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: 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: For lipid raft staining, lymphocytes were previously incubated with cholera toxin subunit B (Fisher Scientific).

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