Journal: Cellular and Molecular Life Sciences: CMLS

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

doi: 10.1007/s00018-026-06202-4

Figure Lengend Snippet: 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 antibodies were used to label endogenous Kv1.3 (green) and CD3 for the plasma membrane (yellow). The PLA signal (magenta) shows palmitoylated Kv1.3. The merged panels show triple colocalization in white. Scale bars represent 5 μm. (D) Kv1.3 is palmitoylated at the immunological synapse (IS) between Kv1.3 YFP-electroporated CD4 + T cells and Raji B cells. Representative confocal images of the PLA in the presence (+ Alk-C16) or absence (-Alk-C16) of palmitic acid. Following the PLA reaction, specific antibodies against Kv1.3 were used to identify endogenous channels (green), CD3 for T lymphocytes (yellow), and CD19 for B cells (white). The PLA signal (magenta) shows palmitoylated Kv1.3. The merged panels show triple colocalization in white. Scale bars represent 5 μm. Right panel, quantification of the PLA signal measured inside and outside the IS. (E) Representative TIRF images showing the marker distribution in synapses formed by CD4 + T cells on the SLB and a cartoon of the SMAC compartments. Anti-CD3 (a-CD3) in yellow accumulates at the central SMAC (cSMAC), and ICAM1 in blue forms a ring at the peripheral SMAC (pSMAC). Interference reflection microscopy (IRM) image showing the cell-SLB contact and delimits the distal SMAC (cSMAC) in gray. (F) Representative TIRF images of CD4 + T cells electroporated with Kv1.3 YFP forming synapses on the SLB. PLA assays revealed palmitoylated channels forming a ring in the presence (+ Alk-C16) but not in the absence (-Alk-C16) of clickable palmitic acid. Scale bars represent 5 μm. Right plot, quantification of Kv1.3 palmitoylation on the basis of the PLA signal intensity. The data were normalized to the average of the + AlkC16 condition. The plot shows the mean ± SE of n > 50 cells from 3 independent blood donors. ****p < 0.0001 by Student’s t test. (G) Orthogonal views of a CD4 + T-cell forming an IS with an SLB and imaged by 3D Airyscan microscopy. Magenta, palmitoylated Kv1.3 (PLA signal); green, total Kv1.3. Note the internalization of the palmitoylated channel in the YZ and XZ planes

Article Snippet: Primary human CD4 + T cells were incubated with 100 μM sonicated 15-hexadecynoic palmitic acid (15-yne, Avanti Polar Lipids) for 18 h at 37 °C, enabling Alk-C16 protein palmitoylation.

Techniques: Positive Control, Control, Proximity Ligation Assay, Labeling, Incubation, Clinical Proteomics, Membrane, Marker, Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

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

doi: 10.1007/s00018-026-06202-4

Figure Lengend Snippet: 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 the IS regions were manually identified using the CD3 and CD19 masks. Right panel, Manders overlap coefficient (MOC) between Kv1.3 and ZDHHC21 inside and outside the IS. Data are presented as the mean ± SE of 5 cells from the same donor. **p < 0.01 by Student’s t test. (E) Representative Western blot of ZDHHC21-silenced CD4 + T cells. Lymphocytes were electroporated with ZDHHC21 siRNA, and after a 48-h incubation, whole-cell lysates were analyzed by Western blotting. β-actin was used as a loading control. CTRL: control, untreated cells. (F) Representative Western blot of Kv1.3 palmitoylation in CTRL- or ZDHHC21-knockdown (siRNA) CD4 + T cells. Flotillin was used as a positive control. SM: starting materials, PD: pull-down, HA − : negative control. (G) Quantification of Kv1.3 palmitoylation under basal conditions (CTRL) and after ZDHHC21 silencing (siRNA). The data were normalized to those of the CTRL. The data are presented as the means ± SEs of 3 independent donors. ***p < 0.001 by Student’s t test

Article Snippet: Primary human CD4 + T cells were incubated with 100 μM sonicated 15-hexadecynoic palmitic acid (15-yne, Avanti Polar Lipids) for 18 h at 37 °C, enabling Alk-C16 protein palmitoylation.

