Myosin VI plays a role in the maintenance of Golgi morphology and in exocytosis. In a yeast 2-hybrid screen we identified optineurin as a binding partner for myosin VI at the Golgi complex and confirmed this interaction in a range of protein interaction studies. Both proteins colocalize at the Golgi complex and in vesicles at the plasma membrane. When optineurin is depleted from cells using RNA interference, myosin VI is lost from the Golgi complex, the Golgi is fragmented and exocytosis of vesicular stomatitis virus G-protein to the plasma membrane is dramatically reduced. Two further binding partners for optineurin have been identified: huntingtin and Rab8. We show that myosin VI and Rab8 colocalize around the Golgi complex and in vesicles at the plasma membrane and overexpression of constitutively active Rab8-Q67L recruits myosin VI onto Rab8-positive structures. These results show that optineurin links myosin VI to the Golgi complex and plays a central role in Golgi ribbon formation and exocytosis.

Myosin VI is involved in membrane traf®c and dynamics and is the only myosin known to move towards the minus end of actin ®laments. Splice variants of myosin VI with a large insert in the tail domain were speci®cally expressed in polarized cells containing microvilli. In these polarized cells, endogenous myosin VI containing the large insert was concentrated at the apical domain co-localizing with clathrincoated pits/vesicles. Using full-length myosin VI and deletion mutants tagged with green¯uorescent protein (GFP) we have shown that myosin VI associates and co-localizes with clathrin-coated pits/vesicles by its C-terminal tail. Myosin VI, precipitated from whole cytosol, was present in a protein complex containing adaptor protein (AP)-2 and clathrin, and enriched in puri®ed clathrin-coated vesicles. Over-expression of the tail domain of myosin VI containing the large insert in ®broblasts reduced transferrin uptake in transiently and stably transfected cells by >50%. Myosin VI is the ®rst motor protein to be identi®ed associated with clathrin-coated pits/vesicles and shown to modulate clathrin-mediated endocytosis.

Autophagy targets pathogens, damaged organelles and protein aggregates for lysosomal degradation. These ubiquitinated cargoes are recognised by specific autophagy receptors, which recruit LC3-positive membranes to form autophagosomes. Subsequently, autophagosomes fuse with endosomes and lysosomes, thus facilitating degradation of their content, however, the machinery that targets and mediates fusion of these organelles with autophagosomes remains to be established. Here we demonstrate that myosin VI, in concert with its adaptor proteins NDP52, optineurin, T6BP and Tom1, plays a crucial role in autophagy. We identify Tom1 as a myosin VI binding partner on endosomes and demonstrate that their loss reduces autophagosomal delivery of endocytic cargo and causes a block in autophagosome-lysosome fusion. We propose that myosin VI delivers endosomal membranes containing Tom1 to autophagosomes by docking to NDP52, T6BP and optineurin thereby promoting autophagosome maturation and thus driving fusion with lysosomes.

Myosin VI, an actin-based motor protein, and Disabled 2 (Dab2), a molecule involved in endocytosis and cell signalling, have been found to bind together using yeast and mammalian two-hybrid screens. In polarised epithelial cells, myosin VI is known to be associated with apical clathrin-coated vesicles and is believed to move them towards the minus end of actin filaments, away from the plasma membrane and into the cell. Dab2 belongs to a group of signal transduction proteins that bind in vitro to the FXNPXY sequence found in the cytosolic tails of members of the low-density lipoprotein receptor family. The central region of Dab2, containing two DPF motifs, binds to the clathrin adaptor protein AP-2, whereas a C-terminal region contains the binding site for myosin VI. This site is conserved in Dab1, the neuronal counterpart of Dab2. The interaction between Dab2 and myosin VI was confirmed by in vitro binding assays and coimmunoprecipitation and by their colocalisation in clathrin-coated pits/vesicles concentrated at the apical domain of polarised cells. These results suggest that the myosin VI-Dab2 interaction may be one link between the actin cytoskeleton and receptors undergoing endocytosis.

