GASTROENTEROLOGY 1997;113:649 – 658 Myosin I Is Associated With Zymogen Granule Membranes in the Rat Pancreatic Acinar Cell SIRIA POUCELL – HATTON, PENNY SUE PERKINS, THOMAS J. DEERINCK, MARK H. ELLISMAN, WILLIAM G. M. HARDISON, and STEPHEN J. PANDOL Departments of Medicine and Pathology and Department of Neurosciences, San Diego Microscopy and Imaging Resource, University of California San Diego, San Diego; and Department of Veterans Affairs Medical Center, La Jolla, California Background & Aims: The mechanisms whereby intracel- different myosins I and their isoforms. One distinguish- lular messengers mediate zymogen granule transport ing characteristic of two of the most extensively studied and exocytosis in the pancreatic acinar cell are not well myosins I is their ability to attach to phospholipid mem- defined. Electron microscopy has shown a periluminal branes. A. castellanii myosins IA and IB bind directly to network of actin in the acinar cell, suggesting a role for NaOH-extracted membranes isolated from this species actin and myosin in the transport process. The possible as well as to phospholipid vesicles containing phosphati- involvement of two types of myosin in the secretory dylserine or phosphatidylinositol 4,5-biphosphate. 8–11 process was investigated, and their distribution in aci- The binding of the tail to membranes may allow this nar cells was determined. Methods: Antibodies specific motor molecule to act in organellar movement, phagocy- to myosin I or to myosin II were used for immunocyto- tosis, and cell movement. 10,12 The myosins I from A. chemistry and Western blot analysis. Ultrastructural castellanii have an ATP-independent actin-binding site studies were also performed. Results: Western blot in the tail as well as the ATP-sensitive actin filament analysis showed that myosin I and myosin II were pres- ent in total pancreatic homogenate but that only myo- binding site in the head. The tail-binding site may be sin I was present on isolated zymogen granules and important for anchoring to actin filaments. The tails of their membranes. By immunocytochemistry, myosin I myosins are thought to contain targeting signals for was shown in the apical aspect of acinar cells colocal- membrane subdomains and to determine the type of ized with glycoprotein 2, a marker for zymogen gran- transport required. Some myosins I have been localized ules, and actin. By immunocytochemistry, myosin I was to specific membranous structures within cells. 1,5,13–16 also localized on isolated zymogen granules. Conclu- Several Acanthamoeba myosin I isoforms have been sions: The immunolocalization of myosin I to zymogen identified by immunolocalization and show unique cyto- granule membranes and its close association with per- plasmic patterns. 17 Myosin IA occurs almost exclusively iluminal actin suggest that myosin I plays a direct role in the cytoplasm, in the cortex beneath phagocytic cups, in the process of transport and exocytosis of zymogen and in association with small cytoplasmic vesicles. Myo- granules in the pancreatic acinar cell. sin IB is the main isoform associated with the plasma membrane, large vacuole membranes, and phagocytic A membranes. Myosin IC is associated with the plasma ll myosins are characterized by an 80-kilodalton head domain that binds actin filaments, hydrolyzes membrane and large vacuole membranes and is the only adenosine triphosphate (ATP), and translocates along ac- isoform associated with the contractile vacuole mem- tin filaments. 1 In 1973, Pollard and Korn 2,3 reported a brane. These data indicate that myosin IA may function class of myosins in Acanthamoeba castellanii termed myo- in cytoplasmic vesicle transport and cortical contraction, sins I, 4,5 which are characterized as mechanochemical mo- myosin IB in pseudopod extension and phagocytosis, and tors showing actin-activated adenosine triphosphatase ac- myosin IC in contractile vacuole function. tivity. Myosins I contain a single-headed domain and a The first characterized myosin I from vertebrates is tail that differs markedly from that of heavy myosin the avian intestinal brush border myosin I (BBMI). 18,19 (myosin II). Myosins II are double headed and have a molecular weight of approximately 200 kilodaltons, Abbreviations used in this paper: BBMI, brush border myosin I; whereas myosins I are approximately 100 – 130 kilodal- CY5, cyanogen 5; FITC, fluorescein isothiocyanate; GP-2, glycopro- tons. 5 Unlike myosin II, myosin I monomers do not tein 2. polymerize into bipolar filaments. 5–7  1997 by the American Gastroenterological Association 0016-5085/97/$3.00 The structure of the carboxyterminal tails varies for / 5e1f$$0026 07-09-97 17:29:49 gasas WBS-Gastro