Susan D. Arden

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 isoform localized to clathrin‐coated vesicles with a role in clathrin‐mediated endocytosis

Myosin VI is involved in membrane traffic and dynamics and is the only myosin known to move towards the minus end of actin filaments. Splice variants of myosin VI with a large insert in the tail domain were specifically 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 clathrin‐ coated pits/vesicles. Using full‐length myosin VI and deletion mutants tagged with green fluorescent 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 purified clathrin‐coated vesicles. Over‐expression of the tail domain of myosin VI containing the large insert in fibroblasts reduced transferrin uptake in transiently and stably transfected cells by >50%. Myosin VI is the first motor protein to be identified associated with clathrin‐coated pits/vesicles and shown to modulate clathrin‐mediated endocytosis.

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.

Imogen 38: a novel 38-kD islet mitochondrial autoantigen recognized by T cells from a newly diagnosed type 1 diabetic patient.

Cell-mediated autoimmune attack directed against islet proteins of approximately 38 kD in size has been associated with type 1 diabetes. A novel murine cDNA encoding an antigen of this size was cloned using a screening procedure based on the proliferative response of a human diabetic T cell clone (1C6) to a recombinant antigen epitope library. Membrane preparations from COS 7 cells transfected with the full-length 1,267-bp cDNA elicited a proliferative response from the reporter T cells comparable to that of the defined peptide epitope and native insulinoma antigen. In vitro translation and transfection experiments suggested that the protein is initially synthesized as a 44-kD protein and then processed to the native 38-kD form through the proteolytic removal of a 54-aa NH2-terminal mitochondrial targeting sequence. Differential centrifugation, Percoll density gradient centrifugation, and immunofluorescence studies confirmed localization of the antigen to mitochondria. Northern blot, Western blot, and 1C6 T cell proliferation assays showed that, although imogen 38 was more highly expressed in beta cell than alpha cell lines, it was also present in other tissues. It is concluded that imogen 38 may be a target for bystander autoimmune attack in diabetes rather than a primary autoantigen.

T-Cell Reactivity to β-Cell Membrane Antigens Associated With β-Cell Destruction in IDDM

Insulin-dependent diabetes mellitus (IDDM) results from a T-cell-mediated destruction of the insulin-producing β-cells. In this study, we designed a sensitive assay to detect and identify islet cell-reactive T-cells in patients with newly diagnosed IDDM. The relation between T-cell recognition of β-cell antigens with IDDM and the pathogenesis of the disease (the β-cell destruction process) was tested in a large group of IDDM patients and compared with T-cell responses in nondiabetic children with other chronic inflammations and in immunologically normal, age-matched control subjects. The results demonstrate that peripheral blood T-cells reacting with a β-cell membrane preparation enriched for insulin-secretory granule antigen were detectable in the majority of newly diagnosed IDDM patients (27 of 40 [67%]; mean stimulation index [SI] 37.0). Such reactivity was reduced postonset in IDDM patients proportionally to the duration of the disease (11 of 30 [37%]; mean SI 8.7). Nondiabetic age-matched control subjects showed no responses or moderate responses to the granule preparation (4 of 48 [8%]; mean SI 3.4). The magnitude of the T-cell response was significantly greater in newly diagnosed IDDM patients than in IDDM patients tested at least 2 years postonset (P < 0.001). Two children in remission for insulin dependency (so-called honeymoon period) displayed exceptionally high proliferative responses to insulin-secretory granules (mean SI 86.7). These results imply that T-cell recognition of insulin-secretory granule antigens is associated with IDDM and in particular with the immune-mediated process of β-cell destruction.

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.

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.

Overexpression of myosin VI in prostate cancer cells enhances PSA and VEGF secretion, but has no effect on endocytosis

Tissue expression microarrays, employed to determine the players and mechanisms leading to prostate cancer development, have consistently shown that myosin VI, a unique actin-based motor, is upregulated in medium-grade human prostate cancers. Thus, to understand the role of myosin VI in prostate cancer development, we have characterized its intracellular localization and function in the prostate cancer cell line LNCaP. Using light and electron microscopy, we identified myosin VI on Rab5-positive early endosomes, as well as on recycling endosomes and the trans-Golgi network. Intracellular targeting seems to involve two myosin VI-interacting proteins, GIPC and LMTK2, both of which can be co-immunoprecipitated with myosin VI from LNCaP cells. The absence of Disabled-2 (Dab2), a tumour suppressor and myosin VI-binding partner, inhibits recruitment of myosin VI to endocytic structures at the plasma membrane in LNCaP cells, but interestingly has no effect on endocytosis. Small interfering RNA-mediated downregulation of myosin VI expression results in a significant reduction in prostate-specific antigen (PSA) and vascular endothelial growth factor (VEGF) secretion in LNCaP cells. Our results suggest that in prostate cancer cells, myosin VI regulates protein secretion, but the overexpression of myosin VI has no major impact on clathrin-mediated endocytosis.

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 post-translational processing and intracellular sorting of carboxypeptidase H in the islets of Langerhans

The post-translational processing and intracellular sorting of the proinsulin-converting enzyme carboxypeptidase H (CPH) was studied in isolated rat islets of Langerhans. Pulse-chase-radiolabelling experiments using sequence-specific antisera showed that CPH was synthesized initially as a 57-kDa glycoprotein which was processed to a 54-kDa mature form by proteolytic processing at the N-terminus. Processing of the CPH precursor occurred rapidly (t(1/2) = 30) after an initial delay of 15-30 min and the enzyme was secreted in parallel with the insulin-related peptides in response to glucose-stimulation within 1 h after radiolabelling. This indicated that the proteins were packaged into nascent secretory granules at approximately the same rate following synthesis. Conversion of proinsulin and the 57-kDa form was inhibited markedly by chase incubation of islets at 20 degrees C, indicating that maturation of both proteins occurs in a post-Golgi compartment. Affinity purification of the enzyme from insulinoma subcellular fractions showed that the 57-kDa form was associated with endoplasmic reticulum or Golgi elements, and the 54-kDa form was present in secretory granules. Structural analysis showed that the granule form of the enzyme had an N-terminal amino acid sequence beginning at residue 42 of rat CPH, thereby implicating cleavage of the precursor after the fourth Arg in a site containing five consecutive Arg residues. These findings indicate that post-translational processing of CPH is mediated by an endoprotease which cleaves at sites containing multiple basic amino acid residues upon segregation of the enzyme to the secretory granules.