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Articles by Elizabeth A. Miller in JoVE
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Aktivasyon ve İnsan monosit türevli dendritik hücreler, IL-1β kullanarak NLRP3 Inflammasome Aktivitenin Ölçümü
Melissa V. Fernandez1, Elizabeth A. Miller2, Nina Bhardwaj3
1Department of Pathology, New York University School of Medicine, 2Division of Infectious Diseases, Department of Medicine, Mount Sinai Medical Center, 3Division of Hematology and Oncology, Hess Center for Science and Medicine, Mount Sinai Medical Center
Dendritik hücreler (DC'ler) ile nigerisin ile birlikte NLRP3 inflammasome aktivasyonu takiben, sentetik purin, R848 ve TLR8 tanınması, tepki olarak IL-1β salgılar, bu nedenle, IL-1β NLRP3 inflammasome aktivitesini ölçmek için kullanılabilir. Hücre içi sitokin boyama, immunoblotting ve ELISA doğru IL-1β ekspresyonu üzerinden NLRP3 emişli inflammasome ve aktivasyonunu ölçmek için kullanılır.
Other articles by Elizabeth A. Miller on PubMed
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Central Nervous System Consequences of an Unusual Body Disposal Strategy: Case Report and Brief Experimental Investigation
Journal of Forensic Sciences.
Sep, 2003 |
Pubmed ID: 14535685 The body of a 73-year-old man was dismembered by his female companion for the purpose of covert disposal. The method employed included skillful separation of body parts with hacksaw and knife, piecemeal disposal of fragments, and prolonged boiling of the decapitated head. The latter treatment resulted in marked shrinkage of cranial dura mater, separation of dura mater from skull, and extrusion of brain fragments into the resultant enlarged epidural space through a dural defect due to the disproportionately greater shrinkage of dura mater compared to brain parenchyma. This resulted in curd-like brain fragments filling an enlarged epidural space and overlying a shrunken, leathery dura mater. The cranial dura mater, still adherent to the skull base, resembled a "shrunken brain" in contour but contained only the remnants of brain tissue not already extruded through the dural defect. This unusual thermal artifact is rarely illustrated or mentioned in forensic literature. The development of this postmortem artifact likely requires the presence of a specific combination of conditions which must be, but apparently rarely are, simultaneously present.
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ER-Golgi Transport Defects Are Associated with Mutations in the Sed5p-binding Domain of the COPII Coat Subunit, Sec24p
Molecular Biology of the Cell.
Aug, 2005 |
Pubmed ID: 15930124 Selective cargo capture into ER-derived vesicles is driven by the Sec24p subunit of the COPII coat, which contains at least three independent cargo-binding sites. One of these, the "A-site," interacts with a NPF motif found on the SNARE, Sed5p. We have characterized the Sec24p-Sed5p interaction through mutation of the putative ER export motifs of Sed5p and the cargo-binding A-site of Sec24p. Mutational analysis of Sed5p suggests that the NPF motif is the dominant ER export signal. Mutation of the NPF binding pocket on Sec24p led to a dramatic reduction in the capture of Sed5p into COPII vesicles, whereas packaging of other ER-Golgi SNAREs was normal. Of all the cargoes tested, only Sed5p was depleted in vesicles made with Sec24p A-site mutants. Surprisingly, vesicles generated with the mutant Sec24p were unable to fuse with the Golgi apparatus. This inability to fuse was not the result of the lack of Sed5p, because vesicles specifically depleted of Sed5p generated by antibody inhibition targeted and fused normally. We propose that the A-site of Sec24p is a multipurpose cargo-binding site that must recognize additional unidentified cargo proteins, at least one of which is essential at a late stage of vesicle fusion.
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Genomewide Analysis Reveals Novel Pathways Affecting Endoplasmic Reticulum Homeostasis, Protein Modification and Quality Control
Genetics.
Jul, 2009 |
Pubmed ID: 19433630 To gain new mechanistic insight into ER homeostasis and the biogenesis of secretory proteins, we screened a genomewide collection of yeast mutants for defective intracellular retention of the ER chaperone, Kar2p. We identified 87 Kar2p-secreting strains, including a number of known components in secretory protein modification and sorting. Further characterization of the 73 nonessential Kar2p retention mutants revealed roles for a number of novel gene products in protein glycosylation, GPI-anchor attachment, ER quality control, and retrieval of escaped ER residents. A subset of these mutants, required for ER retrieval, included the GET complex and two novel proteins that likely function similarly in membrane insertion of tail-anchored proteins. Finally, the variant histone, Htz1p, and its acetylation state seem to play an important role in maintaining ER retrieval pathways, suggesting a surprising link between chromatin remodeling and ER homeostasis.
