Kv1.3 palmitoylation regulates spatial distribution and channel removal from the immunological synapse

Figures

• 

  Fig. 1. Kv1.3 is palmitoylated at the immunological synapse. (A) Palmitoylation assay (ABE) in CD4+ T cells in the absence (-) or presence (+) of anti-CD3/CD28 dynabeads for 15 min. Flotillin was used as a positive control. SM: starting materials, PD: pull-down. Bottom panel, quantification of Kv1.3 palmitoylation, which was calculated as the PD/SM ratio, and the control was normalized to 1 for better visualization. The data are presented as the mean ± SE of 3 independent blood donors. *p < 0.05 by Student’s t test. (B) Schematic of the click-chemistry and proximity ligation assay (PLA) technique. First, endogenous palmitic acid (green) was replaced by alkynyl palmitic acid (orange) through metabolic labeling. Next, biotin-azide was attached via a click reaction. Finally, specific antibodies against YFP (or Kv1.3 for the endogenous channel) and biotin conjugated with complementary probes were incubated with the samples for the PLA reaction. Therefore, the PLA signal indicates palmitoylated Kv1.3-YFP. (C) Representative confocal images of the PLA signal in human CD4+ T cells in the presence (+ Alk-C16) or absence (-Alk-C16) of clickable palmitic acid. Following the PLA reaction, specific…

• 

  Fig. 2. ZDHHC21 acyltransferase mediates Kv1.3 palmitoylation. (A) Orthogonal views of a representative synapse conjugate between a CD4+ T cell and a Raji B cell immunostained with an anti-ZDHHC21 antibody. Three-dimensional reconstruction revealed the distribution of the acyltransferases in both lymphocytes. (B) Top panel, schematic of the image processing for the visualization of ZDHHC21 at the synaptic platform in (A). In the bottom panel, intensities from the stacks corresponding to 3 µm of the synaptic region were compiled in a single Z projection. Note that, similar to Kv1.3, ZDHHC21 organizes as a distal ring with partial accumulation at the center of the synapse. The scale bar represents 5 μm. (C) Representative confocal images of synapse conjugates between CD4+ T cells and Raji B cells. Immunostaining of endogenous Kv1.3, ZDHHC21, CD3 (T cells), and CD19 (B cells) was performed. Merged panels show colocalization between Kv1.3 and ZDHHC21 (top) and between CD3 and ZDHHC21 (bottom). Colocalized pixels identified by Otsu’s automatic thresholding are displayed to facilitate visualization. The scale bar represents 5 μm. (D) Representative regions of interest (ROIs) inside and outside…

• 

  Fig. 3. Characterization of the Kv1.3 Cysless palmitoylation mutant. (A) Schematic of Kv1.3 highlighting all the intracellular cysteines. (B) Representative Western blot of Kv1.3 palmitoylation in HEK293 cells transfected with wild-type (WT) or palmitoylation mutant (Cysless) Kv1.3 YFP. Flotillin was used as a positive control. (C) Quantification of the palmitoylation of the Kv1.3 channels. The palmitoylated fractions were relativized to the starting materials, and the data were normalized to those of the WT condition. The data are presented as the means ± SEs of 3 independent experiments. **p < 0.01 by Student’s t test. (D) Representative TIRF images of CD4+ T cells forming synapses on the SLB. T cells were either not transfected (no Kv1.3 YFP) or electroporated with WT or Cysless Kv1.3 YFP. T cells were incubated in the absence (-Alk-C16) or presence (+ Alk-C16) of clickable palmitic acid. The PLA signal indicates the palmitoylated channel. The ICAM1 ring indicated the formation of a synapse. The merged panels show colocalization, and the IRM image shows the interference reflection microscopy image. Magenta, PLA; green, Kv1.3; blue, ICAM1. Scale bars represent 5 μm. (E) Pixel-by-pixel…

• 

  Fig. 4. A lack of palmitoylation impairs the lipid raft accumulation of Kv1.3 at immunological synapses and increases channel membrane mobility. (A) Kv1.3 partially localizes to detergent-resistant fractions in Jurkat T lymphocytes. Cell lysates were separated by a sucrose gradient, and low-buoyant fractions from lower (1) to higher (12) densities were analyzed by Western blotting. Clathrin and flotillin were used as nonraft and raft markers, respectively. (B) Confocal images of a CD4+ T-cell showing colocalization between endogenous Kv1.3 (green) and lipid rafts (cholera toxin subunit B (CTxB), magenta). The merged panel shows colocalization in white. The arrows indicate the zoomed-in insets on the right. The scale bar represents 5 μm. (C) Representative TIRF images of CD4+ T cells electroporated with WT or Cysless Kv1.3 YFP (green) to form synapses on the SLB. Lipid rafts were stained with the B subunit of cholera toxin (CTxB, magenta). The ICAM1 ring indicated the formation of a synapse. Merge shows the Kv1.3 and CTxB channels. Colocalization panels show colocalization pixels identified by Otsu’s automatic thresholding for better visualization. Scale bars represent 5 μm. Right panel,…

