Myosin VI and its cargo adaptors – linking endocytosis and autophagy

In vertebrate cells the actin cytoskeleton provides the tensile strength to maintain cell morphology and the necessary mechanical forces and contractility to facilitate cell migration, adhesion, nutrient sensing, wound healing and cytokinesis. Actin filaments also form the tracks for myosin motor proteins to tether and transport various membrane compartments and cargoes during these distinct cellular processes. Actin filaments (F-actin) are polar structures with a fast polymerizing barbed end and a slow polymerizing pointed end, which can be assembled and disassembled in a dynamic equilibrium with actin monomers (G-actin) (Pollard et al., 2000).

Actin filaments and myosin motors are not only important for the steady-state distribution of intracellular organelles but are also intimately involved in shaping and deforming membranes during scission and fusion events, and crucial for the sorting of cargo on endosomes and the bidirectional delivery between membrane compartments of the endocytic pathway (Anitei and Hoflack, 2011; Mooren et al., 2012). Importantly, after vesicle internalisation, cargo sorting and tubular and vesicular carrier formation on endosomes is dependent on spatially restricted actin polymerisation. The force that is required to bud a vesicle is variable for different donor membranes. This might explain why, for example, in yeast, actin and myosin motor proteins are essential for clathrin-mediated endocytosis, but why in mammalian cells actin and myosin VI are only required for endocytosis from the apical but not from the basolateral domain (Buss et al., 2001; Gottlieb et al., 1993; Jackman et al., 1994). In addition, the cortical F-actin network underneath the plasma membrane may provide tracks for the short-range retrograde movement and the transfer of cargo vesicles from the cell periphery to microtubules for their long-distance delivery by microtubule-based motors to areas within the cell.

One specialised and regulated membrane trafficking pathway that requires the actin cytoskeleton, and the coordinated recruitment of membrane and cargo from various subcellular compartments – including fusion with vesicles derived from the endocytic pathway – is autophagy (Axe et al., 2008; Hailey et al., 2010; Ravikumar et al., 2010; Yamamoto et al., 2012; Young et al., 2006). Autophagy, characterised by the formation of a double-membrane autophagosome, is a lysosomal degradation pathway that is required for the clearance of damaged organelles, pathogens and large cytosolic protein complexes, in addition to being a homeostatic process during times of starvation (Yang and Klionsky, 2010). Actin filaments are essential at a very early point during starvation-induced autophagosome biogenesis, at which they are potentially involved in facilitating cargo recognition and aggresome formation; later they also assemble around mature autophagosomes, where they mediate fusion with endocytic and lysosomal compartments by tethering the required components and bringing them into close contact (Aguilera et al., 2012; Lee et al., 2010). Importantly, before an autophagosome is competent to fuse with the lysosome and for its contents to be degraded, it requires influx from the endocytic pathway. It receives input from early and late endosomes as well as from multivesicular bodies (MVB), which are believed to deliver the tethering and adaptor molecules that are necessary for final fusion with the lysosome (Simonsen and Tooze, 2009). One essential component is the ESCRT machinery, which is important for cargo sorting, MVB formation, cytokinesis and viral budding (McCullough et al., 2013). Loss of ESCRT function by disruption of either the ESCRT-I protein TSG101 or the ESCRT-III protein CHMP2B, leads to an accumulation of autophagosomes that is most likely to be linked to a defect in lysosomal fusion (Filimonenko et al., 2007; Lee et al., 2007; Rusten et al., 2007). However, it remains to be tested whether the ESCRT machinery is required at multiple stages during the autophagy pathway, including fusion with endocytic and lysosomal compartments, as well as during the final closure of the double-membrane autophagosome (Rusten and Stenmark, 2009).