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Intragenic Suppressing Mutations Correct the Folding and Intracellular Traffic of Misfolded Mutants of Yor1p, a Eukaryotic Drug Transporter
The Journal of Biological Chemistry.
Nov, 2010 |
Pubmed ID: 20837481 ATP-binding cassette (ABC) transporters play pivotal physiological roles in substrate transport across membranes, and defective assembly of these proteins can cause severe disease associated with improper drug or ion flux. The yeast protein Yor1p is a useful model to study the biogenesis of ABC transporters; deletion of a phenylalanine residue in the first nucleotide-binding domain (NBD1) causes misassembly and retention in the endoplasmic reticulum (ER) of the resulting protein Yor1p-ΔF670, similar to the predominant disease-causing allele in humans, CFTR-ΔF508. Here we describe two novel Yor1p mutants, G278R and I1084P, which fail to assemble and traffic similar to Yor1p-ΔF670. These mutations are located in the two intracellular loops (ICLs) that interface directly with NBD1, and thus disrupt a functionally important structural module. We isolated 2 second-site mutations, F270S and R1168M, which partially correct the folding injuries associated with the G278R, I1084P, and ΔF670 mutants and reinstate their trafficking. The position of both corrective mutations at the cytoplasmic face of a transmembrane helix suggests that they restore biogenesis by influencing the behavior of the transmembrane domains rather than by direct restoration of the ICL1-ICL4-NBD1 structural module. Given the conserved topology of many ABC transporters, our findings provide new understanding of functionally important inter-domain interactions and suggest new potential avenues for correcting folding defects caused by abrogation of those domain interfaces.
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Hph1 and Hph2 Are Novel Components of the Sec63/Sec62 Posttranslational Translocation Complex That Aid in Vacuolar Proton ATPase Biogenesis
Eukaryotic Cell.
Jan, 2011 |
Pubmed ID: 21097665 Hph1 and Hph2 are homologous integral endoplasmic reticulum (ER) membrane proteins required for Saccharomyces cerevisiae survival under environmental stress conditions. To investigate the molecular functions of Hph1 and Hph2, we carried out a split-ubiquitin-membrane-based yeast two-hybrid screen and identified their interactions with Sec71, a subunit of the Sec63/Sec62 complex, which mediates posttranslational translocation of proteins into the ER. Hph1 and Hph2 likely function in posttranslational translocation, as they interact with other Sec63/Sec62 complex subunits, i.e., Sec72, Sec62, and Sec63. hph1Δ hph2Δ cells display reduced vacuole acidification; increased instability of Vph1, a subunit of vacuolar proton ATPase (V-ATPase); and growth defects similar to those of mutants lacking V-ATPase activity. sec71Δ cells exhibit similar phenotypes, indicating that Hph1/Hph2 and the Sec63/Sec62 complex function during V-ATPase biogenesis. Hph1/Hph2 and the Sec63/Sec62 complex may act together in this process, as vacuolar acidification and Vph1 stability are compromised to the same extent in hph1Δ hph2Δ and hph1Δ hph2Δ sec71Δ cells. In contrast, loss of Pkr1, an ER protein that promotes posttranslocation assembly of Vph1 with V-ATPase subunits, further exacerbates hph1Δ hph2Δ phenotypes, suggesting that Hph1 and Hph2 function independently of Pkr1-mediated V-ATPase assembly. We propose that Hph1 and Hph2 aid Sec63/Sec62-mediated translocation of specific proteins, including factors that promote efficient biogenesis of V-ATPase, to support yeast cell survival during environmental stress.
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Test of Recrudescence Hypothesis for Overwintering of Eastern Equine Encephalomyelitis Virus in Gray Catbirds
Journal of Medical Entomology.
