Approaches to Identify and Characterise MYO6-Cargo Interactions.

Given the prevalence and importance of the actin cytoskeleton and the host of associated myosin motors, it comes as no surprise to find that they are linked to a plethora of cellular functions and pathologies. Although our understanding of the biophysical properties of myosin motors has been aided by the high levels of conservation in their motor domains and the extensive work on myosin in skeletal muscle contraction, our understanding of how the nonmuscle myosins participate in such a wide variety of cellular processes is less clear. It is now well established that the highly variable myosin tails are responsible for targeting these myosins to distinct cellular sites for specific functions, and although a number of adaptor proteins have been identified, our current understanding of the cellular processes involved is rather limited. Furthermore, as more adaptor proteins, cargoes and complexes are identified, the importance of elucidating the regulatory mechanisms involved is essential. Ca2+, and now phosphorylation and ubiquitination, are emerging as important regulators of cargo binding, and it is likely that other post-translational modifications are also involved. In the case of myosin VI (MYO6), a number of immediate binding partners have been identified using traditional approaches such as yeast two-hybrid screens and affinity-based pull-downs. However, these methods have only been successful in identifying the cargo adaptors, but not the cargoes themselves, which may often comprise multi-protein complexes. Furthermore, motor-adaptor-cargo interactions are dynamic by nature and often weak, transient and highly regulated and therefore difficult to capture using traditional affinity-based methods. In this chapter we will discuss the various approaches including functional proteomics that have been used to uncover and characterise novel MYO6-associated proteins and complexes and how this work contributes to a fuller understanding of the targeting and function(s) of this unique myosin motor.

Myosins, Actin and Autophagy.

Myosin motor proteins working together with the actin cytoskeleton drive a wide range of cellular processes. In this review, we focus on their roles in autophagy - the pathway the cell uses to ensure homeostasis by targeting pathogens, misfolded proteins and damaged organelles for degradation. The actin cytoskeleton regulated by a host of nucleating, anchoring and stabilizing proteins provides the filament network for the delivery of essential membrane vesicles from different cellular compartments to the autophagosome. Actin networks have also been implicated in structurally supporting the expanding phagophore, moving autophagosomes and enabling efficient fusion with the lysosome. Only a few myosins have so far been shown to play a role in autophagy. Non-muscle myosin IIA functions in the early stages delivering membrane for the initial formation of the autophagosome, whereas myosin IC and myosin VI are involved in the final stages providing specific membranes for autophagosome maturation and its fusion with the lysosome.

Correction: The Autophagy Receptor TAX1BP1 and the Molecular Motor Myosin VI Are Required for Clearance of Salmonella Typhimurium by Autophagy.

[This corrects the article DOI: 10.1371/journal.ppat.1005174.].

The Autophagy Receptor TAX1BP1 and the Molecular Motor Myosin VI Are Required for Clearance of Salmonella Typhimurium by Autophagy.

Autophagy plays a key role during Salmonella infection, by eliminating these pathogens following escape into the cytosol. In this process, selective autophagy receptors, including the myosin VI adaptor proteins optineurin and NDP52, have been shown to recognize cytosolic pathogens. Here, we demonstrate that myosin VI and TAX1BP1 are recruited to ubiquitylated Salmonella and play a key role in xenophagy. The absence of TAX1BP1 causes an accumulation of ubiquitin-positive Salmonella, whereas loss of myosin VI leads to an increase in ubiquitylated and LC3-positive bacteria. Our structural studies demonstrate that the ubiquitin-binding site of TAX1BP1 overlaps with the myosin VI binding site and point mutations in the TAX1BP1 zinc finger domains that affect ubiquitin binding also ablate binding to myosin VI. This mutually exclusive binding and the association of TAX1BP1 with LC3 on the outer limiting membrane of autophagosomes may suggest a molecular mechanism for recruitment of this motor to autophagosomes. The predominant role of TAX1BP1, a paralogue of NDP52, in xenophagy is supported by our evolutionary analysis, which demonstrates that functionally intact NDP52 is missing in Xenopus and mice, whereas TAX1BP1 is expressed in all vertebrates analysed. In summary, this work highlights the importance of TAX1BP1 as a novel autophagy receptor in myosin VI-mediated xenophagy. Our study identifies essential new machinery for the autophagy-dependent clearance of Salmonella typhimurium and suggests modulation of myosin VI motor activity as a potential therapeutic target in cellular immunity.

