RESUMEN
The crosstalk between clathrin-mediated endocytosis (CME) and the autophagy pathway has been reported in mammals; however, the interconnection of CME with autophagy has not been established in plants. Here, we report that the Arabidopsis CLATHRIN LIGHT CHAIN (CLC) subunit 2 and 3 double mutant, clc2-1 clc3-1, phenocopies Arabidopsis AUTOPHAGY-RELATED GENE (ATG) mutants in both autoimmunity and nutrient sensitivity. Accordingly, the autophagy pathway is significantly compromised in the clc2-1 clc3-1 mutant. Interestingly, multiple assays demonstrate that CLC2 directly interacts with ATG8h/ATG8i in a domain-specific manner. As expected, both GFP-ATG8h/GFP-ATG8i and CLC2-GFP are subjected to autophagic degradation, and degradation of GFP-ATG8h is significantly reduced in the clc2-1 clc3-1 mutant. Notably, simultaneous knockout of ATG8h and ATG8i by CRISPR-Cas9 results in enhanced resistance against Golovinomyces cichoracearum, supporting the functional relevance of the CLC2-ATG8h/8i interactions. In conclusion, our results reveal a link between the function of CLCs and the autophagy pathway in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Autofagia , Cadenas Ligeras de Clatrina , Inmunidad de la Planta , Arabidopsis/genética , Arabidopsis/inmunología , Arabidopsis/metabolismo , Cadenas Ligeras de Clatrina/metabolismo , Cadenas Ligeras de Clatrina/genética , Inmunidad de la Planta/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/genéticaRESUMEN
To identify functional differences between vertebrate clathrin light chains (CLCa or CLCb), phenotypes of mice lacking genes encoding either isoform were characterised. Mice without CLCa displayed 50% neonatal mortality, reduced body weight, reduced fertility, and â¼40% of aged females developed uterine pyometra. Mice lacking CLCb displayed a less severe weight reduction phenotype compared with those lacking CLCa and had no survival or reproductive system defects. Analysis of female mice lacking CLCa that developed pyometra revealed ectopic expression of epithelial differentiation markers (FOXA2 and K14) and a reduced number of endometrial glands, indicating defects in the lumenal epithelium. Defects in lumen formation and polarity of epithelial cysts derived from uterine or gut cell lines were also observed when either CLCa or CLCb were depleted, with more severe effects from CLCa depletion. In cysts, the CLC isoforms had different distributions relative to each other, although they converge in tissue. Together, these findings suggest differential and cooperative roles for CLC isoforms in epithelial lumen formation, with a dominant function for CLCa.
Asunto(s)
Quistes , Piómetra , Humanos , Femenino , Animales , Ratones , Cadenas Ligeras de Clatrina/genética , Cadenas Ligeras de Clatrina/metabolismo , Línea Celular , Isoformas de ProteínasRESUMEN
Conformational changes in endocytic proteins are regulators of clathrin-mediated endocytosis. Three clathrin heavy chains associated with clathrin light chains (CLC) assemble into triskelia that link into a geometric lattice that curves to drive endocytosis. Structural changes in CLC have been shown to regulate triskelia assembly in solution, yet the nature of these changes, and their effects on lattice growth, curvature, and endocytosis in cells are unknown. Here, we develop a new correlative fluorescence resonance energy transfer (FRET) and platinum replica electron microscopy method, named FRET-CLEM. With FRET-CLEM, we measure conformational changes in clathrin at thousands of individual morphologically distinct clathrin-coated structures. We discover that the N-terminus of CLC repositions away from the plasma membrane and triskelia vertex as coats curve. Preventing this conformational switch with chemical tools increases lattice sizes and inhibits endocytosis. Thus, a specific conformational switch in the light chain regulates lattice curvature and endocytosis in mammalian cells.
