Rhys C. Roberts

Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis

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 Binds to and Localises with Dab2, Potentially Linking Receptor‐Mediated Endocytosis and the Actin Cytoskeleton

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.

T6BP and NDP52 are myosin VI binding partners with potential roles in cytokine signalling and cell adhesion

Myosin VI has been implicated in many cellular processes including endocytosis, secretion, membrane ruffling and cell motility. We carried out a yeast two-hybrid screen and identified TRAF6-binding protein (T6BP) and nuclear dot protein 52 (NDP52) as myosin VI binding partners. Myosin VI interaction with T6BP and NDP52 was confirmed in vitro and in vivo and the binding sites on each protein were accurately mapped. Immunofluorescence and electron microscopy showed that T6BP, NDP52 and myosin VI are present at the trans side of the Golgi complex, and on vesicles in the perinuclear region. Although the SKICH domain in T6BP and NDP52 does not mediate recruitment into membrane ruffles, loss of T6BP and NDP52 in RNAi knockdown cells results in reduced membrane ruffling activity and increased stress fibre and focal adhesion formation. Furthermore, we observed in these knockdown cells an upregulation of constitutive secretion of alkaline phosphatase, implying that both proteins act as negative regulators of secretory traffic at the Golgi complex. T6BP was also found to inhibit NF-κB activation, implicating it in the regulation of TRAF6-mediated cytokine signalling. Thus myosin VI-T6BP interactions may link membrane trafficking pathways with cell adhesion and cytokine-dependent cell signalling.

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.

Rab8-optineurin-myosin VI: Analysis of interactions and functions in the secretory pathway

The small GTPase Rab8 has been shown to regulate polarized membrane trafficking pathways from the TGN to the cell surface. Optineurin is an effector protein of Rab8 and a binding partner of the actin-based motor protein myosin VI. We used various approaches to study the interactions between myosin VI and its binding partners and to analyze their role(s) in intracellular membrane trafficking pathways. In this chapter, we describe the use of the mammalian two-hybrid assay to demonstrate protein-protein interactions and to identify binding sites. We describe a secretion assay that was used in combination with RNA interference technology to analyze the function of myosin VI, optineurin, and Rab8 in exocytic membrane trafficking pathways.

The Emery–Dreifuss muscular dystrophy associated‐protein emerin is phosphorylated on serine 49 by protein kinase A

Emerin is a ubiquitously expressed inner nuclear membrane protein of unknown function. Mutations in its gene give rise to X‐linked Emery–Dreifuss muscular dystrophy (X‐EDMD), a neuromuscular condition with an associated life‐threatening cardiomyopathy. We have previously reported that emerin is phosphorylated in a cell cycle‐dependent manner in human lymphoblastoid cell lines [Ellis et al. (1998) Aberrant intracellular targeting and cell cycle‐dependent phosphorylation of emerin contribute to the EDMD phenotype. J. Cell Sci. 111, 781–792]. Recently, five residues in human emerin were identified as undergoing cell cycle‐dependent phosphorylation using a Xenopus egg mitotic cytosol model system (Hirano et al. (2005) Dissociation of emerin from BAF is regulated through mitotic phosphorylation of emerin in a Xenopus egg cell‐free system. J. Biol. Chem.280, 39 925–39 933). In the present paper, recombinant human emerin was purified from a baculovirus‐Sf9 heterogeneous expression system, analyzed by protein mass spectrometry and shown to exist in at least four different phosphorylated species, each of which could be dephosphorylated by treatment with alkaline phosphatase. Further analysis identified three phosphopeptides with m/z values of 2191.9 and 2271.7 corresponding to the singly and doubly phosphorylated peptide 158‐DSAYQSITHYRPVSASRSS‐176, and a m/z of 2396.9 corresponding to the phosphopeptide 47‐RLSPPSSSAASSYSFSDLNSTR‐68. Sequence analysis confirmed that residue S49 was phosphorylated and also demonstrated that this residue was phosphorylated in interphase. Using an in vitro protein kinase A assay, we observed two phospho‐emerin species, one of which was phosphorylated at residue S49. Protein kinase A is thus the first kinase that has been identified to specifically phosphorylate emerin. These results improve our understanding of the molecular mechanisms underlying X‐EDMD and point towards possible signalling pathways involved in regulating emerins functions.

Myosin VI: a multifunctional motor

Myosin VI moves towards the minus end of actin filaments unlike all the other myosins so far studied, suggesting that it has unique properties and functions. Myosin VI is present in clathrin-coated pits and vesicles, in membrane ruffles and in the Golgi complex, indicating that it has a wide variety of functions in the cell. To investigate the cellular roles of myosin VI, we have identified a variety of myosin VI-binding partners and characterized their interactions. As an alternative approach, we have studied the in vitro properties of intact myosin VI. Previous studies assumed that myosin VI existed as a dimer but our biochemical characterization and electron microscopy studies reveal that myosin VI is a monomer. Using an optical tweezers force transducer, we showed that monomeric myosin VI is a non-processive motor with a large working stroke of 18 nm. Potential roles for myosin VI in cells are discussed.

