RESUMEN
The Golgi complex comprises a connected ribbon of stacked cisternal membranes localized to the perinuclear region in most vertebrate cells. The position and morphology of this organelle depends upon interactions with microtubules and the actin cytoskeleton. In contrast, we know relatively little about the relationship of the Golgi complex with intermediate filaments (IFs). In this study, we show that the Golgi is in close physical proximity to vimentin IFs in cultured mouse and human cells. We also show that the trans-Golgi network coiled-coil protein GORAB can physically associate with vimentin IFs. Loss of vimentin and/or GORAB had a modest effect upon Golgi structure at the steady state. The Golgi underwent more rapid disassembly upon chemical disruption with brefeldin A or nocodazole, and slower reassembly upon drug washout, in vimentin knockout cells. Moreover, loss of vimentin caused reduced Golgi ribbon integrity when cells were cultured on high-stiffness hydrogels, which was exacerbated by loss of GORAB. These results indicate that vimentin IFs contribute to the structural stability of the Golgi complex and suggest a role for GORAB in this process.
Asunto(s)
Citoesqueleto , Filamentos Intermedios , Ratones , Humanos , Animales , Filamentos Intermedios/metabolismo , Vimentina/metabolismo , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Aparato de Golgi/metabolismo , Mamíferos/metabolismoRESUMEN
During the drug development process, testing potency plays an important role in the quality assessment required for the manufacturing and marketing of biologics. Due to multiple operational and biological factors, higher variability is usually observed in bioassays compared with physicochemical methods. In this paper, we discuss different sources of bioassay variability and how this variability can be statistically estimated. In addition, we propose an algorithm to estimate the variability of reportable results associated with different numbers of runs and their corresponding OOS rates under a given specification. Numerical experiments are conducted on multiple assay formats to elucidate the empirical distribution of bioassay variability.
RESUMEN
Gerodermia osteodysplastica (GO) is characterized by skin laxity and early-onset osteoporosis. GORAB, the responsible disease gene, encodes a small Golgi protein of poorly characterized function. To circumvent neonatal lethality of the GorabNull full knockout, Gorab was conditionally inactivated in mesenchymal progenitor cells (Prx1-cre), pre-osteoblasts (Runx2-cre), and late osteoblasts/osteocytes (Dmp1-cre), respectively. While in all three lines a reduction in trabecular bone density was evident, only GorabPrx1 and GorabRunx2 mutants showed dramatically thinned, porous cortical bone and spontaneous fractures. Collagen fibrils in the skin of GorabNull mutants and in bone of GorabPrx1 mutants were disorganized, which was also seen in a bone biopsy from a GO patient. Measurement of glycosaminoglycan contents revealed a reduction of dermatan sulfate levels in skin and cartilage from GorabNull mutants. In bone from GorabPrx1 mutants total glycosaminoglycan levels and the relative percentage of dermatan sulfate were both strongly diminished. Accordingly, the proteoglycans biglycan and decorin showed reduced glycanation. Also in cultured GORAB-deficient fibroblasts reduced decorin glycanation was evident. The Golgi compartment of these cells showed an accumulation of decorin, but reduced signals for dermatan sulfate. Moreover, we found elevated activation of TGF-ß in GorabPrx1 bone tissue leading to enhanced downstream signalling, which was reproduced in GORAB-deficient fibroblasts. Our data suggest that the loss of Gorab primarily perturbs pre-osteoblasts. GO may be regarded as a congenital disorder of glycosylation affecting proteoglycan synthesis due to delayed transport and impaired posttranslational modification in the Golgi compartment.
