RESUMEN
Genetic screens are a classic approach to dissecting biological pathways including membrane trafficking. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 have enabled the utility of this approach in diploid models, including cultured mammalian cells. Here, we present detailed protocols for generating custom CRISPR libraries. These methods are useful for generating genome-wide libraries for new model organisms that lack an existing genome-wide library, and for generating smaller focused libraries.
Asunto(s)
Sistemas CRISPR-Cas , Pruebas Genéticas , Animales , Sistemas CRISPR-Cas/genética , Línea Celular , Pruebas Genéticas/métodos , Biblioteca Genómica , Mamíferos/genéticaRESUMEN
Diverse gene products contribute to the pathogenesis of Alzheimer's disease (AD). Experimental models have helped elucidate their mechanisms and impact on brain functions. Human amyloid precursor protein (hAPP) transgenic mice from line J20 (hAPP-J20 mice) are widely used to simulate key aspects of AD. However, they also carry an insertional mutation in noncoding sequence of one Zbtb20 allele, a gene involved in neural development. We demonstrate that heterozygous hAPP-J20 mice have reduced Zbtb20 expression in some AD-relevant brain regions, but not others, and that Zbtb20 levels are higher in hAPP-J20 mice than heterozygous Zbtb20 knock-out (Zbtb20+/-) mice. Whereas hAPP-J20 mice have premature mortality, severe deficits in learning and memory, other behavioral alterations, and prominent nonconvulsive epileptiform activity, Zbtb20+/- mice do not. Thus, the insertional mutation in hAPP-J20 mice does not ablate the affected Zbtb20 allele and is unlikely to account for the AD-like phenotype of this model.
Asunto(s)
Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Ratones Transgénicos , Fenotipo , Factores de TranscripciónRESUMEN
Insulin promotes glucose uptake by triggering the translocation of glucose transporter type 4 (GLUT4) from intracellular vesicles to the plasma membrane through exocytosis. GLUT4 exocytosis is a vesicle fusion event involving fusion of GLUT4-containing vesicles with the plasma membrane. For GLUT4 vesicle fusion to occur, GLUT4 vesicles must first be tethered to the plasma membrane. A key tethering factor in exocytosis is a heterooctameric protein complex called the exocyst. The role of the exocyst in GLUT4 exocytosis, however, remains incompletely understood. Here we first systematically analyzed data from a genome-scale CRISPR screen in HeLa cells that targeted virtually all known genes in the human genome, including 12 exocyst genes. The screen recovered only a subset of the exocyst genes, including exocyst complex component 7 (Exoc7/Exo70). Other exocyst genes, however, were not isolated in the screen, likely because of functional redundancy. Our findings suggest that selection of an appropriate exocyst gene is critical for genetic studies of exocyst functions. Next we developed an inducible adipocyte genome editing system that enabled Exoc7 gene deletion in adipocytes without interfering with adipocyte differentiation. We observed that insulin-stimulated GLUT4 exocytosis was markedly inhibited in Exoc7 KO adipocytes. Insulin signaling, however, remained intact in these KO cells. These results indicate that the exocyst plays a critical role in insulin-stimulated GLUT4 exocytosis in adipocytes. We propose that the strategy outlined in this work could be instrumental in genetically dissecting other membrane-trafficking pathways in adipocytes.
Asunto(s)
Exocitosis/efectos de los fármacos , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/farmacología , ARN Guía de Kinetoplastida/metabolismo , Proteínas de Transporte Vesicular/genética , Adipocitos/citología , Adipocitos/metabolismo , Animales , Sistemas CRISPR-Cas/genética , Diferenciación Celular , Edición Génica , Células HeLa , Humanos , Ratones , Transducción de Señal , Proteínas de Transporte Vesicular/deficiencia , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Multimeric adaptors are broadly involved in vesicle-mediated membrane trafficking. AP2 adaptor, in particular, plays a central role in clathrin-mediated endocytosis (CME) by recruiting cargo and clathrin to endocytic sites. It is generally thought that trafficking adaptors such as AP2 adaptor assemble spontaneously. In this work, however, we discovered that AP2 adaptor assembly is an ordered process controlled by alpha and gamma adaptin binding protein (AAGAB), an uncharacterized factor identified in our genome-wide genetic screen of CME. AAGAB guides the sequential association of AP2 subunits and stabilizes assembly intermediates. Without the assistance of AAGAB, AP2 subunits fail to form the adaptor complex, leading to their degradation. The function of AAGAB is abrogated by a mutation that causes punctate palmoplantar keratoderma type 1 (PPKP1), a human skin disease. Since other multimeric trafficking adaptors operate in an analogous manner to AP2 adaptor, their assembly likely involves a similar regulatory mechanism.
