RESUMO
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.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/patologia , Neoplasias Encefálicas/patologia , Tomografia com Microscopia Eletrônica , Encéfalo/patologia , Mitocôndrias/patologiaRESUMO
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.
Assuntos
Microscopia Crioeletrônica , Complexos Endossomais de Distribuição Requeridos para Transporte , Proteínas de Saccharomyces cerevisiae , Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestruturaRESUMO
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.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Pele/metabolismo , Células-Tronco/metabolismo , Cicatrização , Animais , Movimento Celular , Células Cultivadas , Glicogênio Sintase Quinase 3 beta , Camundongos , Camundongos Transgênicos , Fosforilação , Estrutura Terciária de Proteína , Pele/citologia , Células-Tronco/citologiaRESUMO
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.
Assuntos
Conformação Proteica , Proteínas Ribossômicas/ultraestrutura , Ribossomos/fisiologia , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , RNA Ribossômico/metabolismo , RNA Ribossômico/ultraestrutura , Proteínas Ribossômicas/metabolismo , Ribossomos/ultraestruturaRESUMO
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.
Assuntos
Carbono-Nitrogênio Ligases/metabolismo , Glutamatos/metabolismo , Trifosfato de Adenosina/metabolismo , Catálise , Escherichia coli/metabolismo , Ácido Glutâmico/metabolismo , Magnésio/metabolismo , Metilaminas/metabolismo , Microscopia Eletrônica , Rhodovulum/metabolismoRESUMO
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.
Assuntos
Microscopia Crioeletrônica , Vírus da Caxumba , Proteínas Virais , Vírus da Caxumba/genética , Vírus da Caxumba/ultraestrutura , Vírus da Caxumba/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais/ultraestrutura , Proteínas Virais/química , Proteínas Virais/genética , Modelos Moleculares , RNA Viral/metabolismo , RNA Viral/ultraestrutura , RNA Viral/genética , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/ultraestrutura , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Domínios Proteicos , Fosfoproteínas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/ultraestrutura , RNA Polimerase Dependente de RNA/metabolismo , RNA Polimerase Dependente de RNA/ultraestrutura , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , Replicação Viral , Transcrição Gênica , Conformação ProteicaRESUMO
Microsporidia are obligate intracellular parasites that infect a wide variety of hosts including humans. Microsporidian spores possess a unique, highly specialized invasion apparatus involving the polar filament, polaroplast, and posterior vacuole. During spore germination, the polar filament is discharged out of the spore forming a hollow polar tube that transports the sporoplasm components including the nucleus into the host cell. Due to the complicated topological changes occurring in this process, the details of sporoplasm formation are not clear. Our data suggest that the limiting membrane of the nascent sporoplasm is formed by the polaroplast after microsporidian germination. Using electron microscopy and 1,1'-dioctadecyl-3,3,3',3' tetramethyl indocarbocyanine perchlorate staining, we describe that a large number of vesicles, nucleus, and other cytoplasm contents were transported out via the polar tube during spore germination, while the posterior vacuole and plasma membrane finally remained in the empty spore coat. Two Nosema bombycis sporoplasm surface proteins (NbTMP1 and NoboABCG1.1) were also found to localize in the region of the polaroplast and posterior vacuole in mature spores and in the discharged polar tube, which suggested that the polaroplast during transport through the polar tube became the limiting membrane of the sporoplasm. The analysis results of Golgi-tracker green and Golgi marker protein syntaxin 6 were also consistent with the model of the transported polaroplast derived from Golgi transformed into the nascent sporoplasm membrane.IMPORTANCEMicrosporidia, which are obligate intracellular pathogenic organisms, cause huge economic losses in agriculture and even threaten human health. The key to successful infection by the microsporidia is their unique invasion apparatus which includes the polar filament, polaroplast, and posterior vacuole. When the mature spore is activated to geminate, the polar filament uncoils and undergoes a rapid transition into the hollow polar tube that transports the sporoplasm components including the microsporidian nucleus into host cells. Details of the structural difference between the polar filament and polar tube, the process of cargo transport in extruded polar tube, and the formation of the sporoplasm membrane are still poorly understood. Herein, we verify that the polar filament evaginates to form the polar tube, which serves as a conduit for transporting the nucleus and other sporoplasm components. Furthermore, our results indicate that the transported polaroplast transforms into the sporoplasm membrane during spore germination. Our study provides new insights into the cargo transportation process of the polar tube and origin of the sporoplasm membrane, which provide important clarification of the microsporidian infection mechanism.
Assuntos
Microsporídios , Humanos , Esporos Fúngicos , Citoplasma , Microscopia Eletrônica , Membrana Celular , BandagensRESUMO
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.