Asunto(s)
Cadenas Ligeras de Clatrina , Endocitosis , Animales , Cadenas Ligeras de Clatrina/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Cadenas Pesadas de Clatrina/metabolismo , Mamíferos/metabolismoRESUMEN
Non-obstructive azoospermia (NOA) is a serious cause of male infertility. The Sertoli cell responds to androgens and takes on roles supporting spermatogenesis, which may cause infertility. This work aims to enhance the genetic diagnosis of NOA via the discovery of new and hub genes implicated in human NOA and to better assess the odds of successful sperm extraction according to the individual's genotype. Whole exome sequencing (WES) was done on three NOA patients to find key genes involved in NOA. We evaluated genome-wide transcripts (about 50,000 transcripts) by microarray between the Sertoli of non-obstructive azoospermia and normal cells. The microarray analysis of three human cases with different non-obstructive azoospermia revealed that 32 genes were upregulated, and the expressions of 113 genes were downregulated versus the normal case. For this purpose, Enrich Shiny GO, STRING, and Cytoscape online evaluations were applied to predict the functional and molecular interactions of proteins and then recognize the master pathways. The functional enrichment analysis demonstrated that the biological process (BP) terms "inositol lipid-mediated signaling", "positive regulation of transcription by RNA polymerase II", and "positive regulation of DNA-templated transcription" significantly changed in upregulated differentially expressed genes (DEGs). The BP investigation of downregulated DEGs highlighted "mitotic cytokinesis", "regulation of protein-containing complex assembly", "cytoskeleton-dependent cytokinesis", and the "peptide metabolic process". Overrepresented molecular function (MF) terms in upregulated DEGs included "ubiquitin-specific protease binding", "protease binding", "phosphatidylinositol trisphosphate phosphatase activity", and "clathrin light chain binding". Interestingly, the MF analysis of the downregulated DEGs revealed overexpression in "ATPase inhibitor activity", "glutathione transferase activity", and "ATPase regulator activity". Our findings suggest that these genes and their interacting hub proteins could help determine the pathophysiologies of germ cell abnormalities and infertility.
Asunto(s)
Azoospermia , Humanos , Masculino , Azoospermia/metabolismo , Secuenciación del Exoma , ARN Polimerasa II/metabolismo , Cadenas Ligeras de Clatrina/genética , Cadenas Ligeras de Clatrina/metabolismo , Testículo/metabolismo , Semen , Inositol/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Glutatión Transferasa/metabolismo , Fosfatidilinositoles/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Péptido Hidrolasas/metabolismo , Adenosina Trifosfatasas/metabolismo , Péptidos/metabolismo , ADN/metabolismoRESUMEN
BACKGROUND: Giardia lamblia, a parasitic protist of the Metamonada supergroup, has evolved one of the most diverged endocytic compartment systems investigated so far. Peripheral endocytic compartments, currently known as peripheral vesicles or vacuoles (PVs), perform bulk uptake of fluid phase material which is then digested and sorted either to the cell cytosol or back to the extracellular space. RESULTS: Here, we present a quantitative morphological characterization of these organelles using volumetric electron microscopy and super-resolution microscopy (SRM). We defined a morphological classification for the heterogenous population of PVs and performed a comparative analysis of PVs and endosome-like organelles in representatives of phylogenetically related taxa, Spironucleus spp. and Tritrichomonas foetus. To investigate the as-yet insufficiently understood connection between PVs and clathrin assemblies in G. lamblia, we further performed an in-depth search for two key elements of the endocytic machinery, clathrin heavy chain (CHC) and clathrin light chain (CLC), across different lineages in Metamonada. Our data point to the loss of a bona fide CLC in the last Fornicata common ancestor (LFCA) with the emergence of a protein analogous to CLC (GlACLC) in the Giardia genus. Finally, the location of clathrin in the various compartments was quantified. CONCLUSIONS: Taken together, this provides the first comprehensive nanometric view of Giardia's endocytic system architecture and sheds light on the evolution of GlACLC analogues in the Fornicata supergroup and, specific to Giardia, as a possible adaptation to the formation and maintenance of stable clathrin assemblies at PVs.
