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  • A heterologous in-cell assay for investigating intermicrovillar adhesion complex interactions reveals a novel protrusion length-matching mechanism.

A heterologous in-cell assay for investigating intermicrovillar adhesion complex interactions reveals a novel protrusion length-matching mechanism.

The Journal of biological chemistry (2020-10-15)
Meredith L Weck, Scott W Crawley, Matthew J Tyska
ABSTRACT

Solute transporting epithelial cells build arrays of microvilli on their apical surface to increase membrane scaffolding capacity and enhance function potential. In epithelial tissues such as the kidney and gut, microvilli are length-matched and assembled into tightly packed "brush borders," which are organized by ∼50-nm thread-like links that form between the distal tips of adjacent protrusions. Composed of protocadherins CDHR2 and CDHR5, adhesion links are stabilized at the tips by a cytoplasmic tripartite module containing the scaffolds USH1C and ANKS4B and the actin-based motor MYO7B. Because several questions about the formation and function of this "intermicrovillar adhesion complex" remain open, we devised a system that allows one to study individual binary interactions between specific complex components and MYO7B. Our approach employs a chimeric myosin consisting of the MYO10 motor domain fused to the MYO7B cargo-binding tail domain. When expressed in HeLa cells, which do not normally produce adhesion complex proteins, this chimera trafficked to the tips of filopodia and was also able to transport individual complex components to these sites. Unexpectedly, the MYO10-MYO7B chimera was able to deliver CDHR2 and CDHR5 to distal tips in the absence of USH1C or ANKS4B. Cells engineered to localize high levels of CDHR2 at filopodial tips acquired interfilopodial adhesion and exhibited a striking dynamic length-matching activity that aligned distal tips over time. These findings deepen our understanding of mechanisms that promote the distal tip accumulation of intermicrovillar adhesion complex components and also offer insight on how epithelial cells minimize microvillar length variability.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-ANKS4B antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Anti-CDHR2 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Anti-CDHR5 antibody produced in rabbit, Ab1, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Anti-USH1C antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution, ab1
Sigma-Aldrich
Anti-MYO7B antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution