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Guillermo Oliver - One of the best experts on this subject based on the ideXlab platform.
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neuroretina specification in mouse embryos requires six3 mediated suppression of wnt8b in the anterior neural plate
Journal of Clinical Investigation, 2010Co-Authors: Oleg Lagutin, Eric C Swindell, Milan Jamrich, Guillermo OliverAbstract:Retinal degeneration causes vision impairment and blindness in humans. If one day we are to harness the potential of stem cell–based cell replacement therapies to treat these conditions, it is imperative that we better understand normal retina development. Currently, the genes and mechanisms that regulate the specification of the neuroretina during vertebrate eye development remain unknown. Here, we identify sine oculis–related homeobox 3 (Six3) as a crucial player in this process in mice. In Six3 conditional–mutant mouse embryos, specification of the neuroretina was abrogated, but that of the retinal pigmented epithelium was normal. Conditional deletion of Six3 did not affect the initial development of the optic vesicle but did arrest subsequent neuroretina specification. Ectopic rostral expansion of Wnt8b expression was the major response to Six3 deletion and the leading cause for the specific lack of neuroretina, as ectopic Wnt8b expression in transgenic embryos was sufficient to suppress neuroretina specification. Using chromatin immunoprecipitation assays, we identified Six3-responsive elements in the Wnt8b locus and demonstrated that Six3 directly repressed Wnt8b expression in vivo. Our findings provide a molecular framework to the program leading to neuroretina differentiation and may be relevant for the development of novel strategies aimed at characterizing and eventually treating different abnormalities in eye formation.
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regulation of a remote shh forebrain enhancer by the six3 homeoprotein
Nature Genetics, 2008Co-Authors: Yongsu Jeong, Kenia B Eljaick, Anastasia Yocum, Erich Roessler, Maximilian Muenke, Federico Coluccio Leskow, Christèle Dubourg, Xin Geng, Xue Li, Guillermo OliverAbstract:In humans, SHH haploinsufficiency results in holoprosencephaly (HPE), a defect in anterior midline formation. Despite the importance of maintaining SHH transcript levels above a critical threshold, we know little about the upstream regulators of SHH expression in the forebrain. Here we describe a rare nucleotide variant located 460 kb upstream of SHH in an individual with HPE that resulted in the loss of Shh brain enhancer-2 (SBE2) activity in the hypothalamus of transgenic mouse embryos. Using a DNA affinity-capture assay, we screened the SBE2 sequence for DNA-binding proteins and identified members of the Six3 and Six6 homeodomain family as candidate regulators of Shh transcription. Six3 showed reduced binding affinity for the mutant compared to the wild-type SBE2 sequence. Moreover, Six3 with HPE-causing alterations failed to bind and activate SBE2. These data suggest a direct link between Six3 and Shh regulation during normal forebrain development and in the pathogenesis of HPE.
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haploinsufficiency of six3 fails to activate sonic hedgehog expression in the ventral forebrain and causes holoprosencephaly
Developmental Cell, 2008Co-Authors: Xin Geng, Yongsu Jeong, Oleg Lagutin, Adi Inbal, Douglas J Epstein, Lilianna Solnicakrezel, Christina K Speirs, Guillermo OliverAbstract:Holoprosencephaly (HPE), the most common forebrain malformation, is characterized by an incomplete separation of the cerebral hemispheres. Mutations in the homeobox gene SIX3 account for 1.3% of all cases of human HPE. Using zebrafish-based assays, we have now determined that HPE-associated Six3 mutant proteins function as hypomorphs. Haploinsufficiency of Six3 caused by deletion of one allele of Six3 or by replacement of wild-type Six3 with HPE-associated Six3 mutant alleles was sufficient to recapitulate in mouse models most of the phenotypic features of human HPE. We demonstrate that Shh is a direct target of Six3 in the rostral diencephalon ventral midline (RDVM). Reduced amounts of functional Six3 protein fail to activate Shh expression in the mutant RDVM and ultimately lead to HPE. These results identify Six3 as a direct regulator of Shh expression and reveal a crossregulatory loop between Shh and Six3 in the ventral forebrain.