Jul, 2011 |
Pubmed ID: 21845951 Eastern equine encephalitis virus (EEEV; family Togaviridae, genus Alphavirus) epizootics are infrequent, but they can lead to high mortality in infected horses and humans. Despite the importance of EEEV to human and animal health, little is known about how the virus overwinters and reinitiates transmission each spring, particularly in temperate regions where infected adult mosquitoes are unlikely to survive through the winter. One hypothesis to explain the mechanism by which this virus persists from year to year is the spring recrudescence of latent virus in avian reservoir hosts. In this study, we tested the recrudescence hypothesis with gray catbirds (Dumatella carolinensis) captured in northern Ohio (July-August 2007). Birds were experimentally infected with EEEV on 1 October 2007. In January 2008, they were then exposed to exogenous testosterone and/or extended photoperiod to initiate reactivation of latent EEEV infection. All birds became viremic with EEEV, with mean viremia of 6.0 log10 plaque-forming units/ml serum occurring at 1 d postinoculation. One male in the testosterone, long-day treatment group had EEEV viral RNA in a cloacal swab collected on 18 January 2008. Otherwise, no other catbirds exhibited reactivated infections in cloacal swabs or blood. Antibody titers fluctuated over the course of the study, with lowest titers observed in January 2008, which corresponded with the lowest mean weight of the birds. No EEEV viral RNA was detected in the blood, kidney, spleen, brain, liver, and lower intestine upon necropsy at 19 wk postinfection.
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Sec24p and Sec16p Cooperate to Regulate the GTP Cycle of the COPII Coat
The EMBO Journal.
Feb, 2012 |
Pubmed ID: 22157747 Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24-m11) in the cargo-binding subunit, Sec24p, that specifically impacts the GTP-dependent generation of vesicles in vitro. Using a high-throughput approach, we defined genetic interactions between sec24-m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p- and Sec31p-stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p-m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p-m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/disassembly.
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Vesicle-mediated ER Export of Proteins and Lipids
Biochimica Et Biophysica Acta.
Aug, 2012 |
Pubmed ID: 22265716 In eukaryotic cells, the endoplasmic reticulum (ER) is a major site of synthesis of both lipids and proteins, many of which must be transported to other organelles. The COPII coat-comprising Sar1, Sec23/24, Sec13/31-generates transport vesicles that mediate the bulk of protein/lipid export from the ER. The coat exhibits remarkable flexibility in its ability to specifically select and accommodate a large number of cargoes with diverse properties. In this review, we discuss the fundamentals of COPII vesicle production and describe recent advances that further our understanding of just how flexible COPII cargo recruitment and vesicle formation may be. Large or bulky cargo molecules (e.g. collagen rods and lipoprotein particles) exceed the canonical size for COPII vesicles and seem to rely on the additional action of recently identified accessory molecules. Although the bulk of the research has focused on the fate of protein cargo, the mechanisms and regulation of lipid transport are equally critical to cellular survival. From their site of synthesis in the ER, phospholipids, sphingolipids and sterols exit the ER, either accompanying cargo in vesicles or directly across the cytoplasm shielded by lipid-transfer proteins. Finally, we highlight the current challenges to the field in addressing the physiological regulation of COPII vesicle production and the molecular details of how diverse cargoes, both proteins and lipids, are accommodated. This article is part of a Special Issue entitled Lipids and Vesicular Transport.
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A Yeast Phenomic Model for the Gene Interaction Network Modulating CFTR-ΔF508 Protein Biogenesis
Genome Medicine.
Dec, 2012 |
Pubmed ID: 23270647 BACKGROUND: The overall influence of gene interaction in human disease is unknown. In cystic fibrosis (CF) a single allele of the cystic fibrosis transmembrane conductance regulator (CFTR-ΔF508) accounts for most of the disease. In cell models, CFTR-ΔF508 exhibits defective protein biogenesis and degradation rather than proper trafficking to the plasma membrane where CFTR normally functions. Numerous genes function in the biogenesis of CFTR and influence the fate of CFTR-ΔF508. However it is not known whether genetic variation in such genes contributes to disease severity in patients. Nor is there an easy way to study how numerous gene interactions involving CFTR-ΔF would manifest phenotypically. METHODS: To gain insight into the function and evolutionary conservation of a gene interaction network that regulates biogenesis of a misfolded ABC transporter, we employed yeast genetics to develop a 'phenomic' model, in which the CFTR-ΔF508-equivalent residue of a yeast homolog is mutated (Yor1-ΔF670), and where the genome is scanned quantitatively for interaction. We first confirmed that Yor1-ΔF undergoes protein misfolding and has reduced half-life, analogous to CFTR-ΔF. Gene interaction was then assessed quantitatively by growth curves for approximately 5,000 double mutants, based on alteration in the dose response to growth inhibition by oligomycin, a toxin extruded from the cell at the plasma membrane by Yor1. RESULTS: From a comparative genomic perspective, yeast gene interactions influencing Yor1-ΔF biogenesis were representative of human homologs previously found to modulate processing of CFTR-ΔF in mammalian cells. Additional evolutionarily conserved pathways were implicated by the study, and a ΔF-specific pro-biogenesis function of the recently discovered ER membrane complex (EMC) was evident from the yeast screen. This novel function was validated biochemically by siRNA of an EMC ortholog in a human cell line expressing CFTR-ΔF508. The precision and accuracy of quantitative high throughput cell array phenotyping (Q-HTCP), which captures tens of thousands of growth curves simultaneously, provided powerful resolution to measure gene interaction on a phenomic scale, based on discrete cell proliferation parameters. CONCLUSION: We propose phenomic analysis of Yor1-ΔF as a model for investigating gene interaction networks that can modulate cystic fibrosis disease severity. Although the clinical relevance of the Yor1-ΔF gene interaction network for cystic fibrosis remains to be defined, the model appears to be informative with respect to human cell models of CFTR-ΔF. Moreover, the general strategy of yeast phenomics can be employed in a systematic manner to model gene interaction for other diseases relating to pathologies that result from protein misfolding or potentially any disease involving evolutionarily conserved genetic pathways.
