Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line.

Abstract

Myosins I, a ubiquitous monomeric class of myosins that exhibits actin-based motor properties, are associated with plasma and/or vesicular membranes and have been suggested as players for trafficking events between cell surface and intracellular membranous structures. To investigate the function of myosins I, we have transfected a mouse hepatoma cell line (BWTG3) with cDNAs encoding the chicken brush border myosin-I (BBMI) and two variants truncated in the motor domain. One variant is deleted of the first 446 amino acids and thereby lacks the ATP binding site, whereas the other is deleted of the entire motor domain and lacks the ATP and actin binding sites. We have observed (i) that significant amounts of the truncated variants are recovered with membrane fractions after cell fractionation, (ii) that they codistribute with a compartment containing a2-macroglobulin internalized for 30 min as determined by fluorescent microscopy, (iii) that the production of BBMI-truncated variants impairs the distribution of the acidic compartment and ligands internalized for 30 min, and (iv) that the production of the truncated variant containing the actin binding site decreases the rate of a2-macroglobulin degradation whereas the production of the variant lacking the ATP binding site and the actin binding site increases the rate of a2-macroglobulin degradation. These observations indicate that the two truncated variants have a dominant negative effect on the distribution and the function of the endocytic compartments. We propose that an unidentified myosin-I might contribute to the distribution of endocytic compartments in a juxtanuclear position and/or to the regulation of the delivery of ligands to the degradative compartment in BWTG3 cells.

Truncated Brush Border Myosin I Affects Membrane Traffic in Polarized Epithelial Cells

“…DNA transfection with recombinant DNA constructions and isolation of stable transformants was performed as described by Durrbach et al (22). Two Caco-2 cellular clones producing BBMI, three Caco-2 cellular clones producing BBMID446, and three Caco-2 cellular clones producing BBMI Tail were selected, and permanently grown in the culture medium of Caco-2 cells in which penicillin/streptomycin were replaced by 0.7 mg/ml Geneticin (Gibco-BRL Life Technologies).…”

“…MMIa is widely expressed in tissues compared with BBMI and is detected at the plasma membrane and on endosomes (24). We have previously reported that nonfunctional truncated BBMI, which competes with MMIa in an unpolarized hepatoma cell line, affects the delivery of internalized molecules from the late endosomes to lysosomes (22,24). We also showed that actin filaments were required for this step of endocytosis and for protein recycling from pericentriolar recycling endosomes to the plasma membrane (30).…”

“…However, myosin Is can be divided into distinct subclasses based upon sequence homologies in their head and tail domains (18,25,26). It has been suggested that one subclass, which is identified to date only in metazoans and encompasses myosin I alpha (MMIa) and brush border myosin I (BBMI), is involved in endocytosis (22,24). MMIa and BBMI share 78% sequence homology (26)(27)(28).…”

“…Traffic 2000: 1: 411-424 negative effect on endocytosis of unpolarized cells by competing with the endogenous MMIa (22,24) to analyze whether a myosin I might be involved in endocytosis of polarized cells. We expressed truncated non-functional chicken BBMI proteins in Caco-2 cells.…”

Nonpolarized Cells Selectively Sort Apical Proteins from Cell Surface to a Novel Compartment, but Lack Apical Retention Mechanisms

“…Characterization of myosin motors, likely candidates for mediating actin-based vesicle motility, has also suggested that actin regulates recycling (reviewed in Baker and Titus, 1998;Mermall et al, 1998). For example, overexpression of dominant negative myosin 1 dispersed a Tf-positive compartment in hepatoma cells (Durrbach et al, 1996a) and significantly decreased basolateral Tf recycling in Caco2 cells (Durrbach et al, 2000). The expression of mutant Cdc42, an actin-modifying GTPase, also led to decreased Tf recycling in MDCK cells (Kroschewski et al, 1999).…”