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six3 inactivation causes progressive caudalization and aberrant patterning of the mammalian diencephalon
Development, 2008Co-Authors: Alfonso Lavado, Oleg Lagutin, Guillermo OliverAbstract:The homeobox gene Six3 represses Wnt1 transcription. It is also required in the anterior neural plate for the development of the mammalian rostral forebrain. We have now determined that at the 15- to 17-somite stage, the prospective diencephalon is the most-anterior structure in the Six3 -null brain, and Wnt1 expression is anteriorly expanded. Consequently, the brain caudalizes, and at the 22- to 24-somite stage, the prospective thalamic territory is the most-anterior structure. At around E11.0, the pretectum replaces this structure. Analysis of Six3;Wnt1 double-null mice revealed that Six3-mediated repression of Wnt1 is necessary for the formation of the rostral diencephalon and that Six3 activity is required for the formation of the telencephalon. These results provide insight into the mechanisms that establish anteroposterior identity in the developing mammalian brain.
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expression of six3 opposite strand six3os during mouse embryonic development
Gene Expression Patterns, 2007Co-Authors: Xin Geng, Oleg Lagutin, Alfonso Lavado, Guillermo OliverAbstract:Abstract Recently, sequence analyses have identified a large number of opposite strand transcripts in the vertebrate genome. Although the transcripts appear to be spliced and polyadenylated, many of them are predicted to represent noncoding RNAs. High levels of noncoding transcripts of the Six3 Opposite Strand (Six3OS) were recently identified in the embryonic and postnatal retina of the mouse. In this study, we expanded those initial expression analyses, elucidated in detail the developmental expression profile of mouse Six3OS in the brain and visual system, and compared it with that of Six3. Our results show that Six3OS expression overlaps extensively with that of Six3 and is not altered in Six3-null embryos.
Christopher G. Burd - One of the best experts on this subject based on the ideXlab platform.
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Retromer forms low order oligomers on supported lipid bilayers.
The Journal of biological chemistry, 2020Co-Authors: Catherine L. Deatherage, Joerg Nikolaus, Erdem Karatekin, Christopher G. BurdAbstract:Retromer orchestrates the selection and export of integral membrane proteins from the endosome via retrograde and plasma membrane recycling pathways. Long-standing hypotheses regarding the retromer sorting mechanism posit that oligomeric interactions between retromer and associated accessory factors on the endosome membrane drives clustering of retromer-bound integral membrane cargo prior to its packaging into a nascent transport carrier. To test this idea, we examined interactions between components of the sorting nexin 3 (SNX3)–retromer sorting pathway using quantitative single particle fluorescence microscopy in a reconstituted system. This system includes a supported lipid bilayer, fluorescently labeled retromer, SNX3, and two model cargo proteins, RAB7, and retromer-binding segments of the WASHC2C subunit of the WASH complex. We found that the distribution of membrane-associated retromer is predominantly comprised of monomer (∼18%), dimer (∼35%), trimer (∼24%), and tetramer (∼13%). Unexpectedly, neither the presence of membrane-associated cargo nor accessory factors substantially affected this distribution. The results indicate that retromer has an intrinsic propensity to form low order oligomers on a supported lipid bilayer and that neither membrane association nor accessory factors potentiate oligomerization. The results support a model whereby SNX3-retromer is a minimally concentrative coat protein complex adapted to bulk membrane trafficking from the endosomal system.
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Retromer forms low order oligomers on supported lipid bilayers
2020Co-Authors: Catherine L. Deatherage, Joerg Nikolaus, Erdem Karatekin, Christopher G. BurdAbstract:Retromer is a protein sorting device that orchestrates the selection and export of integral membrane proteins from the endosome via retrograde and plasma membrane recycling pathways. Long standing hypotheses regarding the Retromer sorting mechanism posit that oligomeric interactions between Retromer and associated accessory factors on the endosome membrane drives clustering of Retromer-bound integral membrane cargo prior to its packaging into a nascent transport carrier. To test this hypothesis, we examined interactions between the components of the SNX3-Retromer sorting pathway using quantitative single particle fluorescence microscopy of a reconstituted system comprising a supported bilayer, Retromer, a model cargo protein, the accessory proteins SNX3, RAB7, and the Retromer-binding segment of the WASHC2C subunit of the WASH complex. The predominant species of membrane associated Retromer are low order: monomers (~18%), dimers (~35%), trimers (~24%) and tetramers (~24%). Unexpectedly, neither cargo nor accessory factors promote Retromer oligomerization on a supported bilayer. The results indicate that Retromer has an intrinsic propensity to form low order oligomers and that neither membrane association nor accessory factors potentiate oligomerization. Hence, Retromer is a minimally concentrative sorting device adapted to bulk membrane trafficking from the endosomal system.
