The Experts below are selected from a list of 681 Experts worldwide ranked by ideXlab platform
Craig Blackstone - One of the best experts on this subject based on the ideXlab platform.
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Protein Targeting: ER Leads the Way to the Inner Nuclear Envelope
Current Biology, 2017Co-Authors: Craig BlackstoneAbstract:Efficient targeting of newly synthesized membrane proteins from the endoplasmic reticulum to the inner nuclear membrane depends on nucleotide hydrolysis. A new study shows that this dependence reflects critical actions of the Atlastin family of GTPases in maintaining the morphology of the endoplasmic reticulum network.
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Mammalian knock out cells reveal prominent roles for Atlastin GTPases in ER network morphology.
Experimental Cell Research, 2016Co-Authors: Guohua Zhao, Peng-peng Zhu, Benoît Renvoisé, Lymarie Maldonado-báez, Seonghee Park, Craig BlackstoneAbstract:Atlastins are large, membrane-bound GTPases that participate in the fusion of endoplasmic reticulum (ER) tubules to generate the polygonal ER network in eukaryotes. They also regulate lipid droplet size and inhibit bone morphogenetic protein (BMP) signaling, though mechanisms remain unclear. Humans have three Atlastins (ATL1, ATL2, and ATL3), and ATL1 and ATL3 are mutated in autosomal dominant hereditary spastic paraplegia and hereditary sensory neuropathies. Cellular investigations of Atlastin orthologs in most yeast, plants, flies and worms are facilitated by the presence of a single or predominant isoform, but loss-of-function studies in mammalian cells are complicated by multiple, broadly-expressed paralogs. We have generated mouse NIH-3T3 cells lacking all three mammalian Atlastins (Atl1/2/3) using CRISPR/Cas9-mediated gene knockout (KO). ER morphology is markedly disrupted in these triple KO cells, with prominent impairment in formation of three-way ER tubule junctions. This phenotype can be rescued by expression of distant orthologs from Saccharomyces cerevisiae (Sey1p) and Arabidopsis (ROOT HAIR DEFECTIVE3) as well as any one of the three human Atlastins. Minimal, if any, changes are observed in the morphology of mitochondria and the Golgi apparatus. Alterations in BMP signaling and increased sensitivity to ER stress are also noted, though effects appear more modest. Finally, Atlastins appear required for the proper differentiation of NIH-3T3 cells into an adipocyte-like phenotype. These findings have important implications for the pathogenesis of hereditary spastic paraplegias and sensory neuropathies associated with Atlastin mutations.
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Atlastin GTPases are required for Golgi apparatus and ER morphogenesis. Hum Mol Genet 2008;17:1591–1604
2014Co-Authors: Neggy Rismanchi, Peng-peng Zhu, Cynthia Soderblom, Julia Stadler, Craig BlackstoneAbstract:The hereditary spastic paraplegias (SPG1-33) comprise a cluster of inherited neurological disorders characterized principally by lower extremity spasticity and weakness due to a length-dependent, retrograde axonopathy of corticospinal motor neurons. Mutations in the gene encoding the large oligomeric GTPase Atlastin-1 are responsible for SPG3A, a common autosomal dominant hereditary spastic paraplegia. Here we describe a family of human GTPases, Atlastin-2 and-3 that are closely related to Atlastin-1. Interestingly, while Atlastin-1 is predominantly localized to vesicular tubular complexes and cis-Golgi cister-nae, mostly in brain, Atlastin-2 and-3 are localized to the endoplasmic reticulum (ER) and are most enriched in other tissues. Knockdown of Atlastin-2 and-3 levels in HeLa cells using siRNA (small interfering RNA) causes disruption of Golgi morphology, and these Golgi structures remain sensitive to brefeldin A treatment. Interestingly, expression of SPG3A mutant or dominant-negative Atlastin proteins lacking GTPase activity causes prominent inhibition of ER reticularization, suggesting a role for Atlastin GTPases in the formation of three-way junctions in the ER. However, secretory pathway trafficking as assessed using vesicular stoma-titis virus G protein fused to green fluorescent protein (VSVG-GFP) as a reporter was essentially normal in both knockdown and dominant-negative overexpression conditions for all Atlastins. Thus, the Atlastin family of GTPases functions prominently in both ER and Golgi morphogenesis, but they do not appear t
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pharmacologic rescue of axon growth defects in a human ipsc model of hereditary spastic paraplegia spg3a
Human Molecular Genetics, 2014Co-Authors: Kyle R Denton, Tyler Mark Pierson, Xue-jun Li, Craig BlackstoneAbstract:Hereditary spastic paraplegias are a large, diverse group of neurological disorders (SPG1-71) with the unifying feature of prominent lower extremity spasticity, owing to a length-dependent axonopathy of corticospinal motor neurons. The most common early-onset form of pure, autosomal dominant hereditary spastic paraplegia is caused by mutation in the ATL1 gene encoding the Atlastin-1 GTPase, which mediates homotypic fusion of ER tubules to form the polygonal ER network. We have identified a p.Pro342Ser mutation in a young girl with pure SPG3A. This residue is in a critical hinge region of Atlastin-1 between its GTPase and assembly domains, and it is conserved in all known eukaryotic Atlastin orthologs. We produced induced pluripotent stem cells from skin fibroblasts and differentiated these into forebrain neurons to generate a human neuronal model for SPG3A. Axons of these SPG3A neurons showed impaired growth, recapitulating axonal defects in Atlastin-1-depleted rat cortical neurons and impaired root hair growth in loss-of-function mutants of the ATL1 ortholog rhd3 in the plant Arabidopsis. Both the microtubule cytoskeleton and tubular ER are important for mitochondrial distribution and function within cells, and SPG3A neurons showed alterations in mitochondrial motility. Even so, it is not clear whether this change is involved in disease pathogenesis. The SPG3A axon growth defects could be rescued with microtubule-binding agents, emphasizing the importance of tubular ER interactions with the microtubule cytoskeleton in hereditary spastic paraplegia pathogenesis. The prominent alterations in axon growth in SPG3A neurons may represent a particularly attractive target for suppression in screens for novel pharmacologic agents.
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hereditary spastic paraplegia proteins reep1 spastin and Atlastin 1 coordinate microtubule interactions with the tubular er network
Journal of Clinical Investigation, 2010Co-Authors: Seong H. Park, Rell L Parker, Craig BlackstoneAbstract:Hereditary spastic paraplegias (HSPs; SPG1–45) are inherited neurological disorders characterized by lower extremity spastic weakness. More than half of HSP cases result from autosomal dominant mutations in Atlastin-1 (also known as SPG3A), receptor expression enhancing protein 1 (REEP1; SPG31), or spastin (SPG4). The Atlastin-1 GTPase interacts with spastin, a microtubule-severing ATPase, as well as with the DP1/Yop1p and reticulon families of ER-shaping proteins, and SPG3A caused by Atlastin-1 mutations has been linked pathogenically to abnormal ER morphology. Here we investigated SPG31 by analyzing the distribution, interactions, and functions of REEP1. We determined that REEP1 is structurally related to the DP1/Yop1p family of ER-shaping proteins and localizes to the ER in cultured rat cerebral cortical neurons, where it colocalizes with spastin and Atlastin-1. Upon overexpression in COS7 cells, REEP1 formed protein complexes with Atlastin-1 and spastin within the tubular ER, and these interactions required hydrophobic hairpin domains in each of these proteins. REEP proteins were required for ER network formation in vitro, and REEP1 also bound microtubules and promoted ER alignment along the microtubule cytoskeleton in COS7 cells. A SPG31 mutant REEP1 lacking the C-terminal cytoplasmic region did not interact with microtubules and disrupted the ER network. These data indicate that the HSP proteins Atlastin-1, spastin, and REEP1 interact within the tubular ER membrane in corticospinal neurons to coordinate ER shaping and microtubule dynamics. Thus, defects in tubular ER shaping and network interactions with the microtubule cytoskeleton seem to be the predominant pathogenic mechanism of HSP.
James A Mcnew - One of the best experts on this subject based on the ideXlab platform.
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Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays.
Journal of Visualized Experiments, 2019Co-Authors: Miguel A. Betancourt-solis, James A McnewAbstract:Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila Atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila Atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for Atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.
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the Atlastin membrane anchor forms an intramembrane hairpin that does not span the phospholipid bilayer
Journal of Biological Chemistry, 2018Co-Authors: Miguel A Betancourtsolis, Tanvi Desai, James A McnewAbstract:: The endoplasmic reticulum (ER) is composed of flattened sheets and interconnected tubules that extend throughout the cytosol and makes physical contact with all other cytoplasmic organelles. This cytoplasmic distribution requires continuous remodeling. These discrete ER morphologies require specialized proteins that drive and maintain membrane curvature. The GTPase Atlastin is required for homotypic fusion of ER tubules. All Atlastin homologs possess a conserved domain architecture consisting of a GTPase domain, a three-helix bundle middle domain, a hydrophobic membrane anchor, and a C-terminal cytosolic tail. Here, we examined several Drosophila-human Atlastin chimeras to identify functional domains of human Atlastin-1 in vitro Although all chimeras could hydrolyze GTP, only chimeras containing the human C-terminal tail, hydrophobic segments, or both could fuse membranes in vitro We also determined that co-reconstitution of Atlastin with reticulon does not influence GTPase activity or membrane fusion. Finally, we found that both human and Drosophila Atlastin hydrophobic membrane anchors do not span the membrane, but rather form two intramembrane hairpin loops. The topology of these hairpins remains static during membrane fusion and does not appear to play an active role in lipid mixing.
