RESUMO
Transient, regulated binding of globular protein domains to Short Linear Motifs (SLiMs) in disordered regions of other proteins drives cellular signaling. Mapping the energy landscapes of these interactions is essential for deciphering and perturbing signaling networks but is challenging due to their weak affinities. We present a powerful technology (MRBLE-pep) that simultaneously quantifies protein binding to a library of peptides directly synthesized on beads containing unique spectral codes. Using MRBLE-pep, we systematically probe binding of calcineurin (CN), a conserved protein phosphatase essential for the immune response and target of immunosuppressants, to the PxIxIT SLiM. We discover that flanking residues and post-translational modifications critically contribute to PxIxIT-CN affinity and identify CN-binding peptides based on multiple scaffolds with a wide range of affinities. The quantitative biophysical data provided by this approach will improve computational modeling efforts, elucidate a broad range of weak protein-SLiM interactions, and revolutionize our understanding of signaling networks.
Assuntos
Hidrogéis/química , Microesferas , Biblioteca de Peptídeos , Peptídeos/metabolismo , Proteínas/metabolismo , Algoritmos , Sequência de Aminoácidos , Ligação Competitiva , Calcineurina/metabolismo , Humanos , Modelos Teóricos , Fosfoproteínas Fosfatases/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas , Processamento de Proteína Pós-TraducionalRESUMO
Multiplexed bioassays, in which multiple analytes of interest are probed in parallel within a single small volume, have greatly accelerated the pace of biological discovery. Bead-based multiplexed bioassays have many technical advantages, including near solution-phase kinetics, small sample volume requirements, many within-assay replicates to reduce measurement error, and, for some bead materials, the ability to synthesize analytes directly on beads via solid-phase synthesis. To allow bead-based multiplexing, analytes can be synthesized on spectrally encoded beads with a 1:1 linkage between analyte identity and embedded codes. Bead-bound analyte libraries can then be pooled and incubated with a fluorescently-labeled macromolecule of interest, allowing downstream quantification of interactions between the macromolecule and all analytes simultaneously via imaging alone. Extracting quantitative binding data from these images poses several computational image processing challenges, requiring the ability to identify all beads in each image, quantify bound fluorescent material associated with each bead, and determine their embedded spectral code to reveal analyte identities. Here, we present a novel open-source Python software package (the mrbles analysis package) that provides the necessary tools to: (1) find encoded beads in a bright-field microscopy image; (2) quantify bound fluorescent material associated with bead perimeters; (3) identify embedded ratiometric spectral codes within beads; and (4) return data aggregated by embedded code and for each individual bead. We demonstrate the utility of this package by applying it towards analyzing data generated via multiplexed measurement of calcineurin protein binding to MRBLEs (Microspheres with Ratiometric Barcode Lanthanide Encoding) containing known and mutant binding peptide motifs. We anticipate that this flexible package should be applicable to a wide variety of assays, including simple bead or droplet finding analysis, quantification of binding to non-encoded beads, and analysis of multiplexed assays that use ratiometric, spectrally encoded beads.
Assuntos
Bioensaio/métodos , Software , Bioensaio/instrumentação , Bioensaio/estatística & dados numéricos , Interpretação Estatística de Dados , Corantes Fluorescentes , Processamento de Imagem Assistida por Computador , Elementos da Série dos Lantanídeos , Microesferas , Ligação ProteicaRESUMO
Quantitative analysis of bioimaging data is often skewed by both shading in space and background variation in time. We introduce BaSiC, an image correction method based on low-rank and sparse decomposition which solves both issues. In comparison to existing shading correction tools, BaSiC achieves high-accuracy with significantly fewer input images, works for diverse imaging conditions and is robust against artefacts. Moreover, it can correct temporal drift in time-lapse microscopy data and thus improve continuous single-cell quantification. BaSiC requires no manual parameter setting and is available as a Fiji/ImageJ plugin.
