Figure 8. Three-dimensional distribution and colocalization of FMNL1 and F-actin at the immune synapse (IS) 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-labeled SEE-pulsed Raji cells (blue) for 1 hr, 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. (A) The upper row includes top, yx views corresponding to the maximum intensity projection (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 regions of interest (ROIs) (white rectangles), as shown in Figure 8—video 1. In the second row, the enlarged ROIs (2× 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 Figure 8—video 1). The last frame of these videos corresponds to the en face view (interface) (second row in both panels). Mean fluorescence intensity (MFI) profiles along the indicated line (horizontal white arrow) of each separate channel (F-actin in blue, anti-FMNL1 in red) and the colocalization pixels (gray) are shown below the IS interfaces. (B) Same as (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 Figure 8—video 1. At least six synapses of each cell group were analyzed. Results are representative of data from several independent experiments (n=3) with similar results.

Figure 8—figure supplement 1. Colocalization of FMNL1 and anti-phospho-Ser PKC substrate at the immune synapse (IS) 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-labeled SEE-pulsed Raji cells (blue) for 1 hr, 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. (A) The upper row includes representative examples of the top yx views corresponding to the maximum intensity projection (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 regions of interest (ROIs) (white rectangles), as shown in Video 4. In the second row, the enlarged ROIs (2× 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 Video 4). The last frame of this video corresponded to en face view (interface) (second row on each panel). Mean fluorescence intensity (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 (gray) are shown below the IS interfaces. (B) Same as (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 eight synapses of each cell group were analyzed. Results are representative of data from several independent experiments (n=3) with similar results.

Figure 8—figure supplement 2. STED colocalization of FMNL1 and anti-phospho-Ser protein kinase C (PKC) substrate at the immune synapse (IS).

Control C3 clone was challenged with CMAC-labeled SEE-pulsed Raji cells (blue) for 1 hr, 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 Figure 8—figure supplement 1. Results are representative of data from several independent experiments (n=3) with similar results.

Figure 8—video 1. Colocalization of FMNL1 with F-actin at the immune synapse (IS) interface.

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Untransfected C3 clone cells (Control YFP^-) (first left column) or C3 cells transfected with either the bi-cistronic, YFP expression plasmid interfering all FMNL1 isoforms (shFMNL1-HA-YFP) (second column), or FMNL1 interfering and re-expressing interference-resistant YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT) (third and fourth column), YFP-FMNL1βS1086A (fifth column), or YFP-FMNL1βS1086D (sixth column) constructs, were mixed with CMAC-labeled, SEE-pulsed Raji cells (blue) attached to slides, as described in Materials and methods. After 1 hr of culture, synaptic conjugates were fixed and labeled for F-actin (acquired in magenta, changed to blue) and anti-FMNL1 (red). The video (10 fps) shows fixed whole cells (top row) and synapse contact areas (bottom row) that were imaged with confocal microscopy and Z stacks were processed to generate en face, zx views of the synaptic interface, as indicated in Materials and methods. Colocalization pixels (white) of FMNL1 (red) and F-actin (blue) are shown. This video is related to Figure 8.