The spatial organisation of membrane compartments and the temporal regulation of membrane trafficking between intracellular organelles is a coordinated process involving dynamic cytoskeletal remodelling coupled to the action of specific motor protein-adaptor complexes that tether or drive transport along the actin and microtubule cytoskeletons. Dynein and Kinesin motors provide respective minus and plus end directed long distance movement of cargo along microtubules, while the myosin superfamily can anchor organelles to actin filaments and provide short-range movement along the actin cytoskeleton (Peckham, 2011). In humans, the myosin superfamily comprises 39 known myosin genes that are divided into 12 classes, and which are made up of both the conventional and unconventional myosins, whose functions are specified by their unique interactions with various cargo proteins and phospholipids. The conventional myosins, which comprise the non-muscle myosin II family and the skeletal, cardiac and smooth muscle myosins, assemble to form bipolar filaments that mediate sliding and crosslinking of actin filaments to generate contractility and tension. By contrast, unconventional myosins do not form filaments but, rather, function as either monomeric or dimeric cargo transporters, regulators of actin organisation, adjustable tethers for organelles, and/or as load-dependent tension sensors (Bloemink and Geeves, 2011; Hartman et al., 2011). The precise cellular roles of the different classes of unconventional myosins are mainly due to their divergent cargo-binding tail regions that mediate distinct interactions for targeting to various subcellular locations.

In this Commentary, we will focus on myosin of class VI, which has the unique ability, unlike the rest of the myosin family, to translocate towards the pointed or minus end of actin filaments. We will first highlight the structural properties of myosin VI, and the role and function of its distinct cargo adaptor proteins, which target this myosin to specific subcellular compartments to mediate its function (Fig. 1). Next, we will discuss the function of multiple adaptor molecules involved in the recruitment of myosin VI to the early endocytic pathway, where this motor could be required for sorting events directing cargo either to be recycled back to the plasma membrane or delivered to the lysosome for degradation. Finally, we will discuss the role of myosin VI during autophagy and its potential impact on the sorting and delivery of endosomal membranes required for autophagosome maturation, a process that is mediated by the interaction of myosin VI with the adaptor protein target of Myb1 (Tom1) (Tumbarello et al., 2012).

Structurally, myosin VI, like all myosin motors, consists of an N-terminal highly conserved motor domain that can bind ATP and filamentous actin. The conformational changes in the motor domain generated by ATP hydrolysis are transmitted through the converter region into a large movement of the adjacent lever arm. Myosin VI contains a unique insert, the reverse gear, between the converter and the lever arm, which is stabilised by a calmodulin and swings the lever arm in the opposite direction to that of all the other myosins. A second calmodulin is bound through a canonical IQ motif in the neck region. The C-terminal tail contains a central single alpha helix (SAH) domain followed by a globular cargo-binding domain (CBD) (Peckham, 2011). Purified full-length myosin VI exists as a monomer (Lister et al., 2004), unless it is forced in vitro to dimerise artificially when including a leucine zipper motif or adding a myosin II coiled-coil motif.

Intriguingly, myosin VI might have the ability to function either as a monomer or as a processive dimeric motor, which may be regulated by ligand-induced dimerisation (Spudich et al., 2007; Yu et al., 2009); thus cargo-attachment might directly regulate its motor properties. Indeed, binding to the endocytic adaptor disabled-2 (Dab2) might induce myosin VI dimerisation, thereby generating a processive endocytic motor for the transport and clustering of transmembrane receptors in the area of a clathrin-coated pit or even for the short-range transport of internalised vesicles away from the cell surface. However, under certain conditions, for example, when it acts to regulate actin stabilisation during spermatid individualisation, myosin VI can fully function when it lacks the ability to dimerise. Under these circumstances it remains bound to actin for long periods, thus functioning as an actin tether (Noguchi et al., 2009; Noguchi et al., 2006). Furthermore, it has been suggested that, as load is increased on myosin VI, it switches from its transport function to an anchoring function (Altman et al., 2004), such as at the base of microvilli, stereocilia and on vesicles in the terminal synaptic bouton (Hertzano et al., 2008; Kisiel et al., 2011; Sakurai et al., 2011; Seiler et al., 2004). Thus, myosin VI most probably functions as both a weak processive motor and an actin tethering protein; however, further work in vivo is required to determine the conditions, the physiological regulation and the cellular mechanisms involved.

