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Folma Buss - One of the best experts on this subject based on the ideXlab platform.

  • autophagy receptors link myosin vi to autophagosomes to mediate tom1 dependent autophagosome maturation and fusion with the lysosome
    Nature Cell Biology, 2012
    Co-Authors: David A Tumbarello, Susan D. Arden, Bennett J Waxse, Nicholas A Bright, John Kendrickjones, Folma Buss
    Abstract:

    Autophagy targets pathogens, damaged organelles and protein aggregates for lysosomal degradation. These ubiquitylated cargoes are recognized 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 loss of myosin VI and Tom1 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 and its binding partner Optineurin are involved in secretory vesicle fusion at the plasma membrane
    Molecular Biology of the Cell, 2011
    Co-Authors: Lisa M Bond, John Kendrickjones, Andrew A Peden, James R Sellers, Folma Buss
    Abstract:

    During constitutive secretion, proteins synthesized at the endoplasmic reticulum (ER) are transported to the Golgi complex for processing and then to the plasma membrane for incorporation or extracellular release. This study uses a unique live-cell constitutive secretion assay to establish roles for the molecular motor myosin VI and its binding partner Optineurin in discrete stages of secretion. Small interfering RNA-based knockdown of myosin VI causes an ER-to-Golgi transport delay, suggesting an unexpected function for myosin VI in the early secretory pathway. Depletion of myosin VI or Optineurin does not affect the number of vesicles leaving the trans-Golgi network (TGN), indicating that these proteins do not function in TGN vesicle formation. However, myosin VI and Optineurin colocalize with secretory vesicles at the plasma membrane. Furthermore, live-cell total internal reflection fluorescence microscopy demonstrates that myosin VI or Optineurin depletion reduces the total number of vesicle fusion events at the plasma membrane and increases both the proportion of incomplete fusion events and the number of docked vesicles in this region. These results suggest a novel role for myosin VI and Optineurin in regulation of fusion pores formed between secretory vesicles and the plasma membrane during the final stages of secretion.

  • myosin vi and Optineurin are required for polarized egfr delivery and directed migration
    Traffic, 2010
    Co-Authors: Margarita V Chibalina, John Kendrickjones, Alexei Poliakov, Folma Buss
    Abstract:

    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.

  • rab8 Optineurin myosin vi analysis of interactions and functions in the secretory pathway
    Methods in Enzymology, 2008
    Co-Authors: Margarita V Chibalina, Rhys C. Roberts, Susan D. Arden, John Kendrickjones, Folma Buss
    Abstract:

    Abstract 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.

  • Myosin VI is required for sorting of AP-1B–dependent cargo to the basolateral domain in polarized MDCK cells
    The Journal of cell biology, 2007
    Co-Authors: Claudia Puri, John Kendrick-jones, Gudrun Ihrke, Folma Buss
    Abstract:

    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.

Philip Cohen - One of the best experts on this subject based on the ideXlab platform.

  • Optineurin negatively regulates osteoclast differentiation by modulating nf κb and interferon signaling implications for paget s disease
    Cell Reports, 2015
    Co-Authors: Rami Obaid, Philip Cohen, Sachin Wani, Asim Azfer, Toby W Hurd, Ruth Jones, Stuart H Ralston, Omar M E Albagha
    Abstract:

    Paget's disease of bone (PDB) is a common disease characterized by osteoclast activation that leads to various skeletal complications. Susceptibility to PDB is mediated by a common variant at the Optineurin (OPTN) locus, which is associated with reduced levels of mRNA. However, it is unclear how this leads to the development of PDB. Here, we show that OPTN acts as a negative regulator of osteoclast differentiation in vitro and that mice with a loss-of-function mutation in Optn have increased osteoclast activity and bone turnover. Osteoclasts derived from Optn mutant mice have an increase in NF-κB activation and a reduction in interferon beta expression in response to RANKL when compared to wild-type mice. These studies identify OPTN as a regulator of bone resorption and are consistent with a model whereby genetically determined reductions in OPTN expression predispose to PDB by enhancing osteoclast differentiation.

