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How cells establish the interphase genome organization after mitosis is incompletely understood. Using quantitative and super-resolution microscopy, we show that the transition from a Condensin to a Cohesin-based genome organization occurs dynamically over two hours. While a significant fraction of Condensins remains chromatin-bound until early G1, Cohesin-STAG1 and its boundary factor CTCF are rapidly imported into daughter nuclei in telophase, immediately bind chromosomes as individual complexes and are sufficient to build the first interphase TAD structures. By contrast, the more abundant Cohesin-STAG2 accumulates on chromosomes only gradually later in G1, is responsible for compaction inside TAD structures and forms paired complexes upon completed nuclear import. Our quantitative time-resolved mapping of mitotic and interphase loop extruders in single cells reveals that the nested loop architecture formed by sequential action of two Condensins in mitosis is seamlessly replaced by a less compact, but conceptually similar hierarchically nested loop architecture driven by sequential action of two Cohesins.
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Faithful chromosome segregation during cell division requires accurate mitotic spindle formation. As mitosis occurs rapidly within the cell cycle, the proteins involved in mitotic spindle assembly undergo rapid changes, including their interactions with other proteins. The proper localization of the HURP protein on the kinetochore fibers, in close proximity to chromosomes, is crucial for ensuring accurate congression and segregation of chromosomes. In this study, we employ photoactivation and FRAP experiments to investigate the impact of alterations in microtubule flux and phosphorylation of HURP at the Ser627 residue on its dynamics. Furthermore, through immunoprecipitations assays, we demonstrate the interactions of HURP with various proteins, such as TPX2, Aurora A, Eg5, Dynein, Kif5B, and Importin ß, in mammalian cells during mitosis. We also find that phosphorylation of HURP at Ser627 regulates its interaction with these partners during mitosis. Our findings suggest that HURP participates in at least two distinct complexes during metaphase to ensure its proper localization in close proximity to chromosomes, thereby promoting the bundling and stabilization of kinetochore fibers.
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Photosynthetic microalgae are responsible for an important fraction of CO2 fixation and O2 production on Earth. Three-dimensional (3D) ultrastructural characterization of these organisms in their natural environment can contribute to a deeper understanding of their cell biology. However, the low throughput of volume electron microscopy (vEM) methods along with the complexity and heterogeneity of environmental samples pose great technical challenges. In the present study, we used a workflow based on a specific electron microscopy sample preparation method compatible with both light and vEM imaging in order to target one cell among a complex natural community. This method revealed the 3D subcellular landscape of a photosynthetic dinoflagellate, which we identified as Ensiculifera tyrrhenica, with quantitative characterization of multiple organelles. We show that this cell contains a single convoluted chloroplast and show the arrangement of the flagellar apparatus with its associated photosensitive elements. Moreover, we observed partial chromatin unfolding, potentially associated with transcription activity in these organisms, in which chromosomes are permanently condensed. Together with providing insights in dinoflagellate biology, this proof-of-principle study illustrates an efficient tool for the targeted ultrastructural analysis of environmental microorganisms in heterogeneous mixes.
Assuntos
Imageamento Tridimensional , Microscopia Eletrônica de Varredura , Imageamento Tridimensional/métodosRESUMO
Subnuclear compartmentalization has been proposed to play an important role in gene regulation by segregating active and inactive parts of the genome in distinct physical and biochemical environments. During X chromosome inactivation (XCI), the noncoding Xist RNA coats the X chromosome, triggers gene silencing and forms a dense body of heterochromatin from which the transcription machinery appears to be excluded. Phase separation has been proposed to be involved in XCI, and might explain the exclusion of the transcription machinery by preventing its diffusion into the Xist-coated territory. Here, using quantitative fluorescence microscopy and single-particle tracking, we show that RNA polymerase II (RNAPII) freely accesses the Xist territory during the initiation of XCI. Instead, the apparent depletion of RNAPII is due to the loss of its chromatin stably bound fraction. These findings indicate that initial exclusion of RNAPII from the inactive X reflects the absence of actively transcribing RNAPII, rather than a consequence of putative physical compartmentalization of the inactive X heterochromatin domain.
