RESUMEN
Glioblastomas (GBMs) are the most lethal primary brain tumors with limited survival, even under aggressive treatments. The current therapeutics for GBMs are flawed due to the failure to accurately discriminate between normal proliferating cells and distinctive tumor cells. Mitochondria are essential to GBMs and serve as potential therapeutical targets. Here, we utilize cryo-electron tomography to quantitatively investigate nanoscale details of randomly sampled mitochondria in their native cellular context of GBM cells. Our results show that compared with cancer-free brain cells, GBM cells own more inter-mitochondrial junctions of several types for communications. Furthermore, our tomograms unveil microtubule-dependent mitochondrial nanotunnel-like bridges in the GBM cells as another inter-mitochondrial structure. These quantified inter-mitochondrial features, together with other mitochondria-organelle and intra-mitochondrial ones, are sufficient to distinguish GBM cells from cancer-free brain cells under scrutiny with predictive modeling. Our findings decipher high-resolution inter-mitochondrial structural signatures and provide clues for diagnosis and therapeutic interventions for GBM and other mitochondria-related diseases.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/patología , Neoplasias Encefálicas/patología , Tomografía con Microscopio Electrónico , Encéfalo/patología , Mitocondrias/patologíaRESUMEN
The endosomal sorting complexes required for transport (ESCRTs) are responsible for membrane remodeling in many cellular processes, such as multivesicular body biogenesis, viral budding, and cytokinetic abscission. ESCRT-III, the most abundant ESCRT subunit, assembles into flat spirals as the primed state, essential to initiate membrane invagination. However, the three-dimensional architecture of ESCRT-III flat spirals remained vague for decades due to highly curved filaments with a small diameter and a single preferred orientation on the membrane. Here, we unveiled that yeast Snf7, a component of ESCRT-III, forms flat spirals on the lipid monolayers using cryogenic electron microscopy. We developed a geometry-constrained Euler angle-assigned reconstruction strategy and obtained moderate-resolution structures of Snf7 flat spirals with varying curvatures. Our analyses showed that Snf7 subunits recline on the membrane with N-terminal motifs α0 as anchors, adopt an open state with fused α2/3 helices, and bend α2/3 gradually from the outer to inner parts of flat spirals. In all, we provide the orientation and conformations of ESCRT-III flat spirals on the membrane and unveil the underlying assembly mechanism, which will serve as the initial step in understanding how ESCRTs drive membrane abscission.
Asunto(s)
Microscopía por Crioelectrón , Complejos de Clasificación Endosomal Requeridos para el Transporte , Proteínas de Saccharomyces cerevisiae , Membrana Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestructuraRESUMEN
Homeostasis and wound healing rely on stem cells (SCs) whose activity and directed migration are often governed by Wnt signaling. In dissecting how this pathway integrates with the necessary downstream cytoskeletal dynamics, we discovered that GSK3ß, a kinase inhibited by Wnt signaling, directly phosphorylates ACF7, a > 500 kDa microtubule-actin crosslinking protein abundant in hair follicle stem cells (HF-SCs). We map ACF7's GSK3ß sites to the microtubule-binding domain and show that phosphorylation uncouples ACF7 from microtubules. Phosphorylation-refractile ACF7 rescues overall microtubule architecture, but phosphorylation-constitutive mutants do not. Neither mutant rescues polarized movement, revealing that phospho-regulation must be dynamic. This circuitry is physiologically relevant and depends upon polarized GSK3ß inhibition at the migrating front of SCs/progeny streaming from HFs during wound repair. Moreover, only ACF7 and not GSKß-refractile-ACF7 restore polarized microtubule-growth and SC-migration to ACF7 null skin. Our findings provide insights into how this conserved spectraplakin integrates signaling, cytoskeletal dynamics, and polarized locomotion of somatic SCs.
Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas de Microfilamentos/metabolismo , Microtúbulos/metabolismo , Piel/metabolismo , Células Madre/metabolismo , Cicatrización de Heridas , Animales , Movimiento Celular , Células Cultivadas , Glucógeno Sintasa Quinasa 3 beta , Ratones , Ratones Transgénicos , Fosforilación , Estructura Terciaria de Proteína , Piel/citología , Células Madre/citologíaRESUMEN
Researchers commonly anneal metals, alloys, and semiconductors to repair defects and improve microstructures via recrystallization. Theoretical studies indicate that simulated annealing on biological macromolecules helps predict the final structures with minimum free energy. Experimental validation of this homogenizing effect and further exploration of its applications are fascinating scientific questions that remain elusive. Here, we chose the apo-state 70S ribosome from Escherichia coli as a model, wherein the 30S subunit undergoes a thermally driven intersubunit rotation and exhibits substantial structural flexibility as well as distinct free energy. We experimentally demonstrate that annealing at a fast cooling rate enhances the 70S ribosome homogeneity and improves local resolution on the 30S subunit. After annealing, the 70S ribosome is in a nonrotated state with respect to corresponding intermediate structures in unannealed or heated ribosomes. Manifold-based analysis further indicates that the annealed 70S ribosome takes a narrow conformational distribution and exhibits a minimum-energy state in the free-energy landscape. Our experimental results offer a facile yet robust approach to enhance protein stability, which is ideal for high-resolution cryogenic electron microscopy. Beyond structure determination, annealing shows great potential for synchronizing proteins on a single-molecule level and can be extended to study protein folding and explore conformational and energy landscapes.
Asunto(s)
Conformación Proteica , Proteínas Ribosómicas/ultraestructura , Ribosomas/fisiología , Microscopía por Crioelectrón , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , ARN Ribosómico/metabolismo , ARN Ribosómico/ultraestructura , Proteínas Ribosómicas/metabolismo , Ribosomas/ultraestructuraRESUMEN
Monomethylamine (MMA) is an important climate-active oceanic trace gas and ubiquitous in the oceans. γ-Glutamylmethylamide synthetase (GmaS) catalyzes the conversion of MMA to γ-glutamylmethylamide, the first step in MMA metabolism in many marine bacteria. The gmaS gene occurs in â¼23% of microbial genomes in the surface ocean and is a validated biomarker to detect MMA-utilizing bacteria. However, the catalytic mechanism of GmaS has not been studied because of the lack of structural information. Here, the GmaS from Rhodovulum sp. 12E13 (RhGmaS) was characterized, and the crystal structures of apo-RhGmaS and RhGmaS with different ligands in five states were solved. Based on structural and biochemical analyses, the catalytic mechanism of RhGmaS was explained. ATP is first bound in RhGmaS, leading to a conformational change of a flexible loop (Lys287-Ile305), which is essential for the subsequent binding of glutamate. During the catalysis of RhGmaS, the residue Arg312 participates in polarizing the γ-phosphate of ATP and in stabilizing the γ-glutamyl phosphate intermediate; Asp177 is responsible for the deprotonation of MMA, assisting the attack of MMA on γ-glutamyl phosphate to produce a tetrahedral intermediate; and Glu186 acts as a catalytic base to abstract a proton from the tetrahedral intermediate to finally generate glutamylmethylamide. Sequence analysis suggested that the catalytic mechanism of RhGmaS proposed in this study has universal significance in bacteria containing GmaS. Our results provide novel insights into MMA metabolism, contributing to a better understanding of MMA catabolism in global carbon and nitrogen cycles.
Asunto(s)
Ligasas de Carbono-Nitrógeno/metabolismo , Glutamatos/metabolismo , Adenosina Trifosfato/metabolismo , Catálisis , Escherichia coli/metabolismo , Ácido Glutámico/metabolismo , Magnesio/metabolismo , Metilaminas/metabolismo , Microscopía Electrónica , Rhodovulum/metabolismoRESUMEN
The viral polymerase complex, comprising the large protein (L) and phosphoprotein (P), is crucial for both genome replication and transcription in non-segmented negative-strand RNA viruses (nsNSVs), while structures corresponding to these activities remain obscure. Here, we resolved two L-P complex conformations from the mumps virus (MuV), a typical member of nsNSVs, via cryogenic-electron microscopy. One conformation presents all five domains of L forming a continuous RNA tunnel to the methyltransferase domain (MTase), preferably as a transcription state. The other conformation has the appendage averaged out, which is inaccessible to MTase. In both conformations, parallel P tetramers are revealed around MuV L, which, together with structures of other nsNSVs, demonstrates the diverse origins of the L-binding X domain of P. Our study links varying structures of nsNSV polymerase complexes with genome replication and transcription and points to a sliding model for polymerase complexes to advance along the RNA templates.