Asunto(s)
Enfermedades Óseas/congénito , Enanismo/metabolismo , Osteoblastos/patología , Proteoglicanos/metabolismo , Enfermedades Cutáneas Genéticas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Enfermedades Óseas/metabolismo , Enfermedades Óseas/patología , Diferenciación Celular , Decorina/metabolismo , Dermatán Sulfato/metabolismo , Modelos Animales de Enfermedad , Enanismo/patología , Femenino , Fracturas Óseas/genética , Glicosilación , Proteínas de la Matriz de Golgi , Células Madre Mesenquimatosas/patología , Células Madre Mesenquimatosas/fisiología , Ratones Endogámicos C57BL , Ratones Transgénicos , Osteoblastos/metabolismo , Transducción de Señal , Enfermedades Cutáneas Genéticas/patología , Proteínas de Transporte Vesicular/genéticaRESUMEN
MicroRNA (miRNA)-mediated crosstalk between coding and non-coding RNAs of various types is known as the competing endogenous RNA (ceRNA) concept. Here, we propose that there is a specific variant of the ceRNA language that takes advantage of simple sequence repeat (SSR) wording. We applied bioinformatics tools to identify human transcripts that may be regarded as repeat-associated ceRNAs (raceRNAs). Multiple protein-coding transcripts, transcribed pseudogenes, long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) showing this potential were identified, and numerous miRNAs were predicted to bind to SSRs. We propose that simple repeats expanded in various hereditary neurological diseases may act as sponges for miRNAs containing complementary repeats that would affect raceRNA crosstalk. Based on the representation of specific SSRs in transcripts, expression data for SSR-binding miRNAs and expression profiling data from patients, we determined that raceRNA crosstalk is most likely to be perturbed in the case of myotonic dystrophy type 1 (DM1) and type 2 (DM2).
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MicroARNs/genética , Repeticiones de Microsatélite/genética , Distrofia Miotónica/genética , ARN Mensajero/genética , Regulación de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Humanos , Distrofia Miotónica/patología , ARN/genética , ARN Circular , ARN Largo no Codificante/genéticaRESUMEN
The fundamental role of microRNAs (miRNAs) in the regulation of gene expression has been well-established, but many miRNA-driven regulatory mechanisms remain elusive. In the present study, we demonstrate that miRNAs regulate the expression of DMPK, the gene mutated in myotonic dystrophy type 1 (DM1), and we provide insight regarding the concerted effect of the miRNAs on the DMPK target. Specifically, we examined the binding of several miRNAs to the DMPK 3' UTR using luciferase assays. We validated the interactions between the DMPK transcript and the conserved miR-206 and miR-148a. We suggest a possible cooperativity between these two miRNAs and discuss gene targeting by miRNA pairs that vary in distance between their binding sites and expression profiles. In the same luciferase reporter system, we showed miR-15b/16 binding to the non-conserved CUG repeat tract present in the DMPK transcript and that the CUG-repeat-binding miRNAs might also act cooperatively. Moreover, we detected miR-16 in cytoplasmic foci formed by exogenously expressed RNAs with expanded CUG repeats. Therefore, we propose that the expanded CUGs may serve as a target for concerted regulation by miRNAs and may also act as molecular sponges for natural miRNAs with CAG repeats in their seed regions, thereby affecting their physiological functions.
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Regiones no Traducidas 3' , Regulación de la Expresión Génica , MicroARNs/metabolismo , Proteína Quinasa de Distrofia Miotónica/genética , Animales , Sitios de Unión , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Transgénicos , Proteína Quinasa de Distrofia Miotónica/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Repeticiones de TrinucleótidosRESUMEN
The deep-sequencing of small RNAs has revealed that different numbers and proportions of miRNA variants called isomiRs are formed from single miRNA genes and that this effect is attributable mainly to imprecise cleavage by Drosha and Dicer. Factors that influence the degree of cleavage precision of Drosha and Dicer are under investigation, and their identification may improve our understanding of the mechanisms by which cells modulate the regulatory potential of miRNAs. In this study, we focused on the sequences and structural determinants of Drosha and Dicer cleavage sites, which may explain the generation of homogeneous miRNAs (in which a single isomiR strongly predominates) as well as the generation of heterogeneous miRNAs. Using deep-sequencing data for small RNAs, we demonstrate that the generation of homogeneous miRNAs requires more sequence constraints at the cleavage sites than the formation of heterogeneous miRNAs. Additionally, our results indicate that specific Drosha cleavage sites have more sequence determinants in miRNA precursors than specific cleavage sites for Dicer and that secondary structural motifs in the miRNA precursors influence the precision of Dicer cleavage. Together, we present the sequence and structural features of Drosha and Dicer cleavage sites that influence the heterogeneity of the released miRNAs.