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Giardia lamblia , Clatrina/metabolismo , Cadenas Pesadas de Clatrina/genética , Cadenas Pesadas de Clatrina/metabolismo , Cadenas Ligeras de Clatrina/metabolismo , Endocitosis , Giardia lamblia/genética , Giardia lamblia/metabolismo , FilogeniaRESUMEN
PIN-FORMED (PIN)-dependent directional auxin transport is crucial for plant development. Although the redistribution of auxin mediated by the polarization of PIN3 plays key roles in modulating hypocotyl cell expansion, how PIN3 becomes repolarized to the proper sites within hypocotyl cells is poorly understood. We previously generated the clathrin light chain clc2-1 clc3-1 double mutant in Arabidopsis thaliana and found that it has an elongated hypocotyl phenotype compared to the wild type. Here, we performed genetic, cell biology, and pharmacological analyses combined with live-cell imaging to elucidate the molecular mechanism underlying the role of clathrin light chains in hypocotyl elongation. Our analyses indicated that the defects of the double mutant enhanced auxin maxima in epidermal cells, thus, promoting hypocotyl elongation. PIN3 relocated to the lateral sides of hypocotyl endodermal cells in clc2-1 clc3-1 mutants to redirect auxin toward the epidermal cell layers. Moreover, the loss of function of PIN3 largely suppressed the long hypocotyl phenotype of the clc2-1 clc3-1 double mutant, as did treatment with auxin transport inhibitors. Based on these data, we propose that clathrin modulates PIN3 abundance and polarity to direct auxin flux and inhibit cell elongation in the hypocotyl, providing novel insights into the regulation of hypocotyl elongation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cadenas Ligeras de Clatrina/metabolismo , Hipocótilo/crecimiento & desarrollo , Ácidos Indolacéticos/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Cadenas Ligeras de Clatrina/genética , Hipocótilo/metabolismo , Epidermis de la Planta/metabolismoRESUMEN
Orchestration of a complex network of protein interactions drives clathrin-mediated endocytosis (CME). A central role for the AP2 adaptor complex beyond cargo recognition and clathrin recruitment has emerged in recent years. It is now apparent that AP2 serves as a pivotal hub for protein interactions to mediate clathrin coated pit maturation, and couples lattice formation to membrane deformation. As a key driver for clathrin assembly, AP2 complements the attenuating role of clathrin light chain subunits, which enable dynamic lattice rearrangement needed for budding. This review summarises recent insights into AP2 function with respect to CME dynamics and biophysics, and its relationship to the role of clathrin light chains in clathrin assembly.
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Complejo 2 de Proteína Adaptadora , Cadenas Ligeras de Clatrina , Complejo 2 de Proteína Adaptadora/metabolismo , Clatrina/metabolismo , Cadenas Ligeras de Clatrina/metabolismo , Endocitosis , Unión ProteicaRESUMEN
One of the most fundamental processes of the cell is the uptake of molecules from the surrounding environment. Clathrin-mediated endocytosis (CME) is the best-described uptake pathway and regulates nutrient uptake, protein and lipid turnover at the plasma membrane (PM), cell signaling, cell motility and cell polarity. The main protein in CME is clathrin, which assembles as a triskelion-looking building block made of three clathrin heavy chains and three clathrin light chains. Compared to clathrin heavy chains (CHCs), the role of the two isoforms of clathrin light chains (CLCA and CLCB) is poorly understood. Here, we confirm that the simultaneous deletion of both CLCA/B causes abnormal actin structures at the ventral PM and we describe them, for the first time, as functional invadopodia rather than disorganized actin-cytoskeleton assembly sites. Their identification is based on the occurrence of common invadopodia markers as well as functional invadopodia activity characterized by an increased local proteolytic activity of the extracellular matrix proteins. We demonstrate that CLCA/B deletion impacts the intracellular trafficking and recovery of the matrix metalloproteinase 14 (MMP14) leading to its accumulation at the plasma membrane and induction of invadopodia formation. Importantly, we show that invadopodia formation can be prevented by depletion of MMP14. As such, we propose that CLCA/B regulate invadopodia formation by regulating MMP14 delivery to the plasma membrane.
