RESUMEN
Integrin endocytosis is essential for many fundamental cellular processes. Whether and how the internalization impacts cellular mechanics remains elusive. Whereas previous studies reported the contribution of the integrin activator, talin, in force development, the involvement of inhibitors is less documented. We identified ICAP-1 as an integrin inhibitor involved in mechanotransduction by co-working with NME2 to control clathrin-mediated endocytosis of integrins at the edge of focal adhesions (FA). Loss of ICAP-1 enables ß3-integrin-mediated force generation independently of ß1 integrin. ß3-integrin-mediated forces were associated with a decrease in ß3 integrin dynamics stemming from their reduced diffusion within adhesion sites and slow turnover of FA. The decrease in ß3 integrin dynamics correlated with a defect in integrin endocytosis. ICAP-1 acts as an adaptor for clathrin-dependent endocytosis of integrins. ICAP-1 controls integrin endocytosis by interacting with NME2, a key regulator of dynamin-dependent clathrin-coated pits fission. Control of clathrin-mediated integrin endocytosis by an inhibitor is an unprecedented mechanism to tune forces at FA.
Asunto(s)
Clatrina , Endocitosis , Adhesiones Focales , Integrina beta1 , Integrina beta3 , Clatrina/metabolismo , Endocitosis/fisiología , Integrina beta1/genética , Mecanotransducción Celular , Talina/genéticaRESUMEN
The nuclear pore complex (NPC) is the major conduit for nucleocytoplasmic transport and serves as a platform for gene regulation and DNA repair. Several nucleoporins undergo ubiquitylation and SUMOylation, and these modifications play an important role in nuclear pore dynamics and plasticity. Here, we perform a detailed analysis of these post-translational modifications of yeast nuclear basket proteins under normal growth conditions as well as upon cellular stresses, with a focus on SUMOylation. We find that the balance between the dynamics of SUMOylation and deSUMOylation of Nup60 and Nup2 at the NPC differs substantially, particularly in G1 and S phase. While Nup60 is the unique target of genotoxic stress within the nuclear basket that probably belongs to the SUMO-mediated DNA damage response pathway, both Nup2 and Nup60 show a dramatic increase in SUMOylation upon osmotic stress, with Nup2 SUMOylation being enhanced in Nup60 SUMO-deficient mutant yeast strains. Taken together, our data reveal that there are several levels of crosstalk between nucleoporins, and that the post-translational modifications of the NPC serve in sensing cellular stress signals.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sumoilación , Transporte Activo de Núcleo Celular , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Reparación del ADN , Poro Nuclear/ultraestructura , Proteínas de Complejo Poro Nuclear/genética , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
In vivo, the primary molecular mechanotransductive events mechanically initiating cell differentiation remain unknown. Here we find the molecular stretching of the highly conserved Y654-ß-catenin-D665-E-cadherin binding site as mechanically induced by tissue strain. It triggers the increase of accessibility of the Y654 site, target of the Src42A kinase phosphorylation leading to irreversible unbinding. Molecular dynamics simulations of the ß-catenin/E-cadherin complex under a force mimicking a 6 pN physiological mechanical strain predict a local 45% stretching between the two α-helices linked by the site and a 15% increase in accessibility of the phosphorylation site. Both are quantitatively observed using FRET lifetime imaging and non-phospho Y654 specific antibody labelling, in response to the mechanical strains developed by endogenous and magnetically mimicked early mesoderm invagination of gastrulating Drosophila embryos. This is followed by the predicted release of 16% of ß-catenin from junctions, observed in FRAP, which initiates the mechanical activation of the ß-catenin pathway process.
Asunto(s)
Proteínas del Dominio Armadillo/metabolismo , Cadherinas/metabolismo , Diferenciación Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas del Dominio Armadillo/química , Sitios de Unión , Cadherinas/química , Proteínas de Drosophila/química , Transferencia Resonante de Energía de Fluorescencia , Mecanotransducción Celular , Simulación de Dinámica Molecular , Fosforilación , Unión Proteica , Conformación Proteica , Proteínas Proto-Oncogénicas pp60(c-src)/química , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Homología de Secuencia , Factores de Transcripción/químicaRESUMEN
During mesenchymal cell motility, various actin regulators are recruited to the leading edge with exquisite precision in time and space to generate protrusion and retraction cycles. We present here an automated method, named CorRecD (from Correlation Recruitment Dynamics), which quantifies cell edge dynamics, protein recruitment and analyze their cross-correlation. The Wave Regulatory Complex (WRC), a master driver of protrusions, is used as a case-of-study. This biologist-friendly method relies on free software tools and can be applied to any fluorescently tagged protein of interest.