Loss of cargo binding in the human myosin VI deafness mutant (R1166X) leads to increased actin filament binding.

Mutations in myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising myosin VI vesicle-binding ability by expressing myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this myosin in vitro and in vivo As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of myosin VI, but our in vitro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the myosin VI tail. In conclusion, our characterisation of the human myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave myosin VI in a primed/activated state with an increased actin-binding ability.

Myosins: Domain Organisation, Motor Properties, Physiological Roles and Cellular Functions.

Myosins are cytoskeletal motor proteins that use energy derived from ATP hydrolysis to generate force and movement along actin filaments. Humans express 38 myosin genes belonging to 12 classes that participate in a diverse range of crucial activities, including muscle contraction, intracellular trafficking, cell division, motility, actin cytoskeletal organisation and cell signalling. Myosin malfunction has been implicated a variety of disorders including deafness, hypertrophic cardiomyopathy, Usher syndrome, Griscelli syndrome and cancer. In this chapter, we will first discuss the key structural and kinetic features that are conserved across the myosin family. Thereafter, we summarise for each member in turn its unique functional and structural adaptations, cellular roles and associated pathologies. Finally, we address the broad therapeutic potential for pharmacological interventions that target myosin family members.

Myosin VI and its cargo adaptors - linking endocytosis and autophagy.

The coordinated trafficking and tethering of membrane cargo within cells relies on the function of distinct cytoskeletal motors that are targeted to specific subcellular compartments through interactions with protein adaptors and phospholipids. The unique actin motor myosin VI functions at distinct steps during clathrin-mediated endocytosis and the early endocytic pathway - both of which are involved in cargo trafficking and sorting - through interactions with Dab2, GIPC, Tom1 and LMTK2. This multifunctional ability of myosin VI can be attributed to its cargo-binding tail region that contains two protein-protein interaction interfaces, a ubiquitin-binding motif and a phospholipid binding domain. In addition, myosin VI has been shown to be a regulator of the autophagy pathway, because of its ability to link the endocytic and autophagic pathways through interactions with the ESCRT-0 protein Tom1 and the autophagy adaptor proteins T6BP, NDP52 and optineurin. This function has been attributed to facilitating autophagosome maturation and subsequent fusion with the lysosome. Therefore, in this Commentary, we discuss the relationship between myosin VI and the different myosin VI adaptor proteins, particularly with regards to the spatial and temporal regulation that is required for the sorting of cargo at the early endosome, and their impact on autophagy.

Myo1c regulates lipid raft recycling to control cell spreading, migration and Salmonella invasion.

A balance between endocytosis and membrane recycling regulates the composition and dynamics of the plasma membrane. Internalization and recycling of cholesterol- and sphingolipid-enriched lipid rafts is an actin-dependent process that is mediated by a specialized Arf6-dependent recycling pathway. Here, we identify myosin1c (Myo1c) as the first motor protein that drives the formation of recycling tubules emanating from the perinuclear recycling compartment. We demonstrate that the single-headed Myo1c is a lipid-raft-associated motor protein that is specifically involved in recycling of lipid-raft-associated glycosylphosphatidylinositol (GPI)-linked cargo proteins and their delivery to the cell surface. Whereas Myo1c overexpression increases the levels of these raft proteins at the cell surface, in cells depleted of Myo1c function through RNA interference or overexpression of a dominant-negative mutant, these tubular transport carriers of the recycling pathway are lost and GPI-linked raft markers are trapped in the perinuclear recycling compartment. Intriguingly, Myo1c only selectively promotes delivery of lipid raft membranes back to the cell surface and is not required for recycling of cargo, such as the transferrin receptor, which is mediated by parallel pathways. The profound defect in lipid raft trafficking in Myo1c-knockdown cells has a dramatic impact on cell spreading, cell migration and cholesterol-dependent Salmonella invasion; processes that require lipid raft transport to the cell surface to deliver signaling components and the extra membrane essential for cell surface expansion and remodeling. Thus, Myo1c plays a crucial role in the recycling of lipid raft membrane and proteins that regulate plasma membrane plasticity, cell motility and pathogen entry.