Techniques: Immunostaining, Western Blot, Incubation, Control, Knockdown, Positive Control, Negative Control

Journal: Cellular and Molecular Life Sciences: CMLS

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

doi: 10.1007/s00018-026-06202-4

Figure Lengend Snippet: Characterization of the Kv1.3 Cys less 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 (Cys less ) 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 Cys less 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 analysis of the PLA (magenta), Kv1.3 (green), and ICAM1 (blue) signals highlighted by arrows from the images in (D). (F) Quantification of the PLA signal intensity, which corresponds to Kv1.3 palmitoylation. Data were normalized to the average of the WT + Alk-C16 condition. Data are presented as the means ± SEs of n > 50 cells from 3 different blood donors. ****p < 0.0001 according to one-way ANOVA with a post hoc Tukey test

Article Snippet: Primary human CD4 + T cells were incubated with 100 μM sonicated 15-hexadecynoic palmitic acid (15-yne, Avanti Polar Lipids) for 18 h at 37 °C, enabling Alk-C16 protein palmitoylation.

Techniques: Mutagenesis, Western Blot, Transfection, Positive Control, Incubation, Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

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

doi: 10.1007/s00018-026-06202-4

Figure Lengend Snippet: 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 Cys less 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, Manders overlapping coefficient (MOC) of Kv1.3 inside CTxB regions. Data are presented as the means ± SEs of n > 40 cells from 3 independent blood donors. ***p < 0.001 by Student’s t test. (D) Live CD4 + T cells transfected with wild-type (WT) or Cys less Kv1.3 YFP were incubated with SLB at 37 °C and imaged for 1 min at 0.3 s/frame using TIRFM. Single-frame images of Kv1.3 and a time projection of all the frames recorded are shown. Single-particle tracking was performed. The track color indicates the mean speed of the track. The color scale bar on the bottom indicates the minimum values in blue and the maximum values in red in μm/s. Scale bars represent 5 μm. Right violin plots depict the quantification of the mean track speed (average velocity across all the links of the track, μm/s), total displacement (net distance traveled, μm), and confinement ratio (net displacement divided by total distance, 0 = fully confined, 1 = straight path). Data are the means ± SEs (n > 4000 trajectories) from 3 independent blood donors. ***p < 0.001 by Student’s t test

Article Snippet: Primary human CD4 + T cells were incubated with 100 μM sonicated 15-hexadecynoic palmitic acid (15-yne, Avanti Polar Lipids) for 18 h at 37 °C, enabling Alk-C16 protein palmitoylation.

Techniques: Membrane, Western Blot, Staining, Transfection, Incubation, Single-particle Tracking

Journal: Cellular and Molecular Life Sciences: CMLS

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

doi: 10.1007/s00018-026-06202-4

Figure Lengend Snippet: 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 Cys less 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 from 3 independent blood donors. *p < 0.05, ****p < 0.0001 by Student’s t test. (E) A PLA was performed using anti-ubiquitin and anti-Kv1.3 antibodies to detect ubiquitinated Kv1.3. Orthogonal views from a representative confocal image of ubiquitinated Kv1.3 (PLA signal, magenta) in a synapse conjugate between a human CD4 + T cell and a Raji B cell. CD19 (gray) was used as a B-cell-specific marker, and phalloidin (cyan) was used to stain the actin filaments. The PLA signal detected within the synaptic contact is located at the center of the synapse (XZ plane). (F) Ubiquitination assay in HEK293 cells transfected with either WT or Cys less Kv1.3 YFP. Cells were incubated in the absence (-) or presence (+) of phorbol 12-myristate 13-acetate (PMA) for 30 min to induce protein internalization as a positive control. Cell lysates were immunoprecipitated (IP) for Kv1.3 and immunoblotted (IB) for both Kv1.3 and ubiquitin (Ubq). SM: starting materials, IP: immunoprecipitation, IP − : negative control in the absence of antibody. (G) Quantification of channel ubiquitination. The data are presented as the means ± SEs of at least 2 independent experiments. **p < 0.01 by Student’s t test

Article Snippet: Primary human CD4 + T cells were incubated with 100 μM sonicated 15-hexadecynoic palmitic acid (15-yne, Avanti Polar Lipids) for 18 h at 37 °C, enabling Alk-C16 protein palmitoylation.