Asunto(s)
Movimiento Celular/fisiología , Membranas/metabolismo , Actinas/metabolismo , Línea Celular , HumanosRESUMEN
BACKGROUND: Characterizing membrane dynamics is a key issue to understand cell exchanges with the extra-cellular medium. Total internal reflection fluorescence microscopy (TIRFM) is well suited to focus on the late steps of exocytosis at the plasma membrane. However, it is still a challenging task to quantify (lateral) diffusion and estimate local dynamics of proteins. RESULTS: A new model was introduced to represent the behavior of cargo transmembrane proteins during the vesicle fusion to the plasma membrane at the end of the exocytosis process. Two biophysical parameters, the diffusion coefficient and the release rate parameter, are automatically estimated from TIRFM image sequences, to account for both the lateral diffusion of molecules at the membrane and the continuous release of the proteins from the vesicle to the plasma membrane. Quantitative evaluation on 300 realistic computer-generated image sequences demonstrated the efficiency and accuracy of the method. The application of our method on 16 real TIRFM image sequences additionally revealed differences in the dynamic behavior of Transferrin Receptor (TfR) and Langerin proteins. CONCLUSION: An automated method has been designed to simultaneously estimate the diffusion coefficient and the release rate for each individual vesicle fusion event at the plasma membrane in TIRFM image sequences. It can be exploited for further deciphering cell membrane dynamics.
Asunto(s)
Antígenos CD/metabolismo , Membrana Celular/metabolismo , Modelos Moleculares , Receptores de Transferrina/metabolismo , Algoritmos , Animales , Difusión , Exocitosis , Microscopía FluorescenteRESUMEN
Coordination between membrane trafficking and actin polymerization is fundamental in cell migration, but a dynamic view of the underlying molecular mechanisms is still missing. The Rac1 GTPase controls actin polymerization at protrusions by interacting with its effector, the Wave regulatory complex (WRC). The exocyst complex, which functions in polarized exocytosis, has been involved in the regulation of cell motility. Here, we show a physical and functional connection between exocyst and WRC. Purified components of exocyst and WRC directly associate in vitro, and interactions interfaces are identified. The exocyst-WRC interaction is confirmed in cells by co-immunoprecipitation and is shown to occur independently of the Arp2/3 complex. Disruption of the exocyst-WRC interaction leads to impaired migration. By using time-lapse microscopy coupled to image correlation analysis, we visualized the trafficking of the WRC towards the front of the cell in nascent protrusions. The exocyst is necessary for WRC recruitment at the leading edge and for resulting cell edge movements. This direct link between the exocyst and WRC provides a new mechanistic insight into the spatio-temporal regulation of cell migration.
Asunto(s)
Movimiento Celular , Extensiones de la Superficie Celular/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Células HEK293 , Humanos , Unión Proteica , Subunidades de Proteína/metabolismoRESUMEN
Fluorescence lifetime is usually defined as the average nanosecond-scale delay between excitation and emission of fluorescence. It has been established that lifetime measurements yield numerous indications on cellular processes such as interprotein and intraprotein mechanisms through fluorescent tagging and Förster resonance energy transfer. In this area, frequency-domain fluorescence lifetime imaging microscopy is particularly appropriate to probe a sample noninvasively and quantify these interactions in living cells. The aim is then to measure the fluorescence lifetime in the sample at each location in space from fluorescence variations observed in a temporal sequence of images obtained by phase modulation of the detection signal. This leads to a sensitivity of lifetime determination to other sources of fluorescence variations such as intracellular motion. In this paper, we propose a robust statistical method for lifetime estimation for both background and small moving structures with a focus on intracellular vesicle trafficking.
