The Experts below are selected from a list of 2436 Experts worldwide ranked by ideXlab platform
Richard G Gardner - One of the best experts on this subject based on the ideXlab platform.
-
A model for the interplay of Cyc8 SUMOylation and inclusion formation during adaptation to hyperosmotic stress.
2019Co-Authors: Cory M Nadel, Timothy D. Mackie, Richard G GardnerAbstract:Under iso-osmotic conditions, the Cyc8-Tup1 corepressor complex is diffusely localized across the nucleus, facilitating repression of target genes. Hyperosmotic stress induces rapid assembly of Cyc8-Tup1 inclusions and SUMOylation of the complex, which assists in orienting the complex at the promoter of specific genes like GPD1. Expression of GPD1 and subsequent glycerol accumulation facilitates adaptation to hyperosmotic stress, upon which Cyc8-Tup1 are deSUMOylated and inclusions are resolved.
-
RESEARCH ARTICLE Dynamic Sumoylation of a Conserved Transcription Corepressor Prevents Persistent Inclusion Formation during Hyperosmotic Stress
2016Co-Authors: Michelle L Oeser, Triana Amen, Cory M Nadel, Amanda I Bradley, Janani Gopalan, Ramon D. Jones, Daniel Kaganovich, J. Reed, Richard G GardnerAbstract:Cells are often exposed to physical or chemical stresses that can damage the structures of essential biomolecules. Stress-induced cellular damage can become deleterious if not man-aged appropriately. Rapid and adaptive responses to stresses are therefore crucial for cell survival. In eukaryotic cells, different stresses trigger post-translational modification of pro-teins with the small ubiquitin-like modifier SUMO. However, the specific regulatory roles of sumoylation in each stress response are not well understood. Here, we examined the sumoylation events that occur in budding yeast after exposure to hyperosmotic stress. We discovered by proteomic and biochemical analyses that hyperosmotic stress incurs the rapid and transient sumoylation of Cyc8 and Tup1, which together form a conserved tran-scription corepressor complex that regulates hundreds of genes. Gene expression and cell biological analyses revealed that sumoylation of each protein directs distinct outcomes. In particular, we discovered that Cyc8 sumoylation prevents the persistence of hyperosmotic stress-induced Cyc8-Tup1 inclusions, which involves a glutamine-rich prion domain in Cyc8. We propose that sumoylation protects against persistent inclusion formation durin
-
dynamic sumoylation of a conserved transcription corepressor prevents persistent inclusion formation during hyperosmotic stress
PLOS Genetics, 2016Co-Authors: Michelle L Oeser, Triana Amen, Cory M Nadel, Amanda I Bradley, Janani Gopalan, Ramon D. Jones, Benjamin J Reed, Daniel Kaganovich, Richard G GardnerAbstract:Cells are often exposed to physical or chemical stresses that can damage the structures of essential biomolecules. Stress-induced cellular damage can become deleterious if not managed appropriately. Rapid and adaptive responses to stresses are therefore crucial for cell survival. In eukaryotic cells, different stresses trigger post-translational modification of proteins with the small ubiquitin-like modifier SUMO. However, the specific regulatory roles of sumoylation in each stress response are not well understood. Here, we examined the sumoylation events that occur in budding yeast after exposure to hyperosmotic stress. We discovered by proteomic and biochemical analyses that hyperosmotic stress incurs the rapid and transient sumoylation of Cyc8 and Tup1, which together form a conserved transcription corepressor complex that regulates hundreds of genes. Gene expression and cell biological analyses revealed that sumoylation of each protein directs distinct outcomes. In particular, we discovered that Cyc8 sumoylation prevents the persistence of hyperosmotic stress-induced Cyc8-Tup1 inclusions, which involves a glutamine-rich prion domain in Cyc8. We propose that sumoylation protects against persistent inclusion formation during hyperosmotic stress, allowing optimal transcriptional function of the Cyc8-Tup1 complex.
Stephanie A Hagstrom - One of the best experts on this subject based on the ideXlab platform.
