RESUMEN
Biological systems are the sum of their dynamic three-dimensional (3D) parts. Therefore, it is critical to study biological structures in 3D and at high resolution to gain insights into their physiological functions. Electron microscopy of metal replicas of unroofed cells and isolated organelles has been a key technique to visualize intracellular structures at nanometer resolution. However, many of these methods require specialized equipment and personnel to complete them. Here, we present novel accessible methods to analyze biological structures in unroofed cells and biochemically isolated organelles in 3D and at nanometer resolution, focusing on Arabidopsis clathrin-coated vesicles (CCVs). While CCVs are essential trafficking organelles, their detailed structural information is lacking due to their poor preservation when observed via classical electron microscopy protocols experiments. First, we establish a method to visualize CCVs in unroofed cells using scanning transmission electron microscopy tomography, providing sufficient resolution to define the clathrin coat arrangements. Critically, the samples are prepared directly on electron microscopy grids, removing the requirement to use extremely corrosive acids, thereby enabling the use of this method in any electron microscopy lab. Secondly, we demonstrate that this standardized sample preparation allows the direct comparison of isolated CCV samples with those visualized in cells. Finally, to facilitate the high-throughput and robust screening of metal replicated samples, we provide a deep learning analysis method to screen the "pseudo 3D" morphologies of CCVs imaged with 2D modalities. Collectively, our work establishes accessible ways to examine the 3D structure of biological samples and provide novel insights into the structure of plant CCVs.
Asunto(s)
Cáusticos , Vesículas Cubiertas por Clatrina , Cáusticos/análisis , Clatrina , Vesículas Cubiertas por Clatrina/química , Vesículas Cubiertas por Clatrina/ultraestructura , Endocitosis/fisiología , Imagenología TridimensionalRESUMEN
In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein assembly polypeptide-1 (AP-1) complex operates as part of the secretory pathway at the trans-Golgi network (TGN), while the AP-2 complex and the TPLATE complex jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched TGN/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis (Arabidopsis thaliana) cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.
Asunto(s)
Arabidopsis , Vesículas Cubiertas por Clatrina , Arabidopsis/genética , Arabidopsis/metabolismo , Clatrina/metabolismo , Vesículas Cubiertas por Clatrina/química , Vesículas Cubiertas por Clatrina/metabolismo , Endocitosis , Proteoma/metabolismo , Proteómica , Factor de Transcripción AP-1/análisis , Factor de Transcripción AP-1/metabolismoRESUMEN
In non-neuronal cells, clathrin has established roles in endocytosis, with clathrin cages enclosing plasma membrane infoldings, followed by rapid disassembly and reuse of monomers. However, in neurons, clathrin is conveyed in slow axonal transport over days to weeks, and the underlying transport/targeting mechanisms, mobile cargo structures, and even its precise presynaptic localization and physiologic role are unclear. Combining live imaging, photobleaching/conversion, mass spectrometry, electron microscopy, and super-resolution imaging, we found that unlike in dendrites, where clathrin cages rapidly assemble and disassemble, in axons, clathrin and related proteins organize into stable "transport packets" that are unrelated to endocytosis and move intermittently on microtubules, generating an overall slow anterograde flow. At synapses, multiple clathrin packets abut synaptic vesicle (SV) clusters, and clathrin packets also exchange between synaptic boutons in a microtubule-dependent "superpool." Within synaptic boundaries, clathrin is surprisingly dynamic, continuously exchanging between local clathrin assemblies, and its depletion impairs SV recycling. Our data provide a conceptual framework for understanding clathrin trafficking and presynaptic targeting that has functional implications.