Assuntos
RNA Polimerase II , RNA Longo não Codificante , RNA Polimerase II/metabolismo , Heterocromatina , Cromossomo X/genética , Cromossomo X/metabolismo , Inativação do Cromossomo X , Cromatina , RNA não Traduzido/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Proteins that enter the secretory pathway are transported from their place of synthesis in the endoplasmic reticulum to the Golgi complex by COPII-coated carriers. The networks of proteins that regulate these components in response to extracellular cues have remained largely elusive. Using high-throughput microscopy, we comprehensively screened 378 cytoskeleton-associated and related proteins for their functional interaction with the coat protein complex II (COPII) components SEC23A and SEC23B. Among these, we identified a group of proteins associated with focal adhesions (FERMT2, MACF1, MAPK8IP2, NGEF, PIK3CA, and ROCK1) that led to the downregulation of SEC23A when depleted by siRNA. Changes in focal adhesions induced by plating cells on ECM also led to the downregulation of SEC23A and decreases in VSVG transport from ER to Golgi. Both the expression of SEC23A and the transport defect could be rescued by treatment with a focal adhesion kinase inhibitor. Altogether, our results identify a network of cytoskeleton-associated proteins connecting focal adhesions and ECM-related signaling with the gene expression of the COPII secretory machinery and trafficking.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório , Matriz Extracelular , Adesões Focais , Complexo de Golgi , Proteínas de Transporte Vesicular , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Matriz Extracelular/metabolismo , Adesões Focais/genética , Adesões Focais/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Transporte Proteico , Via Secretória , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Development of multicellular organisms requires the generation of gene expression patterns that determines cell fate and organ shape. Groups of genetic interactions known as Gene Regulatory Networks (GRNs) play a key role in the generation of such patterns. However, how the topology and parameters of GRNs determine patterning in vivo remains unclear due to the complexity of most experimental systems. To address this, we use the zebrafish notochord, an organ where coin-shaped precursor cells are initially arranged in a simple unidimensional geometry. These cells then differentiate into vacuolated and sheath cells. Using newly developed transgenic tools together with in vivo imaging, we identify jag1a and her6/her9 as the main components of a Notch GRN that generates a lateral inhibition pattern and determines cell fate. Making use of this experimental system and mathematical modeling we show that lateral inhibition patterning is promoted when ligand-receptor interactions are stronger within the same cell than in neighboring cells. Altogether, we establish the zebrafish notochord as an experimental system to study pattern generation, and identify and characterize how the properties of GRNs determine self-organization of gene patterning and cell fate.
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Notocorda , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Notocorda/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
In eukaryotic cells, nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes and mediate nucleocytoplasmic exchange. They are made of 30 different nucleoporins and form a cylindrical architecture around an aqueous central channel. This architecture is highly dynamic in space and time. Variations in NPC diameter have been reported, but the physiological circumstances and the molecular details remain unknown. Here, we combined cryoelectron tomography with integrative structural modeling to capture a molecular movie of the respective large-scale conformational changes in cellulo. Although NPCs of exponentially growing cells adopted a dilated conformation, they reversibly constricted upon cellular energy depletion or conditions of hypertonic osmotic stress. Our data point to a model where the nuclear envelope membrane tension is linked to the conformation of the NPC.