In the Snell's waltzer myosin VI knockout mouse – derived from a spontaneous deletion that resulted in a frameshift mutation and premature stop codon (Avraham et al., 1995) – and also in flies, the loss of functional myosin VI is associated with a wide spectrum of phenotypes, which suggest that myosin VI is a constituent of multiple protein complexes that function in a variety of different pathways (Fig. 1). In mammalian cells and also in Drosophila a substantial number of direct binding partners have been identified (Buss and Kendrick-Jones, 2011; Finan et al., 2011). However, additional transient or low-affinity myosin VI cargo adaptor molecules as well as larger protein complexes may be identified in the future, by using in situ crosslinking or spatially-restricted enzymatic tagging in living cells or tissues (Rhee et al., 2013). Although the ability of myosin VI to associate with multiple cargo adaptors under strict spatial and temporal control is very intriguing, it is not unique to this class of motor protein and highlights the mammalian paradigm that one motor can bind to a number of different cargoes. For example, cytoplasmic dynein – like myosin VI – is a unique motor due to its reverse directionality along microtubules, and transports a variety of cargoes – from endocytic and exocytic transport carriers, to organelles such as mitochondria and ribonucleoproteins (Allan, 2011). In yeast, the two classes of myosin V bind to a number of different receptors to drive movement of peroxisomes, mitochondria, ER, exocytic vesicles and the Golgi complex (Akhmanova and Hammer, 2010). The spatial and temporal regulation and coordination of motor-cargo attachment is likely to involve several mechanisms, including tissue-specific expression of motor protein isoforms and the stoichiometric concentration of adaptor proteins.

In the CBD of myosin VI, at least seven adaptor proteins compete to interact with two binding interfaces and/or subdomains that contain the amino acid motifs RRL or WWY – as suggested by the CBD susceptibility to tryptic digestion leading to two stable soluble peptides (Spudich et al., 2007; Yu et al., 2009) (Table 1). For example, the region containing the RRL motif interacts with nuclear dot protein 52 (NDP52), Traf6-binding protein (T6BP), optineurin and GAIP-interacting protein C-terminus (GIPC), and the region encompassing the WWY motif is required for binding to Tom1, Dab2 and lemur tyrosine kinase-2 (LMTK2) (Bunn et al., 1999; Chibalina et al., 2007; Morris et al., 2002; Morriswood et al., 2007; Sahlender et al., 2005; Spudich et al., 2007; Tumbarello et al., 2012). For other myosin VI adaptor molecules, such as SAP97 (Wu et al., 2002), otoferlin (Heidrych et al., 2009), phospholipase Cδ3 (Sakurai et al., 2011) and Dock7 (Majewski et al., 2012), the exact binding domain has not yet been determined. The two subdomains containing the RRL and WWY protein–protein interaction sites are linked by a phospholipid binding motif that interacts specifically and with high affinity to PtdIns(4,5)P2 (Spudich et al., 2007). The CBD also contains a conserved ubiquitin-binding motif (Penengo et al., 2006); however, ubiquitylated proteins that specifically bind to myosin VI have not yet been identified.