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The Atlastin membrane anchor forms an intramembrane hairpin that does not span the phospholipid bilayer.
Journal of Biological Chemistry, 2018Co-Authors: Miguel A. Betancourt-solis, Tanvi Desai, James A McnewAbstract:The endoplasmic reticulum (ER) is composed of flattened sheets and interconnected tubules that extend throughout the cytosol and makes physical contact with all other cytoplasmic organelles. This cytoplasmic distribution requires continuous remodeling. These discrete ER morphologies require specialized proteins that drive and maintain membrane curvature. The GTPase Atlastin is required for homotypic fusion of ER tubules. All Atlastin homologs possess a conserved domain architecture consisting of a GTPase domain, a three-helix bundle middle domain, a hydrophobic membrane anchor, and a C-terminal cytosolic tail. Here, we examined several Drosophila-human Atlastin chimeras to identify functional domains of human Atlastin-1 in vitro Although all chimeras could hydrolyze GTP, only chimeras containing the human C-terminal tail, hydrophobic segments, or both could fuse membranes in vitro We also determined that co-reconstitution of Atlastin with reticulon does not influence GTPase activity or membrane fusion. Finally, we found that both human and Drosophila Atlastin hydrophobic membrane anchors do not span the membrane, but rather form two intramembrane hairpin loops. The topology of these hairpins remains static during membrane fusion and does not appear to play an active role in lipid mixing.
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The effects of ER morphology on synaptic structure and function in Drosophila melanogaster.
Journal of Cell Science, 2016Co-Authors: James B. Summerville, Diana Pendin, Andrea Daga, Joseph E. Faust, Ethan Fan, Joseph Formella, Michael E. Stern, James A McnewAbstract:Hereditary spastic paraplegia (HSP) is a set of genetic diseases caused by mutations in one of 72 genes that results in age-dependent corticospinal axon degeneration accompanied by spasticity and paralysis. Two genes implicated in HSPs encode proteins that regulate endoplasmic reticulum (ER) morphology. Atlastin 1 (ATL1, also known as SPG3A) encodes an ER membrane fusion GTPase and reticulon 2 (RTN2, also known as SPG12) helps shape ER tube formation. Here, we use a new fluorescent ER marker to show that the ER within wild-type Drosophila motor nerve terminals forms a network of tubules that is fragmented and made diffuse upon loss of the Atlastin 1 ortholog atl. atl or Rtnl1 loss decreases evoked transmitter release and increases arborization. Similar to other HSP proteins, Atl inhibits bone morphogenetic protein (BMP) signaling, and loss of atl causes age-dependent locomotor deficits in adults. These results demonstrate a crucial role for ER in neuronal function, and identify mechanistic links between ER morphology, neuronal function, BMP signaling and adult behavior.
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The Atlastin C-terminal Tail Is an Amphipathic Helix That Perturbs the Bilayer Structure during Endoplasmic Reticulum Homotypic Fusion
Journal of Biological Chemistry, 2015Co-Authors: Joseph E. Faust, Tyler J Moss, Idil Ulengin, Miguel A. Betancourt-solis, Tanvi Desai, Avani Verma, Tzu-lin Sun, Huey W. Huang, Tina Lee, James A McnewAbstract:Fusion of tubular membranes is required to form three-way junctions found in reticular subdomains of the endoplasmic reticulum. The large GTPase Atlastin has recently been shown to drive endoplasmic reticulum membrane fusion and three-way junction formation. The mechanism of Atlastin-mediated membrane fusion is distinct from SNARE-mediated membrane fusion, and many details remain unclear. In particular, the role of the amphipathic C-terminal tail of Atlastin is still unknown. We found that a peptide corresponding to the Atlastin C-terminal tail binds to membranes as a parallel α helix, induces bilayer thinning, and increases acyl chain disorder. The function of the C-terminal tail is conserved in human Atlastin. Mutations in the C-terminal tail decrease fusion activity in vitro, but not GTPase activity, and impair Atlastin function in vivo. In the context of unstable lipid bilayers, the requirement for the C-terminal tail is abrogated. These data suggest that the C-terminal tail of Atlastin locally destabilizes bilayers to facilitate membrane fusion.
