Dynamic multimerization of Dab2–Myosin VI complexes regulates cargo processivity while minimizing cortical actin reorganization

Duha Vang,Michael Ritt,Ashim Rai,Sivaraj Sivaramakrishnan

doi:10.1074/jbc.ra120.012703

Abstract

Myosin VI ensembles on endocytic cargo facilitate directed transport through a dense cortical actin network. Myosin VI is recruited to clathrin-coated endosomes via the cargo adaptor Dab2. Canonically, it has been assumed that the interactions between a motor and its cargo adaptor are stable. However, it has been demonstrated that the force generated by multiple stably attached motors disrupts local cytoskeletal architecture, potentially compromising transport. In this study, we demonstrate that dynamic multimerization of myosin VI-Dab2 complexes facilitates cargo processivity without significant reorganization of cortical actin networks. Specifically, we find that Dab2 myosin interacting region (MIR) binds myosin VI with a moderate affinity (184 nM) and single-molecule kinetic measurements demonstrate a high rate of turnover (1 s−1) of the Dab2 MIR–myosin VI interaction. Single-molecule motility shows that saturating Dab2-MIR concentration (2 μM) promotes myosin VI homodimerization and processivity with run lengths comparable with constitutive myosin VI dimers. Cargo-mimetic DNA origami scaffolds patterned with Dab2 MIR-myosin VI complexes are weakly processive, displaying sparse motility on single actin filaments and “stop-and-go” motion on a cellular actin network. On a minimal actin cortex assembled on lipid bilayers, unregulated processive movement by either constitutive myosin V or VI dimers results in actin remodeling and foci formation. In contrast, Dab2 MIR–myosin VI interactions preserve the integrity of a minimal cortical actin network. Taken together, our study demonstrates the importance of dynamic motor–cargo association in enabling cargo transportation without disrupting cytoskeletal organization.

Highlights

Myosin VI ensembles on endocytic cargo facilitate directed transport through a dense cortical actin network
Adaptor interactions change the trajectory of myosin VI on cellular actin networks Using the same nanoarchitecture, we examined the effects of Dab2 myosin interacting region (MIR)–myosin VI interactions in the context of cortical actin networks derived from fish-scale keratocytes (Fig. 5, A–B) [28, 29]
We find that Dab2 binds weakly to myosin VI and facilitates dimerization at saturating concentrations, leading to sparse processivity

Summary

Results

The myosin VI binding interface for the endocytic adaptor proteins Dab has been well characterized [4, 22]. The run length of Dab cargo movement significantly scales up from one-site to foursite nanostructures, similar to the constitutively processive HMM dimer (Fig. 4C) Together, these results demonstrate that Dab is able to drive sparse processive motility of myosin VI cargo at both single and ensemble motor levels. While in complex with Dab MIR, full-length myosin VI was observed to generate foci, significantly fewer than observed with dimeric myosin VI (Fig. 7, B–C) Taken together, these data suggest that the dynamic interaction of myosin VI with Dab results in an equilibrium between monomeric and dimeric states of myosin VI that allows for processive motor movement on a dense actin network without significant remodeling of the underlying cytoskeletal network (Fig. 8)

Discussion

Experimental procedures

Myosin VI FL-SNAP

Myosin VI CBD constructs