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Buy Ray Ban Sunglasses Online - One of the best experts on this subject based on the ideXlab platform.

Richard T. Swank - One of the best experts on this subject based on the ideXlab platform.

  • A role for Rab27b in NF-E2-dependent pathways of platelet formation
    Blood, 2003
    Co-Authors: Sanjay Tiwari, Duarte C. Barral, Emilie H. Mules, Miguel C. Seabra, Edward K. Novak, Richard T. Swank, Joseph E. Italiano, Ramesh A. Shivdasani
    Abstract:

    Megakaryocytes release platelets by reorganizing the cytoplasm into proplatelet extensions. Fundamental to this process is the need to coordinate transport of products and organelles in the appropriate abundance to nascent platelets. The importance of the Rab family of small GTPases (guanosine 5'-triphosphatases) in platelet biogenesis is revealed in Gunmetal (gm/gm) mice, which show deficient Rab isoprenylation and macrothrombocytopenia with few granules and abnormal megakaryocyte morphology. Although some Rab proteins are implicated in vesicle and organelle transport along microtubules or actin, the role of any Rab protein in platelet biogenesis is unknown. The limited number of Rab proteins with defective membrane association in gm/gm megakaryocytes prominently includes Rab27a and Rab27b. Normal expression of Rab27b is especially increased with terminal megakaryocyte differentiation and dependent on nuclear factor-erythroid 2 (NF-E2), a transcription factor required for thrombopoiesis. Chromatin immunoprecipitation demonstrates recruitment of NF-E2 to the putative Rab27B promoter. Inhibition of endogenous Rab27 function in primary megakaryocytes causes severe quantitative and qualitative defects in proplatelet formation that mimic findings in gm/gm cells. Rab27b localizes to alpha and dense granules in megakaryocytes. These results establish a role for Rab27 in platelet synthesis and suggest that Rab27b in particular may coordinate proplatelet formation with granule transport, possibly by recruiting specific effector pathways.

  • Cell-specific abnormal prenylation of Rab proteins in platelets and melanocytes of the Gunmetal mouse
    British journal of haematology, 2002
    Co-Authors: Qing Zhang, Edward K. Novak, Lijie Zhen, Lucy M. Collinson, Elliott K. Jang, Richard J. Haslam, Rosemary W. Elliott, Richard T. Swank
    Abstract:

    Summary. The mutant Gunmetal mouse exhibits reduced rates of platelet synthesis, abnormalities of platelet alpha and dense granules and hypopigmentation. Several of these features resemble those of human alpha/delta platelet storage pool disease, grey platelet syndrome and Hermansky‐ Pudlak syndrome. Gunmetal mice have reduced levels of Rab geranylgeranyltransferase (RabGGTase), which adds lipophilic prenyl groups to the carboxyl terminus of Rab proteins. The degree of prenylation and the subcellular distribution of several Rab proteins were evaluated in mutant platelets, melanocytes and other tissues. Significant deficits in prenylation and membrane binding of most Rabs were observed in platelets and melanocytes. In contrast, minimal alterations in Rab prenylation were apparent in several other Gunmetal tissues despite the fact that RabGGTase activity was equally diminished in these tissues. The mutant tissue-specific effects are probably due to increased concentrations of Rab proteins in platelets and melanocytes. These experiments show that Rab proteins are differentially sensitive to levels of RabGGTase activity and that normal platelet synthesis, platelet organelle function and normal pigmentation are highly sensitive to the degree of prenylation and membrane association of Rab proteins. Further, the tissue-specific effects of the Gunmetal mutation suggest that RabGGTase is a potential target for therapy of thrombocytosis.