Evan Reid - One of the best experts on this subject based on the ideXlab platform.
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paraplegia, are
2016Co-Authors: Christopher M. S, James W Connell, Thomas L Edwards, Simon Duley, Paul J Luzio, Nicholas A, A Thompson, Evan ReidAbstract:and Atlastin, two proteins mutated in autosomal dominant hereditary spasti
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four novel spg3a Atlastin mutations identified in autosomal dominant hereditary spastic paraplegia kindreds with intra familial variability in age of onset and complex phenotype
Clinical Genetics, 2009Co-Authors: Bradley N. Smith, Simon Bevan, Caroline Vance, P Renwick, P Wilkinson, Christos Proukakis, F Squitieri, Alfredo Berardelli, Thomas T. Warner, Evan ReidAbstract:Mutation of the Atlastin gene (SPG3A) is responsible for ∼10% of autosomal dominant hereditary spastic paraplegia (AD-HSP) cases. The goal of this study was to identify novel disease causing Atlastin mutations. Atlastin nucleotide variations were detected by direct sequencing of all 14 exons in 70 autosomal dominant (AD), 16 single sibship and 14 sporadic spastic paraplegia patients. Six mis-sense mutations (four of which were novel) were identified in six unrelated AD-HSP kindreds in exons 4, 7 and 8 of the Atlastin gene. One kindred with a novel mutation showed variability in clinical phenotype and age of onset. Mutations are predicted to decrease GTPase activity, cause morphological abnormalities of the endoplasmic reticulum and prevent maturation of the Golgi complex resulting in impaired vesicle trafficking. Our study significantly adds to the spectrum of mutations and clinical phenotype of SPG3A. We advocate that all spastin mutation negative AD-HSP kindreds should be screened for pathogenic Atlastin mutations regardless of age of onset or phenotypic complexity.
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Four novel SPG3A/Atlastin mutations identified in autosomal dominant hereditary spastic paraplegia kindreds with intra-familial variability in age of onset and complex phenotype.
Clinical Genetics, 2009Co-Authors: Bradley N. Smith, Simon Bevan, Caroline Vance, P Renwick, P Wilkinson, Christos Proukakis, F Squitieri, Alfredo Berardelli, Thomas T. Warner, Evan ReidAbstract:Mutation of the Atlastin gene (SPG3A) is responsible for ∼10% of autosomal dominant hereditary spastic paraplegia (AD-HSP) cases. The goal of this study was to identify novel disease causing Atlastin mutations. Atlastin nucleotide variations were detected by direct sequencing of all 14 exons in 70 autosomal dominant (AD), 16 single sibship and 14 sporadic spastic paraplegia patients. Six mis-sense mutations (four of which were novel) were identified in six unrelated AD-HSP kindreds in exons 4, 7 and 8 of the Atlastin gene. One kindred with a novel mutation showed variability in clinical phenotype and age of onset. Mutations are predicted to decrease GTPase activity, cause morphological abnormalities of the endoplasmic reticulum and prevent maturation of the Golgi complex resulting in impaired vesicle trafficking. Our study significantly adds to the spectrum of mutations and clinical phenotype of SPG3A. We advocate that all spastin mutation negative AD-HSP kindreds should be screened for pathogenic Atlastin mutations regardless of age of onset or phenotypic complexity.
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spastin and Atlastin two proteins mutated in autosomal dominant hereditary spastic paraplegia are binding partners
Human Molecular Genetics, 2006Co-Authors: Christopher M Sanderson, James W Connell, Thomas L Edwards, Nicholas A Bright, Simon Duley, Amanda Thompson, Paul J Luzio, Evan ReidAbstract:The pure hereditary spastic paraplegias (HSPs) are a group of conditions in which there is progressive length-dependent degeneration of the distal ends of the corticospinal tract axons, resulting in spastic paralysis of the legs. Pure HSPs are most frequently inherited in an autosomal dominant pattern and are commonly caused by mutations in either the SPG4 gene spastin or in the SPG3A gene Atlastin. To identify binding partners for spastin, we carried out a yeast two-hybrid screen on a brain cDNA library, using spastin as bait. Remarkably, nearly all of the positive interacting prey clones coded for Atlastin. We have verified the physiological relevance of this interaction using co-immunoprecipitation, GST-pull down and intracellular co-localisation experiments. We show that the spastin domain required for binding to Atlastin lies within the N-terminal 80 residues of the protein, a region that is only present in the predominantly cytoplasmic full-length spastin isoform. These data suggest that spastin and Atlastin function in the same biochemical pathway and that it is the cytoplasmic function of spastin which is important for the pathogenesis of HSP. They also provide further evidence for a physiological and pathological role for spastin in membrane dynamics.
