examples of each transfected cell group. In the second row, the same examples are displayed, showing the relative MTOC position of each transfected cell group and MTOC PI is indicated in white types. Vertical white arrows indicate the direction to visualize the en face views of the IS (IS interface) enclosed by the ROIs (white rectangles), as shown in Suppl. Video 1. Subsequently, in the third row the en face zx views were generated by merging the indicated channels in the second row of each panel (F-actin in blue merged to anti-γ-tubulin in red), on the IS interfaces of the synaptic areas (generated as shown in Suppl. Video 1). The last frame of this video corresponds to en face view (interface) (third row). MFI profiles along the indicated line (horizontal white arrow) of each separate channel (F-actin in blue, anti-γ-tubulin in red) are shown below the IS interfaces. This figure is related to Fig. 4 and 7. Results are representative of data from several independent experiments (n=3) with similar results.

T cell en face analysis of F-actin and MVB distribution at the immune synapse interface.

C3 control clone was untransfected (Control YFP–) (first column) or transfected with FMNL1-interfering (shFMNL1-HA-YFP) (second column), or FMNL1-interfering expressing interference-resistant YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT) (third column), YFP-FMNL1βS1086A (fourth column), or YFP-FMNL1βS1086D (fifth column) constructs. Subsequently, cells were challenged with CMAC-labelled SEE-pulsed Raji cells (blue) for 1 h, fixed, stained with phalloidin (magenta) and anti-CD63 (red). The corresponding shFMNL1 constructions are in yellow, and cell conjugates were imaged by confocal fluorescence microscopy. Please realize that F-actin (acquired in magenta) was changed to blue color. The upper row includes the top, yx views corresponding to the MIP images of representative examples of each transfected cell group. In the second row, the same examples are displayed, showing the MVB position in each transfected cell group and MVB PI is indicated in white types. Vertical white arrows indicate the direction to visualize the en face views of the IS (IS interface) enclosed by the ROIs (white rectangles), as shown in Suppl. Video 2. The white cross represents the position of the geometric center of the Jurkat cell (CellC), while the white square represents the position of the center of mass of the distribution of MVB (MVBC) that it is an unbiased parameter that mirrors MVB center of mass polarization. Subsequently, in the third row, the en face zx views were generated by merging the indicated channels in the second row of each panel (F-actin in blue merged to anti-CD63 in red), on the IS interfaces of the synaptic areas (generated as shown in Suppl. Video 2). The last frame of these videos corresponds to en face views (interface) (third row). MFI profiles along the indicated line (horizontal white arrow) of each separate channel (Factin in blue, anti-CD63 in red) are shown below the IS interfaces. When MVB are not polarized, some MVB can still be observed at cIS because they are scattered throughout the cell. For instance, the YFP-FMNL1βWT transfected cell (third column) shows a pronounced MVB concentration within the synaptic area (white rectangle), whereas the YFP-FMNL1βS1086A transfected cell (fourth column), as it presents a scattered distribution of MVB throughout the cell, also exhibits some MVB (but only a small proportion of the total cellular MVB) in the synaptic area (second row). This figure is related to Fig. 4 and 7. Results are representative of the data from several independent experiments (n=3) with similar results.

Correlation between MTOC and MVB polarization indexes.

C3 control untransfected (Control YFP–) and C3 transfected with the FMNL1-interfering plasmid (shFMNL1-HA-YFP) were challenged with CMAC-labelled SEE-pulsed Raji cells for 1 h, fixed, stained with anti-γ-tubulin AF647 to label MTOC and anti-CD63 AF546, to label MVB and imaged by confocal fluorescence microscopy. Subsequently, MTOC and MVB PI were calculated for the synapses established by both cell groups, as described in Materials and Methods and shown in schematic representation in Suppl. Fig. S2A. Linear correlation analyses between the MTOC and MVB PI for both cell groups are represented, as well as the corresponding linear correlation analysis data and the Pearson’s coefficients. Dashed lines represent the adjusted, regression line. This figure is related to Fig. 4. Results are representative of data from several independent experiments (n=3) with similar results.

FMNL1 is located at the immune synapse developed by primary T lymphoblasts and dual CAR T cells recognizing CD19/CD22.