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Opposing Activities of the SNX3-Retromer Complex and ESCRT Proteins Mediate Regulated Cargo Sorting at a Common Endosome
Molecular biology of the cell, 2008Co-Authors: Todd I. Strochlic, David J Katzmann, Briana C. Schmiedekamp, Jacqueline R. E. Lee, Christopher G. BurdAbstract:Endocytosed proteins are either delivered to the lysosome to be degraded or are exported from the endosomal system and delivered to other organelles. Sorting of the Saccharomyces cerevisiae reductive iron transporter, composed of the Fet3 and Ftr1 proteins, in the endosomal system is regulated by available iron; in iron-starved cells, Fet3-Ftr1 is sorted by SNX3/Grd19 and retromer into a recycling pathway that delivers it back to the plasma membrane, but when starved cells are exposed to iron, Fet3-Ftr1 is targeted to the lysosome-like vacuole and is degraded. We report that iron-induced endocytosis of Fet3-Ftr1 is independent of Fet3-Ftr1 ubiquitylation, and after endocytosis, degradation of Fet3-Ftr1 is mediated by the multivesicular body (MVB) sorting pathway. In mutant cells lacking any component of the ESCRT protein-dependent MVB sorting machinery, the Rsp5 ubiquitin ligase, or in wild-type cells expressing Fet3-Ftr1 lacking cytosolic lysyl ubiquitin acceptor sites, Fet3-Ftr1 is constitutively sorted into the recycling pathway independent of iron status. In the presence and absence of iron, Fet3-Ftr1 transits an endosomal compartment where a subunit of the MVB sorting receptor (Vps27), SNX3/Grd19, and retromer proteins colocalize. We propose that this endosome is where Rsp5 ubiquitylates Fet3-Ftr1 and where the recycling and degradative pathways diverge.
Yan Wang - One of the best experts on this subject based on the ideXlab platform.
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the homeotic protein six3 suppresses carcinogenesis and metastasis through recruiting the lsd1 nurd mta3 complex
Theranostics, 2018Co-Authors: Yu Zheng, Shuai Leng, Dandan Feng, Yi Zeng, Wei Huang, Shuang Wang, Yang Yang, Yan WangAbstract:: The homeodomain transcription factor SIX3 was recently reported to be a negative regulator of the Wnt pathway and has an emerging role in cancer. However, how SIX3 contributes to tumorigenesis and metastasis is poorly understood. METHODS: We employed affinity purification and mass spectrometry (MS) to identify the proteins physically associated with SIX3. Genome-wide analysis of the SIX3/LSD1/NuRD(MTA3) complex using a chromatin immunoprecipitation-on-chip approach identified a cohort of target genes including WNT1 and FOXC2, which are critically involved in cell proliferation and epithelial-to-mesenchymal transition. Also, we used flow cytometry, growth curve analysis, EdU incorporation assay, colony formation assays, trans-well invasion assays, immunohistochemical staining and in vivo bioluminescence assay to investigate the function of SIX3 in tumorigenesis. RESULTS: We demonstrate that the SIX3/LSD1/NuRD(MTA3) complex inhibits carcinogenesis in breast cancer cells and suppresses metastasis in breast cancer. SIX3 expression is downregulated in various human cancers and high SIX3 is correlated with improved prognosis. CONCLUSION: Our study revealed an important mechanistic link between the loss of function of SIX3 and tumor progression, identified a molecular basis for the opposing actions of MTA1 and MTA3, and may provide new potential prognostic indicators and targets for cancer therapy.