Whereas myosin VI is widely expressed in most tissues, it exists in multiple tissue-specific splice isoforms with either a large insert (31 amino acids), a small insert (8 amino acids), both inserts, or no insert in the cargo-binding tail domain (Buss et al., 2001; Buss et al., 1998; Hasson and Mooseker, 1994). The tissue-specific expression of the four different myosin VI splice variants is most likely to be a further mechanism to control motor–cargo interactions and their subsequent subcellular localisation and function. In addition, the intracellular targeting of myosin VI might also depend on the stoichiometric expression level of its adaptors (Dance et al., 2004). However, the exact mechanism that regulates adaptor binding and cargo attachment is likely to be complex, involving a combination of different factors. Whereas the isoform with the large insert is preferentially expressed in polarised epithelia, the small and no-insert isoforms are found in non-polarised tissues (Buss et al., 2001). Interestingly, these alternatively spliced isoforms give myosin VI the ability to target to different intracellular locations through a yet undefined mechanism. Myosin VI that contains the large insert (LI) is recruited to clathrin-coated structures at the apical domain of polarised epithelial cells, where this motor is required for clathrin-dependent receptor-mediated endocytosis (Ameen and Apodaca, 2007; Buss et al., 2001). By contrast, myosin VI that lacks this insert (no insert isoform; NI) localises to uncoated vesicles near the cell periphery, where it is recruited by GIPC and the ESCRT-0 protein Tom1, and is suggested to either facilitate the movement of vesicles through the peripheral actin cytoskeleton towards the early endosome and/or to regulate a signalling platform for newly endocytosed receptors (Aschenbrenner et al., 2003; Dance et al., 2004; Naccache et al., 2006; Tumbarello et al., 2012). Myosin VI, however, is not only an endocytic motor but also functions in the exocytic pathway, in order to maintain Golgi morphology and facilitate the fusion of secretory vesicles at the plasma membrane (Bond et al., 2011; Sahlender et al., 2005; Warner et al., 2003). In addition, myosin VI loss-of-function affects the structural integrity of the stereocilia of the sensory hair cells of the inner ear (Self et al., 1999), the stability of cell–cell contacts in epithelia, efficient progression of cytokinesis (Arden et al., 2007) and directed cell migration (Chibalina et al., 2010; Geisbrecht and Montell, 2002). Further studies are required to show whether any of these complex cellular phenotypes that are caused by the loss-of-function of myosin VI are linked to trafficking defects in the endocytic or exocytic pathway.

The tissue-specific expression of the different myosin VI isoforms enables this motor to function in at least two separate stages along the endocytic pathway because the isoforms differ in their intracellular targeting. In cell types that express myosin VI LI, several physiological phenotypes have been identified that demonstrate the role of myosin VI in apical endocytosis. For example, defects identified in the myosin VI knockout mouse show that myosin VI regulates endocytosis of the cystic fibrosis transmembrane conductance regulator from the apical domain of enterocytes. Also, in renal proximal tubule cells, myosin VI mediates the movement of two prominent sodium phosphate co-transporters, NaPi2a and NaPi2c, down the microvilli, and their subsequent removal from the brush-border membrane in response to parathyroid hormone stimulation (Ameen and Apodaca, 2007; Blaine et al., 2009; Lanzano et al., 2011). In polarised epithelial cells, myosin VI LI specifically associates – and can be coimmunoprecipitated – with the tumour suppressor Dab2, an endocytic adaptor protein that functions in cell signalling and the degradation of cell surface receptors (Inoue et al., 2002; Morris et al., 2002). Structurally, Dab2 contains a phosphotyrosine-binding region, which mediates its interaction with cell-surface receptors and phospholipids, multiple clathrin binding motifs as well as binding motifs for the clathrin adaptor AP-2; in addition it has a proline-rich region that interacts with the SH3 domain of Grb2 and a SYF motif that binds myosin VI (Mishra et al., 2002; Morris and Cooper, 2001; Xu et al., 1998). Thus, owing to the presence of binding motifs for AP-2, clathrin and myosin VI, Dab2 functions as an adaptor that links myosin VI LI to clathrin-mediated receptor endocytosis and degradation.