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Spastin and Atlastin, two proteins mutated in autosomal-dominant hereditary spastic paraplegia, are binding partners
Human Molecular Genetics, 2005Co-Authors: Christopher M Sanderson, James W Connell, Thomas L Edwards, Nicholas A Bright, Simon Duley, Amanda Thompson, J. Paul Luzio, Evan ReidAbstract:The pure hereditary spastic paraplegias (HSPs) are a group of conditions in which there is a progressive length-dependent degeneration of the distal ends of the corticospinal tract axons, resulting in spastic paralysis of the legs. Pure HSPs are most frequently inherited in an autosomal-dominant pattern and are commonly caused by mutations either in the SPG4 gene spastin or in the SPG3A gene Atlastin. To identify binding partners for spastin, we carried out a yeast two-hybrid screen on a brain cDNA library, using spastin as bait. Remarkably, nearly all of the positive interacting prey clones coded for Atlastin. We have verified the physiological relevance of this interaction using co-immunoprecipitation, glutathione S-transferase pull-down and intracellular co-localization experiments. We show that the spastin domain required for binding to Atlastin lies within the N-terminal 80 residues of the protein, a region that is only present in the predominantly cytoplasmic, full-length spastin isoform. These data suggest that spastin and Atlastin function in the same biochemical pathway and that it is the cytoplasmic function of spastin which is important for the pathogenesis of HSP. They also provide further evidence for a physiological and pathological role of spastin in membrane dynamics.
Peng-peng Zhu - One of the best experts on this subject based on the ideXlab platform.
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The Atlastin ER-shaping proteins facilitate Zika virus replication
Journal of Virology, 2019Co-Authors: Blandine Monel, Peng-peng Zhu, Maaran Michael Rajah, Mohamed-lamine Hafirassou, Samy Sid-ahmed, Julien Burlaud-gaillard, Quentin Nevers, Julian Buchrieser, Françoise Porrot, Cécile MeunierAbstract:The endoplasmic reticulum (ER) is the site for Zika virus (ZIKV) replication and is central to the cytopathic effects observed in infected cells. ZIKV induces the formation of ER-derived large cytoplasmic vacuoles followed by “implosive” cell death. Little is known about the nature of the ER factors that regulate flavivirus replication. Atlastins (ATL1, -2, and -3) are dynamin-related GTPases that control the structure and the dynamics of the ER membrane. We show here that ZIKV replication is significantly decreased in the absence of ATL proteins. The appearance of infected cells is delayed, the levels of intracellular viral proteins and released virus are reduced, and the cytopathic effects are strongly impaired. We further show that ATL3 is recruited to viral replication sites and interacts with the nonstructural viral proteins NS2A and NS2B3. Thus, proteins that shape and maintain the ER tubular network ensure efficient ZIKV replication. IMPORTANCE Zika virus (ZIKV) is an emerging virus associated with Guillain-Barré syndrome, and fetal microcephaly as well as other neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We found that endoplasmic reticulum (ER)-shaping Atlastin proteins (ATL1, -2, and -3), which induce ER membrane fusion, facilitate ZIKV replication. We show that ATL3 is recruited to the viral replication site and colocalize with the viral proteins NS2A and NS2B3. The results provide insights into host factors used by ZIKV to enhance its replication.
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Atlastin Endoplasmic Reticulum-Shaping Proteins Facilitate Zika Virus Replication.