Asunto(s)
Transporte Axonal/fisiología , Vesículas Cubiertas por Clatrina/metabolismo , Clatrina/metabolismo , Hipocampo/metabolismo , Sinapsis/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Clatrina/química , Vesículas Cubiertas por Clatrina/química , Hipocampo/química , Hipocampo/citología , Ratones , Transporte de Proteínas/fisiología , Ratas , Ratas Wistar , Sinapsis/química , Imagen de Lapso de Tiempo/métodosRESUMEN
Myosin VI ensembles on endocytic cargo facilitate directed transport through a dense cortical actin network. Myosin VI is recruited to clathrin-coated endosomes via the cargo adaptor Dab2. Canonically, it has been assumed that the interactions between a motor and its cargo adaptor are stable. However, it has been demonstrated that the force generated by multiple stably attached motors disrupts local cytoskeletal architecture, potentially compromising transport. In this study, we demonstrate that dynamic multimerization of myosin VI-Dab2 complexes facilitates cargo processivity without significant reorganization of cortical actin networks. Specifically, we find that Dab2 myosin interacting region (MIR) binds myosin VI with a moderate affinity (184 nM) and single-molecule kinetic measurements demonstrate a high rate of turnover (1 s-1) of the Dab2 MIR-myosin VI interaction. Single-molecule motility shows that saturating Dab2-MIR concentration (2 µM) promotes myosin VI homodimerization and processivity with run lengths comparable with constitutive myosin VI dimers. Cargo-mimetic DNA origami scaffolds patterned with Dab2 MIR-myosin VI complexes are weakly processive, displaying sparse motility on single actin filaments and "stop-and-go" motion on a cellular actin network. On a minimal actin cortex assembled on lipid bilayers, unregulated processive movement by either constitutive myosin V or VI dimers results in actin remodeling and foci formation. In contrast, Dab2 MIR-myosin VI interactions preserve the integrity of a minimal cortical actin network. Taken together, our study demonstrates the importance of dynamic motor-cargo association in enabling cargo transportation without disrupting cytoskeletal organization.
Asunto(s)
Citoesqueleto de Actina/genética , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Reguladoras de la Apoptosis/química , Complejos Multiproteicos/química , Cadenas Pesadas de Miosina/química , Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestructura , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/ultraestructura , Vesículas Cubiertas por Clatrina/química , Vesículas Cubiertas por Clatrina/genética , Citoesqueleto/química , Citoesqueleto/genética , Citoesqueleto/ultraestructura , Endocitosis/genética , Endosomas/genética , Humanos , Cinética , Complejos Multiproteicos/genética , Complejos Multiproteicos/ultraestructura , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/ultraestructura , Fosfatidilserinas/genética , Unión Proteica/genética , Multimerización de Proteína/genética , Imagen Individual de MoléculaRESUMEN
Formation of clathrin-coated vesicles (CCVs) in receptor-mediated endocytosis is a mechanistically well-established process, in which clathrin, the adaptor protein complex AP-2, and the large GTPase dynamin play crucial roles. In order to obtain more mechanistic insight into this process, here we established a giant unilamellar vesicle (GUV)-based in vitro CCV reconstitution system with chemically defined components and the full-length recombinant proteins clathrin, AP-2, epsin-1, and dynamin-2. Our results support the predominant model in which hydrolysis of GTP by dynamin is a prerequisite to generate CCVs. Strikingly, in this system at near physiological concentrations of reagents, epsin-1 alone does not have the propensity for scission but is required for bud formation, whereas AP-2 and clathrin are not sufficient. Thus, our study reveals that epsin-1 is an important factor for the maturation of clathrin coated buds, a prerequisite for vesicle generation.
Asunto(s)
Complejo 2 de Proteína Adaptadora , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Vesículas Cubiertas por Clatrina/química , Vesículas Cubiertas por Clatrina/metabolismo , Endocitosis , Complejo 2 de Proteína Adaptadora/metabolismo , Animales , Línea Celular , Dinamina I/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Liposomas/metabolismo , Ratas , Proteínas Recombinantes/metabolismoRESUMEN
In plants, clathrin mediated endocytosis (CME) represents the major route for cargo internalisation from the cell surface. It has been assumed to operate in an evolutionary conserved manner as in yeast and animals. Here we report characterisation of ultrastructure, dynamics and mechanisms of plant CME as allowed by our advancement in electron microscopy and quantitative live imaging techniques. Arabidopsis CME appears to follow the constant curvature model and the bona fide CME population generates vesicles of a predominantly hexagonal-basket type; larger and with faster kinetics than in other models. Contrary to the existing paradigm, actin is dispensable for CME events at the plasma membrane but plays a unique role in collecting endocytic vesicles, sorting of internalised cargos and directional endosome movement that itself actively promote CME events. Internalized vesicles display a strongly delayed and sequential uncoating. These unique features highlight the independent evolution of the plant CME mechanism during the autonomous rise of multicellularity in eukaryotes.