Myosin VI NI is predominantly associated with a specific subpopulation of early endosomes underneath the plasma membrane that are characterised by the presence of the small GTPase Rab5 and its effector APPL1 (Chibalina et al., 2007; Tumbarello et al., 2012). These APPL1-positive early endosomes do not contain the early endosomal antigen 1 (EEA1) but, interestingly, recruit myosin VI together with its adaptor proteins GIPC and Tom1 (Chibalina et al., 2007; Tumbarello et al., 2012). During endosomal maturation, the synthesis of phosphatidylinositol 3-phosphate [PtdIns(3)P] and the recruitment of the PtdIns(3)P-binding protein EEA1 causes the loss of APPL1 from endosomes (Zoncu et al., 2009). APPL1 is a multifunctional adaptor protein containing a pleckstrin homology (PH) domain, a phosphotyrosine binding (PTB) domain and a leucine zipper motif. It binds through its PH domain to Rab5 and interacts through its very C-terminus with the PDZ-domain-containing protein GIPC (Miaczynska et al., 2004). In addition, it is targeted to lipid membranes through its BAR and PH domains, and associates through its PTB domain with the cytosolic domain of a diverse set of membrane receptors. The C-terminal PTB domain of APPL1 also mediates its direct interaction with signalling proteins, such as the serine/threonine kinase Akt and the phosphatidylinositol 3-kinase catalytic subunit p110α (PIK3CA). Thus, APPL1, by binding to several membrane receptors as well as signalling proteins, may regulate downstream signalling events on these specialised endosomes (Mitsuuchi et al., 1999). Thus, this distinct APPL1-positive early endocytic compartment may form a specialised class of signalling endosomes that act as a platform to regulate downstream signal transduction following receptor-mediated endocytosis; this, in turn, may then impact on various cellular functions, such as growth, migration, or homeostasis (Broussard et al., 2012; Cheng et al., 2012; Lin et al., 2006). However, little is known with regard to the mechanisms that determine which receptors are trafficked through this subset of endosomes, and/or the sorting processes that critically influence the lifetime of signalling events at this intracellular compartment.

The myosin VI adaptor protein GIPC, which was first identified as an interacting partner of the GTPase-activating protein RGS-GAIP for G-protein-coupled receptor subunit Gαi, can indirectly link myosin VI to APPL1 (De Vries et al., 1998; Varsano et al., 2006). GIPC acts as a scaffold to regulate receptor-mediated trafficking (Naccache et al., 2006; Varsano et al., 2006; Yi et al., 2007) and, after receptor internalisation, GIPC transiently associates with a pool of endocytic vesicles close to the plasma membrane (De Vries et al., 1998) before this compartment matures into an early endosome that is marked by EEA1. Thus, GIPC, through interaction with myosin VI, facilitates the retrograde movement of endosomes through the peripheral actin cytoskeleton to the early endosome (Aschenbrenner et al., 2003; Naccache et al., 2006; Spudich et al., 2007). This vesicle movement requires the motor activity of myosin VI, because expression of a myosin VI mutant that binds more tightly to actin (a rigor mutant) inhibits trafficking of peripheral vesicles (Aschenbrenner et al., 2004). Interestingly, both myosin VI and GIPC are recruited to the cleavage furrow in dividing cells and, like many endosomal proteins, are required during cytokinesis (Arden et al., 2007).