Myosin VI targeting to clathrin-coated structures and dimerization is mediated by binding to Disabled-2 and PtdIns(4,5)P2.

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 particularly intriguing because they are the only class that has been shown to move 'backwards' towards the minus end of actin filaments. Myosin VI is found in distinct intracellular locations and implicated in processes such as endocytosis, exocytosis, maintenance of Golgi morphology and cell movement. We have shown that the carboxy-terminal tail is the key targeting region and have identified three binding sites: a WWY motif for Disabled-2 (Dab2) binding, a RRL motif for glucose-transporter binding protein (GIPC) and optineurin binding and a site that binds specifically and with high affinity (Kd = 0.3 microM) to PtdIns(4,5)P2-containing liposomes. This is the first demonstration that myosin VI binds lipid membranes. Lipid binding induces a large structural change in the myosin VI tail (31% increase in helicity) and when associated with lipid vesicles, it can dimerize. In vivo targeting and recruitment of myosin VI to clathrin-coated structures (CCSs) at the plasma membrane is mediated by Dab2 and PtdIns(4,5)P2 binding.

Motor domain phosphorylation increases nucleotide exchange and turns MYO6 into a faster and stronger motor.

Myosin motors perform many fundamental functions in eukaryotic cells by providing force generation, transport or tethering capacity. Motor activity control within the cell involves on/off switches, however, few examples are known of how myosins regulate speed or processivity and fine-tune their activity to a specific cellular task. Here, we describe a phosphorylation event for myosins of class VI (MYO6) in the motor domain, which accelerates its ATPase activity leading to a 4-fold increase in motor speed determined by actin-gliding assays, single molecule mechanics and stopped flow kinetics. We demonstrate that the serine/threonine kinase DYRK2 phosphorylates MYO6 at S267 in vitro. Single-molecule optical-tweezers studies at low load reveal that S267-phosphorylation results in faster nucleotide-exchange kinetics without change in the working stroke of the motor. The selective increase in stiffness of the acto-MYO6 complex when proceeding load-dependently into the nucleotide-free rigor state demonstrates that S267-phosphorylation turns MYO6 into a stronger motor. Finally, molecular dynamic simulations of the nucleotide-free motor reveal an alternative interaction network within insert-1 upon phosphorylation, suggesting a molecular mechanism, which regulates insert-1 positioning, turning the S267-phosphorylated MYO6 into a faster motor.

Dynamic exchange of myosin VI on endocytic structures.

The actin-based molecular motor myosin VI functions in the endocytic uptake pathway, both during the early stages of clathrin-mediated uptake and in later transport to/from early endosomes. This study uses fluorescence recovery after photobleaching (FRAP) to examine the turnover rate of myosin VI during endocytosis. The results demonstrate that myosin VI turns over dynamically on endocytic structures with a characteristic half-life common to both the large insert isoform of myosin VI on clathrin-coated structures and the no-insert isoform on early endosomes. This half-life is shared by the myosin VI-binding partner Dab2 and is identical for full-length myosin VI and the cargo-binding tail region. The 4-fold slower half-life of an artificially dimerized construct of myosin VI on clathrin-coated structures suggests that wild type myosin VI does not function as a stable dimer, but either as a monomer or in a monomer/dimer equilibrium. Taken together, these FRAP results offer insight into both the basic turnover dynamics and the monomer/dimer nature of myosin VI.