Asunto(s)
Complejo 2 de Proteína Adaptadora/genética , Proteínas Adaptadoras del Transporte Vesicular/genética , Endocitosis/genética , Secuencia de Aminoácidos/genética , Membrana Celular/genética , Clatrina/genética , Humanos , Queratodermia Palmoplantar/genética , Queratodermia Palmoplantar/patología , Unión Proteica/genética , Transporte de Proteínas/genética , ProteolisisRESUMEN
About one-third of cellular proteins in eukaryotic cells are localized to membrane-enclosed organelles in the endomembrane system. Trafficking of these membrane proteins (including soluble lumenal proteins) among the organelles is mediated by small sac-like vesicles. Vesicle-mediated membrane trafficking regulates a broad range of biological processes, many of which are still poorly understood at the molecular level. A powerful approach to dissect a vesicle-mediated membrane trafficking pathway is unbiased genome-wide genetic screening, which only recently became possible in mammalian cells with the isolation of haploid human cell lines and the development of CRISPR-Cas9 genome editing. Here, we describe a FACS-based method to select populations of live mutant cells based on the surface levels of endogenous proteins or engineered reporters. Collection of these mutant populations enables subsequent deep sequencing and bioinformatics analysis to identify genes that regulate the trafficking pathway. This method can be readily adapted to genetically dissect a broad range of mammalian membrane trafficking processes using haploid genetics or CRISPR-Cas9 screens. © 2018 by John Wiley & Sons, Inc.
Asunto(s)
Transporte Biológico/genética , Membrana Celular/metabolismo , Citometría de Flujo , Pruebas Genéticas , Animales , Células Eucariotas/citología , Células Eucariotas/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismoRESUMEN
Sec1/Munc18 (SM) proteins promote intracellular vesicle fusion by binding to N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). A key SNARE-binding mode of SM proteins involves the N-terminal peptide (N-peptide) motif of syntaxin, a SNARE subunit localized to the target membrane. In in vitro membrane fusion assays, inhibition of N-peptide motif binding previously has been shown to abrogate the stimulatory function of Munc18-1, a SM protein involved in synaptic exocytosis in neurons. The physiological role of the N-peptide-binding mode, however, remains unclear. In this work, we addressed this key question using a "clogged" Munc18-1 protein, in which an ectopic copy of the syntaxin N-peptide motif was directly fused to Munc18-1. We found that the ectopic N-peptide motif blocks the N-peptide-binding pocket of Munc18-1, preventing the latter from binding to the native N-peptide motif on syntaxin-1. In a reconstituted system, we observed that clogged Munc18-1 is defective in promoting SNARE zippering. When introduced into induced neuronal cells (iN cells) derived from human pluripotent stem cells, clogged Munc18-1 failed to mediate synaptic exocytosis. As a result, both spontaneous and evoked synaptic transmission was abolished. These genetic findings provide direct evidence for the crucial role of the N-peptide-binding mode of Munc18-1 in synaptic exocytosis. We suggest that clogged SM proteins will also be instrumental in defining the physiological roles of the N-peptide-binding mode in other vesicle-fusion pathways.
Asunto(s)
Exocitosis , Proteínas Munc18/química , Proteínas Munc18/metabolismo , Péptidos/metabolismo , Sinapsis/metabolismo , Secuencias de Aminoácidos , Humanos , Proteínas Munc18/genética , Neuronas/química , Neuronas/metabolismo , Péptidos/química , Unión Proteica , Transporte de Proteínas , Transducción de Señal , Sinapsis/química , Sinapsis/genética , Transmisión Sináptica , Sintaxina 1/química , Sintaxina 1/genética , Sintaxina 1/metabolismoRESUMEN
Diabetes is a leading cause of death worldwide and results in over 3 million annual deaths. While insulin manages the disease well, many patients fail to comply with injection schedules, and despite significant investment, a more convenient oral formulation of insulin is still unavailable. Studies suggest that glycosylation may stabilize peptides for oral delivery, but the demanding production of homogeneously glycosylated peptides has hampered transition into the clinic. We report here the first total synthesis of homogeneously glycosylated insulin. After characterizing a series of insulin glycoforms with systematically varied O-glycosylation sites and structures, we demonstrate that O-mannosylation of insulin B-chain Thr27 reduces the peptide's susceptibility to proteases and self-association, both critical properties for oral dosing, while maintaining full activity. This work illustrates the promise of glycosylation as a general mechanism for regulating peptide activity and expanding its therapeutic use.
