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Robert S Molday - One of the best experts on this subject based on the ideXlab platform.
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proteomics of photoreceptor outer segments identifies a subset of snare and rab proteins implicated in Membrane Vesicle Trafficking and fusion
Molecular & Cellular Proteomics, 2008Co-Authors: Michael C M Kwok, Juha M Holopainen, Laurie L Molday, Leonard J Foster, Robert S MoldayAbstract:The outer segment is a specialized compartment of vertebrate rod and cone photoreceptor cells where phototransduction takes place. In rod cells it consists of an organized stack of disks enclosed by a separate plasma Membrane. Although most proteins involved in phototransduction have been identified and characterized, little is known about the proteins that are responsible for outer segment structure and renewal. In this study we used a tandem mass spectrometry-based proteomics approach to identify proteins in rod outer segment preparations as an initial step in defining their roles in photoreceptor structure, function, renewal, and degeneration. Five hundred and sixteen proteins were identified including 41 proteins that function in rod and cone phototransduction and the visual cycle and most proteins previously shown to be involved in outer segment structure and metabolic pathways. In addition, numerous proteins were detected that have not been previously reported to be present in outer segments including a subset of Rab and SNARE proteins implicated in Vesicle Trafficking and Membrane fusion. Western blotting and immunofluorescence microscopy confirmed the presence of Rab 11b, Rab 18, Rab 1b, and Rab GDP dissociation inhibitor in outer segments. The SNARE proteins, VAMP2/3, syntaxin 3, N-ethylmaleimide-sensitive factor, and Munc 18 detected in outer segment preparations by mass spectrometry and Western blotting were also observed in outer segments by immunofluorescence microscopy. Syntaxin 3 and N-ethylmaleimide- sensitive factor had a restricted localization at the base of the outer segments, whereas VAMP2/3 and Munc 18 were distributed throughout the outer segments. These results suggest that Rab and SNARE proteins play a role in Vesicle Trafficking and Membrane fusion as part of the outer segment renewal process. The data set generated in this study is a valuable resource for further analysis of photoreceptor outer segment structure and function.
Michael A. Frohman - One of the best experts on this subject based on the ideXlab platform.
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physiological and pathophysiological roles for phospholipase d
Journal of Lipid Research, 2015Co-Authors: Rochelle K Nelson, Michael A. FrohmanAbstract:: Individual members of the mammalian phospholipase D (PLD) superfamily undertake roles that extend from generating the second messenger signaling lipid, phosphatidic acid, through hydrolysis of the Membrane phospholipid, phosphatidylcholine, to functioning as an endonuclease to generate small RNAs and facilitating Membrane Vesicle Trafficking through seemingly nonenzymatic mechanisms. With recent advances in genome-wide association studies, RNA interference screens, next-generation sequencing approaches, and phenotypic analyses of knockout mice, roles for PLD family members are being uncovered in autoimmune, infectious neurodegenerative, and cardiovascular disease, as well as in cancer. Some of these disease settings pose opportunities for small molecule inhibitory therapeutics, which are currently in development.
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5 fluoro 2 indolyl des chlorohalopemide fipi a phospholipase d pharmacological inhibitor that alters cell spreading and inhibits chemotaxis
Molecular Pharmacology, 2009Co-Authors: Oladapo Yeku, Srinivas Olepu, Alyssa Genna, Jaesook Park, Hongmei Ren, Michael H Gelb, Andrew J Morris, Michael A. FrohmanAbstract:The signaling enzyme phospholipase D (PLD) and the lipid second messenger it generates, phosphatidic acid (PA), are implicated in many cell biological processes, including Ras activation, cell spreading, stress fiber formation, chemotaxis, and Membrane Vesicle Trafficking. PLD production of PA is inhibited by the primary alcohol 1-butanol, which has thus been widely employed to identify PLD/PA-driven processes. However, 1-butanol does not always effectively reduce PA accumulation, and its use may result in PLD-independent deleterious effects. Consequently, identification of potent specific small-molecule PLD inhibitors would be an important advance for the field. We examine one such here, 5-fluoro-2-indolyl des-chlorohalopemide (FIPI), which was identified recently in an in vitro chemical screen for PLD2 inhibitors, and show that it rapidly blocks in vivo PA production with subnanomolar potency. We were surprised to find that several biological processes blocked by 1-butanol are not affected by FIPI, suggesting the need for re-evaluation of proposed roles for PLD. However, FIPI does inhibit PLD regulation of F-actin cytoskeleton reorganization, cell spreading, and chemotaxis, indicating potential utility for it as a therapeutic for autoimmunity and cancer metastasis.