Localized mutations in the gene encoding the cytoskeletal protein filamin A cause diverse malformations in humans.

Remodeling of the cytoskeleton is central to the modulation of cell shape and migration. Filamin A, encoded by the gene FLNA, is a widely expressed protein that regulates re-organization of the actin cytoskeleton by interacting with integrins, transmembrane receptor complexes and second messengers. We identified localized mutations in FLNA that conserve the reading frame and lead to a broad range of congenital malformations, affecting craniofacial structures, skeleton, brain, viscera and urogenital tract, in four X-linked human disorders: otopalatodigital syndrome types 1 (OPD1; OMIM 311300) and 2 (OPD2; OMIM 304120), frontometaphyseal dysplasia (FMD; OMIM 305620) and Melnick-Needles syndrome (MNS; OMIM 309350). Several mutations are recurrent, and all are clustered into four regions of the gene: the actin-binding domain and rod domain repeats 3, 10 and 14/15. Our findings contrast with previous observations that loss of function of FLNA is embryonic lethal in males but manifests in females as a localized neuronal migration disorder, called periventricular nodular heterotopia (PVNH; refs. 3-6). The patterns of mutation, X-chromosome inactivation and phenotypic manifestations in the newly described mutations indicate that they have gain-of-function effects, implicating filamin A in signaling pathways that mediate organogenesis in multiple systems during embryonic development.

Myosin VI and optineurin are required for polarized EGFR delivery and directed migration.

The polarized trafficking of membrane proteins into the leading edge of the cell is an integral requirement for cell migration. Myosin VI and its interacting protein optineurin have previously been shown to operate in anterograde trafficking pathways, especially for the polarized delivery of cargo to the basolateral domain in epithelial cells. Here we show that in migratory cells ablation of myosin VI or optineurin inhibits the polarized delivery of the epidermal growth factor receptor (EGFR) into the leading edge and leads to profound defects in lamellipodia formation. Depletion of either myosin VI or optineurin, however, does not impair the overall ability of cells to migrate in a random migration assay, but it dramatically reduces directed migration towards a growth factor stimulus. In summary, we identified a specific role for myosin VI and optineurin in directionally persistent cell migration, which involves the polarized delivery of vesicles containing EGFR into the leading edge of the cell.

Mechanism and specificity of pentachloropseudilin-mediated inhibition of myosin motor activity.

Here, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC(50) values are in the range from 1 to 5 µm for mammalian class-1 myosins and greater than 90 µm for class-2 and class-5 myosins, and no inhibition was observed with class-6 and class-7 myosins. We show that in mammalian cells, PClP selectively inhibits myosin-1c function. To elucidate the structural basis for PClP-induced allosteric coupling and isoform-specific differences in the inhibitory potency of the compound, we used a multifaceted approach combining direct functional, crystallographic, and in silico modeling studies. Our results indicate that allosteric inhibition by PClP is mediated by the combined effects of global changes in protein dynamics and direct communication between the catalytic and allosteric sites via a cascade of small conformational changes along a conserved communication pathway.

Mistargeting of SH3TC2 away from the recycling endosome causes Charcot-Marie-Tooth disease type 4C.