Vesicle transport is essential for the movement of proteins, lipids and other molecules between membrane compartments within the cell. The role of the class VI myosins in vesicular transport is especially intriguing because they are the only class that has been shown to move "backwards" towards the minus end of actin filaments1. Myosin VI is found in distinct intracellular locations and implicated in processes such as endocytosis2,3, exocytosis, maintenance of Golgi morphology4,5 and cell movement6. We have shown that the C-terminal tail is the key targeting region and have identified three binding sites: a WWY motif for Dab2 binding, a RRL motif for GIPC/Optineurin binding and a site that binds specifically and with high affinity (K d 0.3 μM) to PIP 2 -containing liposomes. This is the first demonstration that myosin VI binds lipid membranes. Lipid binding induces a large structural change in the tail (31% increase in helicity) and when associated with lipid vesicles it can dimerise. In vivo targeting and recruitment of myosin VI to clathrin-coated structures (CCSs) at the plasma membrane is mediated by Dab2 and PIP 2 binding.Dab2 is a myosin VI binding partner present on endocytic CCSs at the plasma membrane7,8. To establish whether binding to Dab2 is involved in targeting myosin VI to CCSs we tested an extensive series of myosin VI tail deletion fragments and point mutants using the mammalian 2-hybrid assay7. A relatively conservative single amino acid change from a tryptophan to a leucine (W1184L, WWY→WLY) was found to abolish myosin VI binding to Dab2 (Fig. 1a). 'Pull down' experiments using GST-tagged wild type myosin VI tail or tail containing the WWY→WLY mutation together with in vitro translated Dab2 confirmed the identity of the Dab2 binding site (Fig. 1c). To check whether the Dab2 binding site was essential for targeting myosin VI to CCSs in vivo we over-expressed GFPtagged mutant tail constructs in HeLa cells. We observed that myosin VI containing a mutated Dab2 binding site (WWY→WLY) was not targeted to CCSs (Fig. 1d,e). It was previously shown2,8 that the presence of a large insert just before the globular C-terminal domain (Fig. S1) in conjunction with Dab2 binding was also required for targeting myosin VI to CCSs at the plasma membrane.GIPC is another myosin VI binding partner9 that is found in both clathrin-coated10 and uncoated endocytic vesicles3. To test the involvement of GIPC in targeting myosin VI to CCSs we mapped the GIPC binding site on the myosin VI tail by deletion and alanine scanning mutagenesis. We observed that the mutation RRL to AAA in the C-terminal tail Correspondence should be addressed to: J.K-J (e-mail: jkj@mrc-lmb.cam.ac.uk). (Fig. 1b) abolished GIPC binding, but had no effect on Dab2 binding (Fig. 1a,b). Optineurin, a myosin VI binding protein associated with the Golgi complex and secretion, also specifically binds to the RRL binding site in the myosin VI tail4. Although phosphorylation of the threonines in the TINT sequence 15 residues upstream of RRL (Fig. S1b...

Myosin VI is an unconventional myosin that may play a role in vesicular membrane traffic through actin rich regions of the cytoplasm in eukaryotic cells. In this study we have cloned and sequenced a cDNA encoding a chicken intestinal brush border myosin VI. Polyclonal antisera were raised to bacterially expressed fragments of this myosin VI. The affinity purified antibodies were highly specific for myosin VI by immunoblotting and immunoprecipitation and were used to study the localization of the protein by immunofluorescence and immunoelectron microscopy. It was found that in NRK and A431 cells, myosin VI was associated with both the Golgi complex and the leading, ruffling edge of the cell as well as being present in a cytosolic pool. In A431 cells in which cell surface ruffling was stimulated by EGF, myosin VI was phosphorylated and recruited into the newly formed ruffles along with ezrin and myosin V. In vitro experiments suggested that a p21-activated kinase (PAK) might be the kinase responsible for phosphorylation in the motor domain. These results strongly support a role for myosin VI in membrane traffic on secretory and endocytic pathways.

Filopodia are thin, spike-like cell surface protrusions containing bundles of parallel actin filaments. So far, filopodial dynamics has mainly been studied in the context of cell motility on coverslipadherent filopodia by using fluorescence and differential interference contrast (DIC) microscopy. In this study, we used an optical trap and interferometric particle tracking with nanometer precision to measure the three-dimensional dynamics of macrophage filopodia, which were not attached to flat surfaces. We found that filopodia act as cellular tentacles: a few seconds after binding to a particle, filopodia retract and pull the bound particle toward the cell. We observed F-actin-dependent stepwise retraction of filopodia with a mean step size of 36 nm, suggesting molecular motor activity during filopodial pulling. Remarkably, this intracellular stepping motion, which was measured at counteracting forces of up to 19 pN, was transmitted to the extracellular tracked particle via the filopodial F-actin bundle and the cell membrane. The pulling velocity depended strongly on the counteracting force and ranged between 600 nm/s at forces <1 pN and Ϸ40 nm/s at forces >15 pN. This result provides an explanation of the significant differences in filopodial retraction velocities previously reported in the literature. The measured filopodial retraction force-velocity relationship is in agreement with a model for force-dependent multiple motor kinetics.actin filaments ͉ interferometric three-dimensional particle tracking ͉ molecular motors ͉ nanomechanics ͉ optical trapping

Myosin VI is involved in a wide variety of intracellular processes such as endocytosis, secretion and cell migration. Unlike almost all other myosins so far studied, it moves towards the minus end of actin filaments and is therefore likely to have unique cellular properties. However, its mechanism of force production and movement is not understood. Under our experimental conditions, both expressed full-length and native myosin VI are monomeric. Electron microscopy using negative staining revealed that the addition of ATP induces a large conformational change in the neck/tail region of the expressed molecule. Using an optical tweezers-based force transducer we found that expressed myosin VI is nonprocessive and produces a large working stroke of 18 nm. Since the neck region of myosin VI is short (it contains only a single IQ motif), it is difficult to reconcile the 18 nm working stroke with the classical 'lever arm mechanism', unless other structures in the molecule contribute to the effective lever. A possible model to explain the large working stroke of myosin VI is presented.