Journal: bioRxiv
Article Title: Kv1.3 palmitoylation regulates spatial distribution and channel removal from the immunological synapse
doi: 10.64898/2026.01.19.700329
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: Next, the cells were labeled with primary anti-CD3 (Alexa Fluor 647; Biolegend), anti-CD19 (Brilliant Violet 421; Biolegend), anti-ZDHHC21 (NSJ Bioreagents) and anti-Kv1.3 (FITC; Alomone) antibodies in 1% BSA for 1 h at RT.
Techniques: Ubiquitin Proteomics, Expressing, Microscopy, Activation Assay, Marker, Staining, Transfection, Incubation, Positive Control, Immunoprecipitation, Negative Control
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