Dynamic localization of myosin-I to endocytic structures in Acanthamoeba.
We used fluorescence microscopy of live Acanthamoeba to follow the time course of the concentration of myosin-I next to the plasma membrane at sites of macropinocytosis and phagocytosis. We marked myosin-I with a fluorescently labeled monoclonal antibody (Cy3-M1.7) introduced into the cytoplasm by syringe loading. M1.7 binds myosin-IA and -IC without affecting their activities, but does not bind myosin-IB. Cy3-M1.7 concentrates at two different macropinocytic structures: large circular membrane ruffles that fuse to create macropinosomes, and smaller endocytic structures that occur at the end of stalk-like pseudopodia. These dynamic structures enclose macropinosomes every 30-60 s. Cy3-M1.7 accumulates rapidly as these endocytic structures form and dissipate rapidly after they internalize. Double labeling fixed cells with Cy3-M1.7 and polyclonal antibodies specific for myosin-IA, -IB, or -IC revealed that all three myosin-I isoforms associate with macropinocytic structures, but individual structures vary in their myosin-I isoform composition. Myosin-I and actin also concentrate transiently at sites where amoebae ingest yeast or the pseudopodia of neighboring cells (heterophagy) by the process of phagocytosis. Within 3 min of yeast attachment to the amoeba, myosin-I concentrates around the phagocytic cup, yeast are internalized, and myosin-I de-localizes. Despite known differences in the regulation of macropinocytosis and phagocytosis, the morphology, protein composition, and dynamics of phagocytosis and macropinocytosis are similar, indicating that they share common structural properties and contractile mechanisms.
Ostap
E Michael
EM
The Pennsylvania Muscle Institute and The Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6085, USA. ostap@mail.med.upenn.edu
Maupin
Pamela
P
Doberstein
Steven K
SK
Baines
Ivan C
IC
Korn
Edward D
ED
Pollard
Thomas D
TD
D000048:Acanthamoeba / Q000737:chemistry* / Q000648:ultrastructure; D000199:Actins / Q000032:analysis / Q000648:ultrastructure; D000818:Animals; D002465:Cell Movement; D014644:Genetic Variation; D015151:Immunoblotting; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009218:Myosins / Q000032:analysis* / Q000648:ultrastructure; D010587:Phagocytosis / Q000502:physiology*; D010873:Pinocytosis / Q000502:physiology*; D020033:Protein Isoforms / Q000032:analysis / Q000648:ultrastructure; D014617:Vacuoles
D000199:Actins; D020033:Protein Isoforms; D009218:Myosins