Assuntos
Microscopia/métodos , Software , Algoritmos , Artefatos , Processamento de Imagem Assistida por Computador/métodos , Imagem com Lapso de TempoRESUMO
Genetically encoded fluorescent tags are protein sequences that can be fused to a protein of interest to render it fluorescent. These tags have revolutionized cell biology by allowing nearly any protein to be imaged by light microscopy at submicrometer spatial resolution and subsecond time resolution in a live cell or organism. They can also be used to measure protein abundance in thousands to millions of cells using flow cytometry. Here I provide an introduction to the different genetic tags available, including both intrinsically fluorescent proteins and proteins that derive their fluorescence from binding of either endogenous or exogenous fluorophores. I discuss their optical and biological properties and guidelines for choosing appropriate tags for an experiment. Tools for tagging nucleic acid sequences and reporter molecules that detect the presence of different biomolecules are also briefly discussed.
Assuntos
Corantes Fluorescentes/química , Citometria de Fluxo/métodos , Fluorescência , Proteínas de Fluorescência Verde/química , Humanos , Microscopia de Fluorescência/métodosRESUMO
The protein α-synuclein has a central role in the pathogenesis of Parkinson's disease. Like that of other proteins that accumulate in neurodegenerative disease, however, the function of α-synuclein remains unknown. Localization to the nerve terminal suggests a role in neurotransmitter release, and overexpression inhibits regulated exocytosis, but previous work has failed to identify a clear physiological defect in mice lacking all three synuclein isoforms. Using adrenal chromaffin cells and neurons, we now find that both overexpressed and endogenous synuclein accelerate the kinetics of individual exocytotic events, promoting cargo discharge and reducing pore closure ('kiss-and-run'). Thus, synuclein exerts dose-dependent effects on dilation of the exocytotic fusion pore. Remarkably, mutations that cause Parkinson's disease abrogate this property of α-synuclein without impairing its ability to inhibit exocytosis when overexpressed, indicating a selective defect in normal function.
Assuntos
Exocitose/fisiologia , alfa-Sinucleína/fisiologia , Animais , Células Cultivadas , Células Cromafins/fisiologia , Dilatação , Humanos , Cinética , Camundongos , Camundongos Knockout , Mutação , Neurônios/fisiologia , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Cultura Primária de Células , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Vesículas Secretórias/metabolismo , Vesículas Sinápticas/fisiologia , alfa-Sinucleína/biossínteseRESUMO
In humans, immunity to Plasmodium sp. generally takes the form of protection from symptomatic malaria (i.e., 'clinical immunity') rather than infection ('sterilizing immunity'). In contrast, mice infected with Plasmodium develop sterilizing immunity, hindering progress in understanding the mechanistic basis of clinical immunity. Here we present a novel model in which mice persistently infected with P. chabaudi exhibit limited clinical symptoms despite sustaining patent parasite burdens for many months. Characterization of immune responses in persistently infected mice revealed development of CD4+ T cell exhaustion, increased production of IL-10, and expansion of B cells with an atypical surface phenotype. Additionally, persistently infected mice displayed a dramatic increase in circulating nonclassical monocytes, a phenomenon that we also observed in humans with both chronic Plasmodium exposure and asymptomatic infection. Following pharmacological clearance of infection, previously persistently infected mice could not control a secondary challenge, indicating that persistent infection disrupts the sterilizing immunity that typically develops in mouse models of acute infection. This study establishes an animal model of asymptomatic, persistent Plasmodium infection that recapitulates several central aspects of the immune response in chronically exposed humans. As such, it provides a novel tool for dissection of immune responses that may prevent development of sterilizing immunity and limit pathology during infection.
Assuntos
Modelos Animais de Doenças , Parasitemia/parasitologia , Plasmodium chabaudi/isolamento & purificação , Animais , Criança , Pré-Escolar , Doença Crônica , Humanos , Lactente , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum.