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A role for phospholipase D1 in neurotransmitter release
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Yann Humeau, Nicolas Vitale, Sylvette Chasserot-golaz, Jean-luc Dupont, Michael A. Frohman, Marie-france Bader, Bernard PoulainAbstract:Phosphatidic acid produced by phospholipase D (PLD) as a result of signaling activity is thought to play a role in Membrane Vesicle Trafficking, either as an intracellular messenger or as a cone-shaped lipid that promotes Membrane fusion. We recently described that, in neuroendocrine cells, plasma Membrane-associated PLD1 operates at a stage of Ca2+-dependent exocytosis subsequent to cytoskeletal-mediated recruitment of secretory granules to exocytotic sites. We show here that PLD1 also plays a crucial role in neurotransmitter release. Using purified rat brain synaptosomes subjected to hypotonic lysis and centrifugation, we found that PLD1 is associated with the particulate fraction containing the plasma Membrane. Immunostaining of rat cerebellar granule cells confirmed localization of PLD1 at the neuronal plasma Membrane in zones specialized for neurotransmitter release (axonal neurites, varicosities, and growth cone-like structures). To determine the potential involvement of PLD1 in neurotransmitter release, we microinjected catalytically inactive PLD1(K898R) into Aplysia neurons and analyzed its effects on evoked acetylcholine (ACh) release. PLD1(K898R) produced a fast and potent dose-dependent inhibition of ACh release. By analyzing paired-pulse facilitation and postsynaptic responses evoked by high-frequency stimulations, we found that the exocytotic inhibition caused by PLD1(K898R) was not the result of an alteration in stimulus-secretion coupling or in vesicular Trafficking. Analysis of the fluctuations in amplitude of the postsynaptic responses revealed that the PLD1(K898R) blocked ACh release by reducing the number of active presynaptic-releasing sites. Our results provide evidence that PLD1 plays a major role in neurotransmission, most likely by controlling the fusogenic status of presynaptic release sites.
Michael C M Kwok - One of the best experts on this subject based on the ideXlab platform.
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proteomics of photoreceptor outer segments identifies a subset of snare and rab proteins implicated in Membrane Vesicle Trafficking and fusion
Molecular & Cellular Proteomics, 2008Co-Authors: Michael C M Kwok, Juha M Holopainen, Laurie L Molday, Leonard J Foster, Robert S MoldayAbstract:The outer segment is a specialized compartment of vertebrate rod and cone photoreceptor cells where phototransduction takes place. In rod cells it consists of an organized stack of disks enclosed by a separate plasma Membrane. Although most proteins involved in phototransduction have been identified and characterized, little is known about the proteins that are responsible for outer segment structure and renewal. In this study we used a tandem mass spectrometry-based proteomics approach to identify proteins in rod outer segment preparations as an initial step in defining their roles in photoreceptor structure, function, renewal, and degeneration. Five hundred and sixteen proteins were identified including 41 proteins that function in rod and cone phototransduction and the visual cycle and most proteins previously shown to be involved in outer segment structure and metabolic pathways. In addition, numerous proteins were detected that have not been previously reported to be present in outer segments including a subset of Rab and SNARE proteins implicated in Vesicle Trafficking and Membrane fusion. Western blotting and immunofluorescence microscopy confirmed the presence of Rab 11b, Rab 18, Rab 1b, and Rab GDP dissociation inhibitor in outer segments. The SNARE proteins, VAMP2/3, syntaxin 3, N-ethylmaleimide-sensitive factor, and Munc 18 detected in outer segment preparations by mass spectrometry and Western blotting were also observed in outer segments by immunofluorescence microscopy. Syntaxin 3 and N-ethylmaleimide- sensitive factor had a restricted localization at the base of the outer segments, whereas VAMP2/3 and Munc 18 were distributed throughout the outer segments. These results suggest that Rab and SNARE proteins play a role in Vesicle Trafficking and Membrane fusion as part of the outer segment renewal process. The data set generated in this study is a valuable resource for further analysis of photoreceptor outer segment structure and function.