-
Photoreceptor Compartment-Specific TULP1 Interactomes
'MDPI AG', 2021Co-Authors: Lindsey A Ebke, Gayle J T Pauer, Satyabrata Sinha, Stephanie A HagstromAbstract:Photoreceptors are highly compartmentalized cells with large amounts of proteins synthesized in the inner segment (IS) and transported to the outer segment (OS) and synaptic terminal. TULP1 is a photoreceptor-specific protein localized to the IS and synapse. In the absence of TULP1, several OS-specific proteins are mislocalized and synaptic vesicle recycling is impaired. To better understand the involvement of TULP1 in protein trafficking, our approach in the current study was to physically isolate TULP1-containing photoreceptor compartments by serial tangential sectioning of retinas and to identify compartment-specific TULP1 binding partners by immunoprecipitation followed by liquid chromatography tandem mass spectrometry. Our results indicate that TULP1 has two distinct interactomes. We report the identification of: (1) an IS-specific interaction between TULP1 and the motor protein Kinesin family member 3a (Kif3a), (2) a synaptic-specific interaction between TULP1 and the scaffold protein Ribeye, and (3) an interaction between TULP1 and the cytoskeletal protein microtubule-associated protein 1B (MAP1B) in both compartments. Immunolocalization studies in the wild-type retina indicate that TULP1 and its binding partners co-localize to their respective compartments. Our observations are compatible with TULP1 functioning in protein trafficking in multiple photoreceptor compartments, likely as an adapter molecule linking vesicles to molecular motors and the cytoskeletal scaffold
-
Co-localization of mutant TULP1 protein with the ER-resident protein Calnexin.
2016Co-Authors: Glenn P. Lobo, Philip D. Kiser, Stephanie A HagstromAbstract:Immunofluorescent localization of GFP-fused TULP1 (green) and Calnexin (red) in WT and mutant TULP1 expressing cells. Nuclei were stained using DAPI (blue) (A) WT-TULP1 expressing cells displayed predominant plasma membrane and some cytoplasmic staining patterns. (B-E) In contrast all four mutant TULP1 expressing cell lines (D94Y, R420P, I459K and F491L) displayed punctate co-localization patterns with the ER-resident marker Calnexin (merge: yellow). Scale bar = 10 μM. Approximately 100 cells per transfection were counted. Experiments were repeated twice.
-
Co-localization and induction of UPR markers in mutant TULP1 expressing photoreceptors.
2016Co-Authors: Glenn P. Lobo, Philip D. Kiser, Stephanie A HagstromAbstract:P1 WT mice retinas were co-transfected with GFP-fused (green) plasmids and a mCherry tagged ER reporter (mCh-Sec61b: red). Retinas were harvested and sectioned at P30 (A) Confocal images of retinas show co-localization (merge: yellow) of GFP-fused mutant D94Y-TULP1 and F491L-TULP1 with mCh-Sec61b, the ER resident protein, in the inner segments. Scale bar = 10μM. (B-C) Activation of the ER-UPR stress markers in transgenic mice expressing mutant TULP1 protein. qRT-PCR indicates a 1.3–5 fold increase of multiple ER-UPR stress markers in mutant D94Y-TULP1 (B) or mutant F491L-TULP1 (C) retinas vs WT-TULP1 injected retinas. qRT-PCR analysis was normalized to GAPDH (as a mRNA quantity control) and GFP (as a transfection efficiency control). The ΔΔCt method was employed to calculate fold changes. Samples were assayed in triplicates for each ER-UPR marker and qRT-PCR experiments were repeated twice. Statistical significance was assessed by using the two-tailed Student’s t-test. * = p
-
Induction of the ER-UPR complex in cells expressing mutant TULP1.
2016Co-Authors: Glenn P. Lobo, Philip D. Kiser, Stephanie A HagstromAbstract:(A) Western blot analysis using antibodies against BiP, phosphorylated PERK (pPERK), XBP1 and XBP1s, showed induction of UPR stress proteins in cells expressing mutant forms of TULP1. Actin was used a protein loading control. Western blot experiments were repeated twice. (B) Quantification of ER-stress protein markers was performed using densitometry in mutant TULP1 expressing cells compared to WT-TULP1 expressing cells. The relative band intensity of each protein was normalized to Actin. Data is presented as arbitrary units from two separate western blot densitometry analyses. Statistical significance was calculated using the two-tailed Student’s t-test. * = p< 0.001. Since the untransfected or WT-TULP1 expressing cells revealed no activation of pPERK protein, the pPERK densitometry value was arbitrary set to 1 (arbitrary units/ pixel intensity) for statistical calculations.