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Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
Genetics.
Feb, 2013 |
Pubmed ID: 23396477 The secretory pathway is responsible for the synthesis, folding, and delivery of a diverse array of cellular proteins. Secretory protein synthesis begins in the endoplasmic reticulum (ER), which is charged with the tasks of correctly integrating nascent proteins and ensuring correct post-translational modification and folding. Once ready for forward traffic, proteins are captured into ER-derived transport vesicles that form through the action of the COPII coat. COPII-coated vesicles are delivered to the early Golgi via distinct tethering and fusion machineries. Escaped ER residents and other cycling transport machinery components are returned to the ER via COPI-coated vesicles, which undergo similar tethering and fusion reactions. Ultimately, organelle structure, function, and cell homeostasis are maintained by modulating protein and lipid flux through the early secretory pathway. In the last decade, structural and mechanistic studies have added greatly to the strong foundation of yeast genetics on which this field was built. Here we discuss the key players that mediate secretory protein biogenesis and trafficking, highlighting recent advances that have deepened our understanding of the complexity of this conserved and essential process.
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Vesicle-mediated Export from the ER: COPII Coat Function and Regulation
Biochimica Et Biophysica Acta.
Nov, 2013 |
Pubmed ID: 23419775 Vesicle trafficking from the endoplasmic reticulum (ER) is a vital cellular process in all eukaryotes responsible for moving secretory cargoes from the ER to the Golgi apparatus. To accomplish this feat, the cell employs a set of conserved cytoplasmic coat proteins - the coat protein II (COPII) complex - that recruit cargo into nascent buds and deform the ER membrane to drive vesicle formation. While our understanding of COPII coat mechanics has developed substantially since its discovery, we have only recently begun to appreciate the factors that regulate this complex and, in turn, ER-to-Golgi trafficking. Here, we describe these factors and their influences on COPII vesicle formation. Properties intrinsic to the GTP cycle of the coat, as well as coat structure, have critical implications for COPII vesicle trafficking. Extrinsic factors in the cytosol can modulate COPII activity through direct interaction with the coat or with scaffolding components, or by changing composition of the ER membrane. Further, lumenal and membrane-bound cargoes and cargo receptors can influence COPII-mediated trafficking in equally profound ways. Together, these factors work in concert to ensure proper cargo movement in this first step of the secretory pathway. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.
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Intestinal Parasites of the Gopher Tortoise (Gopherus Polyphemus) from Eight Populations in Georgia
Parasitology Research.
Dec, 2013 |
Pubmed ID: 24072334 The gopher tortoise (Gopherus polyphemus), one of five tortoise species endemic in the USA, was recently classified as a candidate for federal listing as a threatened species. Fecal samples collected from 117 tortoises from eight sites in Georgia were examined for endoparasites using a combination of sedimentation and flotation. Samples from an island population were examined for parasitic oocysts and ova only by flotation, protozoan cysts by trichrome-stained direct smear, and Cryptosporidium by direct immunofluorescence assay and ProSpecT rapid assay. A total of 99 tortoises (85, range 0-100%) was infected with pinworms (Alaeuris spp.), 47 (40, 0-86%) with cestodes (Oochorstica sp.), 34 (41, 0-74%) with Chapiniella spp., 2 (3, 0-33%) with Eimeria paynei, and a single tortoise each with a capillarid and ascarid (1%). On the island, Entamoeba was detected in one tortoise (2%) while Cryptosporidium oocysts were detected in eight (17%). In conclusion, at least eight species of parasites were detected including Cryptosporidium, a possible pathogen of tortoises. Interestingly, we detected spatial variation in the distribution of several parasites among populations suggesting additional work should be conducted across a gradient of tortoise densities, land use, and habitat characteristics.
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