title: Myosin-1A targets to microvilli using multiple membrane binding motifs in the tail homology 1 (TH1) domain.
abstract: One of the most abundant components of the enterocyte brush border is the actin-based monomeric motor, myosin-1a (Myo1a). Within brush border microvilli, Myo1a carries out a number of critical functions at the interface between membrane and actin cytoskeleton. Proper physiological function of Myo1a depends on its ability to bind to microvillar membrane, an interaction mediated by a C-terminal tail homology 1 (TH1) domain. However, little is known about the mechanistic details of the Myo1a-TH1/membrane interaction. Structure-function analysis of Myo1a-TH1 targeting in epithelial cells revealed that an N-terminal motif conserved among class I myosins and a C-terminal motif unique to Myo1a-TH1 are both required for steady state microvillar enrichment. Purified Myo1a bound to liposomes composed of phosphatidylserine and phosphoinositol 4,5-bisphosphate, with moderate affinity in a charge-dependent manner. Additionally, peptides of the N- and C-terminal regions required for targeting were able to compete with Myo1a for binding to highly charged liposomes in vitro. Single molecule total internal reflection fluorescence microscopy showed that these motifs are also necessary for slowing the membrane detachment rate in cells. Finally, Myo1a-TH1 co-localized with both lactadherin-C2 (a phosphatidylserine-binding protein) and PLCδ1-PH (a phosphoinositol 4,5-bisphosphate-binding protein) in microvilli, but only lactaderin-C2 expression reduced brush border targeting of Myo1a-TH1. Together, our results suggest that Myo1a targeting to microvilli is driven by membrane binding potential that is distributed throughout TH1 rather than localized to a single motif. These data highlight the diversity of mechanisms that enable different class I myosins to target membranes in distinct biological contexts.
authors.affiliation: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
authors.forename: Jessica N
authors.initials: JN
authors.lastname: Mazerik
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Tyska
mesh_terms: D000143:Acids / Q000378:metabolism; D000818:Animals; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024461:Myosin Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013552:Swine
chemical_list: D000143:Acids; D008081:Liposomes; C062291:MYO1A protein, human; D008565:Membrane Proteins; D010743:Phospholipids; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D024461:Myosin Type I; D018995:Myosin Heavy Chains