Asunto(s)
Cadenas Ligeras de Clatrina/metabolismo , Podosomas/metabolismo , Actinas/metabolismo , Biomarcadores/metabolismo , Membrana Celular/metabolismo , Endocitosis , Células HEK293 , Humanos , Metaloproteinasa 14 de la Matriz/metabolismo , Modelos Biológicos , Transporte de Proteínas , Proteolisis , Regulación hacia ArribaRESUMEN
Clathrin light chain (CLC) subunits in vertebrates are encoded by paralogous genes CLTA and CLTB, and both gene products are alternatively spliced in neurons. To understand how this CLC diversity influences neuronal clathrin function, we characterized the biophysical properties of clathrin comprising individual CLC variants for correlation with neuronal phenotypes of mice lacking either CLC-encoding gene. CLC splice variants differentially influenced clathrin knee conformation within assemblies, and clathrin with neuronal CLC mixtures was more effective in membrane deformation than clathrin with single neuronal isoforms nCLCa or nCLCb. Correspondingly, electrophysiological recordings revealed that neurons from mice lacking nCLCa or nCLCb were both defective in synaptic vesicle replenishment. Mice with only nCLCb had a reduced synaptic vesicle pool and impaired neurotransmission compared to WT mice, while nCLCa-only mice had increased synaptic vesicle numbers, restoring normal neurotransmission. These findings highlight differences between the CLC isoforms and show that isoform mixing influences tissue-specific clathrin activity in neurons, which requires their functional balance.
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Cadenas Ligeras de Clatrina , Vesículas Sinápticas/química , Vesículas Sinápticas/metabolismo , Animales , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/metabolismo , Células Cultivadas , Cadenas Ligeras de Clatrina/química , Cadenas Ligeras de Clatrina/genética , Cadenas Ligeras de Clatrina/metabolismo , Ratones , Ratones Noqueados , Neuronas/citología , Neuronas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMEN
Clathrin light chains (CLCa and CLCb) are major constituents of clathrin-coated vesicles. Unique functions for these evolutionary conserved paralogs remain elusive, and their role in clathrin-mediated endocytosis in mammalian cells is debated. Here, we find and structurally characterize a direct and selective interaction between CLCa and the long isoform of the actin motor protein myosin VI, which is expressed exclusively in highly polarized tissues. Using genetically-reconstituted Caco-2 cysts as proxy for polarized epithelia, we provide evidence for coordinated action of myosin VI and CLCa at the apical surface where these proteins are essential for fission of clathrin-coated pits. We further find that myosin VI and Huntingtin-interacting protein 1-related protein (Hip1R) are mutually exclusive interactors with CLCa, and suggest a model for the sequential function of myosin VI and Hip1R in actin-mediated clathrin-coated vesicle budding.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Cadenas Ligeras de Clatrina/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Proteínas de Microfilamentos/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Actinas/metabolismo , Células CACO-2 , Técnicas de Cultivo de Célula , Cadenas Ligeras de Clatrina/ultraestructura , Quistes , Endocitosis , Humanos , Espectroscopía de Resonancia Magnética , Cadenas Pesadas de Miosina/ultraestructura , Unión Proteica , Conformación Proteica , Isoformas de ProteínasRESUMEN
Clathrins, composed of clathrin heavy chains (CHCs) and clathrin light chains (CLCs), are usually hijacked by viruses for infection. However, the role of CLCs, especially in regulating fish virus infection, remains poorly understood. Here, two isoforms of CLCs were cloned from the red-spotted grouper (Epinephelus akaara) (EaCLCa and EaCLCb). Both EaCLC transcripts were expressed in all examined tissues, and the expression of EaCLCa was much higher than that of EaCLCb. Over-expressing EaCLCa-W119R mutant significantly reduced Singapore grouper iridovirus (SGIV) infectivity. However, no effect of EaCLCb-W122R on SGIV infection was observed. The detailed steps were further studied, mainly including virus attachment, entry and the following transport to early endosomes. EaCLCa-W119R mutant notably inhibited internalization of SGIV particles with no effect on SGIV attachment. Furthermore, EaCLCa-W119R mutant obviously impaired the delivery of SGIV to early endosomes after virus internalization. In addition, the EaCLCa-W119R mutant markedly reduced the colocalization of SGIV and actin. However, EaCLCb is not required for such events during SGIV infection. Taken together, these results demonstrate for the first time that EaCLCa and EaCLCb exerted different impacts on iridovirus infection, providing a better understanding of the mechanisms of SGIV infection and opportunities for the design of new antiviral strategies.
