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Vol. 17, Issue 2, 977-989, February 2006

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Syntaxins 3 and 4 Are Concentrated in Separate Clusters on the Plasma Membrane before the Establishment of Cell Polarity

Seng Hui Low ^* , Amit Vasanji , Jayasri Nanduri  ^||, Min He , Nikunj Sharma ^* ¶, Michelle Koo , Judith Drazba , and Thomas Weimbs ^* 

^* Department of Molecular, Cellular, and Developmental Biology; Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA 93106-9610; Imaging Facility, Lerner Research Institute, The Cleveland Clinic, Cleveland, OH 44195; Department of Cell Biology, Lerner Research Institute, The Cleveland Clinic, Cleveland, OH 44195; and ^¶ Department of Biological, Geological, and Environmental Sciences, Cleveland State University, Cleveland, OH 44115

Submitted May 31, 2005; Revised October 27, 2005; Accepted November 29, 2005
Monitoring Editor: Sean Munro

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.

^|| Present address: Department of Pathology, Case Western Reserve University, Cleveland, OH 44106.

Address correspondence to: Thomas Weimbs (weimbs{at}lifesci.ucsb.edu).

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