Figure 3: Differences in the length of the TMDs contribute to segregation of syntaxin 1 and 4 to distinct clusters in the plasma membrane.

(a) Two-colour STED microscopy of PC12 cell sheets immunostained for syntaxin 1 and syntaxin 4 shows segregation of endogenous proteins into separate clusters. (b) Reduced co-clustering of sx-1TM and sx-4TM in membranes composed of a mixture of PC with different acyl-chain lengths. FRET assay is similar to Fig. 1c, but now measuring clustering of TMDs in liposomes composed of either C18:1 PC or an equimolar mixture of C14:1, C16:1, C18:1 and C20:1 PC. All liposomes contained 1 mol% PI(4,5)P₂ and 30 mol% cholesterol (± range from two independent reconstitutions, three technical repeats each). (c) Same as in a but now using PC12 cell sheets derived from cells expressing truncation mutants of syntaxin 1 and 4 (sx-1TM; sx-4TM) that are fused to mCherry and EGFP, respectively, and immunostained with antibodies against mCherry and EGFP. (d) Control experiment of PC12 cells co-expressing sx-1TM tagged with either mCherry or EGFP, showing co-localization (Scale bar, 2 μm). (e) Overlap of clusters in membrane sheets from PC12 cells transfected with various sx-1TM and sx-4TM mutants (all mCherry and EGFP tagged, respectively) measured by determining the Pearson-correlation coefficient. Sx-1FL and sx-4FL; full length constructs of syntaxin 1 and syntaxin 4, respectively (from a). Each analysis included at least 10 sheets from 3 independent transfections (***P<0.001, two-sided, unpaired t-test; error bars show s.e.m.). Scale bars, 2 μm.