Cholinergic stimulation of lacrimal acinar cells promotes redistribution of membrane-associated kinesin and the secretory protein, beta-hexosaminidase, and increases kinesin motor activity.
The role of the microtubule-based motor, kinesin, in membrane trafficking has been investigated in resting and stimulated acinar cells from rabbit lacrimal gland, a cholinergically controlled secretory tissue. Microtubule-dependent motors from extracts of control and carbachol-treated acini were isolated by microtubule-affinity purification and their activity was determined using a video-enhanced differential interference contrast microscopy assay for microtubule gliding. The observation that carbachol treatment resulted in a 2.2-fold stimulation of the frequency of GTP-dependent microtubule gliding in fractions isolated by microtubule-affinity purification and GTP release suggested that kinesin was a target of carbachol-induced stimulation. Resolution of membranes from resting cells by fractionation on a sorbitol density gradient followed by partitioning analysis in a dextran-polyethyleneglycol two-phase system revealed that membrane-associated kinesin codistributed with Golgi-derived membranes, a post-Golgi secretory compartment designated Hex1, membranes from a trans Golgi network-like compartment, endoplasmic reticulum and a group of putative lysosomal membranes containing cathepsin B. Comparable fractionation of carbachol-treated acini showed that stimulation caused redistributions of membrane-associated kinesin, the secretory enzyme beta-hexosaminidase, and galactosyltransferase that appeared to reflect both a reorganization within the Golgi complex and a return of material to the Golgi complex from the secretory pathway. Our findings that carbachol promotes activation of lacrimal acinar kinesin as well as major shifts in kinesin-membrane association within the secretory pathway suggests that kinesin plays a major role in secretory vesicle assembly, apical secretion, and/or secretory vesicle membrane recycling in the lacrimal gland.
Hamm-Alvarez
S F
SF
Department of Pharmaceutical Sciences, University of Southern California, Los Angeles 90033, USA.
Da Costa
S
S
Yang
T
T
Wei
X
X
Gierow
J P
JP
Mircheff
A K
AK
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured / Q000187:drug effects / Q000648:ultrastructure; D002642:Chick Embryo; D005260:Female; D005700:Galactosyltransferases / Q000187:drug effects; D007425:Intracellular Membranes / Q000737:chemistry / Q000648:ultrastructure; D016547:Kinesins / Q000187:drug effects* / Q000302:isolation & purification / Q000502:physiology; D007765:Lacrimal Apparatus / Q000737:chemistry / Q000166:cytology / Q000187:drug effects*; D008870:Microtubules / Q000502:physiology; D008916:Miotics / Q000494:pharmacology; D011817:Rabbits; D013347:Subcellular Fractions / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure; D001619:beta-N-Acetylhexosaminidases / Q000187:drug effects* / Q000302:isolation & purification
D008916:Miotics; D002217:Carbachol; D005700:Galactosyltransferases; D001619:beta-N-Acetylhexosaminidases; D016547:Kinesins