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

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.

Multimodal regulation of myosin VI ensemble transport by cargo adaptor protein GIPC

“…An N-terminal mCitrine version of adaptor MIRs with a FLAG tag was used for bimolecular FRET assays. A GFP nanobody construct with C-terminal SNAP and FLAG tags in pBiex1 vector were used to bind GFP-tagged proteins as reported by us previously (17,37,38).…”

Nanosurfer assay dissects β-cardiac myosin and cardiac myosin-binding protein C interactions

“…The C0-C1f fragment contained the C0 and C1 domains with the respective unstructured Pro-Ala-rich linker region, as well as the first 17 amino acids of the M-domain, as described previously ( 30 ). The proteins were then transiently transfected (pBiex-1; Escort IV, Sigma-Aldrich, St. Louis, MO) into Sf9 cells, expressed and affinity purified similarly to previously published protocols ( 35 , 39 ). Briefly, cells were lysed at 72 h in lysis buffer: 0.5% IGEPAL, 4 mM MgCl 2 , 200 mM NaCl, 7% sucrose, 20 mM imidazole (pH 7.5), 0.5 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), 1 μg/ml phenylmethylsulfonyl fluoride (PMSF), 10 μg/ml aprotinin, and 10 μg/ml leupeptin.…”

How myosin VI traps its off-state, is activated and dimerizes

“…The configuration of the Myo6 active state, the nature of its Lever arm and its oligomeric state can make critical differences in the way the force produced by the motor is used 16 , 30 , 31 . In fact, Myo6 is well adapted to transport as well as to anchor, depending on the load it is working against, according to single molecule assays 32 .…”

“…In fact, Myo6 is well adapted to transport as well as to anchor, depending on the load it is working against, according to single molecule assays 32 . Although the capacity of partners to favor either monomeric, dimeric or oligomeric assemblies has been described 13 , 27 – 29 , 31 , 33 – 35 , the active configuration required to perform the different cellular roles of Myo6 is unknown. In vitro studies have identified a proximal dimerization region 11 , 17 , but its role in the cellular function of Myo6 is not established, nor is the structure of this region.…”