  • Melanosome morphologies in murine models of hermansky-pudlak syndrome reflect blocks in organelle development.
    The Journal of investigative dermatology, 2002
    Co-Authors: Thuyen Nguyen, Edward K. Novak, Richard T. Swank, Maryam Kermani, Joachim W. Fluhr, Luanne L. Peters, Maria L. Wei
    Abstract:

    Hermansky-Pudlak syndrome is an autosomal recessive disease characterized by pigment dilution and prolonged bleeding time. At least 15 mutant mouse strains have been classified as models of Hermansky-Pudlak syndrome. Some of the genes are implicated in intracellular vesicle trafficking: budding, targeting, and secretion. Many of the Hermansky-Pudlak syndrome genes remain uncharacterized and their functions are unknown. Clues to the functions of these genes can be found by analyzing the physiologic and cellular phenotypes. Here we have examined the morphology of the melanosomes in the skin of 10 of the mutant mouse Hermansky-Pudlak syndrome strains by transmission electron microscopy. We demonstrate that the morphologies reflect inhibition of organelle maturation or transfer. The Hermansky-Pudlak syndrome strains are classified into morphologic groups characterized by the step at which melanosome biogenesis or transfer to keratinocytes is inhibited, with the cappuccino strain observed to be blocked at the earliest step and Gunmetal blocked at the latest step. We show that all Hermansky-Pudlak syndrome mutant strains except Gunmetal have an increase in unpigmented or hypopigmented immature melanosomal forms, leading to the hypopigmented coat colors seen in these strains. In contrast, the hypopigmentation seen in the Gunmetal strain is due to the retention of melanosomes in melanocytes, and inefficient transfer into keratinocytes.

  • Abnormal vesicular trafficking in mouse models of Hermansky-Pudlak syndrome.
    Pigment Cell Research, 2000
    Co-Authors: Richard T. Swank, Edward K. Novak, Michael P. Mcgarry, Qing Zhang, Yuke Zhang, Lijun Feng
    Abstract:

    Hermansky-Pudlak Syndrome (HPS) is a group of related multigenic recessively inherited disorders which causes abnormalities in the biosynthesis and/or function of three related organelles; melanosomes, platelet-dense granules and lysosomes. These lead, in turn, to hypopigmentation, prolonged bleeding and ceroid deposition. Positional cloning strategies have identified five mouse HPS genes. Two orthologous human diseases (HPS1 and HPS2) have likewise been identified. At least four of the five mouse genes encode proteins involved in the regulation of intracellular vesicle trafficking. The pearl (HPS2) and mocha genes encode the beta3A and delta subunits, respectively, of the AP-3 adaptor complex, which captures organelle membrane proteins at the trans-Golgi apparatus. The protein products of the pallid and Gunmetal genes are also important components of the vesicular trafficking machinery. The former interacts with a t-SNARE, syntaxin13, and the latter is the alpha subunit of Rab geranylgeranyltransferase, which renders Rab proteins sufficiently lipophilic to function at their target membranes. The pale ear (HPS1) gene encodes a ubiquitously expressed protein of unknown function. Recent physiological studies have shown that mouse HPS mutants, like their human HPS counterparts, have variably reduced lifespans and may have lung abnormalities.

  • 5′-UTR Structural Organization, Transcript Expression, and Mutational Analysis of the Human Rab Geranylgeranyl Transferase α-Subunit (RABGGTA) Gene
    Molecular genetics and metabolism, 2000
    Co-Authors: John C. Detter, Edward K. Novak, Qing Zhang, Harvey J. Weiss, Elisabeth M. Cramer, Rémi Favier, Stephen F. Kingsmore, Richard T. Swank
    Abstract:

    Abstract Hermansky-Pudlak syndrome (HPS) is a recessively inherited disease with dysfunction of several related subcellular organelles including platelet-dense granules, melanosomes, and lysosomes. Our recent identification of the mutation in murine Rab geranylgeranyl transferase α-subunit gene ( Rabggta ) in one mouse model of HPS, the Gunmetal mouse, suggested that human patients with similar phenotypes might have mutations in the human orthologous RABGGTA gene. This prompted reanalysis of the 5′-untranslated structure of the human RABGGTA gene in normal individuals and in patients with deficiencies of platelet-dense granules (αδ-SPD), alpha granules (α-SPD or gray platelet syndrome, GPS) or alpha plus dense granules (αδ-SPD). We report the complete sequence of intron α of RABGGTA and demonstrate that exon α is immediately upstream of intron α. The exon/intron structural organization of the 5′-untranslated region (UTR) of human RABGGTA was found to be similar to that of the mouse Rabggta gene. However, exons α and introns α are not homologous between mouse and human. Features of the 5′-UTR of RABGGTA suggest it is a housekeeping gene. While obvious disease-causing mutations of human RABGGTA were not found in our existing SPD patients by sequencing its entire coding region, several polymorphisms of RABGGTA including a putative cryptic splicing mutation in intron 4 were identified. Knowledge of the 5′-UTR structure of RABGGTA and its common polymorphisms will be useful for mutation screening or linkage analysis in patients with albinism, thrombocytopenia, or platelet SPD.

Edward K. Novak - One of the best experts on this subject based on the ideXlab platform.

  • A role for Rab27b in NF-E2-dependent pathways of platelet formation
    Blood, 2003
    Co-Authors: Sanjay Tiwari, Duarte C. Barral, Emilie H. Mules, Miguel C. Seabra, Edward K. Novak, Richard T. Swank, Joseph E. Italiano, Ramesh A. Shivdasani
    Abstract:

    Megakaryocytes release platelets by reorganizing the cytoplasm into proplatelet extensions. Fundamental to this process is the need to coordinate transport of products and organelles in the appropriate abundance to nascent platelets. The importance of the Rab family of small GTPases (guanosine 5'-triphosphatases) in platelet biogenesis is revealed in Gunmetal (gm/gm) mice, which show deficient Rab isoprenylation and macrothrombocytopenia with few granules and abnormal megakaryocyte morphology. Although some Rab proteins are implicated in vesicle and organelle transport along microtubules or actin, the role of any Rab protein in platelet biogenesis is unknown. The limited number of Rab proteins with defective membrane association in gm/gm megakaryocytes prominently includes Rab27a and Rab27b. Normal expression of Rab27b is especially increased with terminal megakaryocyte differentiation and dependent on nuclear factor-erythroid 2 (NF-E2), a transcription factor required for thrombopoiesis. Chromatin immunoprecipitation demonstrates recruitment of NF-E2 to the putative Rab27B promoter. Inhibition of endogenous Rab27 function in primary megakaryocytes causes severe quantitative and qualitative defects in proplatelet formation that mimic findings in gm/gm cells. Rab27b localizes to alpha and dense granules in megakaryocytes. These results establish a role for Rab27 in platelet synthesis and suggest that Rab27b in particular may coordinate proplatelet formation with granule transport, possibly by recruiting specific effector pathways.

  • Cell-specific abnormal prenylation of Rab proteins in platelets and melanocytes of the Gunmetal mouse
    British journal of haematology, 2002
    Co-Authors: Qing Zhang, Edward K. Novak, Lijie Zhen, Lucy M. Collinson, Elliott K. Jang, Richard J. Haslam, Rosemary W. Elliott, Richard T. Swank
    Abstract:

    Summary. The mutant Gunmetal mouse exhibits reduced rates of platelet synthesis, abnormalities of platelet alpha and dense granules and hypopigmentation. Several of these features resemble those of human alpha/delta platelet storage pool disease, grey platelet syndrome and Hermansky‐ Pudlak syndrome. Gunmetal mice have reduced levels of Rab geranylgeranyltransferase (RabGGTase), which adds lipophilic prenyl groups to the carboxyl terminus of Rab proteins. The degree of prenylation and the subcellular distribution of several Rab proteins were evaluated in mutant platelets, melanocytes and other tissues. Significant deficits in prenylation and membrane binding of most Rabs were observed in platelets and melanocytes. In contrast, minimal alterations in Rab prenylation were apparent in several other Gunmetal tissues despite the fact that RabGGTase activity was equally diminished in these tissues. The mutant tissue-specific effects are probably due to increased concentrations of Rab proteins in platelets and melanocytes. These experiments show that Rab proteins are differentially sensitive to levels of RabGGTase activity and that normal platelet synthesis, platelet organelle function and normal pigmentation are highly sensitive to the degree of prenylation and membrane association of Rab proteins. Further, the tissue-specific effects of the Gunmetal mutation suggest that RabGGTase is a potential target for therapy of thrombocytosis.