RESUMEN
Dissemination of carcinoma cells requires the pericellular degradation of the extracellular matrix, which is mediated by membrane type 1-matrix metalloproteinase (MT1-MMP). In this article, we report a co-up-regulation and colocalization of MT1-MMP and atypical protein kinase C iota (aPKCι) in hormone receptor-negative breast tumors in association with a higher risk of metastasis. Silencing of aPKC in invasive breast-tumor cell lines impaired the delivery of MT1-MMP from late endocytic storage compartments to the surface and inhibited matrix degradation and invasion. We provide evidence that aPKCι, in association with MT1-MMP-containing endosomes, phosphorylates cortactin, which is present in F-actin-rich puncta on MT1-MMP-positive endosomes and regulates cortactin association with the membrane scission protein dynamin-2. Thus, cell line-based observations and clinical data reveal the concerted activity of aPKC, cortactin, and dynamin-2, which control the trafficking of MT1-MMP from late endosome to the plasma membrane and play an important role in the invasive potential of breast-cancer cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Isoenzimas/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Proteína Quinasa C/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adulto , Anciano , Transporte Biológico Activo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Línea Celular Tumoral , Cortactina/metabolismo , Gránulos Citoplasmáticos/metabolismo , Progresión de la Enfermedad , Dinamina II/metabolismo , Endosomas/metabolismo , Matriz Extracelular/metabolismo , Femenino , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Metaloproteinasa 14 de la Matriz/genética , Persona de Mediana Edad , Invasividad Neoplásica , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , ARN Interferente Pequeño/genética , Regulación hacia ArribaRESUMEN
Particle tracking is of key importance for quantitative analysis of intracellular dynamic processes from time-lapse microscopy image data. Because manually detecting and following large numbers of individual particles is not feasible, automated computational methods have been developed for these tasks by many groups. Aiming to perform an objective comparison of methods, we gathered the community and organized an open competition in which participating teams applied their own methods independently to a commonly defined data set including diverse scenarios. Performance was assessed using commonly defined measures. Although no single method performed best across all scenarios, the results revealed clear differences between the various approaches, leading to notable practical conclusions for users and developers.
Asunto(s)
Interpretación de Imagen Asistida por Computador , Microscopía Fluorescente/métodos , Interpretación de Imagen Asistida por Computador/normas , Microscopía Fluorescente/normasRESUMEN
Early endosomes consist of vacuolar sorting and tubular recycling domains that segregate components fated for degradation in lysosomes or reuse by recycling to the plasma membrane or Golgi. The tubular transport intermediates that constitute recycling endosomes function in cell polarity, migration, and cytokinesis. Endosomal tubulation and fission require both actin and intact microtubules, but although factors that stabilize recycling endosomal tubules have been identified, those required for tubule generation from vacuolar sorting endosomes (SEs) remain unknown. We show that the microtubule motor KIF13A associates with recycling endosome tubules and controls their morphogenesis. Interfering with KIF13A function impairs the formation of endosomal tubules from SEs with consequent defects in endosome homeostasis and cargo recycling. Moreover, KIF13A interacts and cooperates with RAB11 to generate endosomal tubules. Our data illustrate how a microtubule motor couples early endosome morphogenesis to its motility and function.
Asunto(s)
Endocitosis , Endosomas/metabolismo , Cinesinas/metabolismo , Morfogénesis , Endosomas/ultraestructura , Humanos , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Unión Proteica , Transporte de Proteínas , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Loss-of-function mutations in PARK2/PARKIN and PINK1 cause early-onset autosomal recessive Parkinson disease (PD). The cytosolic E3 ubiquitin-protein ligase PARK2 cooperates with the mitochondrial kinase PINK1 to maintain mitochondrial quality. A loss of mitochondrial transmembrane potential (ΔΨ) leads to the PINK1-dependent recruitment of PARK2 to the outer mitochondrial membrane (OMM), followed by the ubiquitination and proteasome-dependent degradation of OMM proteins, and by the autophagy-dependent clearance of mitochondrial remnants. We showed here that blockade of mitochondrial protein import triggers the recruitment of PARK2, by PINK1, to the TOMM machinery. PD-causing PARK2 mutations weakened or disrupted the molecular interaction between PARK2 and specific TOMM subunits: the surface receptor, TOMM70A, and the channel protein, TOMM40. The downregulation of TOMM40 or its associated core subunit, TOMM22, was sufficient to trigger OMM protein clearance in the absence of PINK1 or PARK2. However, PARK2 was required to promote the degradation of whole organelles by autophagy. Furthermore, the overproduction of TOMM22 or TOMM40 reversed mitochondrial clearance promoted by PINK1 and PARK2 after ΔΨ loss. These results indicated that the TOMM machinery is a key molecular switch in the mitochondrial clearance program controlled by the PINK1-PARK2 pathway. Loss of functional coupling between mitochondrial protein import and the neuroprotective degradation of dysfunctional mitochondria may therefore be a primary pathogenic mechanism in autosomal recessive PD.
