Syntaxins 3 and 4 are concentrated in separate clusters on the plasma membrane before the establishment of cell polarity.
Syntaxins 3 and 4 localize to the apical and basolateral plasma membrane, respectively, of epithelial cells where they mediate vesicle fusion. Here, we report that before establishment of cell polarity, syntaxins 3 and 4 are confined to mutually exclusive, submicron-sized clusters. Syntaxin clusters are remarkably uniform in size, independent of expression levels, and are distinct from caveolae and clathrin-coated pits. SNAP-23 partially colocalizes with both syntaxin 3 and 4 clusters. Deletion of the apical targeting signal of syntaxin 3 does not prevent sorting into clusters away from syntaxin 4. Syntaxin 3 and 4 cluster formation depends on different mechanisms because the integrity of syntaxin 3 clusters depends on intact microtubules, whereas syntaxin 4 clusters depend on intact actin filaments. Cholesterol depletion causes dispersion of syntaxin 3 but not syntaxin 4 clusters. In migrating cells, syntaxin clusters polarize to the leading edge, suggesting a role in polarized exocytosis. These results suggest that exocytosis occurs at small fusion sites exhibiting high local concentrations of SNARE proteins that may be required for efficient membrane fusion. The establishment of separate clusters for each syntaxin suggests that the plasma membrane is inherently polarized on an ultrastructural level even before the establishment of true cell polarity.
Low
Seng Hui
SH
Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara 93106-9610, USA.
Vasanji
Amit
A
Nanduri
Jayasri
J
He
Min
M
Sharma
Nikunj
N
Koo
Michelle
M
Drazba
Judith
J
Weimbs
Thomas
T
D000199:Actins / Q000502:physiology / Q000648:ultrastructure; D000818:Animals; D021941:Caveolae / Q000648:ultrastructure; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry* / Q000648:ultrastructure; D016764:Cell Polarity; D002784:Cholesterol / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000648:ultrastructure; D004285:Dogs; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D008870:Microtubules / Q000502:physiology / Q000648:ultrastructure; D050765:Qa-SNARE Proteins / Q000032:analysis* / Q000378:metabolism; D050766:Qb-SNARE Proteins / Q000032:analysis; D050767:Qc-SNARE Proteins / Q000032:analysis; D017384:Sequence Deletion; D050827:Syntaxin 1 / Q000032:analysis
D000199:Actins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; C100493:SNAP23 protein, human; D050827:Syntaxin 1; D002784:Cholesterol