Primary T lymphoblasts and dual CAR T cells recognizing CD19/CD22 growing in the presence of recombinant human IL-2 cells were challenged with SEE plus SEB-pulsed Raji cells for 30 min (panel A), or Raji cells for 1 h (panel B), respectively. After the indicated coculture times, cell conjugates were fixed, and stained with phalloidinAF488 (green), anti-pericentrin (magenta) and anti-FMNL1 (red). Synapses were imaged with confocal fluorescence microscopy and colocalization pixels between FMNL1 (red) and F-actin (green) are represented in white (Coloc). Representative MIP and optical sections of synapses formed by both cell types are shown in the left side columns. The effector T lymphocytes are labelled with a yellow arrow in these figures. The F-actin/FMNL1 colocalization coefficients measured in the IS ROI are indicated in the second column. In the right-side columns, top views and IS interface views of the IS are included, as well as the F-actin IS interface architecture and MTOC position. The multifocal nature of the synapse developed by CAR T cells (71) is evident. This figure is related to Fig. 6 and Fig. 8. The percentage of synapse conjugates substantiating endogenous FMNL1 at the IS was 15% for lymphoblasts and 12% for CAR T, respectively, with at least 120 synapses analysed per condition. Results are representative of data from several independent experiments (n=3) with similar results.

YFP-FMNL1βS1086A and S1086D mutants are recruited to the immune synapse.

C3 control cells were transfected with FMNL1-interfering expressing interferenceresistant YFP-FMNL1βS1086A or S1086D (shFMNL1-HA-YFP-FMNL1βS1086A or shFMNL1-HA-YFP-FMNL1βS1086D) plasmids. Subsequently, transfected cells were simultaneously challenged with CMAC-labelled, SEE-pulsed Raji cells (blue) attached to slides and time-lapse acquisition of emerging synapses was performed as indicated in Materials and Methods. The videos (7 fps) (Suppl. Video 4) were captured and in the left side, representative frames from each video are shown. White arrows indicate accumulations of YFP-FMNL1βS1086A or S1086D at the IS. In the right side, YFP-FMNL1βS1086A or S1086D MFI within the cell ROI (grey line) and the IS ROI (red line) are represented. The inserts in the diagrams include the cell ROI (white) and the IS ROI (red) used for the time-lapse measurements on representative frames for both FMNL1β mutants. This figure is related to Fig. 6 and Suppl. Video 4. At least 6 synapses of each cell group were analysed. Results are representative of data from several independent experiments (n=3) with similar results.

FMNL1 and FMNL1β accumulation at the immune synapse revealed by epifluorescence microscopy.

C3 control clone was untransfected (Control YFP–) (first row) or transfected with FMNL1-interfering expressing interference-resistant YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT) (second row) or YFP-FMNL1βS1086A (third row) or YFP-FMNL1βS1086D (fourth row) constructs. Subsequently, cells were challenged with CMAC-labelled SEE-pulsed Raji cells (blue) for 1 h, fixed, stained with anti-pericentrin (magenta) and anti-FMNL1 (red) and imaged by epifluorescence microscopy. The corresponding FMNL1β constructions are in yellow, whose signal is observed in both the first and third columns. The first and second columns represent those individual optical sections in which the accumulation of FMNL1 or FMNL1β variant in the IS formed by each cell group is better visualized, while the third and fourth columns represent MIPs of the same selected cells. FMNL1 or FMNL1β variant accumulation at the IS can be visualized in each cell group in red or yellow, respectively. This figure is related to Fig. 6. Results are representative of the data from several independent experiments (n=3) with similar results.

YFP-FMNL1β variants accumulation at the immune synapse revealed by confocal fluorescence microscopy.