-
Retention of mutant TULP1 in the ER of fractionated cells.
2016Co-Authors: Glenn P. Lobo, Philip D. Kiser, Stephanie A HagstromAbstract:(A) Overview of the subcellular fractionation procedure used for the isolation of ER microsomes. (B) Western blot analysis of ER microsomes isolated from GFP-fused WT or mutant TULP1 expressing hTERT-RPE-1 cells. Antibodies against Calnexin and Calreticulin were used to determine the ER fractions; whereas antibodies against Golgin97 and COX IV were used to determine the presence of Golgi or mitochondria, respectively. Expression of mutant TULP1 proteins (D94Y, R420P, I459K and F491L) was retained within the ER. Total cell lysate from untransfected HEK293T or hTERT-RPE-1 cells were used as controls. Actin was used a protein loading control.
Craig Vierra - One of the best experts on this subject based on the ideXlab platform.
-
Conserved C-terminal domain of spider tubuliform spidroin 1 contributes to extensibility in synthetic fibers.
Biomacromolecules, 2012Co-Authors: Eric Gnesa, Yang Hsia, Warner S. Weber, Joan Lin-cereghino, Geoff P. Lin-cereghino, Simon Y. Tang, Kimiko Agari, Jeffery L. Yarger, Craig VierraAbstract:Spider silk is renowned for its extraordinary mechanical properties, having a balance of high tensile strength and extensibility. To date, the majority of studies have focused on the production of dragline silks from synthetic spider silk gene products. Here we report the first mechanical analysis of synthetic egg case silk fibers spun from the Latrodectus hesperus tubuliform silk proteins, TuSp1 and ECP-2. We provide evidence that recombinant ECP-2 proteins can be spun into fibers that display mechanical properties similar to other synthetic spider silks. We also demonstrate that silks spun from recombinant thioredoxin-TuSp1 fusion proteins that contain the conserved C-terminal domain exhibit increased extensibility and toughness when compared to the identical fibers spun from fusion proteins lacking the C-terminus. Mechanical analyses reveal that the properties of synthetic tubuliform silks can be modulated by altering the postspin draw ratios of the fibers. Fibers subject to increased draw ratios showe...
-
araneoid egg case silk a fibroin with novel ensemble repeat units from the black widow spider latrodectus hesperus
Biochemistry, 2005Co-Authors: Xiaoyi Hu, Anne M F Moore, Barbara Lawrence, Kristin Kohler, Erin Mcmullen, Patrick R Jones, Arnold M Falick, Craig VierraAbstract:Araneoid spiders use specialized abdominal glands to manufacture up to seven different protein-based silks/glues that have diverse physical properties. The fibroin sequences that encode egg case fibers (cover silk for the egg case sac) and the secondary structure of these threads have not been previously determined. In this study, MALDI tandem TOF mass spectrometry (MS/MS) and reverse genetics were used to isolate the first egg case fibroin, named tubuliform spidroin 1 (TuSp1), from the black widow spider, Latrodectus hesperus. Real-time quantitative PCR analysis demonstrates TuSp1 is selectively expressed in the tubuliform gland. Analysis of the amino acid composition of raw egg case silk closely aligns with the predicted amino acid composition from the primary sequence of TuSp1, which supports the assertion that TuSp1 represents a major component of egg case fibers. TuSp1 is composed of highly homogeneous repeats that are 184 amino acids in length. The long stretches of polyalanine and glycine-alanine s...
Michelle L Oeser - One of the best experts on this subject based on the ideXlab platform.
