The MYO6 interactome reveals adaptor complexes coordinating early endosome and cytoskeletal dynamics

Abstract

The intracellular functions of myosin motors requires a number of adaptor molecules, which control cargo attachment, but also fine‐tune motor activity in time and space. These motor–adaptor–cargo interactions are often weak, transient or highly regulated. To overcome these problems, we use a proximity labelling‐based proteomics strategy to map the interactome of the unique minus end‐directed actin motor MYO6. Detailed biochemical and functional analysis identified several distinct MYO6‐adaptor modules including two complexes containing RhoGEFs: the LIFT (LARG‐Induced F‐actin for Tethering) complex that controls endosome positioning and motility through RHO‐driven actin polymerisation; and the DISP (DOCK7‐Induced Septin disPlacement) complex, a novel regulator of the septin cytoskeleton. These complexes emphasise the role of MYO6 in coordinating endosome dynamics and cytoskeletal architecture. This study provides the first in vivo interactome of a myosin motor protein and highlights the power of this approach in uncovering dynamic and functionally diverse myosin motor complexes.

The MYO6 interactome: selective motor‐cargo complexes for diverse cellular processes

“…Indeed, in vitro experiments using purified proteins have shown that two adaptor proteins such as TOM1 and NDP52 can bind simultaneously directly to the MYO6 ABD1 and the ABD2 . Interestingly, in situ proximity labelling of MYO6 NI CBD has indicated that the mutation of the RRL motif also leads to the loss of TOM1, and its partner TOLLIP . These results suggest that although the interaction between TOM1 and MYO6 occurs directly via the WWY motif, in vivo the binding between MYO6 and TOM1 may also be indirect via ubiquitin using the ubiquitin‐binding TOM1 VHS domain and the MYO6 MyUb domain.…”

“…Point mutations of the tryptophan of either binding motif, W 1202 of WWY or W 1262 of IWE, disrupt Dab2 binding to MYO6 and abolish cellular localization to clathrin-coated structures at the plasma membrane in vivo [52,76]. The importance of site I was confirmed by in situ proximity labelling, which demonstrated the loss of Dab2 and other proteins associated with clathrin-mediated endocytosis upon mutation of the WWY site [18].…”

OPTN translocates to an ATG9A-positive compartment to regulate innate immune signalling and cytokine secretion

“…GFP-OPTN E50K and E478G pLXIN mutants were generated by site-directed mutagenesis. The myc-BirA*-OPTN pLXIN vector was created by subcloning OPTN into the myc-BirA* pLXIN plasmid described previously [55]. using the Δ Δ Ct method [57].…”

“…48 hours post-transfection, cells were lysed with 1% NP-40 lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40) containing complete protease inhibitor cocktail (Roche), passed repeatedly through a 25G needle to homogenise and clarified by centrifugation at 20,000 x g for 10 minutes at 4°C. Subsequently, clarified lysates were incubated with 10 µl of GFP-nanobody Affi-gel resin [55] for 3 hours with mixing. Beads were washed with 1% NP-40 buffer, then TBS and were eluted in SDS sample loading buffer at 95°C.…”

“…BioID experiments were performed as described previously [55]. Data was processed using the online tool at CRAPome.org using the default settings and a threshold of ≥ 3 FC-B was established to determine candidate OPTN interacting proteins.…”