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ARN Helicasas DEAD-box/genética , MicroARNs/genética , División del ARN/genética , Isoformas de ARN/genética , Ribonucleasa III/genética , Línea Celular , Células HEK293 , Humanos , Precursores del ARN/genética , Análisis de Secuencia de ARN/métodosRESUMEN
Idiopathic Pulmonary Fibrosis (IPF) is a devastating form of respiratory disease with a life expectancy of 3-4 years. Inflammation, epithelial injury and myofibroblast proliferation have been implicated in disease initiation and, recently, epithelial-fibroblastic crosstalk has been identified as a central driver. However, the ability to interrogate this crosstalk is limited due to the absence of in vitro models that mimic physiological conditions. To investigate IPF dysregulated cross-talk, primary normal human bronchial epithelial (NHBE) cells and primary normal human lung fibroblasts (NHLF) or diseased human lung fibroblasts (DHLF) from IPF patients, were co-cultured in direct contact at the air-liquid interface (ALI). Intercellular crosstalk was assessed by comparing cellular phenotypes of co-cultures to respective monocultures, through optical, biomolecular and electrical methods. A co-culture-dependent decrease in epithelium thickness, basal cell mRNA (P63, KRT5) and an increase in transepithelial electrical resistance (TEER) was observed. This effect was significantly enhanced in DHLF co-cultures and lead to the induction of epithelial to mesenchymal transition (EMT) and increased mRNA expression of TGFß-2, ZO-1 and DN12. When stimulated with exogenous TGFß, NHBE and NHLF monocultures showed a significant upregulation of EMT (COL1A1, FN1, VIM, ASMA) and senescence (P21) markers, respectively. In contrast, direct NHLF/NHBE co-culture indicated a protective role of epithelial-fibroblastic cross-talk against TGFß-induced EMT, fibroblast-to-myofibroblast transition (FMT) and inflammatory cytokine release (IL-6, IL-8, IL-13, IL-1ß, TNF-α). DHLF co-cultures showed no significant phenotypic transition upon stimulation, likely due to the constitutively high expression of TGFß isoforms prior to any exogenous stimulation. The model developed provides an alternative method to generate IPF-related bronchial epithelial phenotypes in vitro, through the direct co-culture of human lung fibroblasts with NHBEs. These findings highlight the importance of fibroblast TGFß signaling in EMT but that monocultures give rise to differential responses compared to co-cultures, when exposed to this pro-inflammatory stimulus. This holds implications for any translation conclusions drawn from monoculture studies and is an important step in development of more biomimetic models of IPF. In summary, we believe this in vitro system to study fibroblast-epithelial crosstalk, within the context of IPF, provides a platform which will aid in the identification and validation of novel targets.
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Transición Epitelial-Mesenquimal , Fibrosis Pulmonar Idiopática , Humanos , Transición Epitelial-Mesenquimal/fisiología , Fibrosis Pulmonar Idiopática/metabolismo , Fibroblastos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , ARN Mensajero/metabolismoRESUMEN
We have systematically investigated six compendial nonionic detergents as potential replacements for Triton ×-100 in bioprocessing applications. Use of compendial raw materials in cGMP bioprocessing is advantageous for a variety of reasons including material specifications developed to meet stringent pharmaceutical product quality requirements, regulatory familiarity and comfort, and availability from vendors experienced supplying the biopharmaceutical industry. We first examine material properties of the detergents themselves including melting point and viscosity. Process performance and product contact in real-world bioprocess applications are then investigated. Lastly, we test the detergents in virus inactivation (VI) experiments with recombinant proteins and adeno-associated virus. Two of the detergents tested, PEG 9 Lauryl Ether and PEG 6 Caprylic/Capric Glycerides, showed favorable properties that make them attractive for use as potential Triton X-100 replacements. Process performance testing indicated negligible impact of the detergents on product yield, purity, and activity compared to a control with no detergent. Importantly, both PEG 9 Lauryl Ether and PEG 6 Caprylic/Capric Glycerides demonstrated very fast VI kinetics with complete inactivation of XMuLV observed in less than 1 min at a target 1% detergent concentration. Potential advantages and disadvantages of both candidate detergents for use in cGMP bioprocessing are summarized and discussed.
