Actin and non-muscle myosin II facilitate apical exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells.
The acinar epithelial cells of the lacrimal gland exocytose the contents of mature secretory vesicles containing tear proteins at their apical membranes in response to secretagogues. Here we use time-lapse confocal fluorescence microscopy and fluorescence recovery after photobleaching to investigate the changes in actin filaments located beneath the apical membrane during exocytosis evoked by the muscarinic agonist, carbachol (100 microM). Time-lapse confocal fluorescence microscopy of apical actin filaments in reconstituted rabbit lacrimal acini transduced with replication-deficient adenovirus containing GFP-actin revealed a relatively quiescent apical actin array in resting acini. Carbachol markedly increased apical actin filament turnover and also promoted transient actin assembly around apparent fusion intermediates. Fluorescence recovery after photobleaching measurements revealed significant (P< or =0.05) increases and decreases, respectively, in mobile fraction (Mf) and turnover times (t1/2) for apical actin filaments in carbachol-stimulated acini relative to untreated acini. The myosin inhibitors, 2,3-butanedione monoxime (BDM, 10 mM, 15 minutes) and ML-7 (40 microM, 15 minutes), significantly decreased carbachol-stimulated secretion of bulk protein and the exogenous secretory vesicle marker, syncollin-GFP; these agents also promoted accumulation of actin-coated structures which were enriched, in transduced acini, in syncollin-GFP, confirming their identity as fusion intermediates. Actin-coated fusion intermediates were sized consistent with incorporation of multiple rather than single secretory vesicles; moreover, BDM and ML-7 caused a shift towards formation of multiple secretory vesicle aggregates while significantly increasing the diameter of actin-coated fusion intermediates. Our findings suggest that the increased turnover of apical actin filaments and the interaction of actin with non-muscle myosin II assembled around aggregates of secretory vesicles facilitate exocytosis in lacrimal acinar epithelial cells.
Jerdeva
Galina V
GV
Department of Pharmaceutical Sciences, University of Southern California, 1985 Zonal Avenue, PSC 406A, Los Angeles, CA 90033, USA.
Wu
Kaijin
K
Yarber
Francie A
FA
Rhodes
Christopher J
CJ
Kalman
Daniel
D
Schechter
Joel E
JE
Hamm-Alvarez
Sarah F
SF
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D000199:Actins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D003931:Diacetyl / Q000031:analogs & derivatives / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism*; D005089:Exocytosis; D005136:Eye Proteins / Q000378:metabolism*; D005260:Female; D036681:Fluorescence Recovery After Photobleaching; D007765:Lacrimal Apparatus / Q000166:cytology* / Q000187:drug effects / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D009281:Naphthalenes / Q000494:pharmacology; D024507:Nonmuscle Myosin Type IIA / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D022142:Secretory Vesicles / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines
D000199:Actins; D001381:Azepines; D019086:Bridged Bicyclo Compounds, Heterocyclic; D005136:Eye Proteins; D008565:Membrane Proteins; D009281:Naphthalenes; D011993:Recombinant Fusion Proteins; D013844:Thiazoles; D053778:Thiazolidines; C042434:tear proteins; C070571:ML 7; C004717:diacetylmonoxime; D002217:Carbachol; D024507:Nonmuscle Myosin Type IIA; D003931:Diacetyl; C037068:latrunculin B