Asunto(s)
Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Regulación hacia Abajo , Células HEK293 , Células HeLa , Humanos , Membranas Mitocondriales/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Mitofagia , Modelos Biológicos , Mutación/genética , Enfermedad de Parkinson/genética , Unión Proteica , Transporte de Proteínas , Transducción de SeñalRESUMEN
In eukaryotes, mRNA export involves many evolutionarily conserved factors that carry the nascent transcript to the nuclear pore complex (NPC). The THO/TREX complex couples transcription to mRNA export and recruits the mRNA export receptor NXF1 for the transport of messenger ribonucleoprotein particles (mRNP) to the NPC. The transcription and export complex 2 (TREX-2) was suggested to interact with NXF1 and to shuttle between transcription sites and the NPC. Here, we characterize the dynamics of human TREX-2 and show that it stably associates with the NPC basket. Moreover, the association of TREX-2 with the NPC requires the basket nucleoporins NUP153 and TPR, but is independent of transcription. Differential profiles of mRNA nuclear accumulation reveal that TREX-2 functions similarly to basket nucleoporins, but differently from NXF1. Thus, our results show that TREX-2 is an NPC-associated complex in mammalian cells and suggest that it is involved in putative NPC basket-related functions.
Asunto(s)
Exodesoxirribonucleasas/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Fosfoproteínas/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Exodesoxirribonucleasas/genética , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transporte de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Transcripción Genética/fisiologíaRESUMEN
Regulation of the cellular volume is fundamental for cell survival and function. Deviations from equilibrium trigger dedicated signaling and transcriptional responses that mediate water homeostasis and volume recovery. Cells are densely packed with proteins, and molecular crowding may play an important role in cellular processes. Indeed, increasing molecular crowding has been shown to modify the kinetics of biochemical reactions in vitro; however, the effects of molecular crowding in living cells are mostly unexplored. Here, we report that, in yeast, a sudden reduction in cellular volume, induced by severe osmotic stress, slows down the dynamics of several signaling cascades, including the stress-response pathways required for osmotic adaptation. We show that increasing osmotic compression decreases protein mobility and can eventually lead to a dramatic stalling of several unrelated signaling and cellular processes. The rate of these cellular processes decreased exponentially with protein density when approaching stalling osmotic compression. This suggests that, under compression, the cytoplasm behaves as a soft colloid undergoing a glass transition. Our results shed light on the physical mechanisms that force cells to cope with volume fluctuations to maintain an optimal protein density compatible with cellular functions.
Asunto(s)
Adaptación Fisiológica/fisiología , Citoplasma/química , Proteínas Fúngicas/análisis , Presión Osmótica/fisiología , Transducción de Señal/fisiología , Levaduras/citología , Biofisica , Western Blotting , Electroforesis en Gel de Poliacrilamida , Recuperación de Fluorescencia tras Fotoblanqueo , Homeostasis/fisiología , Cinética , Modelos Biológicos , Agua/metabolismoRESUMEN
The CD1e protein participates in the presentation of lipid antigens in dendritic cells. Its transmembrane precursor is transported to lysosomes where it is cleaved into an active soluble form. In the presence of bafilomycin, which inhibits vacuolar ATPase and consequently the acidification of endosomal compartments, CD1e associates with a 27 kD protein. In this work, we identified this molecular partner as LAPTM5. The latter protein and CD1e colocalize in trans-Golgi and late endosomal compartments. The quantity of LAPTM5/CD1e complexes increases when the cells are treated with bafilomycin, probably due to the protection of LAPTM5 from lysosomal proteases. Moreover, we could demonstrate that LAPTM5/CD1e association occurs under physiological conditions. Although LAPTM5 was previously shown to act as a platform recruiting ubiquitin ligases and facilitating the transport of receptors to lysosomes, we found no evidence that LATPM5 controls either CD1e ubiquitination or the generation of soluble lysosomal CD1e proteins. Notwithstanding these last observations, the interaction of LAPTM5 with CD1e and their colocalization in antigen processing compartments both suggest that LAPTM5 might influence the role of CD1e in the presentation of lipid antigens.
