The Distribution and Binding Partners of Kinesin II in Rabbit lacrimal acini

Abstract

Abstract: : Purpose: : In stimulated lacrimal acini, p150^Glued and dynein translocate recruitable secretory transport vesicles toward microtubule plus–ends located at the apical membrane. Kinesin II, a member of the kinesin superfamily, drives the plus–end directed transport of different membrane organelles along cellular microtubules in different cell types. The goal of this research was to determine the role of kinesin II in lacrimal acinar membrane trafficking by focusing on its cargo interactions and binding partners. Methods: Primary rabbit lacrimal acinar cells were cultured for 2–3 days in a medium favoring reconstitution of acinus–like structures, then stimulated with or without 100 µM carbachol (CCH) for 15 min. Kinesin II distribution and colocalization with rab6, rab11 and p150^Glued were evaluated by confocal fluorescence microscopy using appropriate primary and secondary antibodies. Subcellular membrane compartments from resting and CCH–stimulated acini were isolated by isopycnic centrifugation over sorbitol density gradients, and markers of interest evaluated by Western blotting and infrared fluorescence imaging. Kinesin II–p150^Glued interactions were evaluated by co–immunoprecipitation from acinar lysates and Western blotting. Results: Confocal fluorescence microscopy of resting acini revealed kinesin II immunofluorescence in a punctate pattern beneath the apical membrane and throughout the cytoplasm with partial colocalization with rab6 (Golgi), rab11 (endosomes) and p150^Glued, a dynein effector. CCH stimulation increased both the intensity of kinesin II immunofluorescence beneath the apical membrane and the extent of its colocalization with rab11 and p150^Glued. Density gradient analysis showed kinesin II associated with diverse membrane compartments. CCH stimulation caused a relative increase in kinesin II enrichment in fractions containing the recycling endosome and trans–Golgi network elements. Kinesin II monoclonal antibodies co–immunoprecipitated traces of p150^Glued from resting cell lysates and increased amounts from CCH–stimulated cell lysates. The apparent increase in kinesin II–p150^Glued association in CCH–stimulated acini was confirmed in co–immunoprecipitation experiments with antibodies to p150^Glued. Conclusions: Coupled retrieval and recycling of exocytosed secretory vesicle membrane by kinesin II may be mediated by binding to p150^Glued following displacement of dynein.

Keywords: lacrimal gland • cytoskeleton • cell membrane/membrane specializations