C3 control clone was transfected with FMNL1-interfering expressing interferenceresistant YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT) (first and second rows), YFP-FMNL1βS1086A (third and fourth rows) or YFP-FMNL1βS1086D (fifth and sixth rows) constructs. Subsequently, cells were challenged with CMAC-labelled SEE-pulsed Raji cells (blue) for 1 h, fixed, stained with phalloidin (magenta) and anti-FMNL1 (red). The corresponding shFMNL1 constructions are in yellow, and cells were imaged by confocal fluorescence microscopy. Panel A), top views corresponding to representative examples of each transfected cell group are shown. The first column includes those individual optical sections in which the accumulation of each FMNL1β variant in the IS formed by each cell group is better visualized. Please realize that, since colocalization in NIS-AR only works with red, green and blue channels, F-actin (acquired in magenta) was changed to blue color, and YFP (acquired in yellow) was changed to green in the second column of each panel, where colocalization pixels between anti-FMNL1 or YFP-FMNL1β and F-actin in the same optical section are shown in white. The third column displays the ROIs used to measure the indicated colocalization coefficients. The Pearson and Manders colocalization coefficients for each of the selected cells are displayed. Panel B), the MIPs of the same cells displayed in panel A) are shown. At the right side, the relative MFI quantification of the accumulation at the IS of YFP-FMNL1β variants and FMNL1 compared to the signal of the entire cell is shown. This figure is related to Fig. 6. Results are representative of data from several independent experiments (n=3) with similar results.

Colocalization of FMNL1 and anti-phospho-Ser PKC substrate at the immune synapse interface.

Untransfected, control C3 clone (Control YFP–) (first column) and C3 clone transfected with either FMNL1-interfering (shFMNL1-HA-YFP) (second column), or FMNL1-interfering and expressing interference-resistant YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT) (third column) constructs were challenged with CMAC-labelled SEE-pulsed Raji cells (blue) for 1 h, fixed, stained with anti-Phospho-Ser PKC substrate (magenta) and anti-FMNL1 (red), and imaged by confocal fluorescence microscopy. Please realize that interface colocalization in NIS-AR only works with red, green and blue channels, therefore anti-Phospho-Ser PKC substrate (acquired in magenta) was changed to blue color, and YFP (acquired in yellow) was changed to green, for this purpose. Panel A), The upper row includes representative examples of the top yx views corresponding to the MIP images of the indicated, merged channels of each cell group. The white arrows indicate the direction to visualize the en face zx views of the IS (IS interface) enclosed by the ROIs (white rectangles), as shown in Suppl. Video 7. In the second row, the enlarged ROIs (2x zoom) used to generate the IS interface images of each cell group are shown. Subsequently, the colocalization pixels (white) along all the Z stacks were generated by merging the indicated channels in the second row of each panel (anti-phospho-Ser PKC substrate in blue merged to anti-FMNL1 in red), on the IS interfaces of the synaptic areas (generated as shown in Suppl. Video 7). The last frame of this video corresponded to en face view (interface) (second row on each panel). MFI profile along the indicated line (horizontal white arrow) of each separate channel (anti-phospho-Ser PKC substrate in blue and anti-FMNL1 in red) and the colocalization pixels (grey) are shown below the IS interfaces. Panel B), same as panel A), but both the top views and the IS interfaces were generated and stained with anti-phospho-Ser PKC substrate (blue) merged to YFP (green). At least 8 synapses of each cell group were analysed. Results are representative of data from several independent experiments (n=3) with similar results.

STED colocalization of FMNL1 and anti-phospho-Ser PKC substrate at the immune synapse.

Control C3 clone was challenged with CMAC-labelled SEE-pulsed Raji cells (blue) for 1 h, fixed, stained with anti-Phospho-Ser PKC substrate (magenta) and anti-FMNL1 (red), and imaged by confocal and STED fluorescence microscopy and two representative examples are shown. Please realize that colocalization in NIS-AR only works with red, green and blue channels, therefore anti-Phospho-Ser PKC substrate (acquired in magenta) was changed to green color, and FMNL1 maintains red color. Subsequently, the colocalization pixels (white) along an optical section were generated by merging the indicated channels in the fourth column of each example (anti-phospho-Ser PKC substrate in green merged to anti-FMNL1 in red). In both cases, colocalization is specifically detected in the synaptic zone. First column, confocal Images. Right columns, STED colocalization. The FMNL1/Phospho-PKC colocalization coefficients are indicated in the right-side column. This figure is related to Suppl. Fig. S11. Results are representative of data from several independent experiments (n=3) with similar results.

P-values from tukey’s method (post hoc).

P-values after applying tukey’s method to the indicated pairwise comparisons from the indicated figures are indicated