Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line.
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 1, 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 alpha2-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 alpha2-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.
Durrbach
A
A
Morphogenése et Signalisation Cellulaires, Centre National de la Recherche Scientifique, UMR 144, Paris, France.
Collins
K
K
Matsudaira
P
P
Louvard
D
D
Coudrier
E
E
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D002645:Chickens; D004705:Endocytosis; D014644:Genetic Variation; D006801:Humans; D007700:Kinetics; D008114:Liver Neoplasms, Experimental; D051379:Mice; D008871:Microvilli / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D014168:Transferrin / Q000378:metabolism; D014407:Tumor Cells, Cultured; D000511:alpha-Macroglobulins / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014168:Transferrin; D000511:alpha-Macroglobulins; D000255:Adenosine Triphosphate; D009218:Myosins