Asunto(s)
Cadenas Ligeras de Clatrina/metabolismo , Iridovirus/enzimología , Iridovirus/fisiología , Perciformes/virología , Secuencia de Aminoácidos , Animales , Cadenas Ligeras de Clatrina/química , Cadenas Ligeras de Clatrina/genética , Endosomas/metabolismo , Regulación Enzimológica de la Expresión Génica , Espacio Intracelular/metabolismo , Iridovirus/genética , Mutación , Transporte de Proteínas , Análisis de Secuencia , Internalización del VirusRESUMEN
Clathrin heavy chain is the structural component of the clathrin triskelion, but unique functions for the two distinct and highly conserved clathrin light chains (CLCa and CLCb, also known as CLTA and CLTB, respectively) have been elusive. Here, we show that following detachment and replating, CLCa is uniquely responsible for promoting efficient cell spreading and migration. Selective depletion of CLCa, but not of CLCb, reduced the initial phase of isotropic spreading of HeLa, H1299 and HEK293 cells by 60-80% compared to siRNA controls, and wound closure and motility by â¼50%. Surface levels of ß1-integrins were unaffected by CLCa depletion. However, CLCa was required for effective targeting of FAK (also known as PTK2) and paxillin to the adherent surface of spreading cells, for integrin-mediated activation of Src, FAK and paxillin, and for maturation of focal adhesions, but not their microtubule-based turnover. Depletion of CLCa also blocked the interaction of clathrin with the nucleation-promoting factor WAVE complex, and altered actin distribution. Furthermore, preferential recruitment of CLCa to budding protrusions was also observed. These results comprise the first identification of CLCa-specific functions, with implications for normal and neoplastic integrin-based signaling and cell migration.
Asunto(s)
Cadenas Ligeras de Clatrina/metabolismo , Adhesiones Focales/metabolismo , Línea Celular Tumoral , Movimiento Celular/fisiología , Humanos , Transducción de SeñalRESUMEN
BACKGROUND: Magnetic nanoparticles (MNPs) have been widely used in biomedical applications. Proper control of the duration of MNPs in circulation promises to improve further their applications, in particularly drug delivery. It is known that the uptake of tissue-associated MNPs is mainly carried out by macrophages. Yet, the molecular mechanism to control MNPs internalization in macrophages remains to be elusive. Missing-in-metastasis (MIM) is a scaffolding protein that is highly expressed in macrophages and regulates receptor-mediated endocytosis. We hypothesize that uptake of MNPs may also involve the function of MIM. METHODS: We investigated the effect of MIM expression on the intracellular trafficking of MNPs by transmission electronic microscopy, flow cytometry, o-phenanthroline photometric analysis, Perl's staining, immunofluorescence microscopy and co-immunoprecipitation. To explore the molecular events in MIM-mediated MNPs uptake, we examined the effect of MNPs on the interaction of MIM with clathrin, Rab5 and Rab7. RESULTS: Uptake of MNPs was significantly enhanced in cells overexpressing MIM. Upon exposure to MNPs, MIM was associated with clathrin light chain in endocytic vesicles and Rab7, a protein that regulates late endosomes. However, MNPs caused dissociation of MIM with Rab5, an early endosome-associated protein. CONCLUSIONS: MIM regulates internalization of MNPs via promoting their trafficking from plasma membrane to late endosomes. GENERAL SIGNIFICANCE: Our data unveiled a novel pathway which MNPs internalization and intracellular trafficking in macrophages. This new pathway may allow us to control the uptake of MNPs within cells by targeting MIM, thereby improving their medical applications.
