# Live-cell single-molecule fluorescence microscopy for protruding organelles reveals regulatory mechanisms of MYO7A-driven cargo transport in stereocilia of inner ear hair cells

## TL;DR

This paper introduces a new live-cell microscopy method to study how MYO7A proteins transport cargo in stereocilia of inner ear hair cells.

## Contribution

A novel live-cell single-molecule fluorescence microscopy method using diSPIM to study organelle protrusions in hair cells.

## Key findings

- MYO7A traffics as a dimer in stereocilia.
- MYO7A movement is restricted by interactions with the plasma membrane and F-actin.
- The methodology enables detailed analysis of cargo transport in protruding organelles.

## Abstract

Stereocilia are unidirectional F-actin-based cylindrical protrusions on the apical surface of inner ear hair cells and function as biological mechanosensors of sound and acceleration. Development of functional stereocilia requires motor activities of unconventional myosins to transport proteins necessary for elongating the F-actin cores and to assemble the mechanoelectrical transduction (MET) channel complex. However, how each myosin localizes in stereocilia using the energy from ATP hydrolysis is only partially understood. In this study, we develop a methodology for live-cell single-molecule fluorescence microscopy of organelles protruding from the apical surface using a dual-view light-sheet microscope, diSPIM. We demonstrate that MYO7A, a component of the MET machinery, traffics as a dimer in stereocilia. Movements of MYO7A are restricted when scaffolded by the plasma membrane and F-actin as mediated by MYO7A’s interacting partners. Here, we discuss the technical details of our methodology and its future applications including analyses of cargo transportation in various organelles.