Asunto(s)
Microscopía por Crioelectrón , Virus de la Parotiditis , Proteínas Virales , Virus de la Parotiditis/genética , Virus de la Parotiditis/ultraestructura , Virus de la Parotiditis/metabolismo , Proteínas Virales/metabolismo , Proteínas Virales/ultraestructura , Proteínas Virales/química , Proteínas Virales/genética , Modelos Moleculares , ARN Viral/metabolismo , ARN Viral/ultraestructura , ARN Viral/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/ultraestructura , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Dominios Proteicos , Fosfoproteínas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/ultraestructura , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/ultraestructura , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Replicación Viral , Transcripción Genética , Conformación ProteicaRESUMEN
White spot syndrome virus (WSSV) is one of the largest DNA viruses and the major pathogen responsible for white spot syndrome in crustaceans. The WSSV capsid is critical for genome encapsulation and ejection and exhibits the rod-shaped and oval-shaped structures during the viral life cycle. However, the detailed architecture of the capsid and the structural transition mechanism remain unclear. Here, using cryo-electron microscopy (cryo-EM), we obtained a cryo-EM model of the rod-shaped WSSV capsid and were able to characterize its ring-stacked assembly mechanism. Furthermore, we identified an oval-shaped WSSV capsid from intact WSSV virions and analyzed the structural transition mechanism from the oval-shaped to rod-shaped capsids induced by high salinity. These transitions, which decrease internal capsid pressure, always accompany DNA release and mostly eliminate the infection of the host cells. Our results demonstrate an unusual assembly mechanism of the WSSV capsid and offer structural insights into the pressure-driven genome release.
Asunto(s)
Cápside , Virus del Síndrome de la Mancha Blanca 1 , Cápside/química , Microscopía por Crioelectrón , Proteínas de la Cápside/químicaRESUMEN
In the periplasm of Escherichia coli, DegP (also known as HtrA), which has both chaperone-like and proteolytic activities, prevents the accumulation of toxic misfolded and unfolded polypeptides. In solution, upon binding to denatured proteins, DegP forms large cage-like structures. Here, we show that DegP forms a range of bowl-shaped structures, independent of substrate proteins, each with a 4-, 5-, or 6-fold symmetry and all with a DegP trimer as the structural unit, on lipid membranes. These membrane-bound DegP assemblies have the capacity to recruit and process substrates in the bowl chamber, and they exhibit higher proteolytic and lower chaperone-like activities than DegP in solution. Our findings imply that DegP might regulate its dual roles during protein quality control, depending on its assembly state in the narrow bacterial envelope.
Asunto(s)
Membrana Celular/enzimología , Escherichia coli/enzimología , Proteínas de Choque Térmico/química , Proteínas Periplasmáticas/química , Serina Endopeptidasas/química , Membrana Celular/ultraestructura , Escherichia coli/citología , Escherichia coli/ultraestructura , Proteínas de Choque Térmico/ultraestructura , Lípidos/química , Chaperonas Moleculares/metabolismo , Proteínas Periplasmáticas/ultraestructura , Procesamiento Proteico-Postraduccional , Estructura Cuaternaria de Proteína , Serina Endopeptidasas/ultraestructura , Especificidad por SustratoRESUMEN
All paramyxoviruses, which include the mumps virus, measles virus, Nipah virus, Newcastle disease virus, and Sendai virus, have non-segmented single-stranded negative-sense RNA genomes. These RNA genomes are enwrapped throughout the viral life cycle by nucleoproteins, forming helical nucleocapsids. In addition to these helical structures, recombinant paramyxovirus nucleocapsids may occur in other assembly forms such as rings, clam-shaped structures, and double-headed nucleocapsids; the latter two are composed of two single-stranded helices packed in a back-to-back pattern. In all of these assemblies, the neighboring nucleoprotein protomers adopt the same domain-swapping mode via the N-terminal arm, C-terminal arm, and recently disclosed N-hole. An intrinsically disordered region in the C-terminal domain of the nucleoproteins, called the N-tail, plays an unexpected role in regulating the transition among the different assembly forms that occurs with other viral proteins, especially phosphoprotein. These structures, together with the helical nucleocapsids, significantly enrich the structural diversity of the paramyxovirus nucleocapsids and help explain the functions of these diverse assemblies, including RNA genome protection, transcription, and replication, as well as encapsulation.