Asunto(s)
Cadenas Ligeras de Clatrina/metabolismo , Nanopartículas de Magnetita/química , Proteínas de Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Membrana Celular/metabolismo , Células Cultivadas , Endosomas/metabolismo , Ratones , Tamaño de la Partícula , Células RAW 264.7 , Propiedades de Superficie , Proteínas de Unión a GTP rab7RESUMEN
Alternative splicing is a regulatory mechanism by which multiple mRNA isoforms are generated from single genes. Numerous genes that encode membrane trafficking proteins are alternatively spliced. However, there is limited information about the functional consequences that result from these splicing transitions. Here, we developed appropriate tools to study the functional impact of alternative splicing in development within the most in vivo context. Secondly, we provided evidence of the physiological implications of splicing regulation during muscle development. Our previous work in mouse heart development identified three trafficking genes that are regulated by alternative splicing between birth and adulthood: the clathrin heavy chain, the clathrin light chain-a, and the trafficking kinesin binding protein-1. Here, we demonstrated that alternative splicing regulation of these three genes is tissue- and developmental stage-specific. To identify the functional consequences of splicing regulation in vivo, we used genome editing to block the neonatal-to-adult splicing transitions. We characterized the phenotype of one of these mouse lines and demonstrated that when splicing regulation of the clathrin heavy chain gene is prevented mice exhibit an increase in body and muscle weights which is due to an enlargement in myofiber size. The significance of this work has two components. First, we revealed novel roles of the clathrin heavy chain in muscle growth and showed that its regulation by alternative splicing contributes to muscle development. Second, the new mouse lines will provide a useful tool to study how splicing regulation of three trafficking genes affects tissue identity acquisition and maturation in vivo.
Asunto(s)
Empalme Alternativo , Edición Génica , Músculo Esquelético/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cadenas Pesadas de Clatrina/antagonistas & inhibidores , Cadenas Pesadas de Clatrina/genética , Cadenas Pesadas de Clatrina/metabolismo , Cadenas Ligeras de Clatrina/antagonistas & inhibidores , Cadenas Ligeras de Clatrina/genética , Cadenas Ligeras de Clatrina/metabolismo , Femenino , Homocigoto , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Músculo Esquelético/crecimiento & desarrollo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
Clathrin light chains (CLCs) control selective uptake of a range of G protein-coupled receptors (GPCRs), although the mechanism by which this occurs has remained elusive thus far. In particular, site-specific phosphorylation of CLCb controls the uptake of the purinergic GPCR P2Y12, but it is dispensable for the constitutive uptake of the transferrin receptor (TfR). We demonstrate that phosphorylation of CLCb is required for the maturation of clathrin-coated pits (CCPs) through the transition of flat lattices into invaginated buds. This transition is dependent on efficient clathrin exchange regulated by CLCb phosphorylation and mediated through auxilin. Strikingly, this rearrangement is required for the uptake of P2Y12 but not TfR. These findings link auxilin-mediated clathrin exchange to early stages of CCP invagination in a cargo-specific manner. This supports a model in which CCPs invaginate with variable modes of curvature depending on the cargo they incorporate.