• 

  Fig. 5. Palmitoylation facilitates the ubiquitination and central accumulation of the channel at the immunological synapse. (A) Representative TIRF images of CD4+ T cells electroporated with WT or Cysless Kv1.3 YFP to form synapses on the SLB. The ICAM1 ring indicates the formation of a synapse. The merged image shows Kv1.3 (green) and ICAM1 (blue) expression. IRM, interference reflection microscopy. Scale bars represent 5 μm. (B) Quantification of Kv1.3 intensity at the immunological synapse normalized to the average of the WT for each donor. Data are presented as the means ± SEs of n > 300 cells from 3 independent blood donors. **p < 0.01 by Student’s t test. (C) ROIs of each supramolecular activation complex (SMAC) in (A). IRM images were used to define the total cell contact area. The ICAM1 ring indicates the pSMAC. IRM regions outside or inside the ICAM1 ring were classified as dSMAC or cSMAC, respectively. (D) Kv1.3 intensity was quantified within each ROI corresponding to the cSMAC, pSMAC, dSMAC, and the entire synapse. The percentage of Kv1.3 intensity in each SMAC was calculated with respect to the total number of synapses. Data are presented as the means ± SEs of n > 50 cells…

• 

  Fig. 6. Palmitoylation facilitates Kv1.3 endocytosis. (A) Representative confocal images of HEK293 cells transfected with either WT or Cysless Kv1.3 YFP. The cells were incubated in the absence (-PMA) or presence (+ PMA) of PMA for 30 min, after which early endosomes (EEA1) were identified. The plasma membrane (PM) and nuclei were stained with wheat germ agglutinin (WGA) and DAPI, respectively. The merged panels show the indicated colocalizations in white. Green, Kv1.3 channels; magenta, cell markers (EEA1 or PM). Scale bars represent 10 μm. (B) Manders overlapping coefficients between Kv1.3 and EEA1 (top panel) and between Kv1.3 and the PM (bottom panel). Data are presented as the means ± SEs of n > 30 cells from 3 independent experiments. ***p < 0.001 by one-way ANOVA with post hoc Tukey test. (C) Membrane protein biotinylation assay of HEK293 cells transfected with either WT or Cysless Kv1.3 YFP and incubated in the absence (-PMA) or presence (+ PMA) of PMA for 30 min to trigger endocytosis. Calnexin was used as a negative control because it is a transmembrane protein located in the ER and is absent at the PM, and β-actin was used as a loading control. (D) Quantification of Kv1.3…

• 

  Fig. 7. The absence of palmitoylation altered channel‒protein interactions with plasma membrane-stabilizing proteins in HEK293 cells. (A) Illustration of the C-terminal domain of human Kv1.3 highlighting several known scaffold protein binding domains, cysteine residues, and anterograde traffic signatures. (B) Representative confocal images of HEK293 cells transfected with either WT or Cysless Kv1.3 YFP. Cells were incubated in the absence (-PMA) or presence (+ PMA) of PMA for 30 min to induce endocytosis. Cells were stained for PSD95 and the B subunit of cholera toxin (CTxB, a lipid raft marker). Nuclei were stained with DAPI. The merged panels show colocalization in white, as magnified in the enlarged inset. Green, Kv1.3; magenta, PSD95. Scale bars represent 10 μm. The right panel shows the Manders overlap coefficient between Kv1.3 and PSD95. Data are presented as the mean ± SE of n > 20 cells from 3 independent experiments. **p < 0.01, ***p < 0.001 by one-way ANOVA with a post hoc Tukey test. (C) Left panel, representative immunoblot of the coimmunoprecipitation between Kv1.3 and PSD95. HEK293 cells were transfected with either WT or Cysless Kv1.3 YFP. Cell lysates were…

• 

  Fig. 8. Functional consequences of the absence of Kv1.3 palmitoylation. (A) HEK293 cells were transfected with wild-type (WT) and Cysless mutant Kv1.3, and voltage-dependent currents were analyzed by patch clamp. Cells were held at −80 mV, and voltage-gated currents were elicited by 250 ms square pulses at + 60 mV. Current densities were calculated (bottom panel). The data are presented as the means ± SEs of at least 5 independent cells per condition. *p < 0.05 by Student’s t test. (B–C) Representative TIRF images of CD4+ T cells electroporated with wild-type (WT) (B) or Cysless (C) Kv1.3 YFP and exposed to SLBs at different times (0, 2, 5, 10, 15, and 30 min). ICAM1 rings identify immunological synapses. pZAP70 staining indicated T-cell activation. The merged panels show colocalization. Green, Kv1.3 channels; magenta, pZAP70; blue, ICAM1. Scale bars represent 5 μm. (D) Quantification of pZAP70 intensity at different times of IS formation. The data were normalized to the average of the 0 min time point for each donor. Data are presented as the means ± SEs of n > 100 cells. ***p < 0.001, ****p < 0.0001 by Student’s t test. (E) Left panel, representative snapshots of Ca2+ signaling…