Asunto(s)
Arabidopsis , Clatrina , Invaginaciones Cubiertas de la Membrana Celular , Endocitosis/fisiología , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiología , Evolución Biológica , Clatrina/química , Clatrina/metabolismo , Clatrina/ultraestructura , Vesículas Cubiertas por Clatrina/química , Vesículas Cubiertas por Clatrina/metabolismo , Vesículas Cubiertas por Clatrina/ultraestructura , Invaginaciones Cubiertas de la Membrana Celular/química , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Microscopía Electrónica , Modelos BiológicosRESUMEN
Background: Clathrin-mediated endocytosis (CME) plays a fundamental role in podocyte health. Genetic ablation of genes implicated in CME has been shown to cause severe proteinuria and foot process effacement in mice. However, little is known about the cargo of clathrin-coated vesicles (CCVs) in podocytes. The goal of this study was to isolate CCVs from podocytes and identify their cargo by proteomic analysis. Methods: Glomeruli isolated from Podocin-Cre Rosa-DTRflox mouse kidneys were seeded and treated with diphtheria toxin to obtain pure primary podocyte cultures. CCVs were isolated by differential gradient ultracentrifugation, and enrichment of CCVs was assessed by immunoblotting and electron microscopy (EM). Liquid chromatography-mass spectrometry (LC-MS) was performed for proteomic analysis. Proteins with higher abundance than transferrin receptor protein 1 were evaluated for CCV cargo potential against previously published literature. Immunofluorescence staining of identified cargo proteins and CCVs was performed in podocytes for further verification. Results: Immunoblotting for multiple protein markers of CME revealed enrichment in the CCV fraction. Enrichment of CCVs among other small vesicles was observed via EM. Proteomics yielded a total of >1200 significant proteins. Multiple-step data analysis revealed 36 CCV-associated proteins, of which 10 represent novel, highly abundant cargo proteins in podocytes. Colocalization of cargo proteins and CCVs on immunostaining was observed. Conclusions: Our identification of podocyte CCV cargo proteins helps to elucidate the importance of endocytic trafficking for podocyte health and maintenance of the glomerular environment.
Asunto(s)
Vesículas Cubiertas por Clatrina , Podocitos , Animales , Vesículas Cubiertas por Clatrina/química , Endocitosis , Glomérulos Renales , Ratones , ProteómicaRESUMEN
Deformation of the plasma membrane into clathrin-coated vesicles is a critical step in clathrin-mediated endocytosis and requires the orchestrated assembly of clathrin and endocytic adaptors into a membrane-associated protein coat. The individual role of these membrane-bending and curvature-stabilizing factors is subject to current debate. As such, it is unclear whether the clathrin coat itself is stiff enough to impose curvature and if so, whether this could be effectively transferred to the membrane by the linking adaptor proteins. We have recently demonstrated that clathrin alone is sufficient to form membrane buds in vitro. Here, we use atomic force microscopy to assess the contributions of clathrin and its membrane adaptor protein 2 (AP2) to clathrin coat stiffness, which determines the mechanics of vesicle formation. We found that clathrin coats are less than 10-fold stiffer than the membrane they enclose, suggesting a delicate balance between the forces harnessed from clathrin coat formation and those required for membrane bending. We observed that clathrin adaptor protein AP2 increased the stiffness of coats formed from native clathrin, but did not affect less-flexible coats formed from clathrin lacking the light chain subunits. We thus propose that clathrin light chains are important for clathrin coat flexibility and that AP2 facilitates efficient cargo sequestration during coated vesicle formation by modulating clathrin coat stiffness.
Asunto(s)
Complejo 2 de Proteína Adaptadora/metabolismo , Encéfalo/metabolismo , Membrana Celular/metabolismo , Vesículas Cubiertas por Clatrina/química , Vesículas Cubiertas por Clatrina/metabolismo , Clatrina/metabolismo , Endocitosis , Animales , Unión Proteica , Sus scrofaRESUMEN
The fibroblast growth factor FGF21 was labeled with molecularly defined gold nanoparticles (AuNPs), applied to human adipocytes, and imaged by cryo-electron tomography (cryo-ET). Most AuNPs were in pairs about 80 Å apart, on the outer cell surface. Pairs of AuNPs were also abundant inside the cells in clathrin-coated vesicles and endosomes. AuNPs were present but no longer paired in multivesicular bodies. FGF21 could thus be tracked along the endocytotic pathway. The methods developed here to visualize signaling coupled to endocytosis can be applied to a wide variety of cargo and may be extended to studies of other intracellular transactions.