Asunto(s)
Proteínas de la Nucleocápside/química , Nucleocápside/química , Paramyxovirinae/química , Modelos Moleculares , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , Paramyxovirinae/clasificación , Paramyxovirinae/genética , Conformación Proteica , Dominios Proteicos , Estructura Cuaternaria de Proteína , Subunidades de Proteína/químicaRESUMEN
Mumps virus (MuV) is a highly contagious human pathogen and frequently causes worldwide outbreaks despite available vaccines. Similar to other mononegaviruses such as Ebola and rabies, MuV uses a single-stranded negative-sense RNA as its genome, which is enwrapped by viral nucleoproteins into the helical nucleocapsid. The nucleocapsid acts as a scaffold for genome condensation and as a template for RNA replication and transcription. Conformational changes in the MuV nucleocapsid are required to switch between different activities, but the underlying mechanism remains elusive due to the absence of high-resolution structures. Here, we report two MuV nucleoprotein-RNA rings with 13 and 14 protomers, one stacked-ring filament and two nucleocapsids with distinct helical pitches, in dense and hyperdense states, at near-atomic resolutions using cryo-electron microscopy. Structural analysis of these in vitro assemblies indicates that the C-terminal tail of MuV nucleoprotein likely regulates the assembly of helical nucleocapsids, and the C-terminal arm may be relevant for the transition between the dense and hyperdense states of helical nucleocapsids. Our results provide the molecular mechanism for structural plasticity among different MuV nucleocapsids and create a possible link between structural plasticity and genome condensation.
Asunto(s)
Microscopía por Crioelectrón/métodos , Virus de la Parotiditis/metabolismo , Nucleocápside/ultraestructura , Nucleoproteínas/ultraestructura , Proteínas Virales/ultraestructura , Virión/metabolismo , Humanos , Modelos Moleculares , Virus de la Parotiditis/genética , Conformación de Ácido Nucleico , Nucleocápside/química , Nucleoproteínas/química , Conformación Proteica , ARN Viral/química , ARN Viral/genética , ARN Viral/metabolismo , Proteínas Virales/química , Virión/genéticaRESUMEN
Paramyxoviruses, including the mumps virus, measles virus, Nipah virus and Sendai virus (SeV), have non-segmented single-stranded negative-sense RNA genomes which are encapsidated by nucleoproteins into helical nucleocapsids. Here, we reported a double-headed SeV nucleocapsid assembled in a tail-to-tail manner, and resolved its helical stems and clam-shaped joint at the respective resolutions of 2.9 and 3.9 Å, via cryo-electron microscopy. Our structures offer important insights into the mechanism of the helical polymerization, in particular via an unnoticed exchange of a N-terminal hole formed by three loops of nucleoproteins, and unveil the clam-shaped joint in a hyper-closed state for nucleocapsid dimerization. Direct visualization of the loop from the disordered C-terminal tail provides structural evidence that C-terminal tail is correlated to the curvature of nucleocapsid and links nucleocapsid condensation and genome replication and transcription with different assembly forms.
Asunto(s)
Nucleocápside/ultraestructura , Virus Sendai/ultraestructura , Microscopía por Crioelectrón , Nucleoproteínas/química , Proteínas Virales/químicaRESUMEN
The ocean harbors a variety of bacteria that contain huge protease resources and offer a great potential for industrial and biotechnological applications. Here, we isolated a protease-secreting bacterium Vibrio pomeroyi strain 12613 from Atlantic seawater and purified a protease VP9 from strain 12613. VP9 was identified as a metalloprotease of the M4 family. VP9 could hydrolyze casein and gelatin but not elastin and collagen. With gelatin as the substrate, VP9 showed the highest activity at 40°C and pH 6.0-8.0. It was stable at temperatures of 50°C and less and in the range of pH 5.0-11.0. VP9 also had good tolerance to NaCl, non-ionic detergents, and organic solvent methanol. Unlike other M4 metalloproteases, VP9 has distinct collagen-swelling ability, and its collagen-swelling effect was concentration dependent. The relative expansion volume of collagen increased by approximately eightfold after treatment with 10 µM VP9 at 37°C for 12 h. The collagen-swelling mechanism of VP9 on bovine-insoluble type I collagen was further studied. Atomic force microscopy observation and biochemical analyses showed that VP9 can degrade proteoglycans in collagen fibers, resulting in the release of collagen fibrils from collagen fibers and the swelling of the latter. In addition, VP9 can degrade glycoproteins, a non-collagenous constituent interacting with collagen in the skin. The characteristics of VP9, such as sufficient specificity toward proteoglycans and glycoproteins but no activity toward collagen, suggest its promising potential in the unhairing and fiber-opening processing in leather industry.