  • Melanosome morphologies in murine models of hermansky-pudlak syndrome reflect blocks in organelle development.
    The Journal of investigative dermatology, 2002
    Co-Authors: Thuyen Nguyen, Edward K. Novak, Richard T. Swank, Maryam Kermani, Joachim W. Fluhr, Luanne L. Peters, Maria L. Wei
    Abstract:

    Hermansky-Pudlak syndrome is an autosomal recessive disease characterized by pigment dilution and prolonged bleeding time. At least 15 mutant mouse strains have been classified as models of Hermansky-Pudlak syndrome. Some of the genes are implicated in intracellular vesicle trafficking: budding, targeting, and secretion. Many of the Hermansky-Pudlak syndrome genes remain uncharacterized and their functions are unknown. Clues to the functions of these genes can be found by analyzing the physiologic and cellular phenotypes. Here we have examined the morphology of the melanosomes in the skin of 10 of the mutant mouse Hermansky-Pudlak syndrome strains by transmission electron microscopy. We demonstrate that the morphologies reflect inhibition of organelle maturation or transfer. The Hermansky-Pudlak syndrome strains are classified into morphologic groups characterized by the step at which melanosome biogenesis or transfer to keratinocytes is inhibited, with the cappuccino strain observed to be blocked at the earliest step and Gunmetal blocked at the latest step. We show that all Hermansky-Pudlak syndrome mutant strains except Gunmetal have an increase in unpigmented or hypopigmented immature melanosomal forms, leading to the hypopigmented coat colors seen in these strains. In contrast, the hypopigmentation seen in the Gunmetal strain is due to the retention of melanosomes in melanocytes, and inefficient transfer into keratinocytes.

  • Rab geranylgeranyl transferase α mutation in the Gunmetal mouse reduces Rab prenylation and platelet synthesis
    Proceedings of the National Academy of Sciences of the United States of America, 2000
    Co-Authors: John C. Detter, Emilie H. Mules, Edward K. Novak, Qing Zhang, Vishnu Mishra, Elzbieta B. Mcmurtrie, Velizar T. Tchernev, Margaret R. Wallace, Miguel C. Seabra
    Abstract:

    Few molecular events important to platelet biogenesis have been identified. Mice homozygous for the spontaneous, recessive mutation Gunmetal (gm) have prolonged bleeding, thrombocytopenia, and reduced platelet α- and δ-granule contents. Here we show by positional cloning that gm results from a G→A substitution mutation in a splice acceptor site within the α-subunit of Rab geranylgeranyl transferase (Rabggta), an enzyme that attaches geranylgeranyl groups to Rab proteins. Most Rabggta mRNAs from gm tissues skipped exon 1 and lacked a start codon. Rabggta protein and Rab geranylgeranyl transferase (GGTase) activity were reduced 4-fold in gm platelets. Geranylgeranylation and membrane association of Rab27, a Rab GGTase substrate, were significantly decreased in gm platelets. These findings indicate that geranylgeranylation of Rab GTPases is critical for hemostasis. Rab GGTase inhibition may represent a new treatment for thrombocytosis and clotting disorders.

  • Abnormal vesicular trafficking in mouse models of Hermansky-Pudlak syndrome.
    Pigment Cell Research, 2000
    Co-Authors: Richard T. Swank, Edward K. Novak, Michael P. Mcgarry, Qing Zhang, Yuke Zhang, Lijun Feng
    Abstract:

    Hermansky-Pudlak Syndrome (HPS) is a group of related multigenic recessively inherited disorders which causes abnormalities in the biosynthesis and/or function of three related organelles; melanosomes, platelet-dense granules and lysosomes. These lead, in turn, to hypopigmentation, prolonged bleeding and ceroid deposition. Positional cloning strategies have identified five mouse HPS genes. Two orthologous human diseases (HPS1 and HPS2) have likewise been identified. At least four of the five mouse genes encode proteins involved in the regulation of intracellular vesicle trafficking. The pearl (HPS2) and mocha genes encode the beta3A and delta subunits, respectively, of the AP-3 adaptor complex, which captures organelle membrane proteins at the trans-Golgi apparatus. The protein products of the pallid and Gunmetal genes are also important components of the vesicular trafficking machinery. The former interacts with a t-SNARE, syntaxin13, and the latter is the alpha subunit of Rab geranylgeranyltransferase, which renders Rab proteins sufficiently lipophilic to function at their target membranes. The pale ear (HPS1) gene encodes a ubiquitously expressed protein of unknown function. Recent physiological studies have shown that mouse HPS mutants, like their human HPS counterparts, have variably reduced lifespans and may have lung abnormalities.

The Betty Smithers Design Collection At Staffordshire University - One of the best experts on this subject based on the ideXlab platform.

  • Hitachi RM-2400PR Rechargeable Shaver
    Staffordshire University, 2010
    Co-Authors: Staffordshire University, The Betty Smithers Design Collection At Staffordshire University
    Abstract:

    Compact shaver with tilting head. Gunmetal grey plastic housing. Marked 'Hitachi' and 'Phase 2' On/Off controls in upper half of front, Gunmetal/clear plastic cap to protect foil head. In original box with instructions, electrical charger and cleaning brush.. Maker: Hitachi. Date: 1990 - 1999 - from the The Betty Smithers Design Collection at Staffordshire University

  • Electrolux Airstream 1000 Vacuum Cleaner
    Staffordshire University, 2010
    Co-Authors: Staffordshire University, The Betty Smithers Design Collection At Staffordshire University
    Abstract:

    Vacuum cleaner and attachments in Gunmetal plastic housing. Maker: Electrolux. Date: 1993 - 1996 - from the The Betty Smithers Design Collection at Staffordshire University.

  • Sharp PA-W1410 Word Processor
    Staffordshire University, 2010
    Co-Authors: Staffordshire University, The Betty Smithers Design Collection At Staffordshire University
    Abstract:

    Word processor in Gunmetal grey plastic housing. Large LCD display with backlight. Maker: Sharp. Date: 1995 - 1999 - from the The Betty Smithers Design Collection at Staffordshire University

  • Autolux 300 Slide Projector
    Staffordshire University, 2010
    Co-Authors: Staffordshire University, The Betty Smithers Design Collection At Staffordshire University
    Abstract:

    Slide projector; grey metal housing on Gunmetal grey base with red top.Two switched; one black, one white.. Maker: Autolux. Date: 1960 (circa) - from the The Betty Smithers Design Collection at Staffordshire University

  • Macintosh PowerBook 190 and Case
    Staffordshire University, 2010
    Co-Authors: Staffordshire University, The Betty Smithers Design Collection At Staffordshire University
    Abstract:

    Laptop computer in Gunmetal coloured plastic housing with Apple logo to top. 24cm screen inside top above QWERTY keyboard. Labelled (below screen) McIntosh PowerBook 190. Maker: Apple Computers - from the The Betty Smithers Design Collection at Staffordshire University.

Miguel C. Seabra - One of the best experts on this subject based on the ideXlab platform.

  • The Gunmetal mouse reveals Rab geranylgeranyl transferase to be the major molecular target of phosphonocarboxylate analogues of bisphosphonates
    Bone, 2011
    Co-Authors: Fraser P. Coxon, Miguel C. Seabra, Adam Taylor, Charlotte A. Stewart, Rudi Baron, F. Hal Ebetino, Michael J. Rogers
    Abstract:

    article The described ability of phosphonocarboxylate analogues of bisphosphonates (BPs) to inhibit Rab geranylgeranyl transferase (RGGT) is thought to be the mechanism underlying their cellular effects, including their ability to reduce macrophage cell viability and to inhibit osteoclast-mediated resorption. However, until now the possibility that at least some of the effects of these drugs may be mediated through other targets has not been excluded. Since RGGT is the most distal enzyme in the process of Rab prenylation, it has not proved possible to confirm the mechanism underlying the effects of these drugs by adding back downstream intermediates of the mevalonate pathway, the approach used to demonstrate that bisphosphonates act through this pathway. We now confirm that RGGT is the major pharmacological target of phosphonocarbox- ylates by using several alternative approaches. Firstly, analysis of several different phosphonocarboxylate drugs demonstrates a very good correlation between the ability of these drugs to inhibit RGGT with their ability to: (a) reduce macrophage cell viability; (b) induce apoptosis; and (c) induce vacuolation in rabbit osteoclasts. Secondly, we have found that cells from the Gunmetal (gm/gm) mouse, which bear a homozygous mutation in RGGT that results in ~80% reduced activity of this enzyme compared to wild-type or heterozygous mice, are more sensitive to the effects of active phosphonocarboxylates (including reducing macrophage cell viability, inhibiting osteoclast formation and inhibiting fluid-phase endocytosis), confirming that these effects are mediated through inhibition of RGGT. In conclusion, these data demonstrate that all of the pharmacological effects of phosphonocarboxylates found thus far appear to be mediated through the specific inhibition of RGGT, highlighting the potential therapeutic value of this class of drugs. This article is part of a Special Issue entitled Bisphosphonates.

  • Impaired prenylation of Rab GTPases in the Gunmetal mouse causes defects in bone cell function.
    Small GTPases, 2011
    Co-Authors: Adam Taylor, Emilie H. Mules, Miguel C. Seabra, Miep H. Helfrich, Michael J. Rogers, Fraser P. Coxon
    Abstract:

    Vesicular trafficking is crucial for bone resorption by osteoclasts, in particular for formation of the ruffled border membrane and for removal of the resultant bone degradation products by transcytosis. These processes are regulated by Rab family GTPases, whose activity is dependent on post-translational prenylation by Rab geranylgeranyl transferase (RGGT). Specific pharmacological inhibition of RGGT inhibits bone resorption in vitro and in vivo, illustrating the importance of Rab prenylation for osteoclast function. The Gunmetal (gm/gm) mouse bears a mutation in the catalytic subunit of RGGT, causing a loss of 80% of the activity of this enzyme and hence hypoprenylation of several Rabs in melanocytes, platelets and cytotoxic T cells. We have now found that prenylation of several Rab proteins is also defective in gm/gm osteoclasts. Moreover, while osteoclast formation and cytoskeletal polarization occurs normally, gm/gm osteoclasts exhibit a substantial reduction in resorptive activity in vitro compared ...

  • Multiple factors contribute to inefficient prenylation of Rab27a in Rab prenylation diseases.
    The Journal of biological chemistry, 2003
    Co-Authors: Banafshé Larijani, Alistair N. Hume, Abul K. Tarafder, Miguel C. Seabra
    Abstract:

    Abstract Post-translational geranylgeranylation of Rab GT-Pases is essential for their membrane association and function as regulators of intracellular vesicular transport. The reaction is catalyzed by Rab geranylgeranyltransferase (RGGT) and is assisted by the Rab escort proteins (REP), which form stable complexes with newly synthesized GDP-bound Rabs. Two genetic diseases involve the Rab geranylgeranylation machinery: choroideremia, an X-linked retinal degeneration resulting from loss-of-function mutations in REP1, and Gunmetal, a mouse model of Hermansky-Pudlak syndrome resulting from mutations in the α-subunit of RGGT. A small subset of Rab proteins is selectively under-prenylated in both diseases, most notably Rab27a. Here we analyze why Rab27a is selectively affected in diseases of Rab geranylgeranylation. Semi-quantitative immunoblotting suggests that mass action, i.e. the amount of Rab27a relative to other Rabs, is unlikely to be a factor as the expression level of Rab27a is similar to other Rabs not affected in these diseases. In vitro binding assays and fluorescence resonance energy transfer detected by fluorescence lifetime imaging microscopy in intact cells demonstrate that Rab27a binds equally well to both REP1 and REP2, suggesting differential affinity of Rab27a for REP isoforms is not an important factor. However, steady-state kinetic analysis of the geranylgeranylation reaction indicates that REP2-Rab27a has lower affinity for RGGT compared with REP1-Rab27a. Furthermore, we show that Rab27a has relatively low GTPase activity, presumably decreasing the affinity of the REP interaction in vivo. We suggest that the restricted phenotypes observed in these diseases result from multiple contributing factors.