-
RESEARCH ARTICLE Dynamic Sumoylation of a Conserved Transcription Corepressor Prevents Persistent Inclusion Formation during Hyperosmotic Stress
2016Co-Authors: Michelle L Oeser, Triana Amen, Cory M Nadel, Amanda I Bradley, Janani Gopalan, Ramon D. Jones, Daniel Kaganovich, J. Reed, Richard G GardnerAbstract:Cells are often exposed to physical or chemical stresses that can damage the structures of essential biomolecules. Stress-induced cellular damage can become deleterious if not man-aged appropriately. Rapid and adaptive responses to stresses are therefore crucial for cell survival. In eukaryotic cells, different stresses trigger post-translational modification of pro-teins with the small ubiquitin-like modifier SUMO. However, the specific regulatory roles of sumoylation in each stress response are not well understood. Here, we examined the sumoylation events that occur in budding yeast after exposure to hyperosmotic stress. We discovered by proteomic and biochemical analyses that hyperosmotic stress incurs the rapid and transient sumoylation of Cyc8 and Tup1, which together form a conserved tran-scription corepressor complex that regulates hundreds of genes. Gene expression and cell biological analyses revealed that sumoylation of each protein directs distinct outcomes. In particular, we discovered that Cyc8 sumoylation prevents the persistence of hyperosmotic stress-induced Cyc8-Tup1 inclusions, which involves a glutamine-rich prion domain in Cyc8. We propose that sumoylation protects against persistent inclusion formation durin
-
dynamic sumoylation of a conserved transcription corepressor prevents persistent inclusion formation during hyperosmotic stress
PLOS Genetics, 2016Co-Authors: Michelle L Oeser, Triana Amen, Cory M Nadel, Amanda I Bradley, Janani Gopalan, Ramon D. Jones, Benjamin J Reed, Daniel Kaganovich, Richard G GardnerAbstract:Cells are often exposed to physical or chemical stresses that can damage the structures of essential biomolecules. Stress-induced cellular damage can become deleterious if not managed appropriately. Rapid and adaptive responses to stresses are therefore crucial for cell survival. In eukaryotic cells, different stresses trigger post-translational modification of proteins with the small ubiquitin-like modifier SUMO. However, the specific regulatory roles of sumoylation in each stress response are not well understood. Here, we examined the sumoylation events that occur in budding yeast after exposure to hyperosmotic stress. We discovered by proteomic and biochemical analyses that hyperosmotic stress incurs the rapid and transient sumoylation of Cyc8 and Tup1, which together form a conserved transcription corepressor complex that regulates hundreds of genes. Gene expression and cell biological analyses revealed that sumoylation of each protein directs distinct outcomes. In particular, we discovered that Cyc8 sumoylation prevents the persistence of hyperosmotic stress-induced Cyc8-Tup1 inclusions, which involves a glutamine-rich prion domain in Cyc8. We propose that sumoylation protects against persistent inclusion formation during hyperosmotic stress, allowing optimal transcriptional function of the Cyc8-Tup1 complex.
Rudiger Klein - One of the best experts on this subject based on the ideXlab platform.
-
gulp1 controls eph ephrin trogocytosis and is important for cell rearrangements during development
Journal of Cell Biology, 2019Co-Authors: Jingyi Gong, Thomas N Gaitanos, Louise Gaitanos, Jana Lindner, Yunyun Huang, Rudolf Winklbauer, Rudiger KleinAbstract:Trogocytosis, in which cells nibble away parts of neighboring cells, is an intercellular cannibalism process conserved from protozoa to mammals. Its underlying molecular mechanisms are not well understood and are likely distinct from phagocytosis, a process that clears entire cells. Bi-directional contact repulsion induced by Eph/ephrin signaling involves transfer of membrane patches and full-length Eph/ephrin protein complexes between opposing cells, resembling trogocytosis. Here, we show that the phagocytic adaptor protein Gulp1 regulates EphB/ephrinB trogocytosis to achieve efficient cell rearrangements of cultured cells and during embryonic development. Gulp1 mediates trogocytosis bi-directionally by dynamic engagement with EphB/ephrinB protein clusters in cooperation with the Rac-specific guanine nucleotide exchange factor Tiam2. Ultimately, Gulp1’s presence at the Eph/ephrin cluster is a prerequisite for recruiting the endocytic GTPase dynamin. These results suggest that EphB/ephrinB trogocytosis, unlike other trogocytosis events, uses a phagocytosis-like mechanism to achieve efficient membrane scission and engulfment.