Asunto(s)
Insulina/análogos & derivados , Ingeniería de Proteínas/métodos , Animales , Línea Celular , Diseño de Fármacos , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Insulina/química , Insulina/metabolismo , Insulina/farmacología , Ratones , Relación Estructura-Actividad , Treonina/químicaRESUMEN
Rab GTPases are switched from their GDP-bound inactive conformation to a GTP-bound active state by guanine nucleotide exchange factors (GEFs). The first putative GEFs isolated for Rabs are RABIF (Rab-interacting factor)/MSS4 (mammalian suppressor of Sec4) and its yeast homolog DSS4 (dominant suppressor of Sec4). However, the biological function and molecular mechanism of these molecules remained unclear. In a genome-wide CRISPR genetic screen, we isolated RABIF as a positive regulator of exocytosis. Knockout of RABIF severely impaired insulin-stimulated GLUT4 exocytosis in adipocytes. Unexpectedly, we discovered that RABIF does not function as a GEF, as previously assumed. Instead, RABIF promotes the stability of Rab10, a key Rab in GLUT4 exocytosis. In the absence of RABIF, Rab10 can be efficiently synthesized but is rapidly degraded by the proteasome, leading to exocytosis defects. Strikingly, restoration of Rab10 expression rescues exocytosis defects, bypassing the requirement for RABIF. These findings reveal a crucial role of RABIF in vesicle transport and establish RABIF as a Rab-stabilizing holdase chaperone, a previously unrecognized mode of Rab regulation independent of its GDP-releasing activity. Besides Rab10, RABIF also regulates the stability of two other Rab GTPases, Rab8 and Rab13, suggesting that the requirement of holdase chaperones is likely a general feature of Rab GTPases.
Asunto(s)
Exocitosis/fisiología , Transportador de Glucosa de Tipo 4/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Chaperonas Moleculares/metabolismo , Transporte de Proteínas/fisiología , Adipocitos/metabolismo , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HEK293 , Células HeLa , Humanos , Ratones , Vesículas Transportadoras/fisiología , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Organelles are in constant communication with each other through exchange of proteins (mediated by trafficking vesicles) and lipids [mediated by both trafficking vesicles and lipid transfer proteins (LTPs)]. It has long been known that vesicle trafficking can be tightly regulated by the second messenger Ca(2+), allowing membrane protein transport to be adjusted according to physiological demands. However, it remains unclear whether LTP-mediated lipid transport can also be regulated by Ca(2+) In this work, we show that extended synaptotagmins (E-Syts), poorly understood membrane proteins at endoplasmic reticulum-plasma membrane contact sites, are Ca(2+)-dependent LTPs. Using both recombinant and endogenous mammalian proteins, we discovered that E-Syts transfer glycerophospholipids between membrane bilayers in the presence of Ca(2+) E-Syts use their lipid-accommodating synaptotagmin-like mitochondrial lipid binding protein (SMP) domains to transfer lipids. However, the SMP domains themselves cannot transport lipids unless the two membranes are tightly tethered by Ca(2+)-bound C2 domains. Strikingly, the Ca(2+)-regulated lipid transfer activity of E-Syts was fully recapitulated when the SMP domain was fused to the cytosolic domain of synaptotagmin-1, the Ca(2+)sensor in synaptic vesicle fusion, indicating that a common mechanism of membrane tethering governs the Ca(2+)regulation of lipid transfer and vesicle fusion. Finally, we showed that microsomal vesicles isolated from mammalian cells contained robust Ca(2+)-dependent lipid transfer activities, which were mediated by E-Syts. These findings established E-Syts as a novel class of LTPs and showed that LTP-mediated lipid trafficking, like vesicular transport, can be subject to tight Ca(2+)regulation.