Techniques: Ubiquitin Proteomics, Expressing, Microscopy, Activation Assay, Marker, Staining, Transfection, Incubation, Positive Control, Immunoprecipitation, Negative Control

Journal: Cellular and Molecular Life Sciences: CMLS

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

doi: 10.1007/s00018-026-06202-4

Figure Lengend Snippet: 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 Cys less 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 Cys less Kv1.3 YFP. Cell lysates were immunoprecipitated (IP) against Kv1.3 and immunoblotted (IB) against Kv1.3 and PSD95. SM, starting materials; IP + , positive immunoprecipitation; IP − , negative control in the absence of antibody. Right panel, quantification of the results of the coimmunoprecipitation of Kv1.3-PSD95 by normalizing the coimmunoprecipitation of PSD95 (CoIP) to the immunoprecipitation (IP) of Kv1.3. 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 human CD4 + T cells electroporated with Kv1.3 YFP forming a synapse on an SLB. Phalloidin labeled the actin ring. The merged panel shows colocalization. Green, Kv1.3; magenta, phalloidin. The scale bar represents 5 μm. IRM shows interference reflection microscopy images. (E) Coimmunoprecipitation (CoIP) of Kv1.3 with cortactin. HEK293 cells were transfected with either wild-type (WT) or Cys less Kv1.3. Cell lysates were immunoprecipitated (IP) against Kv1.3 YFP and immunoblotted (IB) against GFP (Kv1.3) and cortactin. SM: starting materials, IP + : positive immunoprecipitation, IP − : negative control in the absence of antibody. Right panel, quantification of the coimmunoprecipitation (CoIP) between Kv1.3 and cortactin by relativizing the CoIP of cortactin to the IP of Kv1.3. The data are presented as the means ± SEs of 2 independent experiments. *p < 0.05 by Student’s t test

Article Snippet: Primary human CD4 + T cells were incubated with 100 μM sonicated 15-hexadecynoic palmitic acid (15-yne, Avanti Polar Lipids) for 18 h at 37 °C, enabling Alk-C16 protein palmitoylation.

Techniques: Clinical Proteomics, Membrane, Protein Binding, Transfection, Incubation, Staining, Marker, Western Blot, Immunoprecipitation, Negative Control, Labeling, Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

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

doi: 10.1007/s00018-026-06202-4

Figure Lengend Snippet: Functional consequences of the absence of Kv1.3 palmitoylation. (A) HEK293 cells were transfected with wild-type (WT) and Cys less 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 Cys less (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 Ca 2+ signaling recordings of CD4 + T cells forming immunological synapses with Raji B cells. T lymphocytes expressing endogenous Kv1.3 (CTRL) or electroporated with either wild-type (WT) or Cys less Kv1.3 YFP were loaded with Calbryte™ 630 AM and incubated with Raji B cells. The calibration bar shows the signal intensity, and the scale bars represent 5 μm. Center panel, representative Ca 2+ traces of the 3 conditions. Right panel, quantification of the maximum peak amplitude calculated as the maximum Ca 2+ intensity relative to the initial signal. Data are presented as the means ± SEs of n > 9 cells. *p < 0.05, **p < 0.01 by Student’s t test

Article Snippet: Primary human CD4 + T cells were incubated with 100 μM sonicated 15-hexadecynoic palmitic acid (15-yne, Avanti Polar Lipids) for 18 h at 37 °C, enabling Alk-C16 protein palmitoylation.

Techniques: Functional Assay, Transfection, Mutagenesis, Patch Clamp, Staining, Activation Assay, Expressing, Incubation