Exclusive expression of the Rab11 effector SH3TC2 in Schwann cells links integrin-α6 and myelin maintenance to Charcot-Marie-Tooth disease type 4C

Charcot-Marie-Tooth disease type 4C (CMT4C) is one of the commonest autosomal recessive inherited peripheral neuropathies and is associated with mutations in the Rab11 effector, SH3TC2. Disruption of the SH3TC2–Rab11 interaction is the molecular abnormality underlying this disease. However, why SH3TC2 mutations cause an isolated demyelinating neuropathy remains unanswered. Here we show that SH3TC2 is an exclusive Schwann cell protein expressed late in myelination and is downregulated following denervation suggesting a functional role in myelin sheath maintenance. We support our data with an evolutionary cell biological analysis showing that the SH3TC2 gene, and its paralogue SH3TC1, are derived from an ancestral homologue, the duplication of which occurred in the common ancestor of jawed vertebrates, coincident with the appearance of Schwann cells and peripheral axon myelination. Furthermore, we report that SH3TC2 associates with integrin-α6, suggesting that aberrant Rab11-dependent endocytic trafficking of this critical laminin receptor in myelinated Schwann cells is connected to the demyelination seen in affected nerves. Our study therefore highlights the inherent evolutionary link between SH3TC2 and peripheral nerve myelination, pointing also towards a molecular mechanism underlying the specific demyelinating neuropathy that characterizes CMT4C.

The Charcot-Marie-Tooth diseases: how can we identify and develop novel therapeutic targets?

Genetic studies have provided significant insights towards understanding inherited neurological diseases. As the most common inherited neuromuscular disorder, Charcot-Marie-Tooth disease (CMT) is a prime example of how the identification during the past 20 years of underlying genetic mutations in patients and their families has led to the realization that we can no longer regard CMT as a single disease but rather a collection of hereditary peripheral neuropathies resulting from pathogenic mutations in more than 40 distinct genes (http//www.molgen.ua.ac.be/CMTMutations/Mutations/MutByGene.cfm). It is no surprise therefore that diagnostic whole-genome sequencing methods were first applied to patients with CMT (Lupski et al ., 2010; Montenegro et al ., 2011). With this wealth of genetic information, a key question is whether all the pathogenic mutations associated with CMT lead to disease by mechanisms converging on a limited number of dysfunctional pathways, or, alternatively, does each genetic mutation lead to peripheral nerve degeneration by a distinct mechanism? The answer will have obvious implications as we attempt to develop effective treatments. Moreover, these questions highlight the fact that the identification of each genetic mutation is only the first step in deciphering molecular mechanisms that fail at the protein and cellular level in patients with CMT. The classification of CMT into ‘demyelinating’ and ‘axonal’ forms, reflecting the presumed main site of pathology, i.e. the Schwann cell or axon, respectively, has proved useful in terms of targeted genetic testing (Reilly et al. , 2011). Although most genes associated with axonal CMT are yet to be identified, many of those mutated in the demyelinating forms are known. Because of the role of the Schwann cells in axon myelination, it is no surprise that among the genes identified as being …

Characterisation of the membrane topology and molecular structure of LITAF to provide insights into the molecular pathogenesis of Charcot-Marie-Tooth disease type 1C

Abstract Background The Charcot-Marie-Tooth (CMT) diseases are the commonest inherited neuromuscular disorders. These disorders are characterised by progressive degenerative peripheral neuropathies and are associated with mutations in over 80 genes. Mutations in LITAF cause CMT type 1C (CMT1C), an autosomal dominant demyelinating subtype of the disease. Why mutations in LITAF cause disease remains unclear, so we aimed to gather data about its molecular structure, which are lacking despite being highly conserved throughout evolution. Methods We applied biochemical and cell biological techniques (differential membrane preparation, semi-permeabilisation and immunofluorescence microscopy, and cell-free protein expression) to determine the membrane topology of LITAF. Micro particle-induced X-ray emission (microPIXE) was used to establish whether LITAF associates with metal ions. We then analysed recombinantly expressed LITAF contructs by solution nuclear magnetic resonance (NMR) spectroscopy to gain structural insights to the LITAF protein, thereby allowing us to use the Rosetta software to generate a structural molecular model. Findings We found that LITAF behaves as an integral membrane protein and that membrane association is dependent on six leucine residues found within the C-terminal LITAF domain. The membrane topology of the LITAF domain was such that both N-terminals and C-terminals were found on the cytosolic surface of endosomes and that these regions coordinated a single zinc atom. NMR spectroscopy was then used to characterise the secondary structural motifs of the LITAF domain, resulting in a correlation spectrum which was over 90% assigned. These data enabled us to create a structural model using NMR restraints and Rosetta. A LITAF construct harbouring a CMT1C-associated pathogenic mutation was then analysed by NMR, revealing marked structural shifts across the LITAF domain. Interpretation We present the first structural model, to our knowledge, of the highly-conserved LITAF domain, which is a membrane-anchoring motif forming a zinc-binding structure on the surface of endosomes. We also show that a CMT1C-associated mutation causes large structural shifts across the LITAF domain, suggesting that structural instability on the surface of Scwann cell endosomes underpins the neuropathy seen in CMT1C. These insights will provide a platform for the development of future treatments. Funding Wellcome Trust.