Assuntos
Análise Mutacional de DNA , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Estresse Fisiológico , Ubiquitina/genética , Ubiquitina/metabolismo , Biologia/educação , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Saccharomyces cerevisiae/fisiologia , Estudantes , UniversidadesRESUMO
Light microscopy is a key tool in modern cell biology. Light microscopy has several features that make it ideally suited for imaging biology in living cells: the resolution is well-matched to the sizes of subcellular structures, a diverse range of available fluorescent probes makes it possible to mark proteins, organelles, and other structures for imaging, and the relatively nonperturbing nature of light means that living cells can be imaged for long periods of time to follow their dynamics. Here I provide a brief introduction to using light microscopy in cell biology, with particular emphasis on factors to be considered when starting microscopy experiments.
Assuntos
Biologia Celular/instrumentação , Microscopia Confocal/métodosRESUMO
Malignancy is associated with altered expression of glycans and glycoproteins that contribute to the cellular glycocalyx. We constructed a glycoprotein expression signature, which revealed that metastatic tumours upregulate expression of bulky glycoproteins. A computational model predicted that these glycoproteins would influence transmembrane receptor spatial organization and function. We tested this prediction by investigating whether bulky glycoproteins in the glycocalyx promote a tumour phenotype in human cells by increasing integrin adhesion and signalling. Our data revealed that a bulky glycocalyx facilitates integrin clustering by funnelling active integrins into adhesions and altering integrin state by applying tension to matrix-bound integrins, independent of actomyosin contractility. Expression of large tumour-associated glycoproteins in non-transformed mammary cells promoted focal adhesion assembly and facilitated integrin-dependent growth factor signalling to support cell growth and survival. Clinical studies revealed that large glycoproteins are abundantly expressed on circulating tumour cells from patients with advanced disease. Thus, a bulky glycocalyx is a feature of tumour cells that could foster metastasis by mechanically enhancing cell-surface receptor function.
Assuntos
Glicocálix/metabolismo , Glicoproteínas/metabolismo , Integrinas/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Mama/citologia , Mama/metabolismo , Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Fibroblastos , Glicocálix/química , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Integrinas/química , Camundongos , Terapia de Alvo Molecular , Mucina-1/metabolismo , Metástase Neoplásica/patologia , Células Neoplásicas Circulantes , Ligação Proteica , Receptores de Superfície CelularRESUMO
Over the course of development, the vertebrate heart undergoes a series of complex morphogenetic processes that transforms it from a simple myocardial epithelium to the complex 3D structure required for its function. One of these processes leads to the formation of trabeculae to optimize the internal structure of the ventricle for efficient conduction and contraction. Despite the important role of trabeculae in the development and physiology of the heart, little is known about their mechanism of formation. Using 3D time-lapse imaging of beating zebrafish hearts, we observed that the initiation of cardiac trabeculation can be divided into two processes. Before any myocardial cell bodies have entered the trabecular layer, cardiomyocytes extend protrusions that invade luminally along neighboring cell-cell junctions. These protrusions can interact within the trabecular layer to form new cell-cell contacts. Subsequently, cardiomyocytes constrict their abluminal surface, moving their cell bodies into the trabecular layer while elaborating more protrusions. We also examined the formation of these protrusions in trabeculation-deficient animals, including erbb2 mutants, tnnt2a morphants, which lack cardiac contractions and flow, and myh6 morphants, which lack atrial contraction and exhibit reduced flow. We found that, compared with cardiomyocytes in wild-type hearts, those in erbb2 mutants were less likely to form protrusions, those in tnnt2a morphants formed less stable protrusions, and those in myh6 morphants extended fewer protrusions per cell. Thus, through detailed 4D imaging of beating hearts, we have identified novel cellular behaviors underlying cardiac trabeculation.