  • Polyubiquitin Binding to Optineurin Is Required for Optimal Activation of TANK-binding Kinase 1 and Production of Interferon β
    The Journal of biological chemistry, 2011
    Co-Authors: Catherine E. Gleason, Alban Ordureau, Robert Gourlay, J. Simon C. Arthur, Philip Cohen
    Abstract:

    TANK-binding kinase (TBK1) is essential for transcription of the interferon (IFN) β gene in response to lipopolysaccharide (LPS) and double-stranded RNA, but the molecular mechanisms that underlie the activation of TBK1 are incompletely understood. Previously, we identified the NF-κB essential modulator (NEMO)-related polyubiquitin-binding protein, Optineurin (OPTN), as a novel binding partner of TBK1. To determine whether the ubiquitin-binding function of OPTN is involved in regulating TBK1 and IFNβ production, we generated a mouse in which wild-type Optineurin was replaced by the polyubiquitin binding-defective mutant, OPTND477N/D477N. In this study, we found that LPS or poly(I:C)-induced TBK1 activity was significantly reduced in bone marrow-derived macrophage (BMDM) from OPTND477N/D477N mice. Consistent with this, the phosphorylation of IFN regulatory factor 3 (IRF3) and the production of IFNβ mRNA and secretion were reduced. Stimulation of BMDMs with LPS triggered the phosphorylation of OPTN, which was reversed by phosphatase treatment and prevented by pharmacological inhibition of both the canonical IκB kinases (IKKα/β) and the IKK-related kinases (TBK1/IKKϵ). In contrast, LPS-stimulated phosphorylation of OPTN(D477N) was markedly reduced in BMDMs from OPTND477N/D477N mice, and inhibition of the canonical IKKs alone prevented phosphorylation, providing further evidence that ubiquitin binding to OPTN contributes to LPS-induced TBK1 activation. TBK1 and IKKβ phosphorylated OPTN preferentially at Ser-177 and Ser-513, respectively, in vitro. In conclusion, our results suggest that OPTN binds to polyubiquitylated species formed in response to LPS and poly(I:C), enhancing the activation of TBK1 that is required for optimal phosphorylation of IRF3 and production of IFNβ.

  • enhanced binding of tbk1 by an Optineurin mutant that causes a familial form of primary open angle glaucoma
    FEBS Letters, 2008
    Co-Authors: Simon Morton, Luke B Hesson, Mark Peggie, Philip Cohen
    Abstract:

    TANK-binding kinase 1 (TBK1) was identified as a binding partner for Optineurin (OPTN) in two-hybrid screens, an interaction confirmed by overexpression/immunoprecipitation experiments in HEK293 cells and by coimmunoprecipitation of endogenous OPTN and TBK1 from cell extracts. A TBK1 binding site was located between residues 1-127 of OPTN, residues 78-121 displaying striking homology to the TBK1-binding domain of TANK. The OPTN-binding domain was localised to residues 601-729 of TBK1, while TBK1[1-688] which cannot bind to TANK, did not interact with OPTN. The OPTN[E50K] mutant associated with Primary Open Angle Glaucoma (POAG) displayed strikingly enhanced binding to TBK1, suggesting that this interaction may contribute to familial POAG caused by this mutation.

Masaki Kamada - One of the best experts on this subject based on the ideXlab platform.

  • mutations of Optineurin in amyotrophic lateral sclerosis
    Nature, 2010
    Co-Authors: Hirofumi Maruyama, Hiroyuki Morino, Hidefumi Ito, Yuishin Izumi, Hidemasa Kato, Yasuhito Watanabe, Yoshimi Kinoshita, Masaki Kamada
    Abstract:

    Amyotrophic lateral sclerosis (ALS) has its onset in middle age and is a progressive disorder characterized by degeneration of motor neurons of the primary motor cortex, brainstem and spinal cord. Most cases of ALS are sporadic, but about 10% are familial. Genes known to cause classic familial ALS (FALS) are superoxide dismutase 1 (SOD1), ANG encoding angiogenin, TARDP encoding transactive response (TAR) DNA-binding protein TDP-43 (ref. 4) and fused in sarcoma/translated in liposarcoma (FUS, also known as TLS). However, these genetic defects occur in only about 20-30% of cases of FALS, and most genes causing FALS are unknown. Here we show that there are mutations in the gene encoding Optineurin (OPTN), earlier reported to be a causative gene of primary open-angle glaucoma (POAG), in patients with ALS. We found three types of mutation of OPTN: a homozygous deletion of exon 5, a homozygous Q398X nonsense mutation and a heterozygous E478G missense mutation within its ubiquitin-binding domain. Analysis of cell transfection showed that the nonsense and missense mutations of OPTN abolished the inhibition of activation of nuclear factor kappa B (NF-kappaB), and the E478G mutation revealed a cytoplasmic distribution different from that of the wild type or a POAG mutation. A case with the E478G mutation showed OPTN-immunoreactive cytoplasmic inclusions. Furthermore, TDP-43- or SOD1-positive inclusions of sporadic and SOD1 cases of ALS were also noticeably immunolabelled by anti-OPTN antibodies. Our findings strongly suggest that OPTN is involved in the pathogenesis of ALS. They also indicate that NF-kappaB inhibitors could be used to treat ALS and that transgenic mice bearing various mutations of OPTN will be relevant in developing new drugs for this disorder.