  • A role for Rab27b in NF-E2-dependent pathways of platelet formation
    Blood, 2003
    Co-Authors: Sanjay Tiwari, Duarte C. Barral, Emilie H. Mules, Miguel C. Seabra, Edward K. Novak, Richard T. Swank, Joseph E. Italiano, Ramesh A. Shivdasani
    Abstract:

    Megakaryocytes release platelets by reorganizing the cytoplasm into proplatelet extensions. Fundamental to this process is the need to coordinate transport of products and organelles in the appropriate abundance to nascent platelets. The importance of the Rab family of small GTPases (guanosine 5'-triphosphatases) in platelet biogenesis is revealed in Gunmetal (gm/gm) mice, which show deficient Rab isoprenylation and macrothrombocytopenia with few granules and abnormal megakaryocyte morphology. Although some Rab proteins are implicated in vesicle and organelle transport along microtubules or actin, the role of any Rab protein in platelet biogenesis is unknown. The limited number of Rab proteins with defective membrane association in gm/gm megakaryocytes prominently includes Rab27a and Rab27b. Normal expression of Rab27b is especially increased with terminal megakaryocyte differentiation and dependent on nuclear factor-erythroid 2 (NF-E2), a transcription factor required for thrombopoiesis. Chromatin immunoprecipitation demonstrates recruitment of NF-E2 to the putative Rab27B promoter. Inhibition of endogenous Rab27 function in primary megakaryocytes causes severe quantitative and qualitative defects in proplatelet formation that mimic findings in gm/gm cells. Rab27b localizes to alpha and dense granules in megakaryocytes. These results establish a role for Rab27 in platelet synthesis and suggest that Rab27b in particular may coordinate proplatelet formation with granule transport, possibly by recruiting specific effector pathways.

  • Rab27a is required for regulated secretion in cytotoxic T lymphocytes.
    The Journal of cell biology, 2001
    Co-Authors: Jane C. Stinchcombe, Duarte C. Barral, Emilie H. Mules, Sarah Booth, Alistair N. Hume, Laura M. Machesky, Miguel C. Seabra, Gillian M. Griffiths
    Abstract:

    Rab27a activity is affected in several mouse models of human disease including Griscelli (ashen mice) and Hermansky-Pudlak (Gunmetal mice) syndromes. A loss of function mutation occurs in the Rab27a gene in ashen (ash), whereas in Gunmetal (gm) Rab27a dysfunction is secondary to a mutation in the α subunit of Rab geranylgeranyl transferase, an enzyme required for prenylation and activation of Rabs. We show here that Rab27a is normally expressed in cytotoxic T lymphocytes (CTLs), but absent in ashen homozygotes (ash/ash). Cytotoxicity and secretion assays show that ash/ash CTLs are unable to kill target cells or to secrete granzyme A and hexosaminidase. By immunofluorescence and electron microscopy, we show polarization but no membrane docking of ash/ash lytic granules at the immunological synapse. In Gunmetal CTLs, we show underprenylation and redistribution of Rab27a to the cytosol, implying reduced activity. Gunmetal CTLs show a reduced ability to kill target cells but retain the ability to secrete hexosaminidase and granzyme A. However, only some of the granules polarize to the immunological synapse, and many remain dispersed around the periphery of the CTLs. These results demonstrate that Rab27a is required in a final secretory step and that other Rab proteins also affected in Gunmetal are likely to be involved in polarization of the granules to the immunological synapse.