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Detergentes , Éter , Detergentes/farmacología , Glicéridos , Octoxinol/farmacología , Inactivación de VirusRESUMEN
COPI is a key mediator of protein trafficking within the secretory pathway. COPI is recruited to the membrane primarily through binding to Arf GTPases, upon which it undergoes assembly to form coated transport intermediates responsible for trafficking numerous proteins, including Golgi-resident enzymes. Here, we identify GORAB, the protein mutated in the skin and bone disorder gerodermia osteodysplastica, as a component of the COPI machinery. GORAB forms stable domains at the trans-Golgi that, via interactions with the COPI-binding protein Scyl1, promote COPI recruitment to these domains. Pathogenic GORAB mutations perturb Scyl1 binding or GORAB assembly into domains, indicating the importance of these interactions. Loss of GORAB causes impairment of COPI-mediated retrieval of trans-Golgi enzymes, resulting in a deficit in glycosylation of secretory cargo proteins. Our results therefore identify GORAB as a COPI scaffolding factor, and support the view that defective protein glycosylation is a major disease mechanism in gerodermia osteodysplastica.
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Proteínas Portadoras/metabolismo , Proteína Coat de Complejo I/metabolismo , Enzimas/metabolismo , Aparato de Golgi/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Enfermedades Óseas/congénito , Enfermedades Óseas/genética , Enfermedades Óseas/metabolismo , Proteínas Portadoras/genética , Células Cultivadas , Proteína Coat de Complejo I/genética , Proteínas de Unión al ADN , Enanismo/genética , Enanismo/metabolismo , Glicosilación , Proteínas de la Matriz de Golgi , Células HEK293 , Células HeLa , Humanos , Mutación , Unión Proteica , Transporte de Proteínas , Interferencia de ARN , Enfermedades Cutáneas Genéticas/genética , Enfermedades Cutáneas Genéticas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Odontochondrodysplasia (ODCD) is an unresolved genetic disorder of skeletal and dental development. Here, we show that ODCD is caused by hypomorphic TRIP11 mutations, and we identify ODCD as the nonlethal counterpart to achondrogenesis 1A (ACG1A), the known null phenotype in humans. TRIP11 encodes Golgi-associated microtubule-binding protein 210 (GMAP-210), an essential tether protein of the Golgi apparatus that physically interacts with intraflagellar transport 20 (IFT20), a component of the ciliary intraflagellar transport complex B. This association and extraskeletal disease manifestations in ODCD point to a cilium-dependent pathogenesis. However, our functional studies in patient-derived primary cells clearly support a Golgi-based disease mechanism. In spite of reduced abundance, residual GMAP variants maintain partial Golgi integrity, normal global protein secretion, and subcellular distribution of IFT20 in ODCD. These functions are lost when GMAP-210 is completely abrogated in ACG1A. However, a similar defect in chondrocyte maturation is observed in both disorders, which produces a cellular achondrogenesis phenotype of different severity, ensuing from aberrant glycan processing and impaired extracellular matrix proteoglycan secretion by the Golgi apparatus.
RESUMEN
Biallelic loss-of-function mutations in TRIP11, encoding the golgin GMAP-210, cause the lethal human chondrodysplasia achondrogenesis 1A (ACG1A). We now find that a homozygous splice-site mutation of the lamin B receptor (LBR) gene results in the same phenotype. Intrigued by the genetic heterogeneity, we compared GMAP-210- and LBR-deficient primary cells to unravel how particular mutations in LBR cause a phenocopy of ACG1A. We could exclude a regulatory interaction between LBR and GMAP-210 in patients' cells. However, we discovered a common disruption of Golgi apparatus architecture that was accompanied by decreased secretory trafficking in both cases. Deficiency of Golgi-dependent glycan processing indicated a similar downstream effect of the disease-causing mutations upon Golgi function. Unexpectedly, our results thus point to a common pathogenic mechanism in GMAP-210- and LBR-related diseases attributable to defective secretory trafficking at the Golgi apparatus.