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Membrana Nuclear/fisiologia , Poro Nuclear/fisiologia , Poro Nuclear/ultraestrutura , Transporte Ativo do Núcleo Celular , Fenômenos Biomecânicos , Microscopia Crioeletrônica , Citoplasma/metabolismo , Metabolismo Energético , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Modelos Biológicos , Membrana Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/química , Pressão Osmótica , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/ultraestrutura , Proteínas de Schizosaccharomyces pombe/química , Estresse FisiológicoRESUMO
Complex traits are characterized by multiple genes and variants acting simultaneously on a phenotype. However, studying the contribution of individual pairs of genes to complex traits has been challenging since human genetics necessitates very large population sizes, while findings from model systems do not always translate to humans. Here, we combine genetics with combinatorial RNAi (coRNAi) to systematically test for pairwise additive effects (AEs) and genetic interactions (GIs) between 30 lipid genome-wide association studies (GWAS) genes. Gene-based burden tests from 240,970 exomes show that in carriers with truncating mutations in both, APOB and either PCSK9 or LPL ("human double knock-outs") plasma lipid levels change additively. Genetics and coRNAi identify overlapping AEs for 12 additional gene pairs. Overlapping GIs are observed for TOMM40/APOE with SORT1 and NCAN. Our study identifies distinct gene pairs that modulate plasma and cellular lipid levels primarily via AEs and nominates putative drug target pairs for improved lipid-lowering combination therapies.
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Estudo de Associação Genômica Ampla/métodos , Pró-Proteína Convertase 9/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Humanos , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial/metabolismo , Neurocam/genética , Neurocam/metabolismo , Pró-Proteína Convertase 9/genéticaRESUMO
A significant challenge for developmental systems biology is balancing throughput with controlled conditions that minimize experimental artifacts. Large-scale developmental screens such as unbiased mutagenesis surveys have been limited in their applicability to embryonic systems, as the technologies for quantifying precise expression patterns in whole animals has not kept pace with other sequencing-based technologies. Here, we outline an open-source semi-automated pipeline to chemically fixate, stain, and 3D-image Drosophila embryos. Central to this pipeline is a liquid handling robot, Flyspresso, which automates the steps of classical embryo fixation and staining. We provide the schematics and an overview of the technology for an engineer or someone equivalently trained to reproduce and further improve upon Flyspresso, and highlight the Drosophila embryo fixation and colorimetric or antibody staining protocols. Additionally, we provide a detailed overview and stepwise protocol for our adaptive-feedback pipeline for automated embryo imaging on confocal microscopes. We demonstrate the efficiency of this pipeline compared to classical techniques, and how it can be repurposed or scaled to other protocols and biological systems. We hope our pipeline will serve as a platform for future research, allowing a broader community of users to build, execute, and share similar experiments.
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Embrião de Mamíferos , Imuno-Histoquímica/métodos , Robótica/métodos , Animais , Automação , Microscopia ConfocalRESUMO
Fibrosis can affect any organ, resulting in the loss of tissue architecture and function with often life-threatening consequences. Pathologically, fibrosis is characterised by the expansion of connective tissue due to excessive deposition of extracellular matrix (ECM) proteins, including the fibrillar forms of collagen. A significant limitation for discovering cures for fibrosis is the availability of suitable human models and techniques to quantify mature fibrillar collagen deposition as close as possible to human physiological conditions.Here we have extensively characterised an ex vivo cultured human lung tissue-derived, precision-cut lung slices (hPCLS) model using label-free second harmonic generation (SHG) light microscopy to quantify fibrillar collagen deposition and mass spectrometry-based techniques to obtain a proteomic and metabolomic fingerprint of hPCLS in ex vivo culture.We demonstrate that hPCLS are viable and metabolically active, with mesenchymal, epithelial, endothelial and immune cell types surviving for at least 2â weeks in ex vivo culture. Analysis of hPCLS-conditioned supernatants showed a strong induction of pulmonary fibrosis-related ECM proteins upon transforming growth factor-ß1 (TGF-ß1) stimulation. This upregulation of ECM proteins was not translated into an increased deposition of fibrillar collagen. In support of this observation, we revealed the presence of a pro-ECM degradation activity in our ex vivo cultures of hPCLS, inhibition of which by a metalloproteinase inhibitor resulted in increased collagen deposition in response to TGF-ß1 stimulation.Together the data show that an integrated approach of measuring soluble pro-fibrotic markers alongside quantitative SHG-based analysis of fibrillar collagen is a valuable tool for studying pro-fibrotic signalling and testing anti-fibrotic agents.