Asunto(s)
Calcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Metabolismo de los Lípidos/fisiología , Sinaptotagminas/metabolismo , Animales , Transporte Biológico Activo/fisiología , Calcio/química , Membrana Celular/química , Retículo Endoplásmico/química , Sinaptotagminas/químicaRESUMEN
The fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane requires two classes of molecules-SNAP receptor (SNARE) and Sec1/Munc18 (SM) protein. Reconstitution studies suggest that the SM protein Munc18-1 promotes the zippering of trans-SNARE complexes and accelerates the kinetics of SNARE-dependent membrane fusion. However, the physiological role of this trans-SNARE-regulating function in synaptic exocytosis remains to be established. Here we first demonstrate that two mutations in the vesicle-anchored v-SNARE selectively impair the ability of Munc18-1 to promote trans-SNARE zippering, whereas other known Munc18-1/SNARE-binding modes are unaffected. In cultured neurons, these v-SNARE mutations strongly inhibit spontaneous as well as evoked neurotransmitter release, providing genetic evidence for the trans-SNARE-regulating function of Munc18-1 in synaptic exocytosis. Finally, we show that the trans-SNARE-regulating function of Munc18-1 is compromised by a mutation associated with Ohtahara Syndrome, a severe form of epilepsy.
Asunto(s)
Exocitosis/genética , Proteínas Munc18/genética , Neuronas/metabolismo , Transmisión Sináptica/genética , Proteína 2 de Membrana Asociada a Vesículas/genética , Animales , Sitios de Unión , Corteza Cerebral/citología , Epilepsia/genética , Immunoblotting , Liposomas/metabolismo , Fusión de Membrana , Ratones , Proteínas Munc18/metabolismo , Mutación , Técnicas de Placa-Clamp , Proteínas SNARE/genética , Proteínas SNARE/metabolismoRESUMEN
Tomosyn negatively regulates SNARE-dependent exocytic pathways including insulin secretion, GLUT4 exocytosis, and neurotransmitter release. The molecular mechanism of tomosyn, however, has not been fully elucidated. Here, we reconstituted SNARE-dependent fusion reactions in vitro to recapitulate the tomosyn-regulated exocytic pathways. We then expressed and purified active full-length tomosyn and examined how it regulates the reconstituted SNARE-dependent fusion reactions. Using these defined fusion assays, we demonstrated that tomosyn negatively regulates SNARE-mediated membrane fusion by inhibiting the assembly of the ternary SNARE complex. Tomosyn recognizes the t-SNARE complex and prevents its pairing with the v-SNARE, therefore arresting the fusion reaction at a pre-docking stage. The inhibitory function of tomosyn is mediated by its C-terminal domain (CTD) that contains an R-SNARE-like motif, confirming previous studies carried out using truncated tomosyn fragments. Interestingly, the N-terminal domain (NTD) of tomosyn is critical (but not sufficient) to the binding of tomosyn to the syntaxin monomer, indicating that full-length tomosyn possesses unique features not found in the widely studied CTD fragment. Finally, we showed that the inhibitory function of tomosyn is dominant over the stimulatory activity of the Sec1/Munc18 protein in fusion. We suggest that tomosyn uses its CTD to arrest SNARE-dependent fusion reactions, whereas its NTD is required for the recruitment of tomosyn to vesicle fusion sites through syntaxin interaction.
Asunto(s)
Membrana Celular/metabolismo , Exocitosis/genética , Fusión de Membrana/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas R-SNARE/metabolismo , Animales , Membrana Celular/química , Etilmaleimida/química , Transportador de Glucosa de Tipo 4/metabolismo , Proteínas del Tejido Nervioso/química , Células PC12 , Mapas de Interacción de Proteínas/genética , Estructura Terciaria de Proteína , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/química , Ratas , Proteínas SNARE/genética , Transmisión Sináptica/genéticaRESUMEN
This protocol describes an EDTA-based passaging procedure to be used with chemically defined E8 medium that serves as a tool for basic and translational research into human pluripotent stem cells (PSCs). In this protocol, passaging one six-well or 10-cm plate of cells takes about 6-7 min. This enzyme-free protocol achieves maximum cell survival without enzyme neutralization, centrifugation or drug treatment. It also allows for higher throughput, requires minimal material and limits contamination. Here we describe how to produce a consistent E8 medium for routine maintenance and reprogramming and how to incorporate the EDTA-based passaging procedure into human induced PSC (iPSC) derivation, colony expansion, cryopreservation and teratoma formation. This protocol has been successful in routine cell expansion, and efficient for expanding large-volume cultures or a large number of cells with preferential dissociation of PSCs. Effective for all culture stages, this procedure provides a consistent and universal approach to passaging human PSCs in E8 medium.