Journal of Virology, 2019Co-Authors: Blandine Monel, Peng-peng Zhu, Maaran Michael Rajah, Mohamed-lamine Hafirassou, Julien Burlaud-gaillard, Quentin Nevers, Julian Buchrieser, Françoise Porrot, Samy Sid Ahmed, Cécile MeunierAbstract:The endoplasmic reticulum (ER) is the site for Zika virus (ZIKV) replication and is central to the cytopathic effects observed in infected cells. ZIKV induces the formation of ER-derived large cytoplasmic vacuoles followed by "implosive" cell death. Little is known about the nature of the ER factors that regulate flavivirus replication. Atlastins (ATL1, -2, and -3) are dynamin-related GTPases that control the structure and the dynamics of the ER membrane. We show here that ZIKV replication is significantly decreased in the absence of ATL proteins. The appearance of infected cells is delayed, the levels of intracellular viral proteins and released virus are reduced, and the cytopathic effects are strongly impaired. We further show that ATL3 is recruited to viral replication sites and interacts with the nonstructural viral proteins NS2A and NS2B3. Thus, proteins that shape and maintain the ER tubular network ensure efficient ZIKV replication.IMPORTANCE Zika virus (ZIKV) is an emerging virus associated with Guillain-Barre syndrome, and fetal microcephaly as well as other neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We found that endoplasmic reticulum (ER)-shaping Atlastin proteins (ATL1, -2, and -3), which induce ER membrane fusion, facilitate ZIKV replication. We show that ATL3 is recruited to the viral replication site and colocalize with the viral proteins NS2A and NS2B3. The results provide insights into host factors used by ZIKV to enhance its replication.
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Mammalian knock out cells reveal prominent roles for Atlastin GTPases in ER network morphology.
Experimental Cell Research, 2016Co-Authors: Guohua Zhao, Peng-peng Zhu, Benoît Renvoisé, Lymarie Maldonado-báez, Seonghee Park, Craig BlackstoneAbstract:Atlastins are large, membrane-bound GTPases that participate in the fusion of endoplasmic reticulum (ER) tubules to generate the polygonal ER network in eukaryotes. They also regulate lipid droplet size and inhibit bone morphogenetic protein (BMP) signaling, though mechanisms remain unclear. Humans have three Atlastins (ATL1, ATL2, and ATL3), and ATL1 and ATL3 are mutated in autosomal dominant hereditary spastic paraplegia and hereditary sensory neuropathies. Cellular investigations of Atlastin orthologs in most yeast, plants, flies and worms are facilitated by the presence of a single or predominant isoform, but loss-of-function studies in mammalian cells are complicated by multiple, broadly-expressed paralogs. We have generated mouse NIH-3T3 cells lacking all three mammalian Atlastins (Atl1/2/3) using CRISPR/Cas9-mediated gene knockout (KO). ER morphology is markedly disrupted in these triple KO cells, with prominent impairment in formation of three-way ER tubule junctions. This phenotype can be rescued by expression of distant orthologs from Saccharomyces cerevisiae (Sey1p) and Arabidopsis (ROOT HAIR DEFECTIVE3) as well as any one of the three human Atlastins. Minimal, if any, changes are observed in the morphology of mitochondria and the Golgi apparatus. Alterations in BMP signaling and increased sensitivity to ER stress are also noted, though effects appear more modest. Finally, Atlastins appear required for the proper differentiation of NIH-3T3 cells into an adipocyte-like phenotype. These findings have important implications for the pathogenesis of hereditary spastic paraplegias and sensory neuropathies associated with Atlastin mutations.
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Atlastin GTPases are required for Golgi apparatus and ER morphogenesis. Hum Mol Genet 2008;17:1591–1604
2014Co-Authors: Neggy Rismanchi, Peng-peng Zhu, Cynthia Soderblom, Julia Stadler, Craig BlackstoneAbstract:The hereditary spastic paraplegias (SPG1-33) comprise a cluster of inherited neurological disorders characterized principally by lower extremity spasticity and weakness due to a length-dependent, retrograde axonopathy of corticospinal motor neurons. Mutations in the gene encoding the large oligomeric GTPase Atlastin-1 are responsible for SPG3A, a common autosomal dominant hereditary spastic paraplegia. Here we describe a family of human GTPases, Atlastin-2 and-3 that are closely related to Atlastin-1. Interestingly, while Atlastin-1 is predominantly localized to vesicular tubular complexes and cis-Golgi cister-nae, mostly in brain, Atlastin-2 and-3 are localized to the endoplasmic reticulum (ER) and are most enriched in other tissues. Knockdown of Atlastin-2 and-3 levels in HeLa cells using siRNA (small interfering RNA) causes disruption of Golgi morphology, and these Golgi structures remain sensitive to brefeldin A treatment. Interestingly, expression of SPG3A mutant or dominant-negative Atlastin proteins lacking GTPase activity causes prominent inhibition of ER reticularization, suggesting a role for Atlastin GTPases in the formation of three-way junctions in the ER. However, secretory pathway trafficking as assessed using vesicular stoma-titis virus G protein fused to green fluorescent protein (VSVG-GFP) as a reporter was essentially normal in both knockdown and dominant-negative overexpression conditions for all Atlastins. Thus, the Atlastin family of GTPases functions prominently in both ER and Golgi morphogenesis, but they do not appear t