Elizabeth Conibear - One of the best experts on this subject based on the ideXlab platform.

  • the palmitoyl acyltransferase hip14 shares a high proportion of interactors with huntingtin implications for a role in the pathogenesis of huntington s disease
    Human Molecular Genetics, 2014
    Co-Authors: Stefanie L. Butland, Mandi E. Schmidt, Seanpatrick Riechers, Dale D O Martin, Erich E. Wanker, Roshni R. Singaraja, Kuljeet Vaid, Shaun S. Sanders, Rona K. Graham, Elizabeth Conibear
    Abstract:

    HIP14 is the most highly conserved of 23 human palmitoyl acyltransferases (PATs) that catalyze the post-translational addition of palmitate to proteins, including huntingtin (HTT). HIP14 is dysfunctional in the presence of mutant HTT (mHTT), the causative gene for Huntington disease (HD), and we hypothesize that reduced palmitoylation of HTT and other HIP14 substrates contributes to the pathogenesis of the disease. Here we describe the yeast two-hybrid (Y2H) interactors of HIP14 in the first comprehensive study of interactors of a mammalian PAT. Unexpectedly, we discovered a highly significant overlap between HIP14 interactors and 370 published interactors of HTT, 4-fold greater than for control proteins (P = 8 × 10−5). Nearly half of the 36 shared interactors are already implicated in HD, supporting a direct link between HIP14 and the disease. The HIP14 Y2H interaction set is significantly enriched for palmitoylated proteins that are candidate substrates. We confirmed that three of them, GPM6A, and the Sprouty domain-containing proteins SPRED1 and SPRED3, are indeed palmitoylated by HIP14; the first enzyme known to palmitoylate these proteins. These novel substrates functions might be affected by reduced palmitoylation in HD. We also show that the vesicular cargo adapter Optineurin, an established HTT-binding protein, co-immunoprecipitates with HIP14 but is not palmitoylated. mHTT leads to mislocalization of Optineurin and aberrant cargo trafficking. Therefore, it is possible that Optineurin regulates trafficking of HIP14 to its substrates. Taken together, our data raise the possibility that defective palmitoylation by HIP14 might be an important mechanism that contributes to the pathogenesis of HD.

  • the palmitoyl acyltransferase hip14 shares a high proportion of interactors with huntingtin implications for a role in the pathogenesis of huntington s disease
    Human Molecular Genetics, 2014
    Co-Authors: Stefanie L. Butland, Mandi E. Schmidt, Seanpatrick Riechers, Dale D O Martin, Erich E. Wanker, Roshni R. Singaraja, Kuljeet Vaid, Shaun S. Sanders, Rona K. Graham, Elizabeth Conibear
    Abstract:

    HIP14 is the most highly conserved of 23 human palmitoyl acyltransferases (PATs) that catalyze the post-translational addition of palmitate to proteins, including huntingtin (HTT). HIP14 is dysfunctional in the presence of mutant HTT (mHTT), the causative gene for Huntington disease (HD), and we hypothesize that reduced palmitoylation of HTT and other HIP14 substrates contributes to the pathogenesis of the disease. Here we describe the yeast two-hybrid (Y2H) interactors of HIP14 in the first comprehensive study of interactors of a mammalian PAT. Unexpectedly, we discovered a highly significant overlap between HIP14 interactors and 370 published interactors of HTT, 4-fold greater than for control proteins (P = 8 × 10−5). Nearly half of the 36 shared interactors are already implicated in HD, supporting a direct link between HIP14 and the disease. The HIP14 Y2H interaction set is significantly enriched for palmitoylated proteins that are candidate substrates. We confirmed that three of them, GPM6A, and the Sprouty domain-containing proteins SPRED1 and SPRED3, are indeed palmitoylated by HIP14; the first enzyme known to palmitoylate these proteins. These novel substrates functions might be affected by reduced palmitoylation in HD. We also show that the vesicular cargo adapter Optineurin, an established HTT-binding protein, co-immunoprecipitates with HIP14 but is not palmitoylated. mHTT leads to mislocalization of Optineurin and aberrant cargo trafficking. Therefore, it is possible that Optineurin regulates trafficking of HIP14 to its substrates. Taken together, our data raise the possibility that defective palmitoylation by HIP14 might be an important mechanism that contributes to the pathogenesis of HD.