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Microscopia , Fibrose Pulmonar , Fibrose , Humanos , Pulmão/patologia , Proteômica , Fibrose Pulmonar/patologia , Fator de Crescimento Transformador beta1RESUMO
Changes in gene regulation underlie much of phenotypic evolution1. However, our understanding of the potential for regulatory evolution is biased, because most evidence comes from either natural variation or limited experimental perturbations2. Using an automated robotics pipeline, we surveyed an unbiased mutation library for a developmental enhancer in Drosophila melanogaster. We found that almost all mutations altered gene expression and that parameters of gene expression-levels, location, and state-were convolved. The widespread pleiotropic effects of most mutations may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse Drosophila larvae revealed apparent biases in the phenotypes influenced by the enhancer. Developmental enhancers may encode a higher density of regulatory information than has been appreciated previously, imposing constraints on regulatory evolution.
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Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Drosophila/genética , Evolução Molecular , Proteínas de Homeodomínio/genética , Larva/genética , Larva/crescimento & desenvolvimento , Mutação , Fenótipo , Fatores de Transcrição/genéticaRESUMO
Arrayed CRISPR-based screens emerge as a powerful alternative to pooled screens making it possible to investigate a wide range of cellular phenotypes that are typically not amenable to pooled screens. Here, we describe a solid-phase transfection platform that enables CRISPR-based genetic screens in arrayed format with flexible readouts. We demonstrate efficient gene knockout upon delivery of guide RNAs and Cas9/guide RNA ribonucleoprotein complexes into untransformed and cancer cell lines. In addition, we provide evidence that our platform can be easily adapted to high-throughput screens and we use this approach to study oncogene addiction in tumor cells. Finally demonstrating that the human primary cells can also be edited using this method, we pave the way for rapid testing of potential targeted therapies.
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Edição de Genes/instrumentação , Neoplasias/genética , RNA Guia de Cinetoplastídeos/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Predisposição Genética para Doença , Ensaios de Triagem em Larga Escala , Humanos , Fenótipo , TransfecçãoRESUMO
Newly synthesized proteins are sorted into COPII-coated transport carriers at the endoplasmic reticulum (ER). Assembly of the COPII coat complex, which occurs at ER exit sites (ERES), is initiated by membrane association and GTP loading of SAR1, followed by the recruitment of the SEC23-SEC24 and SEC13-SEC31 subcomplexes. Both of these two subcomplexes stimulate GTP hydrolysis and coat disassembly. This inherent disassembly capacity of COPII complexes needs to be regulated to allow sufficient time for cargo sorting and transport carrier formation. By performing fluorescence recovery after photobleaching (FRAP) and mathematical modeling, we show that p150(glued) (also known as DCTN1), a component of the dynactin complex, stabilizes the COPII pre-budding complex on ER membranes in a microtubule-independent manner. Concentration of the secretory marker ts-O45-G at ERES is reduced in the presence of a C-terminal p150(glued) fragment that prevents binding of endogenous p150(glued) to SEC23. A similar cargo reduction is observed upon p150(glued) knockdown. Taken together, our data suggest that cargo concentration at ERES is regulated by p150(glued) to coordinate protein sorting and transport carrier formation with the subsequent long-range transport towards the Golgi complex along microtubules.