Asunto(s)
Membrana Celular/química , Endocitosis/genética , Endosomas/química , Factores de Crecimiento de Fibroblastos/química , Movimiento Celular/genética , Vesículas Cubiertas por Clatrina/química , Vesículas Cubiertas por Clatrina/metabolismo , Tomografía con Microscopio Electrónico , Factores de Crecimiento de Fibroblastos/aislamiento & purificación , Oro/química , Humanos , Nanopartículas del Metal/química , Transporte de Proteínas/genética , Transducción de Señal , Propiedades de SuperficieRESUMEN
Clathrin-independent endocytosis (CIE) mediates the cellular uptake of many extracellular ligands, receptors, and pathogens, including several life-threatening bacterial toxins and viruses. So far, our understanding of CIE carrier formation has lagged behind that of clathrin-coated vesicles. Impediments have been the imprecise definition of some CIE pathways, the lack of specific cargoes being transported and of exclusive cytosolic markers and regulators. Notwithstanding these limitations, three distinct molecular mechanisms by which CIE carriers form can be defined. Cargo capture by cytosolic proteins is the main mechanism used by fast endophilin-mediated endocytosis (FEME) and interleukin 2 receptor (IL-2R) endocytosis. Acute signaling-induced membrane remodeling drives macropinocytosis. Finally, extracellular lipid or cargo clustering by the glycolipid-lectin (GL-Lect) hypothesis mediates the uptake of Shiga and cholera toxins and receptors by the CLIC/GEEC pathway. Here, we review these mechanisms and highlight current gaps in knowledge that will need to be addressed to complete our understanding of CIE.
Asunto(s)
Vesículas Cubiertas por Clatrina/genética , Clatrina/genética , Endocitosis/genética , Transporte Biológico/genética , Clatrina/química , Vesículas Cubiertas por Clatrina/química , Humanos , Transducción de Señal/genéticaRESUMEN
Vesicular carriers transport proteins and lipids from one organelle to another, recognizing specific identifiers for the donor and acceptor membranes. Two important identifiers are phosphoinositides and GTP-bound GTPases, which provide well-defined but mutable labels. Phosphatidylinositol and its phosphorylated derivatives are present on the cytosolic faces of most cellular membranes. Reversible phosphorylation of its headgroup produces seven distinct phosphoinositides. In endocytic traffic, phosphatidylinositol-4,5-biphosphate marks the plasma membrane, and phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate mark distinct endosomal compartments. It is unknown what sequence of changes in lipid content confers on the vesicles their distinct identity at each intermediate step. Here we describe 'coincidence-detecting' sensors that selectively report the phosphoinositide composition of clathrin-associated structures, and the use of these sensors to follow the dynamics of phosphoinositide conversion during endocytosis. The membrane of an assembling coated pit, in equilibrium with the surrounding plasma membrane, contains phosphatidylinositol-4,5-biphosphate and a smaller amount of phosphatidylinositol-4-phosphate. Closure of the vesicle interrupts free exchange with the plasma membrane. A substantial burst of phosphatidylinositol-4-phosphate immediately after budding coincides with a burst of phosphatidylinositol-3-phosphate, distinct from any later encounter with the phosphatidylinositol-3-phosphate pool in early endosomes; phosphatidylinositol-3,4-biphosphate and the GTPase Rab5 then appear and remain as the uncoating vesicles mature into Rab5-positive endocytic intermediates. Our observations show that a cascade of molecular conversions, made possible by the separation of a vesicle from its parent membrane, can label membrane-traffic intermediates and determine their destinations.
Asunto(s)
Vesículas Cubiertas por Clatrina/química , Vesículas Cubiertas por Clatrina/metabolismo , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endocitosis , Endosomas/metabolismo , Fosfatidilinositoles/metabolismo , Animales , Auxilinas/metabolismo , Células COS , Línea Celular , Membrana Celular/química , Membrana Celular/metabolismo , Chlorocebus aethiops , Invaginaciones Cubiertas de la Membrana Celular/química , Endosomas/química , Humanos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositoles/análisis , Fosfatidilinositoles/química , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Fosfotransferasas/metabolismo , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.
