title: Actin cytoskeleton controls movement of intracellular organelles in pancreatic duct epithelial cells.
abstract: In most eukaryotic cells, microtubules and filamentous actin (F-actin) provide tracks on which intracellular organelles move using molecular motors. Here we report that cytoplasmic movement of both mitochondria and lysosomes is slowed by F-actin meshwork formation in pancreatic duct epithelial cells (PDEC). Mitochondria and lysosomes were labeled with fluorescent Mitotracker Red CMXRos and Lysotracker Red DND-99, respectively, and their movements were monitored using epi-fluorescence and confocal microscopy. Mitochondria and lysosomes moving actively at rest stopped rapidly within several seconds after an intracellular Ca(2+) rise induced by activation of P2Y(2) purinergic receptors. The 'freezing' of the organelles was inhibited by blocking the Ca(2+) rise or by pretreatment with latrunculin B, an inhibitor of F-actin formation. Indeed, this freezing effect on the organelles was accompanied by the formation of F-actin in the whole cytoplasm as stained with Alexa 488-phalloidin in fixed PDEC. For real-time monitoring of F-actin formation in live cells, we expressed sGFP-fimbrin actin binding domain2 (fABD2) in PDEC. Rapid recruitment of the fluorescent probe near the nucleus and lysosomes suggested dense F-actin formation around intracellular structures. The development of F-actin paralleled that of organelle freezing. We conclude that rapid Ca(2+)-dependent F-actin formation physically restrains intracellular organelles and reduces their mobility non-selectively in PDEC.
authors.affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, 98195, United States.
authors.forename: Seung-Ryoung
authors.initials: SR
authors.lastname: Jung
authors.forename: Jong Bae
authors.initials: JB
authors.lastname: Seo
authors.forename: Donghwan
authors.initials: D
authors.lastname: Shim
authors.forename: Bertil
authors.initials: B
authors.lastname: Hille
authors.forename: Duk-Su
authors.initials: DS
authors.lastname: Koh
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000588:Amines / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003080:Cold Temperature; D017077:Culture Media, Conditioned; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D061252:Feeder Cells; D036681:Fluorescence Recovery After Photobleaching; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D009930:Organic Chemicals / Q000378:metabolism; D010183:Pancreatic Ducts / Q000166:cytology* / Q000378:metabolism; D058473:Receptors, Purinergic P2Y2 / Q000378:metabolism; D014162:Transfection
chemical_list: D000199:Actins; D000588:Amines; D017077:Culture Media, Conditioned; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D058473:Receptors, Purinergic P2Y2; C493330:Red DND-99; C107472:red dye CMXRos; D002118:Calcium