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Retículo Endoplasmático/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Complexo Dinactina , Recuperação de Fluorescência Após Fotodegradação , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/genética , Modelos Biológicos , Ligação ProteicaRESUMO
Focal adhesions are integrin-based structures that link the actin cytoskeleton and the extracellular matrix. They play an important role in various cellular functions such as cell signaling, cell motility and cell shape. To ensure and fine tune these different cellular functions, adhesions are regulated by a large number of proteins. The LIM domain protein zyxin localizes to focal adhesions where it participates in the regulation of the actin cytoskeleton. Because of its interactions with a variety of binding partners, zyxin has been proposed to act as a molecular scaffold. Here, we studied the interaction of zyxin with such a partner: Tes. Similar to zyxin, Tes harbors three highly conserved LIM domains of which the LIM1 domain directly interacts with zyxin. Using different zyxin variants in pull-down assays and ectopic recruitment experiments, we identified the Tes binding site in zyxin and showed that four highly conserved amino acids are crucial for its interaction with Tes. Based upon these findings, we used a zyxin mutant defective in Tes-binding to assess the functional consequences of abrogating the zyxin-Tes interaction in focal adhesions. Performing fluorescence recovery after photobleaching, we showed that zyxin recruits Tes to focal adhesions and modulates its turnover in these structures. However, we also provide evidence for zyxin-independent localization of Tes to focal adhesions. Zyxin increases focal adhesion numbers and reduces focal adhesion lifetimes, but does so independent of Tes. Quantitative analysis showed that the loss of interaction between zyxin and Tes affects the process of cell spreading. We conclude that zyxin influences focal adhesion dynamics, that it recruits Tes and that this interaction is functional in regulating cell spreading.
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Proteínas de Ligação a DNA/metabolismo , Mapeamento de Interação de Proteínas , Zixina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Contagem de Células , Movimento Celular , Sequência Conservada , Proteínas do Citoesqueleto , Adesões Focais/metabolismo , Humanos , Cinética , Camundongos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Relação Estrutura-Atividade , Zixina/químicaRESUMO
BACKGROUND: The majority of human cancer deaths are caused by metastasis. The metastatic dissemination is initiated by the breakdown of epithelial cell homeostasis. During this phenomenon, referred to as epithelial to mesenchymal transition (EMT), cells change their genetic and trancriptomic program leading to phenotypic and functional alterations. The challenge of understanding this dynamic process resides in unraveling regulatory networks involving master transcription factors (e.g. SNAI1/2, ZEB1/2 and TWIST1) and microRNAs. Here we investigated microRNAs regulated by SNAI1 and their potential role in the regulatory networks underlying epithelial plasticity. RESULTS: By a large-scale analysis on epithelial plasticity, we highlighted miR-203 and its molecular link with SNAI1 and the miR-200 family, key regulators of epithelial homeostasis. During SNAI1-induced EMT in MCF7 breast cancer cells, miR-203 and miR-200 family members were repressed in a timely correlated manner. Importantly, miR-203 repressed endogenous SNAI1, forming a double negative miR203/SNAI1 feedback loop. We integrated this novel miR203/SNAI1 with the known miR200/ZEB feedback loops to construct an a priori EMT core network. Dynamic simulations revealed stable epithelial and mesenchymal states, and underscored the crucial role of the miR203/SNAI1 feedback loop in state transitions underlying epithelial plasticity. CONCLUSION: By combining computational biology and experimental approaches, we propose a novel EMT core network integrating two fundamental negative feedback loops, miR203/SNAI1 and miR200/ZEB. Altogether our analysis implies that this novel EMT core network could function as a switch controlling epithelial cell plasticity during differentiation and cancer progression.
