Impact of the Motor and Tail Domains of Class III Myosins on Regulating the Formation and Elongation of Actin Protrusions

Manmeet H Raval,Omar A Quintero,Meredith L Weck,William C Unrath,James W Gallagher,Runjia Cui,Bechara Kachar,Matthew J Tyska,Christopher M Yengo

doi:10.1074/jbc.m116.733741

Abstract

Class III myosins (MYO3A and MYO3B) are proposed to function as transporters as well as length and ultrastructure regulators within stable actin-based protrusions such as stereocilia and calycal processes. MYO3A differs from MYO3B in that it contains an extended tail domain with an additional actin-binding motif. We examined how the properties of the motor and tail domains of human class III myosins impact their ability to enhance the formation and elongation of actin protrusions. Direct examination of the motor and enzymatic properties of human MYO3A and MYO3B revealed that MYO3A is a 2-fold faster motor with enhanced ATPase activity and actin affinity. A chimera in which the MYO3A tail was fused to the MYO3B motor demonstrated that motor activity correlates with formation and elongation of actin protrusions. We demonstrate that removal of individual exons (30–34) in the MYO3A tail does not prevent filopodia tip localization but abolishes the ability to enhance actin protrusion formation and elongation in COS7 cells. Interestingly, our results demonstrate that MYO3A slows filopodia dynamics and enhances filopodia lifetime in COS7 cells. We also demonstrate that MYO3A is more efficient than MYO3B at increasing formation and elongation of stable microvilli on the surface of cultured epithelial cells. We propose that the unique features of MYO3A, enhanced motor activity, and an extended tail with tail actin-binding motif, allow it to play an important role in stable actin protrusion length and ultrastructure maintenance.

Highlights

Removal of THD2 from MYO3A (MYO3A.⌬THD2) abolished its tip localization (Fig. 3J) as shown previously [26]
Role of MYO3 Motor Domain in Regulating Actin Protrusion Dynamics—Our results demonstrate that MYO3A is a faster motor compared with MYO3B (Fig. 2 and Table 1), and an active motor domain is absolutely required for allowing MYO3A to induce and elongate actin protrusions (Fig. 3, K and L)
Based on our results we envision MYO3A as a motorized monomeric actin cross-linker that localizes to the barbed ends of actin filaments and stabilizes growing actin filaments to facilitate length maintenance

Summary

Introduction

Removal of THD2 from MYO3A (MYO3A.⌬THD2) abolished its tip localization (Fig. 3J) as shown previously [26]. MYO3A Requires Enhanced Motor Activity and THD2 to Induce and Elongate Actin Protrusions—MYO3A (kinase domain deleted) demonstrated the highest tip localization, as well as filopodia formation (density) and elongation (length) activity compared with the rest of the constructs (Fig. 3, A–L). It has been shown previously that the motor activity is required for MYO3A tip localization; its impact on actin protrusion formation and elongation has never been examined before.

Results

Conclusion