PV IgG causes redistribution of Dsg3 into raft-containing linear arrays.

<p>(<b>A–C</b>) Dsg3 colocalization with various membrane markers was analyzed using structured illumination microscopy (SIM) in human keratinocytes cultured in high calcium for 16–18 hrs and then treated with PV IgG for 3 hrs. CD59 was detected with FITC conjugated antibody by live labeling for 10 min prior to fixing. Dsg3 was detected with Alexa Fluor 555 conjugated AK15 mAb by live labeling for 10 min prior to fixing for the top two rows. For clathrin colocalization Dsg3 was monitored using PV IgG and secondary antibody detection of human IgG. In response to PV IgG, Dsg3 enters endocytic linear structures (previously termed ‘linear arrays’) that emanate perpendicular from cell-cell borders and extend toward the cell center (Jennings <i>et al.,</i> 2011). (<b>A</b>) Raft markers CD59 and caveolin-1 were enriched in linear arrays and colocalizaed significantly with Dsg3 relative to the non-raft marker clathrin. (<b>B</b>) Fluorescence intensity measurements of lines drawn perpendicularly through linear arrays show alignment of Dsg3 (bottom line) and raft marker (top line) fluorescence. (<b>C</b>) Quantification of Dsg3 colocalization in linear arrays as indicated by Mander’s coefficient (ratio of red in green). Means ± SEM (<i>n</i> = 27–36 arrays per group); *p<0.05. (<b>D</b>) SIM was also used to view Dsg3 colocalization with CD59 in linear arrays in excised normal human epidermis injected with PV IgG. Basal keratinocytes are shown. D, dermis. Scale bar in A and D, 5 µm.