Julie D Atkin - One of the best experts on this subject based on the ideXlab platform.

  • Dysfunction of Optineurin in Amyotrophic Lateral Sclerosis and Glaucoma
    Frontiers Media S.A., 2018
    Co-Authors: Reka P. Toth, Julie D Atkin
    Abstract:

    Neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia, and glaucoma, affect millions of people worldwide. ALS is caused by the loss of motor neurons in the spinal cord, brainstem, and brain, and genetic mutations are responsible for 10% of all ALS cases. Glaucoma is characterized by the loss of retinal ganglion cells and is the most common cause of irreversible blindness. Interestingly, mutations in OPTN, encoding Optineurin, are associated with both ALS and glaucoma. Optineurin is a highly abundant protein involved in a wide range of cellular processes, including the inflammatory response, autophagy, Golgi maintenance, and vesicular transport. In this review, we summarize the role of Optineurin in cellular mechanisms implicated in neurodegenerative disorders, including neuroinflammation, autophagy, and vesicular trafficking, focusing in particular on the consequences of expression of mutations associated with ALS and glaucoma. This review, therefore showcases the impact of Optineurin dysfunction in ALS and glaucoma

  • a novel amyotrophic lateral sclerosis mutation in optn induces er stress and golgi fragmentation in vitro
    Amyotrophic Lateral Sclerosis, 2017
    Co-Authors: Jennifer A Fifita, Vinod Sundaramoorthy, Emily P Mccann, Kelly L Williams, Julie D Atkin, Garth A Nicholson, Ian P Blair
    Abstract:

    AbstractMutations in the Optineurin gene (OPTN) have been identified in a small proportion (  T, p.Val295Phe) in a single familial ALS case. This mutation induced recognized cellular features of ALS pathogenesis including Golgi fragmentation and ER stress in NSC-34 cells. In conclusion, the identification of a novel OPTN mutation in an Aus...

  • defects in Optineurin and myosin vi mediated cellular trafficking in amyotrophic lateral sclerosis
    Human Molecular Genetics, 2015
    Co-Authors: Vinod Sundaramoorthy, Adam K Walker, Vanessa Tan, Jennifer A Fifita, Emily P Mccann, Kelly L Williams, Ian P Blair, Gilles J Guillemin, Manal A Farg, Julie D Atkin
    Abstract:

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder primarily affecting motor neurons. Mutations in Optineurin cause a small proportion of familial ALS cases, and wild-type (WT) Optineurin is misfolded and forms inclusions in sporadic ALS patient motor neurons. However, it is unknown how Optineurin mutation or misfolding leads to ALS. Optineurin acts an adaptor protein connecting the molecular motor myosin VI to secretory vesicles and autophagosomes. Here, we demonstrate that ALS-linked mutations p.Q398X and p.E478G disrupt the association of Optineurin with myosin VI, leading to an abnormal diffuse cytoplasmic distribution, inhibition of secretory protein trafficking, endoplasmic reticulum (ER) stress and Golgi fragmentation in motor neuron-like NSC-34 cells. We also provide further insight into the role of Optineurin as an autophagy receptor. WT Optineurin associated with lysosomes and promoted autophagosome fusion to lysosomes in neuronal cells, implying that it mediates trafficking of lysosomes during autophagy in association with myosin VI. However, either expression of ALS mutant Optineurin or small interfering RNA-mediated knockdown of endogenous Optineurin blocked lysosome fusion to autophagosomes, resulting in autophagosome accumulation. Together these results indicate that ALS-linked mutations in Optineurin disrupt myosin VI-mediated intracellular trafficking processes. In addition, in control human patient tissues, Optineurin displayed its normal vesicular localization, but in sporadic ALS patient tissues, vesicles were present in a significantly decreased proportion of motor neurons. Optineurin binding to myosin VI was also decreased in tissue lysates from sporadic ALS spinal cords. This study therefore links several previously described pathological mechanisms in ALS, including defects in autophagy, fragmentation of the Golgi and induction of ER stress, to disruption of Optineurin function. These findings also indicate that Optineurin-myosin VI dysfunction is a common feature of both sporadic and familial ALS.