Asunto(s)
Cadenas Ligeras de Clatrina/metabolismo , Modelos Biológicos , Receptores Purinérgicos P2Y12/metabolismo , Receptores de Transferrina/metabolismo , Auxilinas/genética , Auxilinas/metabolismo , Cadenas Ligeras de Clatrina/genética , Células HeLa , Humanos , Fosforilación , Receptores Purinérgicos P2Y12/genética , Receptores de Transferrina/genéticaRESUMEN
Labeling proteins with high specificity and efficiency is a fundamental prerequisite for microscopic visualization of subcellular protein structures and interactions. Although the comparatively small size of epitope tags makes them less perturbative to fusion proteins, they require the use of large antibodies that often limit probe accessibility and effective resolution. Here we use the covalent SpyTag-SpyCatcher system as an epitope-like tag for fluorescent labeling of intracellular proteins in fixed cells for both conventional and super-resolution microscopy. We also applied this method to endogenous proteins by gene editing, demonstrating its high labeling efficiency and capability for isoform-specific labeling.
Asunto(s)
Adhesinas Bacterianas/química , Proteínas Portadoras/química , Fragmentos de Péptidos/química , Péptidos/química , Actinas/química , Adhesinas Bacterianas/metabolismo , Carbocianinas/química , Proteínas Portadoras/metabolismo , Cadenas Ligeras de Clatrina/química , Cadenas Ligeras de Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Colorantes Fluorescentes , Edición Génica , Células HeLa , Humanos , Queratinas/química , Microscopía Fluorescente , Fragmentos de Péptidos/metabolismo , Canales de Translocación SEC/química , Canales de Translocación SEC/metabolismo , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Signaling receptors are internalized and regulated by clathrin-mediated endocytosis (CME). Two clathrin light chain isoforms, CLCa and CLCb, are integral components of the endocytic machinery whose differential functions remain unknown. We report that CLCb is specifically upregulated in non-small-cell lung cancer (NSCLC) cells and is associated with poor patient prognosis. Engineered single CLCb-expressing NSCLC cells, as well as "switched" cells that predominantly express CLCb, exhibit increased rates of CME and altered clathrin-coated pit dynamics. This "adaptive CME" resulted from upregulation of dynamin-1 (Dyn1) and its activation through a positive feedback loop involving enhanced epidermal growth factor (EGF)-dependent Akt/GSK3ß phosphorylation. CLCb/Dyn1-dependent adaptive CME selectively altered EGF receptor trafficking, enhanced cell migration in vitro, and increased the metastatic efficiency of NSCLC cells in vivo. We define molecular mechanisms for adaptive CME in cancer cells and a role for the reciprocal crosstalk between signaling and CME in cancer progression.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Cadenas Ligeras de Clatrina/metabolismo , Clatrina/metabolismo , Dinamina I/metabolismo , Endocitosis , Receptores ErbB/metabolismo , Neoplasias Pulmonares/patología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Movimiento Celular , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endosomas/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Neoplasias Pulmonares/genética , Ratones Desnudos , Metástasis de la Neoplasia , Fosforilación , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Riesgo , Transducción de Señal , Análisis de Supervivencia , Regulación hacia Arriba/genéticaRESUMEN
Trypanosomatids are parasitic protozoa with a significant burden on human health. African and American trypanosomes are causative agents of Nagana and Chagas disease respectively, and speciated about 300 million years ago. These parasites have highly distinct life cycles, pathologies, transmission strategies and surface proteomes, being dominated by the variant surface glycoprotein (African) or mucins (American) respectively. In African trypanosomes clathrin-mediated trafficking is responsible for endocytosis and post-Golgi transport, with several mechanistic aspects distinct from higher organisms. Using clathrin light chain (TcCLC) and EpsinR (TcEpsinR) as affinity handles, we identified candidate clathrin-associated proteins (CAPs) in Trypanosoma cruzi; the cohort includes orthologs of many proteins known to mediate vesicle trafficking, but significantly not the AP-2 adaptor complex. Several trypanosome-specific proteins common with African trypanosomes, were also identified. Fluorescence microscopy revealed localisations for TcEpsinR, TcCLC and TcCHC at the posterior region of trypomastigote cells, coincident with the flagellar pocket and Golgi apparatus. These data provide the first systematic analysis of clathrin-mediated trafficking in T. cruzi, allowing comparison between protein cohorts and other trypanosomes and also suggest that clathrin trafficking in at least some life stages of T. cruzi may be AP-2-independent.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Cadenas Ligeras de Clatrina/metabolismo , Clatrina/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Endocitosis , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Inmunoprecipitación , Espectrometría de Masas , Microscopía Fluorescente , Transporte de Proteínas , Proteoma , Trypanosoma brucei brucei/metabolismoRESUMEN
Clathrin facilitates vesicle formation during endocytosis and sorting in the trans-Golgi network (TGN)/endosomal system. Unlike in mammals, yeast clathrin function requires both the clathrin heavy (CHC) and clathrin light (CLC) chain, since Chc1 does not form stable trimers without Clc1. To further delineate clathrin subunit functions, we constructed a chimeric CHC protein (Chc-YR) , which fused the N-terminus of yeast CHC (1-1312) to the rat CHC residues 1318-1675, including the CHC trimerization region. The novel CHC-YR allele encoded a stable protein that fractionated as a trimer. CHC-YR also complemented chc1Δ slow growth and clathrin TGN/endosomal sorting defects. In strains depleted for Clc1 (either clc1Δ or chc1Δ clc1Δ), CHC-YR, but not CHC1, suppressed TGN/endosomal sorting and growth phenotypes. Chc-YR-GFP (green fluorescent protein) localized to the TGN and cortical patches on the plasma membrane, like Chc1 and Clc1. However, Clc1-GFP was primarily cytoplasmic in chc1Δ cells harboring pCHC-YR, indicating that Chc-YR does not bind yeast CLC. Still, some partial phenotypes persisted in cells with Chc-YR, which are likely due either to loss of CLC recruitment or chimeric HC lattice instability. Ultimately, these studies have created a tool to examine non-trimerization roles for the clathrin LC.
Asunto(s)
Cadenas Pesadas de Clatrina/metabolismo , Cadenas Ligeras de Clatrina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Membrana Celular/metabolismo , Cadenas Pesadas de Clatrina/genética , Cadenas Ligeras de Clatrina/genética , Endocitosis/fisiología , Proteínas Fluorescentes Verdes/genética , Unión Proteica , Transporte de Proteínas , Ratas , Proteínas Recombinantes de Fusión/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Red trans-Golgi/metabolismoRESUMEN
In plants, clathrin-mediated endocytosis (CME) is dependent on the function of clathrin and its accessory heterooligomeric adaptor protein complexes, ADAPTOR PROTEIN2 (AP-2) and the TPLATE complex (TPC), and is negatively regulated by the hormones auxin and salicylic acid (SA). The details for how clathrin and its adaptor complexes are recruited to the plasma membrane (PM) to regulate CME, however, are poorly understood. We found that SA and the pharmacological CME inhibitor tyrphostin A23 reduce the membrane association of clathrin and AP-2, but not that of the TPC, whereas auxin solely affected clathrin membrane association, in Arabidopsis (Arabidopsis thaliana). Genetic and pharmacological experiments revealed that loss of AP2µ or AP2σ partially affected the membrane association of other AP-2 subunits and that the AP-2 subunit AP2σ, but not AP2µ, was required for SA- and tyrphostin A23-dependent inhibition of CME Furthermore, we show that although AP-2 and the TPC are both required for the PM recruitment of clathrin in wild-type cells, the TPC is necessary for clathrin PM association in AP-2-deficient cells. These results indicate that developmental signals may differentially modulate the membrane recruitment of clathrin and its core accessory complexes to regulate the process of CME in plant cells.