Asunto(s)
Antígenos CD1/metabolismo , Proteínas de la Membrana/metabolismo , Compartimento Celular/efectos de los fármacos , Línea Celular Tumoral , Dendritas/efectos de los fármacos , Dendritas/metabolismo , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Semivida , Células HeLa , Humanos , Inmunoprecipitación , Macrólidos/farmacología , Melanoma/genética , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Solubilidad/efectos de los fármacos , Transfección , Ubiquitinación/efectos de los fármacos , Red trans-Golgi/efectos de los fármacos , Red trans-Golgi/metabolismoRESUMEN
A large body of knowledge relating to the constitution of Rab GTPase/Rab effector complexes and their impact on both membrane domain organization and overall membrane trafficking has been built up in recent years. However in the context of the live cell there are still many questions that remain to be answered, such as where and when these complexes assemble and where they perform their primary function(s). We describe here the dynamic processes that take place in the final steps of the Rab11A dependent recycling pathway, in the context of the membrane platform constituted by Myosin Vb, Rab11A, and Rab11-FIP2. We first confirm that a series of previously reported observations obtained during the study of a number of trafficking cargoes also apply to langerin. Langerin is a cargo molecule that traffics through Rab11A-positive membrane domains of the endosomal recycling pathway. In order to explore the relative dynamics of this set of partners, we make extensive use of a combinatory approach of Live-FRET, fast FRAP video, fast confocal and TIRF microscopy modalities. Our data show that the Myosin Vb/Rab11A/Rab11-FIP2 platform is spatially involved in the regulation of langerin trafficking at two distinct sites within live cells, first at the sorting site in the endosomal recycling compartment (ERC) where transport vesicles are formed, and subsequently, in a strict time-defined order, at the very late stage of docking/tethering and fusion of these langerin recycling vesicles to the plasma membrane.
Asunto(s)
Antígenos CD/metabolismo , Proteínas Portadoras/metabolismo , Endosomas/metabolismo , Regulación Neoplásica de la Expresión Génica , Lectinas Tipo C/metabolismo , Lectinas de Unión a Manosa/metabolismo , Proteínas de la Membrana/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Melanoma/metabolismo , Microscopía Confocal/métodos , Transporte de Proteínas , Factores de TiempoRESUMEN
In natural, industrial and medical environments, microorganisms mainly live as structured and organised matrix-encased communities known as biofilms. In these communities, microorganisms demonstrate coordinated behaviour and are able to perform specific functions such as dramatic resistance to antimicrobials, which potentially lead to major public health and industrial problems. It is now recognised that the appearance of such specific biofilm functions is intimately related to the three-dimensional organisation of the biological edifice, and results from multifactorial processes. During the last decade, the emergence of innovative optical microscopy techniques such as confocal laser scanning microscopy in combination with fluorescent labelling has radically transformed imaging in biofilm research, giving the possibility to investigate non-invasively the dynamic mechanisms of formation and reactivity of these biostructures. In this chapter, we discuss the contribution of fluorescence analysis and imaging to the study at different timescales of various processes: biofilm development (hours to days), antimicrobial reactivity within the three-dimensional structure (minutes to hours) or molecular diffusion/reaction phenomena (pico- to milliseconds).
Asunto(s)
Biopelículas/crecimiento & desarrollo , Fluorometría/métodos , Microbiología Ambiental , Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Imagenología Tridimensional , Consorcios Microbianos/fisiología , Interacciones Microbianas/fisiología , Fenómenos Microbiológicos , Microscopía Confocal/métodos , Espectrometría de Fluorescencia/métodosRESUMEN
The presence of multiple membrane-bound intracellular compartments is a major feature of eukaryotic cells. Many of the proteins required for formation and maintenance of these compartments share an evolutionary history. Here, we identify the SEA (Seh1-associated) protein complex in yeast that contains the nucleoporin Seh1 and Sec13, the latter subunit of both the nuclear pore complex and the COPII coating complex. The SEA complex also contains Npr2 and Npr3 proteins (upstream regulators of TORC1 kinase) and four previously uncharacterized proteins (Sea1-Sea4). Combined computational and biochemical approaches indicate that the SEA complex proteins possess structural characteristics similar to the membrane coating complexes COPI, COPII, the nuclear pore complex, and, in particular, the related Vps class C vesicle tethering complexes HOPS and CORVET. The SEA complex dynamically associates with the vacuole in vivo. Genetic assays indicate a role for the SEA complex in intracellular trafficking, amino acid biogenesis, and response to nitrogen starvation. These data demonstrate that the SEA complex is an additional member of a family of membrane coating and vesicle tethering assemblies, extending the repertoire of protocoatomer-related complexes.