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Acondroplasia/genética , Proteínas Nucleares/genética , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genética , Acondroplasia/patología , Transporte Biológico Activo/genética , Proliferación Celular , Supervivencia Celular , Colesterol/análisis , Proteínas del Citoesqueleto , Retículo Endoplásmico/ultraestructura , Femenino , Feto , Fibroblastos/patología , Enfermedades Genéticas Congénitas/genética , Aparato de Golgi/fisiología , Aparato de Golgi/ultraestructura , Humanos , Mutación , Linaje , Fenotipo , Análisis de Secuencia de Proteína , Esteroles/análisis , Receptor de Lamina BRESUMEN
The Golgi apparatus occupies a central position within the secretory pathway where it is a hub for vesicle trafficking. Distinct classes of transport vesicles traffic diverse cargoes into and out of this organelle, as well as between the different Golgi subcompartments. A key feature of Golgi trafficking is the specific recognition of transport vesicles at the different regions of the Golgi apparatus, required for the correct cargo delivery. Specificity is ensured by coiled-coil golgins and multi-subunit tethering complexes (MTCs), which act together to capture vesicles and promote their subsequent fusion with the Golgi membrane. In this review we discuss our current understanding of how golgins and MTCs function together to mediate the specific recognition of vesicles at the Golgi apparatus.
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Aparato de Golgi/metabolismo , Vesículas Transportadoras/metabolismo , Animales , Humanos , Proteínas de la Fusión de la Membrana/metabolismo , Transporte de Proteínas , Vías Secretoras , Proteínas de Transporte Vesicular/metabolismoRESUMEN
The golgins are a family of predominantly coiled-coil proteins that are localized to the Golgi apparatus. Golgins are present in all eukaryotes, suggesting an evolutionary conserved function. Golgins are anchored to the Golgi membrane by their carboxy terminus and are predicted to adopt an extended conformation that projects into the surrounding cytoplasm. This arrangement is ideal for the capture or tethering of nearby membranes or cytoskeletal elements. Golgin-mediated tethering is thought to be important for vesicular traffic at the Golgi apparatus, the maintenance of Golgi architecture, as well as the positioning of the Golgi apparatus within cells. In addition to acting as tethers, some golgins can also sequester various factors at the Golgi membrane, allowing for the spatiotemporal regulation of downstream cellular functions. Although it is now established that golgins are membrane and cytoskeleton tethers, the mechanisms underlying tethering remain poorly defined. Moreover, the importance of golgin-mediated tethering in a physiological context remains to be fully explored. This review will describe our current understanding of golgin function, highlighting recent progress that has been made, and goes on to discuss outstanding questions and potential avenues for future research with regard to this family of conserved Golgi-associated proteins.
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The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network.
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Citoesqueleto de Actina/metabolismo , Retículo Endoplásmico/metabolismo , Miosina Tipo I/metabolismo , Actinas/metabolismo , Línea Celular Tumoral , Humanos , Microtúbulos/metabolismo , Miosina Tipo I/química , Fenotipo , Polimerizacion , Unión Proteica , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Protein trafficking within the secretory pathway of mammalian cells is amenable to analysis by biochemical methods. This can be achieved by monitoring posttranslational modifications that occur naturally within the secretory pathway, or by measuring the delivery of cargo to the cell surface or extracellular medium. These approaches can be combined with additional manipulations such as specific temperature blocks that permit analysis of distinct trafficking steps. Biochemical analysis is advantageous in that it permits both a sensitive and quantitative measure of trafficking along the pathway. The methods discussed in this chapter permit the analysis of trafficking of both endogenous cargo proteins and ectopically expressed model cargos, which can be followed using either Western blotting or metabolic pulse-chase approaches. These methods are relatively straightforward and suitable for use in most modern cell biology laboratories. In addition to the well-established methods that we describe here in detail, we also refer to the development of more recent tailored approaches that add further to the arsenal of tools that can be used to assess trafficking in the secretory pathway.