RESUMEN
Non-segmented negative-strand RNA viruses, such as measles, ebola and Newcastle disease viruses (NDV), encapsidate viral genomic RNAs into helical nucleocapsids, which serve as the template for viral replication and transcription. Here, the clam-shaped nucleocapsid structure, where the NDV viral genome is sequestered, was determined at 4.8 Å resolution by cryo-electron microscopy. The clam-shaped structure is composed of two single-turn spirals packed in a back-to-back mode. This tightly packed structure functions as a seed for the assembly of a nucleocapsid from both directions, facilitating the growth of double-headed filaments with two separate RNA strings inside. Disruption of this structure by mutations in its loop interface yielded a single-headed unfunctional filament.
Asunto(s)
Virus de la Enfermedad de Newcastle/fisiología , Virus de la Enfermedad de Newcastle/ultraestructura , Nucleocápside/fisiología , Nucleocápside/ultraestructura , Ensamble de Virus , Microscopía por Crioelectrón , Nucleoproteínas/metabolismo , Unión Proteica , ARN Viral/metabolismoRESUMEN
The lentiviruses HIV and simian immunodeficiency virus (SIV) subvert intracellular membrane traffic as part of their replication cycle. The lentiviral Nef protein helps viruses evade innate and adaptive immune defenses by hijacking the adaptor protein 1 (AP-1) and AP-2 clathrin adaptors. We found that HIV-1 Nef and the guanosine triphosphatase Arf1 induced trimerization and activation of AP-1. Here we report the cryo-electron microscopy structures of the Nef- and Arf1-bound AP-1 trimer in the active and inactive states. A central nucleus of three Arf1 molecules organizes the trimers. We combined the open trimer with a known dimer structure and thus predicted a hexagonal assembly with inner and outer faces that bind the membranes and clathrin, respectively. Hexagons were directly visualized and the model validated by reconstituting clathrin cage assembly. Arf1 and Nef thus play interconnected roles in allosteric activation, cargo recruitment, and coat assembly, revealing an unexpectedly intricate organization of the inner AP-1 layer of the clathrin coat.
Asunto(s)
Factor 1 de Ribosilacion-ADP/química , Complejo 1 de Proteína Adaptadora/química , Antígenos CD/química , Vesículas Cubiertas por Clatrina/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/química , Clatrina/química , Clatrina/ultraestructura , Microscopía por Crioelectrón , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Proteínas Ligadas a GPI/química , Humanos , Conformación Proteica , Multimerización de Proteína , Estabilidad Proteica , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/ultraestructura , Red trans-Golgi/metabolismoRESUMEN
In a recent issue of Cell, Chiaruttini et al. (2015) reveal the mechanical properties of the mysterious spiral filaments formed by the yeast ESCRT-III protein Snf7. The spirals are shown to be springs whose bending drives membrane deformation and perhaps membrane scission.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Complejos de Clasificación Endosomal Requeridos para el Transporte/ultraestructura , Membrana Dobles de Lípidos/química , Modelos Moleculares , Levaduras/metabolismoRESUMEN
Sea ice is one of the most frigid environments for marine microbes. In contrast to other ocean ecosystems, microbes in permanent sea ice are space confined and subject to many extreme conditions, which change on a seasonal basis. How these microbial communities are regulated to survive the extreme sea ice environment is largely unknown. Here, we show that filamentous phages regulate the host bacterial community to improve survival of the host in permanent Arctic sea ice. We isolated a filamentous phage, f327, from an Arctic sea ice Pseudoalteromonas strain, and we demonstrated that this type of phage is widely distributed in Arctic sea ice. Growth experiments and transcriptome analysis indicated that this phage decreases the host growth rate, cell density and tolerance to NaCl and H2O2, but enhances its motility and chemotaxis. Our results suggest that the presence of the filamentous phage may be beneficial for survival of the host community in sea ice in winter, which is characterized by polar night, nutrient deficiency and high salinity, and that the filamentous phage may help avoid over blooming of the host in sea ice in summer, which is characterized by polar day, rich nutrient availability, intense radiation and high concentration of H2O2. Thus, while they cannot kill the host cells by lysing them, filamentous phages confer properties advantageous to host survival in the Arctic sea ice environment. Our study provides a foremost insight into the ecological role of filamentous phages in the Arctic sea ice ecosystem.