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Targeted High-Throughput Sequencing Identifies Mutations in Atlastin-1 as a Cause of Hereditary Sensory Neuropathy Type I
The American Journal of Human Genetics, 2011Co-Authors: Christian Guelly, Peng-peng Zhu, Lea Leonardis, Lea Papić, Janez Zidar, Maria Schabhüttl, Heimo Strohmaier, Joachim Weis, Tim M. Strom, Jonathan BaetsAbstract:Hereditary sensory neuropathy type I (HSN I) is an axonal form of autosomal-dominant hereditary motor and sensory neuropathy distinguished by prominent sensory loss that leads to painless injuries. Unrecognized, these can result in delayed wound healing and osteomyelitis, necessitating distal amputations. To elucidate the genetic basis of an HSN I subtype in a family in which mutations in the few known HSN I genes had been excluded, we employed massive parallel exon sequencing of the 14.3 Mb disease interval on chromosome 14q. We detected a missense mutation (c.1065C>A, p.Asn355Lys) in Atlastin-1 (ATL1), a gene that is known to be mutated in early-onset hereditary spastic paraplegia SPG3A and that encodes the large dynamin-related GTPase Atlastin-1. The mutant protein exhibited reduced GTPase activity and prominently disrupted ER network morphology when expressed in COS7 cells, strongly supporting pathogenicity. An expanded screen in 115 additional HSN I patients identified two further dominant ATL1 mutations (c.196G>C [p.Glu66Gln] and c.976 delG [p.Val326TrpfsX8]). This study highlights an unexpected major role for Atlastin-1 in the function of sensory neurons and identifies HSN I and SPG3A as allelic disorders.
Huanquan Zheng - One of the best experts on this subject based on the ideXlab platform.
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Efficient ER Fusion Requires a Dimerization and a C-Terminal Tail Mediated Membrane Anchoring of RHD3.
Plant Physiology, 2017Co-Authors: Jiaqi Sun, Huanquan ZhengAbstract:The endoplasmic reticulum (ER) is a network of tubules and sheets stretching throughout the eukaryotic cells. The formation of the ER requires homotypic membrane fusion, which is mediated by a family of Dynamin-like Atlastin GTPase proteins. The Arabidopsis (Arabidopsis thaliana) member ROOT HAIR DEFECTIVE3 (RHD3) has been demonstrated to mediate ER membrane fusion, but how exactly RHD3 is involved in the process is still unknown. Here we conducted systemic structure-function analyses of roles of different RHD3 domains in mediating ER fusion. We showed that efficient ER membrane fusion mediated by RHD3 requires a proper dimerization of RHD3 through the GTPase domain (GD) and the first and second three helix bundles (3HBs) in the middle domain. RHD3 has a 3HB-enriched middle domain longer than that of Atlastins, and we revealed that the third and fourth 3HBs are required for the stability of RHD3. The transmembrane segments of RHD3 are essential for targeting and retention of RHD3 in the ER and can also facilitate an oligomerization of RHD3. Furthermore, we showed that an amphipathic helix in the C-terminal cytosolic tail of RHD3 has a membrane anchoring ability that is required for efficient ER membrane fusion mediated by RHD3. This work contributes to a better understanding of a coordinated action of RHD3 in the fusion of ER membranes.
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A GTPase-Dependent Fine ER Is Required for Localized Secretion in Polarized Growth of Root Hairs1.
Plant Physiology, 2016Co-Authors: Jiaqi Sun, Huanquan ZhengAbstract:The endoplasmic reticulum (ER) is a cellular network comprising membrane tubules and sheets stretching throughout the cytoplasm. Atlastin GTPases, including Atlastin-1 in mammals and RHD3 in plants, play a role in the generation of the interconnected tubular ER network by promoting the fusion of ER tubules. Root hairs in rhd3 are short and wavy, a defect reminiscent of axon growth in cells with depleted Atlastin-1. However, how a loss in the ER complexity could lead to a defective polarized cell growth of root hairs or neurons remains elusive. Using live-cell imaging techniques, we reveal that, a fine ER distribution, which is found in the subapical zone of growing root hairs of wild-type plants, is altered to thick bundles in rhd3 The localized secretion to the apical dome as well as the apical localization of root hair growth regulator ROP2 is oscillated in rhd3 Interestingly, the shift of ROP2 precedes the shift of localized secretion as well as the fine ER distribution in rhd3 Our live imaging and pharmacologic modification of root hair growth defects in rhd3 suggest that there is interplay between the ER and microtubules in the polarized cell growth of root hairs. We hypothesize that, under the guidance of ROP2, RHD3, together with the action of microtubules, is required for the formation of a fine ER structure in the subapical zone of growing root hairs. This fine ER structure is essential for the localized secretion to the apical dome in polarized cell growth.
