Filopodia formation and endosome clustering induced by mutant plus-end–directed myosin VI

Abstract

Myosin VI (MYO6) is the only myosin known to move toward the minus end of actin filaments. It has roles in numerous cellular processes, including maintenance of stereocilia structure, endocytosis, and autophagosome maturation. However, the functional necessity of minus-end-directed movement along actin is unclear as the underlying architecture of the local actin network is often unknown. To address this question, we engineered a mutant of MYO6, MYO6+, which undergoes plus-end-directed movement while retaining physiological cargo interactions in the tail. Expression of this mutant motor in HeLa cells led to a dramatic reorganization of cortical actin filaments and the formation of actin-rich filopodia. MYO6 is present on peripheral adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) signaling endosomes and MYO6+ expression causes a dramatic relocalization and clustering of this endocytic compartment in the cell cortex. MYO6+ and its adaptor GAIP interacting protein, C terminus (GIPC) accumulate at the tips of these filopodia, while APPL1 endosomes accumulate at the base. A combination of MYO6+ mutagenesis and siRNA-mediated depletion of MYO6 binding partners demonstrates that motor activity and binding to endosomal membranes mediated by GIPC and PI(4,5)P 2 are crucial for filopodia formation. A similar reorganization of actin is induced by a constitutive dimer of MYO6+, indicating that multimerization of MYO6 on endosomes through binding to GIPC is required for this cellular activity and regulation of actin network structure. This unique engineered MYO6+ offers insights into both filopodia formation and MYO6 motor function at endosomes and at the plasma membrane.is a monomeric motor protein with a large working stroke (1), which takes processive 36-nm steps along actin filaments in vitro when dimerized (2). Uniquely among myosin motors, it takes these steps toward the minus end of actin filaments (3). In cells and tissues, MYO6 has been implicated in an ever-growing list of functions, including secretion, endocytosis, cell migration, stereocilia maintenance, and autophagy (4). It mediates these functions through interactions with a wide array of cargo adaptor proteins that bind to the tail region of MYO6, thereby localizing and potentially activating the motor through conformational change and unfolding of the lever arm (5) mediated by calcium (6). However, how the mechanical properties (for instance step size, duty ratio, stall force, etc.) and in particular the directionality of MYO6 relate to its biological roles is poorly understood.Isolated, purified MYO6 is primarily monomeric (1), but can dimerize in vitro through interactions between the cargo binding domain and adaptor molecules (2, 7). Whether any of these adaptors are capable of inducing functional MYO6 dimers in cells is not known and there has been little definitive evidence as to whether MYO6 functions as a monomer or as a processive dimer in cells and how motor processivity relates to its biological function (8,...

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

“…These results support, and may provide the molecular mechanism for, our recent finding that MYO6 mediates association of APPL1 endosomes with cortical actin filaments 34, 30. Depletion of MYO6 or expression of the reverse MYO6 + affects endosome localisation in the cell cortex.…”

“…MYO6 CBD LI (isoform 1, Q9UM54‐1, amino acids 1037–1285) and MYO6 CBD NI (isoform 5, Q9UM54‐5, amino acids 1036–1253) WT, ΔWWY, ΔRRL and ΔPIP 2 were amplified by PCR from MYO6 FL and tail wild‐type, ΔWWY, ΔRRL and ΔPIP 2 pEGFPC constructs described elsewhere 8, 46, 47, 48 and inserted in‐frame at the 3′ end of the BirA* tag. Mammalian two‐hybrid MYO6 tail wild‐type and mutant pM constructs have been described elsewhere 9, as have GFP‐MYO6 + and GFP‐MYO10 30. HA‐MYO6 CBD was generated by PCR using primers containing the HA tag sequence.…”

“…We next investigated a potential role of the MYO6-GIPC1-LARG-SH3BP4 complex in regulating actin filament organisation, as LARG is a GEF for RHO GTPases and SH3BP4 localised to actin structures including filopodia at the cell surface ( Fig 3D). For this analysis, we used an engineered mutant of MYO6 (MYO6 + ), which moves in the opposite direction to the wild-type protein [30]. As previously shown, this mutant clusters early endosomes and induces actin reorganisation, leading to the formation of filopodia protruding from the cell surface, above a cortical cluster of endosomes, in a GIPC1dependent manner [30].…”

TheMYO6 interactome: selective motor‐cargo complexes for diverse cellular processes

“…In vivo observations support the importance of MYO6 oligomerization induced by GIPC through a mutant MYO6+ that moves towards the plus end of actin filaments. MYO6+ induced filopodia formation only occurs in the presence of GIPC, which accumulates together with MYO6+ at the tips . Interestingly, an artificially dimerized MYO6 without the CBD, can rescue this phenotype, which supports the importance of GIPC‐mediated dimerization/oligomerization for MYO6 functions in vivo .…”

“…The GIPC‐MYO6 complex has been suggested to translocate early endosomes away from the plasma membrane through the cortical actin network, since expression of a nonfunctional MYO6 rigor mutant inhibits endosome movement . A crucial role for MYO6 in the spatial organization of the APPL1‐signalling endosomes is also supported by the findings that the depletion of MYO6 leads to the premature maturation and displacement of APPL1 endosomes from the cell cortex in the perinuclear region of the cell and expression of a mutant MYO6 that moves towards the plus end of actin filaments causes extreme cortical clustering of endosomes . Finally, recent in situ proximity labelling experiments identified a tripartite complex of GIPC1, MYO6 and LARG, which is a GEF for RHO GTPases.…”