The second myosin VI adaptor protein that is present on APPL1-positive signalling endosomes is Tom1 and its close relative Tom1-like 2 (Tom1L2) (Fig. 2). The Drosophila homologue of Tom1/Tom1L2 (CG3529) was first identified as an interacting partner of myosin VI (Jaguar in Drosophila) by using a proteomics approach (Finan et al., 2011). We have confirmed this interaction in mammalian cells and demonstrated that Tom1 is an essential adaptor protein for intracellular targeting of myosin VI to early endosomes. In addition, both proteins function together to facilitate autophagosome maturation to enable autophagosome and lysosome fusion (Tumbarello et al., 2012). Tom1 was first identified as a target gene for the Myb oncogene (Burk et al., 1997) and, later characterised as part of the endosomal sorting complex required for transport (ESCRT type 0 family of proteins), which includes Stam1 and Stam2, and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) (Seroussi et al., 1999). The ESCRT machinery functions as a multi-protein complex in the lysosomal degradation pathway. ESCRT-0 proteins mediate cargo recognition and sorting through interactions with ubiquitin moieties, and work in concert with the other ESCRT components to deform membranes and create intralumenal vesicles to form late endocytic multi-vesicular bodies (Henne et al., 2011). Tom1 is part of a smaller subfamily that includes the paralogues Tom1-like 1 (Tom1L1) and Tom1L2. Structurally, members of the Tom1 family consist of a conserved N-terminal Vps27–Hrs–STAM (VHS) domain, a central GAT domain that binds ubiquitin and a less-conserved C-terminal tail. Unlike the VHS domain in Hrs, Tom1 does not contain a FYVE membrane-interaction domain, but might interact directly with membranes through other potential regions within the VHS or indirectly through binding to its interacting partners endofin and Tollip (Misra et al., 2000; Seet et al., 2004; Yamakami et al., 2003). In addition, for cargo recognition, Tom1 contains two ubiquitin-binding motifs within its GAT domain and interacts directly with clathrin to mediate its recruitment to endosomes (Akutsu et al., 2005; Seet and Hong, 2005). Although all Tom1 family members are capable of interacting with the ESCRT-0 component Hrs, only Tom1L1 interacts with the ESCRT-I component TSG101. Moreover, only Tom1 can associate with endofin, suggesting that the family members have both redundant and unique functions (Puertollano, 2005; Seet et al., 2004; Yanagida-Ishizaki et al., 2008). Further divergence in the Tom1 family occurs within the C-terminal region, in which Tom1 and Tom1L2 are most similar, whereas Tom1L1 shows less similarity (Fig. 2). The C-terminus also contains a conserved IEXWL amino acid motif that is unique to Tom1 and Tom1L2, and mediates binding to myosin VI (F.B. and D.A.T., unpublished observations) (Fig. 2).

Although the cellular functions of Tom1 and Tom1L2 are not completely understood, Tom1 regulates signalling downstream of IL-1 and TNF-α receptors (Yamakami and Yokosawa, 2004) by linking them to the endosomal sorting machinery through its interaction with the membrane adaptor Toll-interaction protein (TOLLIP). The latter interacts with several Toll-like receptors, thereby mediating their lysosomal degradation (Brissoni et al., 2006). In addition, work from our lab has recently highlighted the importance of Tom1 and/or Tom1L2 in targeting myosin VI to early endosomes, and in potentially mediating cargo sorting in the endocytic pathway – which is required during autophagosome maturation (Tumbarello et al., 2012).

The third adaptor molecule associating with myosin VI in the early endocytic pathway is LMTK2, a transmembrane serine/threonine kinase and a binding partner for the protein phosphatase inhibitor 2 (PPP1R2) (Wang and Brautigan, 2002; Wang and Brautigan, 2006); it is phosphorylated and regulated by cyclin-dependent kinase-5 (Cdk5) (Kesavapany et al., 2003). LMTK2 directly binds to the WWY motif of myosin VI and regulates trafficking through the early endocytic pathway towards the endocytic recycling compartment (Chibalina et al., 2007). The open question remains whether myosin VI directs trafficking and targeting of LMTK2 or whether myosin VI activity is regulated through phosphorylation by LMTK2. Interestingly, both myosin VI and LMTK2 have been implicated in prostate cancer: myosin VI is highly overexpressed (Dunn et al., 2006) and genetic risk loci for LMTK2 have been identified (Eeles et al., 2008).