Assuntos
Ventrículos do Coração/anatomia & histologia , Ventrículos do Coração/citologia , Imageamento Tridimensional/métodos , Morfogênese , Miócitos Cardíacos/citologia , Animais , Extensões da Superfície Celular/metabolismo , Ventrículos do Coração/crescimento & desenvolvimento , Miócitos Cardíacos/metabolismo , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Mitochondria have long been implicated in the pathogenesis of Parkinson's disease (PD). Mutations in the mitochondrial kinase PINK1 that reduce kinase activity are associated with mitochondrial defects and result in an autosomal-recessive form of early-onset PD. Therapeutic approaches for enhancing the activity of PINK1 have not been considered because no allosteric regulatory sites for PINK1 are known. Here, we show that an alternative strategy, a neo-substrate approach involving the ATP analog kinetin triphosphate (KTP), can be used to increase the activity of both PD-related mutant PINK1(G309D) and PINK1(WT). Moreover, we show that application of the KTP precursor kinetin to cells results in biologically significant increases in PINK1 activity, manifest as higher levels of Parkin recruitment to depolarized mitochondria, reduced mitochondrial motility in axons, and lower levels of apoptosis. Discovery of neo-substrates for kinases could provide a heretofore-unappreciated modality for regulating kinase activity.
Assuntos
Mitocôndrias/metabolismo , Doença de Parkinson/patologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Sequência de Aminoácidos , Animais , Apoptose , Axônios/metabolismo , Linhagem Celular , Células Cultivadas , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Cinetina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/metabolismo , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Fosforilação , Proteínas Quinases/química , Ratos , Alinhamento de Sequência , Ubiquitina-Proteína Ligases/metabolismo , Proteína bcl-X/metabolismoRESUMO
Fluorescent protein fusions are a powerful tool to monitor the localization and trafficking of proteins. Such studies are particularly easy to carry out in the budding yeast Saccharomyces cerevisiae due to the ease with which tags can be introduced into the genome by homologous recombination. However, the available yeast tagging plasmids have not kept pace with the development of new and improved fluorescent proteins. Here, we have constructed yeast optimized versions of 19 different fluorescent proteins and tested them for use as fusion tags in yeast. These include two blue, seven green, and seven red fluorescent proteins, which we have assessed for brightness, photostability and perturbation of tagged proteins. We find that EGFP remains the best performing green fluorescent protein, that TagRFP-T and mRuby2 outperform mCherry as red fluorescent proteins, and that mTagBFP2 can be used as a blue fluorescent protein tag. Together, the new tagging vectors we have constructed provide improved blue and red fluorescent proteins for yeast tagging and three color imaging.
Assuntos
Proteínas Luminescentes/genética , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Fluorescência , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína Vermelha FluorescenteRESUMO
Mitochondria must grow with the growing cell to ensure proper cellular physiology and inheritance upon division. We measured the physical size of mitochondrial networks in budding yeast and found that mitochondrial network size increased with increasing cell size and that this scaling relation occurred primarily in the bud. The mitochondria-to-cell size ratio continually decreased in aging mothers over successive generations. However, regardless of the mother's age or mitochondrial content, all buds attained the same average ratio. Thus, yeast populations achieve a stable scaling relation between mitochondrial content and cell size despite asymmetry in inheritance.
Assuntos
Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Tamanho Mitocondrial , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/ultraestrutura , Fase G1 , Microscopia Confocal , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
VIDEO ABSTRACT: Some endocytic cargoes control clathrin-coated pit (CCP) maturation, but it is not known how such regulation is communicated. We found that µ-opioid neuropeptide receptors signal to their enclosing CCPs by ubiquitination. Nonubiquitinated receptors delay CCPs at an intermediate stage of maturation, after clathrin lattice assembly is complete but before membrane scission. Receptor ubiquitination relieves this inhibition, effectively triggering CCP scission and producing a receptor-containing endocytic vesicle. The ubiquitin modification that conveys this endocytosis-promoting signal is added to the receptor's first cytoplasmic loop, catalyzed by the Smurf2 ubiquitin ligase, and coordinated with activation-dependent receptor phosphorylation and clustering through Smurf2 recruitment by the endocytic adaptor beta-arrestin. Epsin1 detects the signal at the CCP and is required for ubiquitin-promoted scission. This cargo-to-coat communication system mediates a biochemical checkpoint that ensures appropriate receptor ubiquitination for later trafficking, and it controls specific receptor loading into CCPs by sensing when a sufficient quorum is reached.