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the homeotic protein six3 suppresses carcinogenesis and metastasis through recruiting the lsd1 nurd mta3 complex
Theranostics, 2018Co-Authors: Yu Zheng, Shuai Leng, Dandan Feng, Yi Zeng, Wei Huang, Shuang Wang, Yang Yang, Yan WangAbstract:: The homeodomain transcription factor SIX3 was recently reported to be a negative regulator of the Wnt pathway and has an emerging role in cancer. However, how SIX3 contributes to tumorigenesis and metastasis is poorly understood. METHODS: We employed affinity purification and mass spectrometry (MS) to identify the proteins physically associated with SIX3. Genome-wide analysis of the SIX3/LSD1/NuRD(MTA3) complex using a chromatin immunoprecipitation-on-chip approach identified a cohort of target genes including WNT1 and FOXC2, which are critically involved in cell proliferation and epithelial-to-mesenchymal transition. Also, we used flow cytometry, growth curve analysis, EdU incorporation assay, colony formation assays, trans-well invasion assays, immunohistochemical staining and in vivo bioluminescence assay to investigate the function of SIX3 in tumorigenesis. RESULTS: We demonstrate that the SIX3/LSD1/NuRD(MTA3) complex inhibits carcinogenesis in breast cancer cells and suppresses metastasis in breast cancer. SIX3 expression is downregulated in various human cancers and high SIX3 is correlated with improved prognosis. CONCLUSION: Our study revealed an important mechanistic link between the loss of function of SIX3 and tumor progression, identified a molecular basis for the opposing actions of MTA1 and MTA3, and may provide new potential prognostic indicators and targets for cancer therapy.
Stephan N Witt - One of the best experts on this subject based on the ideXlab platform.
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alpha synuclein inhibits SNX3 retromer mediated retrograde recycling of iron transporters in s cerevisiae and c elegans models of parkinson s disease
Human Molecular Genetics, 2018Co-Authors: Dhaval Patel, Sureshbabu Nagarajan, Zhengchang Liu, Wayne O Hemphill, Runhua Shi, Vladimir N Uversky, Guy A Caldwell, Kim A Caldwell, Stephan N WittAbstract:We probed the role of alpha-synuclein (α-syn) in modulating sorting nexin 3 (SNX3)-retromer-mediated recycling of iron transporters in Saccharomyces cerevisiae and Caenorhabditis elegans. In yeast, the membrane-bound heterodimer Fet3/Ftr1 is the high affinity iron importer. Fet3 is a membrane-bound multicopper ferroxidase, whose ferroxidase domain is orthologous to human ceruloplasmin (Cp), that oxidizes external Fe+2 to Fe+3; the Fe+3 ions then channel through the Ftr1 permease into the cell. When the concentration of external iron is low ( 10 µM), Fet3/Ftr1 is endocytosed and shunted to the vacuole for degradation. We discovered that α-syn expression phenocopies the high iron condition: under the low iron condition (<1 µM), α-syn inhibits SNX3-retromer-mediated recycling of Fet3/Ftr1 and instead shunts Fet3/Ftr1 into the multivesicular body pathway to the vacuole. α-Syn inhibits recycling by blocking the association of SNX3-mCherry molecules with endocytic vesicles, possibly by interfering with the binding of SNX3 to phosphatidylinositol-3-monophosphate. In C. elegans, transgenic worms expressing α-syn exhibit an age-dependent degeneration of dopaminergic neurons that is partially rescued by the iron chelator desferoxamine. This implies that α-syn-expressing dopaminergic neurons are susceptible to changes in iron neurotoxicity with age, whereby excess iron enhances α-syn-induced neurodegeneration. In vivo genetic analysis indicates that α-syn dysregulates iron homeostasis in worm dopaminergic neurons, possibly by inhibiting SNX-3-mediated recycling of a membrane-bound ortholog of Cp (F21D5.3), the iron exporter ferroportin (FPN1.1), or both.