In addition to cargo adaptor proteins, such as Dab2, GIPC, Tom1, Tom1L2 and LMTK2, that link myosin VI to distinct functions along the endocytic pathway, we identified three myosin-VI-interacting proteins – NDP52, T6BP and optineurin – that have recently been characterised as cargo-selective receptors in autophagy (Fig. 3). The targeting of ubiquitylated cargo for autophagy-dependent degradation requires specific adaptors that bind cargo through an ubiquitin-binding domain (UBD) and recruit LC3-associated autophagic membranes through an LC3-interaction region (LIR), thus facilitating autophagosome formation and degradation of its contents. Two of these autophagy receptors, NDP52 and optineurin, have an important function in innate immunity for the autophagy-dependent clearance of ubiquitin-coated Salmonella (Thurston et al., 2009; Wild et al., 2011). The third autophagy receptor that specifically binds to myosin VI is the NDP52-related protein T6BP, which also localises to autophagosomes and is required for autophagosome biogenesis (Tumbarello et al., 2012). Structurally, T6BP and NDP52 are most similar and consist of an N-terminal SKICH domain, followed by a non-canonical LIR, a coiled-coil domain and C-terminal zinc finger domains that mediate ubiquitin-binding (Morriswood et al., 2007; von Muhlinen et al., 2012). Optineurin also consists of multiple coiled-coil regions, a leucine zipper domain and a C-terminal zinc finger domain (Fig. 3). Optineurin contains multiple protein–protein interaction motifs, and binding to ubiquitin involves – like for its close homologue NF-κB essential modulator (NEMO) – a canonical motif within the central coiled-coiled domain together with its C-terminal zinc finger (Cordier et al., 2009; Laplantine et al., 2009). Recently, it has been shown that phosphorylation of optineurin at serine 177, adjacent to its LIR, regulates its interaction with LC3 and its ability to degrade ubiqutin-coated Salmonella (Wild et al., 2011). Recognition and clearance of ubiqutin-coated Salmonella by NDP52 is mediated through its interaction with the specific LC3C isoform (von Muhlinen et al., 2012). Interestingly, each of these three autophagy receptors is targeted sufficiently to autophagosomes under basal conditions and under conditions that induce selective autophagy (Tumbarello et al., 2012). Although this suggests that these myosin VI-associated autophagy adaptors have some redundant overlapping functions, it is also possible that they have distinct selective roles that are mediated by binding to specific LC3-isoforms or by displaying different specificity towards different types of ubiquitin chains, giving further support to a non-redundant function for the clearance of distinct substrates in the autophagy pathway (von Muhlinen et al., 2012; Wild et al., 2011).

We previously identified each of these autophagy adaptors – NDP52, optineurin and T6BP – as direct myosin VI-binding partners, and showed that myosin VI binding is mediated through the RRL motif in its C-terminal cargo-binding tail region (Morriswood et al., 2007; Sahlender et al., 2005). T6BP and NDP52 associate with myosin VI through their respective zinc finger domains, and optineurin through a central region that overlaps with the coiled-coiled- and ubiquitin-binding domains (Fig. 3). Functionally, these adaptors colocalise with myosin VI on autophagosomes, and depletion of the three adaptors inhibits autophagosome biogenesis (Tumbarello et al., 2012).

Myosin VI facilitates the delivery of endocytic membranes that contain the ESCRT-0 component Tom1 to autophagosomes, thereby mediating autophagosome maturation and its fusion with the lysosome (Tumbarello et al., 2012) (Fig. 4). How the ESCRT machinery works in concert with myosin VI in order to facilitate endocytic cargo delivery, vesicle fusion events and/or membrane deformation, is complicated by the fact that the exact temporal role of the ESCRT machinery during autophagosome maturation has not yet been established (Rusten and Stenmark, 2009).

It is possible that docking of Tom1-positive endosomes to autophagosomes indirectly involves myosin VI through its association with the autophagy adaptors T6BP, NDP52 and optineurin. These autophagy receptors might have a dual role by acting not only as adaptors during autophagosome biogenesis to facilitate cargo recognition and recruitment of autophagocytic membranes, but also by decorating the cytosol-facing outer membrane of the autophagosome, where they act as myosin VI receptors, thereby facilitating the delivery and docking of the endocytic membrane, or even fusion events required for its maturation. The ubiquitin-binding region in T6BP, NDP52 and optineurin overlaps with their myosin VI-interaction domain, suggesting that ubiquitylation and myosin VI binding is mutually exclusive, thereby, supporting our model that the autophagy adaptors function as multiple ligand receptors on both the inner and outer autophagocytic membranes (Fig. 4).