Juha M Holopainen - One of the best experts on this subject based on the ideXlab platform.
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proteomics of photoreceptor outer segments identifies a subset of snare and rab proteins implicated in Membrane Vesicle Trafficking and fusion
Molecular & Cellular Proteomics, 2008Co-Authors: Michael C M Kwok, Juha M Holopainen, Laurie L Molday, Leonard J Foster, Robert S MoldayAbstract:The outer segment is a specialized compartment of vertebrate rod and cone photoreceptor cells where phototransduction takes place. In rod cells it consists of an organized stack of disks enclosed by a separate plasma Membrane. Although most proteins involved in phototransduction have been identified and characterized, little is known about the proteins that are responsible for outer segment structure and renewal. In this study we used a tandem mass spectrometry-based proteomics approach to identify proteins in rod outer segment preparations as an initial step in defining their roles in photoreceptor structure, function, renewal, and degeneration. Five hundred and sixteen proteins were identified including 41 proteins that function in rod and cone phototransduction and the visual cycle and most proteins previously shown to be involved in outer segment structure and metabolic pathways. In addition, numerous proteins were detected that have not been previously reported to be present in outer segments including a subset of Rab and SNARE proteins implicated in Vesicle Trafficking and Membrane fusion. Western blotting and immunofluorescence microscopy confirmed the presence of Rab 11b, Rab 18, Rab 1b, and Rab GDP dissociation inhibitor in outer segments. The SNARE proteins, VAMP2/3, syntaxin 3, N-ethylmaleimide-sensitive factor, and Munc 18 detected in outer segment preparations by mass spectrometry and Western blotting were also observed in outer segments by immunofluorescence microscopy. Syntaxin 3 and N-ethylmaleimide- sensitive factor had a restricted localization at the base of the outer segments, whereas VAMP2/3 and Munc 18 were distributed throughout the outer segments. These results suggest that Rab and SNARE proteins play a role in Vesicle Trafficking and Membrane fusion as part of the outer segment renewal process. The data set generated in this study is a valuable resource for further analysis of photoreceptor outer segment structure and function.
Leonard J Foster - One of the best experts on this subject based on the ideXlab platform.
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proteomics of photoreceptor outer segments identifies a subset of snare and rab proteins implicated in Membrane Vesicle Trafficking and fusion
Molecular & Cellular Proteomics, 2008Co-Authors: Michael C M Kwok, Juha M Holopainen, Laurie L Molday, Leonard J Foster, Robert S MoldayAbstract:The outer segment is a specialized compartment of vertebrate rod and cone photoreceptor cells where phototransduction takes place. In rod cells it consists of an organized stack of disks enclosed by a separate plasma Membrane. Although most proteins involved in phototransduction have been identified and characterized, little is known about the proteins that are responsible for outer segment structure and renewal. In this study we used a tandem mass spectrometry-based proteomics approach to identify proteins in rod outer segment preparations as an initial step in defining their roles in photoreceptor structure, function, renewal, and degeneration. Five hundred and sixteen proteins were identified including 41 proteins that function in rod and cone phototransduction and the visual cycle and most proteins previously shown to be involved in outer segment structure and metabolic pathways. In addition, numerous proteins were detected that have not been previously reported to be present in outer segments including a subset of Rab and SNARE proteins implicated in Vesicle Trafficking and Membrane fusion. Western blotting and immunofluorescence microscopy confirmed the presence of Rab 11b, Rab 18, Rab 1b, and Rab GDP dissociation inhibitor in outer segments. The SNARE proteins, VAMP2/3, syntaxin 3, N-ethylmaleimide-sensitive factor, and Munc 18 detected in outer segment preparations by mass spectrometry and Western blotting were also observed in outer segments by immunofluorescence microscopy. Syntaxin 3 and N-ethylmaleimide- sensitive factor had a restricted localization at the base of the outer segments, whereas VAMP2/3 and Munc 18 were distributed throughout the outer segments. These results suggest that Rab and SNARE proteins play a role in Vesicle Trafficking and Membrane fusion as part of the outer segment renewal process. The data set generated in this study is a valuable resource for further analysis of photoreceptor outer segment structure and function.