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Sorting Out the Role of α-Synuclein in Retromer-Mediated Endosomal Protein Sorting
SAGE Publishing, 2018Co-Authors: Dhaval Patel, Stephan N WittAbstract:Retromer is a phylogenetically conserved, multisubunit coat complex that controls endosomal protein trafficking and sorting. Mutations in the retromer gene VPS35 cause late-onset Parkinson disease, suggesting that trafficking defects cause neurodegeneration. Sorting nexins assist retromer to guide cell surface proteins to their assigned destinations, and our interest here is sorting nexin 3 (SNX3). SNX3 binds to membranes via a phox homolog (PX) domain that binds phosphatidylinositol 3-phosphate (PI3P), and in human cells its cargo proteins are the transferrin and Wnt receptors and the divalent metal ion transporter, whereas in yeast the best characterized cargo is the iron permease Ftr1. We recently discovered that α-synuclein inhibits SNX3-retromer recycling of Ftr1 in an unexpected way: α-synuclein, which avidly binds to negatively charged lipids, blocks the association of SNX3 to early endosomes. Here, we discuss mechanisms by which α-synuclein can disrupt SNX3-retromer–mediated recycling
Jean Gruenberg - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation of conserved phosphoinositide binding pocket regulates sorting nexin membrane targeting.
Nature communications, 2018Co-Authors: Marc Lenoir, Jean Gruenberg, Cansel Ustunel, Sandya Rajesh, Jaswant Kaur, Dimitri Moreau, Michael OverduinAbstract:Sorting nexins anchor trafficking machines to membranes by binding phospholipids. The paradigm of the superfamily is sorting nexin 3 (SNX3), which localizes to early endosomes by recognizing phosphatidylinositol 3-phosphate (PI3P) to initiate retromer-mediated segregation of cargoes to the trans-Golgi network (TGN). Here we report the solution structure of full length human SNX3, and show that PI3P recognition is accompanied by bilayer insertion of a proximal loop in its extended Phox homology (PX) domain. Phosphoinositide (PIP) binding is completely blocked by cancer-linked phosphorylation of a conserved serine beside the stereospecific PI3P pocket. This "PIP-stop" releases endosomal SNX3 to the cytosol, and reveals how protein kinases control membrane assemblies. It constitutes a widespread regulatory element found across the PX superfamily and throughout evolution including of fungi and plants. This illuminates the mechanism of a biological switch whereby structured PIP sites are phosphorylated to liberate protein machines from organelle surfaces.
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Phosphorylation of conserved phosphoinositide binding pocket regulates sorting nexin membrane targeting
Nature Publishing Group, 2018Co-Authors: Marc Lenoir, Jean Gruenberg, Cansel Ustunel, Sandya Rajesh, Jaswant Kaur, Dimitri Moreau, Michael OverduinAbstract:Sorting nexin 3 (SNX3) is a phosphatidylinositol 3-phosphate binding protein that localizes to early endosomes. Here the authors use NMR to resolve SNX3′s membrane interactions, revealing that membrane binding is regulated through phosphorylation of a conserved serine by its lipid recognition site
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Hrs and SNX3 functions in sorting and membrane invagination within multivesicular bodies.
PLOS Biology, 2008Co-Authors: Véronique Pons, Pierre-philippe Luyet, Etienne Morel, Robert G. Parton, Laurence Abrami, F. Gisou Goot, Jean GruenbergAbstract:After internalization, ubiquitinated signaling receptors are delivered to early endosomes. There, they are sorted and incorporated into the intralumenal invaginations of nascent multivesicular bodies, which function as transport intermediates to late endosomes. Receptor sorting is achieved by Hrs—an adaptor-like protein that binds membrane PtdIns3P via a FYVE motif—and then by ESCRT complexes, which presumably also mediate the invagination process. Eventually, intralumenal vesicles are delivered to lysosomes, leading to the notion that EGF receptor sorting into multivesicular bodies mediates lysosomal targeting. Here, we report that Hrs is essential for lysosomal targeting but dispensable for multivesicular body biogenesis and transport to late endosomes. By contrast, we find that the PtdIns3P-binding protein SNX3 is required for multivesicular body formation, but not for EGF receptor degradation. PtdIns3P thus controls the complementary functions of Hrs and SNX3 in sorting and multivesicular body biogenesis.