If myosin VI simultaneously binds to Tom1 on endosomal membranes through its tail domain that contains the WWY-motif as well as to autophagy receptors through its second cargo-binding site, the RRL-motif, then myosin VI could bring both of its cargo binding subdomains into close proximity to prepare for SNARE-mediated fusion of endosomes with autophagosomes. Indeed, we have shown previously (Spudich et al., 2007) that the CBD of monomeric myosin VI consists of two subdomains that can be separated: one contains the RRL motif that is bound by optineurin, NDP52 and T6BP; the other contains the WWY motif that is bound by Tom1. In vitro studies have further shown that binding to optineurin and Dab2 can induce dimerisation of myosin VI (Phichith et al., 2009). Therefore, it will be important to test whether monomeric and dimeric myosin VI molecules can bind two different adaptor proteins (cargo) because, if this were the case, it could lead to tethering or positioning and, ultimately, crosslinking of different subcellular compartments. A tethering function might not be regarded as the typical activity for a motor protein because, characteristically, it drives transport of cargo along a cytoskeletal track. However, in vitro motility measurements have shown that myosin VI is a slow motor that moves with a maximum speed of ∼30–60 nm/second. In the cell, it might move even slower. Therefore, consistent with its known motor properties, myosin VI is likely to be involved in short-distance transport, such as positioning and tethering of endosomes around autophagosomes or other vesicular organelles to enhance fusion. In fact, in the final stages of the secretory pathway, myosin VI is required, through interaction with optineurin, for the fusion events that take place between secretory vesicles and the plasma membrane (Bond et al., 2011).

It is clear that myosin VI is a multifunctional motor protein that operates during multiple steps of the endocytic pathway. Myosin VI dysfunction has been predominantly associated with hearing loss due to the degeneration of stereocilia in the inner ear. However, the consequences of loss-of-function of many of its adaptors (Table 1) suggests that a number of other pathologies are associated with myosin VI dysfunction, which results in defects along the endocytic and autophagy-dependent pathways. These defects would be likely to lead to a disruption in protein quality control, cell homeostasis and innate immunity. Furthermore, because myosin VI is overexpressed in ovarian and prostate cancers (Dunn et al., 2006; Yoshida et al., 2004), the identification of associated adaptors and functional consequences of this overexpression on cancer progression is of great importance. Further characterisation of the myosin VI knockout mice will also yield further insights into the physiological effects of myosin VI loss-of-function. It has already been established that the physiological loss-of-function of myosin VI is associated with astrogliosis in the brain, hypertrophic cardiomyopathy, defects in endocytosis in neuronal and epithelial tissues, disruption in the integrity of the intestinal brush border, and various metabolic dysfunctions (Ameen and Apodaca, 2007; Hegan et al., 2012; Mohiddin et al., 2004; Osterweil et al., 2005) (see also ‘myo6’ at the Mouse Resources Portal of the Wellcome Trust Sanger Institute, http://www.sanger.ac.uk/mouseportal/search?query=myo6). Further work is required, however, before we can understand the myosin VI-dependent mechanisms that underlay these pathologies.

Importantly, in order to understand the mechanisms behind the above stated pathologies, we need to apprehend the complex nature of the spatiotemporal regulation of myosin VI with respect to its functions within the cell. This will entail understanding how the CBD of myosin VI interacts with different cargo adaptors and whether there is a further layer of regulation, such as phosphorylation, ubiquitylation or cargo-induced structural modulations. In addition, more cargo adaptors are likely to be identified when new techniques for probing these interactions are developed, as illustrated recently by Finan et al. (Finan et al., 2011). Regardless of whether these new or previously identified cargo-binding adaptors are tissue-specific or regulatory in function, they make the mechanism of how myosin VI functions at distinct phases of membrane trafficking more complex. New and innovative approaches are, therefore, needed to delineate the molecular mechanisms of the interaction between cargo adaptors and myosin VI.