Asunto(s)
Bacteriófagos/fisiología , Cubierta de Hielo/microbiología , Pseudoalteromonas/virología , Agua de Mar/microbiología , Regiones Árticas , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Ecosistema , Peróxido de Hidrógeno/metabolismo , Pseudoalteromonas/crecimiento & desarrollo , Pseudoalteromonas/metabolismo , Estaciones del Año , Cloruro de Sodio/metabolismoRESUMEN
The scission of biological membranes is facilitated by a variety of protein complexes that bind and manipulate lipid bilayers. ESCRT-III (endosomal sorting complex required for transport III) filaments mediate membrane scission during the ostensibly disparate processes of multivesicular endosome biogenesis, cytokinesis, and retroviral budding. However, mechanisms by which ESCRT-III subunits assemble into a polymer remain unknown. Using cryogenic electron microscopy (cryo-EM), we found that the full-length ESCRT-III subunit Vps32/CHMP4B spontaneously forms single-stranded spiral filaments. The resolution afforded by two-dimensional cryo-EM combined with molecular dynamics simulations revealed that individual Vps32/CHMP4B monomers within a filament are flexible and able to accommodate a range of bending angles. In contrast, the interface between monomers is stable and refractory to changes in conformation. We additionally found that the carboxyl terminus of Vps32/CHMP4B plays a key role in restricting the lateral association of filaments. Our findings highlight new mechanisms by which ESCRT-III filaments assemble to generate a unique polymer capable of membrane remodeling in multiple cellular contexts.
Asunto(s)
Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Complejos de Clasificación Endosomal Requeridos para el Transporte/ultraestructura , Subunidades de Proteína/metabolismo , Animales , Cristalografía por Rayos X , Microscopía Electrónica , Simulación de Dinámica Molecular , Polímeros/metabolismo , Conformación Proteica , Multimerización de Proteína , Subunidades de Proteína/biosíntesisRESUMEN
This protocol describes an assay that uses suspended nanomembranes called nanodiscs to analyze fusion events. A nanodisc is a lipid bilayer wrapped by membrane scaffold proteins. Fluorescent lipids and a protein that is part of a fusion machinery, VAMP2 in the example detailed herein, are included in the nanodiscs. Upon fusion of a nanodisc with a nonfluorescent liposome containing cognate proteins (for instance, the VAMP2 cognate syntaxin1/SNAP-25 complex), the fluorescent lipids are dispersed in the liposome and the increase in fluorescence, initially quenched in the nanodisc, is monitored on a plate reader. Because the scaffold proteins restrain pore expansion, the fusion pore eventually reseals. A reducing agent, such as dithionite, which can quench the fluorescence of accessible lipids, can then be used to determine the number of fusion events. A fluorescence-based approach can also be used to monitor the release of encapsulated cargo. From data on the total cargo release and the number of the much faster lipid-mixing events, the researcher may determine the amount of cargo released per fusion event. This assay requires 3 d for preparation and 4 h for data acquisition and analysis.
Asunto(s)
Transporte Biológico , Nanoestructuras , Proteína 25 Asociada a Sinaptosomas/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Liposomas/química , Liposomas/metabolismo , Fusión de Membrana , Microscopía Fluorescente , Modelos BiológicosRESUMEN
The microtubule (MT) and actin cytoskeletons drive many essential cellular processes, yet fairly little is known about how their functions are coordinated. One factor that mediates important cross talk between these two systems is WHAMM, a Golgi-associated protein that utilizes MT binding and actin nucleation activities to promote membrane tubulation during intracellular transport. Using cryoelectron microscopy and other biophysical and biochemical approaches, we unveil the underlying mechanisms for how these activities are coordinated. We find that WHAMM bound to the outer surface of MT protofilaments via a novel interaction between its central coiled-coil region and tubulin heterodimers. Upon the assembly of WHAMM onto MTs, its N-terminal membrane-binding domain was exposed at the MT periphery, where it can recruit vesicles and remodel them into tubular structures. In contrast, MT binding masked the C-terminal portion of WHAMM and prevented it from promoting actin nucleation. These results give rise to a model whereby distinct MT-bound and actin-nucleating populations of WHAMM collaborate during membrane tubulation.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Aparato de Golgi/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SpodopteraRESUMEN
Neurotransmitters are released through nascent fusion pores, which ordinarily dilate after bilayer fusion, preventing consistent biochemical studies. We used lipid bilayer nanodiscs as fusion partners; their rigid protein framework prevents dilation and reveals properties of the fusion pore induced by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor). We found that although only one SNARE per nanodisc is required for maximum rates of bilayer fusion, efficient release of content on the physiologically relevant time scale of synaptic transmission apparently requires three or more SNARE complexes (SNAREpins) and the native transmembrane domain of vesicle-associated membrane protein 2 (VAMP2). We suggest that several SNAREpins simultaneously zippering their SNARE transmembrane helices within the freshly fused bilayers provide a radial force that prevents the nascent pore from resealing during synchronous neurotransmitter release.