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

Meredith L Weck,Scott W Crawley,Matthew J Tyska

doi:10.1074/jbc.ra120.015929

Meredith L Weck, Scott W Crawley + Show 1 more

Open Access

https://doi.org/10.1074/jbc.ra120.015929

Copy DOI

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.

Highlights

All cells use plasma membrane protrusions to physically and biochemically interact with the external environment
To establish a system for studying intermicrovillar adhesion complex (IMAC) component interactions and function, we focused on the filopodia that extend from the surface of HeLa cells
To circumvent this kinetic limitation, we created a chimeric myosin based on the filopodial motor, MYO10; this approach was inspired by previous studies that took advantage of MYO10 motor domain tip targeting potential in a novel protein–protein interaction assay [35]

Summary

Introduction

All cells use plasma membrane protrusions to physically and biochemically interact with the external environment. This synthetic myosin demonstrates robust targeting to the distal tips of HeLa cell filopodia and was able to drive enrichment of all other individual IMAC components: USH1C, ANKS4B, CDHR2, and CDHR5, to these sites.

Results

Conclusion