Asunto(s)
Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Autofagia , Inmunoprecipitación , Membranas Intracelulares/metabolismo , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Proteínas de Complejo Poro Nuclear/química , Fenotipo , Filogenia , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae/química , Homología Estructural de Proteína , Fracciones Subcelulares/metabolismoRESUMEN
Invasion across tissue boundaries by metastatic tumor cells depends on the proteolytic degradation of the extracellular matrix, initiated by the formation of invadopodia, actin-driven membrane protrusions with matrix-degradative activity. Yet, mechanisms underlying invadopodia formation remain largely unknown. In this report, we examined the role of the histone deacetylase HDAC6 in invadopodia formation and invasion by breast cancer cells. Using small interfering RNA silencing of protein expression in highly invasive MDA-MB-231 breast adenocarcinoma cells, we show that HDAC6 is required for two-dimensional matrix proteolysis. In addition, we demonstrate that HDAC6 acts as a tubulin and cortactin deacetylase. We also report that the inhibition of HDAC6 by siRNA or treatment with HDAC inhibitor TSA results in a decreased invasion capacity of a three-dimensional type I collagen matrix by MDA-MB-231 cells. These data identify HDAC6 as a critical component of the invasive apparatus of tumor cells, in both two- and three-dimensional matrices.
Asunto(s)
Adenocarcinoma/enzimología , Adenocarcinoma/patología , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Extensiones de la Superficie Celular/enzimología , Histona Desacetilasas/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/ultraestructura , Membrana Basal/enzimología , Membrana Basal/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/ultraestructura , Línea Celular Tumoral , Extensiones de la Superficie Celular/patología , Colágeno Tipo I/metabolismo , Cortactina/metabolismo , Matriz Extracelular/enzimología , Matriz Extracelular/patología , Femenino , Histona Desacetilasa 6 , Histona Desacetilasas/biosíntesis , Histona Desacetilasas/genética , Humanos , Invasividad Neoplásica , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , TransfecciónRESUMEN
Research about the reactional and structural dynamics of biofilms at the molecular level has made great strides, owing to efficient fluorescence imaging methods in terms of spatial resolution and fast acquisition time but also to noninvasive conditions of observation consistent with in situ biofilm studies. In addition to conventional fluorescence intensity imaging, the fluorescence recovery after photobleaching (FRAP) module can now be routinely implemented on commercial confocal laser scanning microscopes (CLSMs). This method allows measuring of local diffusion coefficients in biofilms and could become an alternative to fluorescence correlation spectroscopy (FCS). We present here an image-based FRAP protocol to improve the accuracy of FRAP measurements inside "live" biofilms and the corresponding analysis. An original kymogram representation allows control of the absence of perturbing bacterial movement during image acquisition. FRAP data analysis takes into account molecular diffusion during the bleach phase and uses the image information to extract molecular diffusion coefficients. The fluorescence spatial intensity profile analysis used here for the first time with biofilms is supported both by our own mathematical model and by a previously published one. This approach was validated to FRAP experiments on fluorescent-dextran diffusion inside Lactococcus lactis and Stenotrophomonas maltophilia biofilms, and the results were compared to previously published FCS measurements.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Lactococcus lactis/fisiología , Microscopía Confocal/métodos , Stenotrophomonas maltophilia/fisiología , Difusión , Procesamiento de Imagen Asistido por Computador/métodos , Lactococcus lactis/crecimiento & desarrollo , Lactococcus lactis/metabolismo , Stenotrophomonas maltophilia/crecimiento & desarrollo , Stenotrophomonas maltophilia/metabolismoRESUMEN
Rab11 and Rab6 guanosine triphosphatases are associated with membranes of the recycling endosomes (REs) and Golgi complex, respectively. Evidence indicates that they sequentially regulate a retrograde transport pathway between these two compartments, suggesting the existence of proteins that must co-ordinate their functions. Here, we report the characterization of two isoforms of a protein, Rab6-interacting protein 1 (R6IP1), originally identified as a Rab6-binding protein. R6IP1 also binds to Rab11A in its GTP-bound conformation. In interphase cells, R6IP1 is targeted to the Golgi in a Rab6-dependent manner but can associate with Rab11-positive compartments when the level of Rab11A is increased within the cells. Fluorescence resonance energy transfer analysis using fluorescence lifetime imaging shows that the overexpression of R6IP1 promotes an interaction between Rab11A and Rab6 in living cells. Accordingly, the REs marked by Rab11 and transferrin receptor are depleted from the cell periphery and accumulate in the pericentriolar area. However, endosomal and Golgi membranes do not appear to fuse with each other. We also show that R6IP1 function is required during metaphase and cytokinesis, two mitotic steps in which a role of Rab6 and Rab11 has been previously documented. We propose that R6IP1 may couple Rab6 and Rab11 function throughout the cell cycle.