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Transição Epitelial-Mesenquimal/fisiologia , MicroRNAs/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , MicroRNAs/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
BACKGROUND: Initially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers. Phosphorylation of L-plastin on residue Ser5 increases its F-actin binding activity and is required for L-plastin-mediated cell invasion. METHODOLOGY/PRINCIPAL FINDINGS: To study the kinetics of L-plastin and the impact of L-plastin Ser5 phosphorylation on L-plastin dynamics and actin turn-over in live cells, simian Vero cells were transfected with GFP-coupled WT-L-plastin, Ser5 substitution variants (S5/A, S5/E) or actin and analyzed by fluorescence recovery after photobleaching (FRAP). FRAP data were explored by mathematical modeling to estimate steady-state reaction parameters. We demonstrate that in Vero cell focal adhesions L-plastin undergoes rapid cycles of association/dissociation following a two-binding-state model. Phosphorylation of L-plastin increased its association rates by two-fold, whereas dissociation rates were unaffected. Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation. In MCF-7 breast carcinoma cells, phorbol 12-myristate 13-acetate (PMA) treatment induced L-plastin translocation to de novo actin polymerization sites in ruffling membranes and spike-like structures and highly increased its Ser5 phosphorylation. Both inhibition studies and siRNA knock-down of PKC isozymes pointed to the involvement of the novel PKC-delta isozyme in the PMA-elicited signaling pathway leading to L-plastin Ser5 phosphorylation. Furthermore, the L-plastin contribution to actin dynamics regulation was substantiated by its association with a protein complex comprising cortactin, which is known to be involved in this process. CONCLUSIONS/SIGNIFICANCE: Altogether these findings quantitatively demonstrate for the first time that L-plastin contributes to the fine-tuning of actin turn-over, an activity which is regulated by Ser5 phosphorylation promoting its high affinity binding to the cytoskeleton. In carcinoma cells, PKC-delta signaling pathways appear to link L-plastin phosphorylation to actin polymerization and invasion.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Algoritmos , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Cortactina/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Adesões Focais/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinética , Glicoproteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteína Quinase C-delta/genética , Proteína Quinase C-delta/metabolismo , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/genética , Serina/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Células VeroRESUMO
A novel mathematical model of the actin dynamics in living cells under steady-state conditions has been developed for fluorescence recovery after photobleaching (FRAP) experiments. As opposed to other FRAP fitting models, which use the average lifetime of actins in filaments and the actin turnover rate as fitting parameters, our model operates with unbiased actin association/dissociation rate constants and accounts for the filament length. The mathematical formalism is based on a system of stochastic differential equations. The derived equations were validated on synthetic theoretical data generated by a stochastic simulation algorithm adapted for the simulation of FRAP experiments. Consistent with experimental findings, the results of this work showed that (1) fluorescence recovery is a function of the average filament length, (2) the F-actin turnover and the FRAP are accelerated in the presence of actin nucleating proteins, (3) the FRAP curves may exhibit both a linear and non-linear behaviour depending on the parameters of actin polymerisation, and (4) our model resulted in more accurate parameter estimations of actin dynamics as compared with other FRAP fitting models. Additionally, we provide a computational tool that integrates the model and that can be used for interpretation of FRAP data on actin cytoskeleton.
Assuntos
Citoesqueleto de Actina/química , Recuperação de Fluorescência Após Fotodegradação , Modelos Biológicos , Citoesqueleto de Actina/metabolismo , Difusão , Cinética , Modelos Lineares , Dinâmica não Linear , Multimerização Proteica , Estrutura Quaternária de ProteínaRESUMO
We report on an advanced universal Monte Carlo simulation model of actin polymerization processes offering a broad application panel. The model integrates major actin-related reactions, such as assembly of actin nuclei, association/dissociation of monomers to filament ends, ATP-hydrolysis via ADP-Pi formation and ADP-ATP exchange, filament branching, fragmentation and annealing or the effects of regulatory proteins. Importantly, these reactions are linked to information on the nucleotide state of actin subunits in filaments (ATP hydrolysis) and the distribution of actin filament lengths. The developed stochastic simulation modelling schemes were validated on: i) synthetic theoretical data generated by a deterministic model and ii) sets of our and published experimental data obtained from fluorescence pyrene-actin experiments. Build on an open-architecture principle, the designed model can be extended for predictive evaluation of the activities of other actin-interacting proteins and can be applied for the analysis of experimental pyrene actin-based or fluorescence microscopy data. We provide a user-friendly, free software package ActinSimChem that integrates the implemented simulation algorithms and that is made available to the scientific community for modelling in silico any specific actin-polymerization system.