Asunto(s)
Citoesqueleto/ultraestructura , Endocitosis , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Orgánulos/ultraestructura , Actinina/análisis , Actinas/análisis , Animales , Línea Celular , Clatrina/análisis , Vesículas Cubiertas por Clatrina/química , Vesículas Cubiertas por Clatrina/ultraestructura , Invaginaciones Cubiertas de la Membrana Celular/química , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Citoesqueleto/química , Citoesqueleto/metabolismo , Endosomas/química , Endosomas/ultraestructura , Aparato de Golgi/ultraestructura , Procesamiento de Imagen Asistido por Computador , Imagenología Tridimensional/instrumentación , Microscopía Fluorescente/instrumentación , Mitocondrias/química , Mitocondrias/ultraestructura , Orgánulos/química , Orgánulos/metabolismo , Proteínas de Unión al GTP rab5/análisisRESUMEN
Clathrin-mediated endocytosis (CME) is a key pathway for transporting cargo into cells via membrane vesicles; it plays an integral role in nutrient import, signal transduction, neurotransmission, and cellular entry of pathogens and drug-carrying nanoparticles. Because CME entails substantial local remodeling of the plasma membrane, the presence of membrane tension offers resistance to bending and hence, vesicle formation. Experiments show that in such high-tension conditions, actin dynamics is required to carry out CME successfully. In this study, we build on these pioneering experimental studies to provide fundamental mechanistic insights into the roles of two key endocytic proteins-namely, actin and BAR proteins-in driving vesicle formation in high membrane tension environment. Our study reveals an actin force-induced "snap-through instability" that triggers a rapid shape transition from a shallow invagination to a highly invaginated tubular structure. We show that the association of BAR proteins stabilizes vesicles and induces a milder instability. In addition, we present a rather counterintuitive role of BAR depolymerization in regulating the shape evolution of vesicles. We show that the dissociation of BAR proteins, supported by actin-BAR synergy, leads to considerable elongation and squeezing of vesicles. Going beyond the membrane geometry, we put forth a stress-based perspective for the onset of vesicle scission and predict the shapes and composition of detached vesicles. We present the snap-through transition and the high in-plane stress as possible explanations for the intriguing direct transformation of broad and shallow invaginations into detached vesicles in BAR mutant yeast cells.
Asunto(s)
Endocitosis , Lípidos de la Membrana/química , Proteínas de la Membrana/química , Citoesqueleto de Actina/química , Actinas/química , Animales , Transporte Biológico , Proteínas Portadoras/química , Membrana Celular/química , Clatrina/química , Vesículas Cubiertas por Clatrina/química , Perros , Sustancias Macromoleculares , Células de Riñón Canino Madin Darby , Modelos Teóricos , Polímeros/química , Estrés MecánicoRESUMEN
The internalization and subsequent endosomal trafficking of proteins and membrane along the endocytic pathway is a fundamental cellular process. Over the last two decades, this pathway has emerged to be subject to extensive regulation by phosphoinositides (PIs), phosphorylated derivatives of the minor membrane phospholipid phosphatidylinositol. Clathrin-mediated endocytosis (CME) is the endocytic mechanism characterized in most detail. It now represents a prime example of a process spatiotemporally organized by the interplay of PI metabolizing enzymes. The most abundant PI, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], serves as a denominator of plasma membrane identity and together with cargo proteins is instrumental for the initiation of clathrin-coated pit (CCP) formation. During later stages of the process, the generation of phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and the dephosphorylation of PI(4,5)P2regulate CCP maturation and vesicle uncoating. Here we provide an overview of the mechanisms by which PIs are made and consumed to regulate CME and other endocytic pathways and how conversion of PIs en route to endosomes may be accomplished. Mutations in PI converting enzymes are linked to multiple diseases ranging from mental retardation and neurodegeneration, to inherited muscle and kidney disorders suggesting that the tight control of PI levels along the endocytic pathway plays a critical role in cell physiology. This article is part of a Special Issue entitled Phosphoinositides.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Clatrina/metabolismo , Endocitosis/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Clatrina/genética , Vesículas Cubiertas por Clatrina/química , Vesículas Cubiertas por Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/química , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endosomas/química , Endosomas/metabolismo , Regulación de la Expresión Génica , Humanos , Fosfatos de Fosfatidilinositol/metabolismo , Transducción de SeñalRESUMEN
Clathrin-mediated endocytosis involves the coordinated assembly of clathrin cages around membrane indentations, necessitating fluid-like reorganization followed by solid-like stabilization. This apparent duality in clathrin's in vivo behavior provides some indication that the physical interactions between clathrin triskelia and the membrane effect a local response that triggers fluid-solid transformations within the clathrin lattice. We develop a computational model to study the response of clathrin protein lattices to spherical deformations of the underlying flexible membrane. These deformations are similar to the shapes assumed during intracellular trafficking of nanoparticles. Through Monte Carlo simulations of clathrin-on-membrane systems, we observe that these membrane indentations give rise to a greater than normal defect density within the overlaid clathrin lattice. In many cases, the bulk surrounding lattice remains in a crystalline phase, and the extra defects are localized to the regions of large curvature. This can be explained by the fact that the in-plane elastic stress in the clathrin lattice are reduced by coupling defects to highly curved regions. The presence of defects brought about by indentation can result in the fluidization of a lattice that would otherwise be crystalline, resulting in an indentation-driven, defect-mediated phase transition. Altering subunit elasticity or membrane properties is shown to drive a similar transition, and we present phase diagrams that map out the combined effects of these parameters on clathrin lattice properties.