Assuntos
Capsídeo , Vírus da Síndrome da Mancha Branca 1 , Capsídeo/química , Microscopia Crioeletrônica , Proteínas do Capsídeo/químicaRESUMO
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.
Assuntos
Membrana Celular/enzimologia , Escherichia coli/enzimologia , Proteínas de Choque Térmico/química , Proteínas Periplásmicas/química , Serina Endopeptidases/química , Membrana Celular/ultraestrutura , Escherichia coli/citologia , Escherichia coli/ultraestrutura , Proteínas de Choque Térmico/ultraestrutura , Lipídeos/química , Chaperonas Moleculares/metabolismo , Proteínas Periplásmicas/ultraestrutura , Processamento de Proteína Pós-Traducional , Estrutura Quaternária de Proteína , Serina Endopeptidases/ultraestrutura , Especificidade por SubstratoRESUMO
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.
Assuntos
Proteínas do Nucleocapsídeo/química , Nucleocapsídeo/química , Paramyxovirinae/química , Modelos Moleculares , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Paramyxovirinae/classificação , Paramyxovirinae/genética , Conformação Proteica , Domínios Proteicos , Estrutura Quaternária de Proteína , Subunidades Proteicas/químicaRESUMO
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.
Assuntos
Microscopia Crioeletrônica/métodos , Vírus da Caxumba/metabolismo , Nucleocapsídeo/ultraestrutura , Nucleoproteínas/ultraestrutura , Proteínas Virais/ultraestrutura , Vírion/metabolismo , Humanos , Modelos Moleculares , Vírus da Caxumba/genética , Conformação de Ácido Nucleico , Nucleocapsídeo/química , Nucleoproteínas/química , Conformação Proteica , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais/química , Vírion/genéticaRESUMO
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.
Assuntos
Nucleocapsídeo/ultraestrutura , Vírus Sendai/ultraestrutura , Microscopia Crioeletrônica , Nucleoproteínas/química , Proteínas Virais/químicaRESUMO
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.
RESUMO
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.
Assuntos
Vírus da Doença de Newcastle/fisiologia , Vírus da Doença de Newcastle/ultraestrutura , Nucleocapsídeo/fisiologia , Nucleocapsídeo/ultraestrutura , Montagem de Vírus , Microscopia Crioeletrônica , Nucleoproteínas/metabolismo , Ligação Proteica , RNA Viral/metabolismoRESUMO
The AAA+ ATPase Vps4 disassembles ESCRT-III and is essential for HIV-1 budding and other pathways. Vps4 is a paradigmatic member of a class of hexameric AAA+ ATPases that disassemble protein complexes without degradation. To distinguish between local displacement versus global unfolding mechanisms for complex disassembly, we carried out hydrogen/deuterium exchange during Saccharomyces cerevisiae Vps4 disassembly of a chimeric Vps24-2 ESCRT-III filament. EX1 exchange behavior shows that Vps4 completely unfolds ESCRT-III substrates on a time scale consistent with the disassembly reaction. The established unfoldase ClpX showed the same pattern, thus demonstrating a common unfolding mechanism. Vps4 hexamers containing a single cysteine residue in the pore loops were cross-linked to ESCRT-III subunits containing unique cysteines within the folded core domain. These data support a mechanism in which Vps4 disassembles its substrates by completely unfolding them and threading them through the central pore.
Assuntos
Adenosina Trifosfatases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Dobramento de Proteína , Transporte ProteicoRESUMO
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.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Bicamadas Lipídicas/química , Modelos Moleculares , Leveduras/metabolismoRESUMO
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.
Assuntos
Fator 1 de Ribosilação do ADP/química , Complexo 1 de Proteínas Adaptadoras/química , Antígenos CD/química , Vesículas Revestidas por Clatrina/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/química , Clatrina/química , Clatrina/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Proteínas Ligadas por GPI/química , Humanos , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Produtos do Gene nef do Vírus da Imunodeficiência Humana/ultraestrutura , Rede trans-Golgi/metabolismoRESUMO
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.
Assuntos
Bacteriófagos/fisiologia , Camada de Gelo/microbiologia , Pseudoalteromonas/virologia , Água do Mar/microbiologia , Regiões Árticas , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Ecossistema , Peróxido de Hidrogênio/metabolismo , Pseudoalteromonas/crescimento & desenvolvimento , Pseudoalteromonas/metabolismo , Estações do Ano , Cloreto de Sódio/metabolismoRESUMO
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.
Assuntos
Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/biossíntese , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Subunidades Proteicas/metabolismo , Animais , Cristalografia por Raios X , Microscopia Eletrônica , Simulação de Dinâmica Molecular , Polímeros/metabolismo , Conformação Proteica , Multimerização Proteica , Subunidades Proteicas/biossínteseRESUMO
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.