Mutations in the functionally uncharacterized protein SH3TC2 are associated with the severe hereditary peripheral neuropathy, Charcot-Marie-Tooth disease type 4C (CMT4C). Similarly, to other proteins mutated in CMT, a role for SH3TC2 in endocytic membrane traffic has been previously proposed. However, recent descriptions of the intracellular localization of SH3TC2 are conflicting. Furthermore, no clear functional pathogenic mechanisms have so far been proposed to explain why both nonsense and missense mutations in SH3TC2 lead to similar clinical phenotypes. Here, we describe our intracellular localization studies, supported by biochemical and functional data, using wild-type and mutant SH3TC2. We show that wild-type SH3TC2 targets to the intracellular recycling endosome by associating with the small GTPase, Rab11, which is known to regulate the recycling of internalized membrane and receptors back to the plasma membrane. Furthermore, we demonstrate that SH3TC2 interacts preferentially with the GTP-bound form of Rab11, identifying SH3TC2 as a novel Rab11 effector. Of clinical pathological relevance, all SH3TC2 constructs harbouring disease-causing mutations are shown to be unable to associate with Rab11 with consequent loss of recycling endosome localization. Moreover, we show that wild-type SH3TC2, but not mutant SH3TC2, influences transferrin receptor dynamics, consistent with a functional role on the endocytic recycling pathway. Our data therefore implicate mistargeting of SH3TC2 away from the recycling endosome as the fundamental molecular defect that leads to CMT4C.

Myosin VI is required for sorting of AP-1B-dependent cargo to the basolateral domain in polarized MDCK cells.

In polarized epithelial cells, newly synthesized membrane proteins are delivered on specific pathways to either the apical or basolateral domains, depending on the sorting motifs present in these proteins. Because myosin VI has been shown to facilitate secretory traffic in nonpolarized cells, we investigated its role in biosynthetic trafficking pathways in polarized MDCK cells. We observed that a specific splice isoform of myosin VI with no insert in the tail domain is required for the polarized transport of tyrosine motif containing basolateral membrane proteins. Sorting of other basolateral or apical cargo, however, does not involve myosin VI. Site-directed mutagenesis indicates that a functional complex consisting of myosin VI, optineurin, and probably the GTPase Rab8 plays a role in the basolateral delivery of membrane proteins, whose sorting is mediated by the clathrin adaptor protein complex (AP) AP-1B. Our results suggest that myosin VI is a crucial component in the AP-1B-dependent biosynthetic sorting pathway to the basolateral surface in polarized epithelial cells.

Myosin motor proteins are involved in the final stages of the secretory pathways.

In eukaryotes, the final steps in both the regulated and constitutive secretory pathways can be divided into four distinct stages: (i) the 'approach' of secretory vesicles/granules to the PM (plasma membrane), (ii) the 'docking' of these vesicles/granules at the membrane itself, (iii) the 'priming' of the secretory vesicles/granules for the fusion process, and, finally, (iv) the 'fusion' of vesicular/granular membranes with the PM to permit content release from the cell. Recent work indicates that non-muscle myosin II and the unconventional myosin motor proteins in classes 1c/1e, Va and VI are specifically involved in these final stages of secretion. In the present review, we examine the roles of these myosins in these stages of the secretory pathway and the implications of their roles for an enhanced understanding of secretion in general.

Load-dependent kinetics of force production by smooth muscle myosin measured with optical tweezers.

Muscle contraction is driven by the cyclical interaction of myosin with actin, coupled with ATP hydrolysis. Myosin attaches to actin, forming a crossbridge that produces force and movement as it tilts or rocks into subsequent bound states before finally detaching. It has been hypothesized that the kinetics of one or more of these mechanical transitions are dependent on load, allowing muscle to shorten quickly under low load, but to sustain tension economically, with slowly cycling crossbridges under high load conditions. The idea that muscle biochemistry depends on mechanical output is termed the 'Fenn effect'. However, the molecular details of how load affects the kinetics of a single crossbridge are unknown. Here, we describe a new technique based on optical tweezers to rapidly apply force to a single smooth muscle myosin crossbridge. The crossbridge produced movement in two phases that contribute 4 nm + 2 nm of displacement. Duration of the first phase depended in an exponential manner on the amplitude of applied load. Duration of the second phase was much less affected by load, but was significantly shorter at high ATP concentration. The effect of load on the lifetime of the bound crossbridge is to prolong binding when load is high, but to accelerate release when load is low or negative.