Asunto(s)
Membrana Celular/química , Vesículas Cubiertas por Clatrina/química , Clatrina/química , Fluidez de la Membrana , Modelos Biológicos , Membrana Celular/metabolismo , Clatrina/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Elasticidad , Endocitosis , Método de Montecarlo , Transición de FaseRESUMEN
HeLa cell lines can be experimentally manipulated using drugs or gene-silencing techniques such as RNA interference. Fractions enriched for clathrin-coated vesicles (CCVs) can be isolated from these cell lines and used to study the effects of these manipulations on the composition of CCVs. This protocol, originally developed in the laboratory of Margaret Robinson (Cambridge, United Kingdom), describes the preparation of a HeLa cell fraction that is enriched for a mixed population of CCVs and is suitable for analysis by mass spectroscopy, western blotting, or electron microscopy.
Asunto(s)
Fraccionamiento Celular/métodos , Vesículas Cubiertas por Clatrina/química , Vesículas Cubiertas por Clatrina/ultraestructura , Western Blotting/métodos , Células HeLa , Humanos , Espectrometría de Masas/métodos , Microscopía ElectrónicaRESUMEN
The characterization of clathrin-coated vesicles (CCVs), including the effects of genetic or biochemical manipulations on their composition, can be studied by mass spectrometry analysis of HeLa cell fractions enriched for CCVs. This protocol describes the preparation of samples by tryptic in-gel digest and peptide extraction followed by analysis in an Orbitrap mass spectrometer.
Asunto(s)
Vesículas Cubiertas por Clatrina/química , Espectrometría de Masas/métodos , Proteoma/análisis , Fraccionamiento Celular , Geles , Células HeLa , Humanos , Proteolisis , Tripsina/metabolismoRESUMEN
The characterization of clathrin-coated vesicles (CCVs), including the effects of genetic or biochemical manipulations on their composition, can be studied by mass spectrometry analysis of HeLa cell fractions enriched for CCVs. This protocol describes the preparation of samples by in-solution proteolytic digest and subsequent peptide fractionation, followed by analysis in a Q Exactive mass spectrometer.
Asunto(s)
Fraccionamiento Celular/métodos , Vesículas Cubiertas por Clatrina/química , Espectrometría de Masas/métodos , Proteoma/análisis , Células HeLa , Humanos , ProteolisisRESUMEN
Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.
Asunto(s)
Clatrina/metabolismo , Endocitosis/fisiología , Microscopía Fluorescente/métodos , Receptores Acoplados a Proteínas G/metabolismo , Clatrina/química , Vesículas Cubiertas por Clatrina/química , Vesículas Cubiertas por Clatrina/metabolismo , Colorantes Fluorescentes/química , Células HEK293 , Humanos , Receptores Acoplados a Proteínas G/químicaRESUMEN
Dynamin is a 100 kDa GTPase that organizes into helical assemblies at the base of nascent clathrin-coated vesicles. Formation of these oligomers stimulates the intrinsic GTPase activity of dynamin, which is necessary for efficient membrane fission during endocytosis. Recent evidence suggests that the transition state of dynamin's GTP hydrolysis reaction serves as a key determinant of productive fission. Here, we present the structure of a transition-state-defective dynamin mutant K44A trapped in a prefission state at 12.5 Å resolution. This structure constricts to 3.7 nm, reaching the theoretical limit required for spontaneous membrane fission. Computational docking indicates that the ground-state conformation of the dynamin polymer is sufficient to achieve this superconstricted prefission state and reveals how a two-start helical symmetry promotes the most efficient packing of dynamin tetramers around the membrane neck. These data suggest a model for the assembly and regulation of the minimal dynamin fission machine.