Assuntos
Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Endocitose , Ubiquitinação , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Células HEK293 , Humanos , Fosforilação , Receptores Opioides mu/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Emerging questions in cell biology necessitate nanoscale imaging in live cells. Here we present scanning angle interference microscopy, which is capable of localizing fluorescent objects with nanoscale precision along the optical axis in motile cellular structures. We use this approach to resolve nanotopographical features of the cell membrane and cytoskeleton as well as the temporal evolution, three-dimensional architecture and nanoscale dynamics of focal adhesion complexes.
Assuntos
Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Células Epiteliais/citologia , Microscopia de Interferência/métodos , Nanotecnologia/métodos , Células Cultivadas , Fibronectinas/metabolismo , Adesões Focais , HumanosRESUMO
In anaphase, sister chromatids separate abruptly and are then segregated by the mitotic spindle. The protease separase triggers sister separation by cleaving the Scc1/Mcd1 subunit of the cohesin ring that holds sisters together. Polo-kinase phosphorylation of Scc1 promotes its cleavage, but the underlying regulatory circuits are unclear. We developed a separase biosensor in Saccharomyces cerevisiae that provides a quantitative indicator of cohesin cleavage in single cells. Separase is abruptly activated and cleaves most cohesin within 1 min, after which anaphase begins. Cohesin near centromeres and telomeres is cleaved at the same rate and time. Protein phosphatase PP2A(Cdc55) inhibits cohesin cleavage by counteracting polo-kinase phosphorylation of Scc1. In early anaphase, the previously described separase inhibition of PP2A(Cdc55) promotes cohesin cleavage. Thus, separase acts directly on Scc1 and also indirectly, through inhibition of PP2A(Cdc55), to stimulate cohesin cleavage, providing a feedforward loop that may contribute to a robust and timely anaphase.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/fisiologia , Endopeptidases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Técnicas Biossensoriais , Endopeptidases/genética , Proteínas de Fluorescência Verde/genética , Microscopia de Fluorescência/métodos , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Separase , CoesinasRESUMO
The functional consequences of signaling receptor endocytosis are determined by the endosomal sorting of receptors between degradation and recycling pathways. How receptors recycle efficiently, in a sequence-dependent manner that is distinct from bulk membrane recycling, is not known. Here, in live cells, we visualize the sorting of a prototypical sequence-dependent recycling receptor, the beta-2 adrenergic receptor, from bulk recycling proteins and the degrading delta-opioid receptor. Our results reveal a remarkable diversity in recycling routes at the level of individual endosomes, and indicate that sequence-dependent recycling is an active process mediated by distinct endosomal subdomains distinct from those mediating bulk recycling. We identify a specialized subset of tubular microdomains on endosomes, stabilized by a highly localized but dynamic actin machinery, that mediate this sorting, and provide evidence that these actin-stabilized domains provide the physical basis for a two-step kinetic and affinity-based model for protein sorting into the sequence-dependent recycling pathway.
Assuntos
Actinas/metabolismo , Endossomos/metabolismo , Transporte Proteico , Linhagem Celular , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Humanos , Cinética , Estrutura Terciária de Proteína , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Receptores Opioides delta/metabolismoRESUMO
Spinning-disk confocal microscopy is an imaging technique that combines the out-of-focus light rejection of confocal microscopy with the high sensitivity of wide-field microscopy. Because of its unique features, it is well suited to high-resolution imaging of yeast and other small cells. Elimination of out-of-focus light significantly improves the image contrast and signal-to-noise ratio, making it easier to resolve and quantitate small, dim structures in the cell. These features make spinning-disk confocal microscopy an excellent technique for studying protein localization and dynamics in yeast. In this review, I describe the rationale behind using spinning-disk confocal imaging for yeast, hardware considerations when assembling a spinning-disk confocal scope, and methods for strain preparation and imaging. In particular, I discuss choices of objective lens and camera, choice of fluorescent proteins for tagging yeast genes, and methods for sample preparation.