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The endoplasmic reticulum: a social network in plant cells.
Journal of integrative plant biology, 2012Co-Authors: Jun Chen, Caitlin Doyle, Huanquan ZhengAbstract:The endoplasmic reticulum (ER) is an interconnected network comprised of ribosome-studded sheets and smooth tubules. The ER plays crucial roles in the biosynthesis and transport of proteins and lipids, and in calcium (Ca2+) regulation in compartmentalized eukaryotic cells including plant cells. To support its well-segregated functions, the shape of the ER undergoes notable changes in response to both developmental cues and outside influences. In this review, we will discuss recent findings on molecular mechanisms underlying the unique morphology and dynamics of the ER, and the importance of the interconnected ER network in cell polarity. In animal and yeast cells, two family proteins, the reticulons and DP1/Yop1, are required for shaping high-curvature ER tubules, while members of the Atlastin family of dynamin-like GTPases are involved in the fusion of ER tubules to make an interconnected ER network. In plant cells, recent data also indicate that the reticulons are involved in shaping ER tubules, while RHD3, a plant member of the Atlastin GTPases, is required for the generation of an interconnected ER network. We will also summarize the current knowledge on how the ER interacts with other membrane-bound organelles, with a focus on how the ER and Golgi interplay in plant cells. [ Huanquan Zheng (Corresponding author)]
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The endoplasmic reticulum: a social network in plant cells.
Journal of integrative plant biology, 2012Co-Authors: Jun Chen, Caitlin Doyle, Huanquan ZhengAbstract:The endoplasmic reticulum (ER) is an interconnected network comprised of ribosome-studded sheets and smooth tubules. The ER plays crucial roles in the biosynthesis and transport of proteins and lipids, and in calcium (Ca(2+) ) regulation in compartmentalized eukaryotic cells including plant cells. To support its well-segregated functions, the shape of the ER undergoes notable changes in response to both developmental cues and outside influences. In this review, we will discuss recent findings on molecular mechanisms underlying the unique morphology and dynamics of the ER, and the importance of the interconnected ER network in cell polarity. In animal and yeast cells, two family proteins, the reticulons and DP1/Yop1, are required for shaping high-curvature ER tubules, while members of the Atlastin family of dynamin-like GTPases are involved in the fusion of ER tubules to make an interconnected ER network. In plant cells, recent data also indicate that the reticulons are involved in shaping ER tubules, while RHD3, a plant member of the Atlastin GTPases, is required for the generation of an interconnected ER network. We will also summarize the current knowledge on how the ER interacts with other membrane-bound organelles, with a focus on how the ER and Golgi interplay in plant cells.
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Arabidopsis RHD3 mediates the generation of the tubular ER network and is required for Golgi distribution and motility in plant cells
Journal of Cell Science, 2011Co-Authors: Jun Chen, Giovanni Stefano, Federica Brandizzi, Huanquan ZhengAbstract:In plant cells, the endoplasmic reticulum (ER) and Golgi apparatus form a unique system in which single Golgi stacks are motile and in close association with the underlying ER tubules. Arabidopsis has three RHD3 (ROOT HAIR DEFECTIVE 3) isoforms that are analogous to the mammalian Atlastin GTPases involved in shaping ER tubules. We used live-cell imaging, genetic complementation, split ubiquitin assays and western blot analyses in Arabidopsis and tobacco to show that RHD3 mediates the generation of the tubular ER network and is required for the distribution and motility of Golgi stacks in root and leaf epidermal cells. We established that RHD3 forms homotypic interactions at ER punctae. In addition, the activity of RHD3 on the tubular ER is specifically correlated with the cellular distribution and motility of Golgi stacks because ER to Golgi as well as Golgi to plasma membrane transport was not affected by RHD3 mutations in the conserved GDP/GTP motifs. We found a possible partial redundancy within the RHD3 isoforms in Arabidopsis. However, yeast Sey1p, a functional Atlastin homologue, and RHD3 are not interchangeable in complementing the respective loss-of-function mutants, suggesting that the molecular mechanisms controlling ER tubular morphology might not be entirely conserved among eukaryotic lineages.