Asunto(s)
Técnicas de Cultivo de Célula , Células Madre Pluripotentes/citología , Animales , Células Cultivadas , Colágeno , Medios de Cultivo/química , Combinación de Medicamentos , Ácido Edético/química , Femenino , Humanos , Laminina , Ratones , Ratones SCID , ProteoglicanosRESUMEN
Fibroblast growth factor (FGF), transforming growth factor (TGF)/Nodal, and Insulin/insulin-like growth factor (IGF) signaling pathways are sufficient to maintain human embryonic stem cells (ESCs) and induced pluripotent stem cells in a proliferative, undifferentiated state. Here, we show that only a few FGF family members (FGF2, FGF4, FGF6, and FGF9) are able to sustain strong extracellular-signal-regulated kinase (ERK) phosphorylation and NANOG expression levels in human ESCs. Surprisingly, FGF1, which is reported to target the same set of receptors as FGF2, fails to sustain ERK phosphorylation and NANOG expression under standard culture conditions. We find that the failure of FGF1 to sustain ES is due to thermal instability of the wild-type protein, not receptor specificity, and that a mutated thermal-stable FGF1 sustains human ESCs and supports both differentiation and reprogramming protocols.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Reprogramación Celular/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/farmacología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Temperatura , Proliferación Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Heparina/metabolismo , Humanos , Proteínas Mutantes/metabolismo , Mutación/genética , Fosforilación/efectos de los fármacos , Células Madre Pluripotentes/enzimología , Estabilidad Proteica/efectos de los fármacos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismoRESUMEN
We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives, and should be applicable to other reprogramming methods.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Medios de Cultivo/química , Células Madre Pluripotentes Inducidas/citología , Animales , Biopsia , Bovinos , Proliferación Celular , Supervivencia Celular , Materiales Biocompatibles Revestidos , Medio de Cultivo Libre de Suero/química , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Fibroblastos/citología , Expresión Génica , Sustancias de Crecimiento , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cariotipificación , Albúmina Sérica Bovina , Piel/citología , VitronectinaRESUMEN
Gene-corrected patient-specific induced pluripotent stem (iPS) cells offer a unique approach to gene therapy. Here, we begin to assess whether the mutational load acquired during gene correction of iPS cells is compatible with use in the treatment of genetic causes of retinal degenerative disease. We isolated iPS cells free of transgene sequences from a patient with gyrate atrophy caused by a point mutation in the gene encoding ornithine-δ-aminotransferase (OAT) and used homologous recombination to correct the genetic defect. Cytogenetic analysis, array comparative genomic hybridization (aCGH), and exome sequencing were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. No abnormalities were detected after standard G-band metaphase analysis. However, aCGH and exome sequencing identified two deletions, one amplification, and nine mutations in protein coding regions in the initial iPS cell clone. Except for the targeted correction of the single nucleotide in the OAT locus and a single synonymous base-pair change, no additional mutations or copy number variation were identified in iPS cells after the two subsequent clonal events. These findings confirm that iPS cells themselves may carry a significant mutational load at initial isolation, but that the clonal events and prolonged cultured required for correction of a genetic defect can be accomplished without a substantial increase in mutational burden.
Asunto(s)
Atrofia Girata/enzimología , Atrofia Girata/genética , Ornitina-Oxo-Ácido Transaminasa/genética , Ornitina-Oxo-Ácido Transaminasa/metabolismo , Células Madre Pluripotentes/enzimología , Células Cultivadas , Marcación de Gen/métodos , Estudio de Asociación del Genoma Completo , Inestabilidad Genómica/genética , Atrofia Girata/patología , Atrofia Girata/terapia , Humanos , Células Madre Pluripotentes/patología , Recombinación GenéticaRESUMEN
Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs exhibit basal-apical polarity, junctional complexes, integrin-dependent matrix adhesion, and E-cadherin-dependent cell-cell adhesion, all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures, programmed cell death is commonly observed. If individualized human ESCs are prevented from reattaching and forming colonies, their viability is significantly reduced. Here, we show that actin-myosin contraction is a critical effector of the cell death response to human ESC dissociation. Inhibition of myosin heavy chain ATPase, downregulation of myosin heavy chain, and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ESCs. ROCK inhibition decreases phosphorylation of myosin light chain, suggesting that inhibition of actin-myosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ESCs.