Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 18.388
Filtrar
1.
Nat Commun ; 11(1): 4514, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908139

RESUMO

The velocity of nerve conduction is moderately enhanced by larger axonal diameters and potently sped up by myelination of axons. Myelination thus allows rapid impulse propagation with reduced axonal diameters; however, no myelin-dependent mechanism has been reported that restricts radial growth of axons. By label-free proteomics, STED-microscopy and cryo-immuno electron-microscopy we here identify CMTM6 (chemokine-like factor-like MARVEL-transmembrane domain-containing family member-6) as a myelin protein specifically localized to the Schwann cell membrane exposed to the axon. We find that disruption of Cmtm6-expression in Schwann cells causes a substantial increase of axonal diameters but does not impair myelin biogenesis, radial sorting or integrity of axons. Increased axonal diameters correlate with accelerated sensory nerve conduction and sensory responses and perturbed motor performance. These data show that Schwann cells utilize CMTM6 to restrict the radial growth of axons, which optimizes nerve function.


Assuntos
Axônios/metabolismo , Proteínas da Mielina/metabolismo , Nervos Periféricos/citologia , Células de Schwann/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Axônios/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Masculino , Camundongos , Camundongos Knockout , Bainha de Mielina/metabolismo , Condução Nervosa , Nervos Periféricos/metabolismo , Nervos Periféricos/ultraestrutura , Proteômica , Células de Schwann/citologia , Células de Schwann/ultraestrutura , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/ultraestrutura
2.
PLoS Pathog ; 16(9): e1008883, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32956394

RESUMO

Infection by human parainfluenza viruses (HPIVs) causes widespread lower respiratory diseases, including croup, bronchiolitis, and pneumonia, and there are no vaccines or effective treatments for these viruses. HPIV3 is a member of the Respirovirus species of the Paramyxoviridae family. These viruses are pleomorphic, enveloped viruses with genomes composed of single-stranded negative-sense RNA. During viral entry, the first step of infection, the viral fusion complex, comprised of the receptor-binding glycoprotein hemagglutinin-neuraminidase (HN) and the fusion glycoprotein (F), mediates fusion upon receptor binding. The HPIV3 transmembrane protein HN, like the receptor-binding proteins of other related viruses that enter host cells using membrane fusion, binds to a receptor molecule on the host cell plasma membrane, which triggers the F glycoprotein to undergo major conformational rearrangements, promoting viral entry. Subsequent fusion of the viral and host membranes allows delivery of the viral genetic material into the host cell. The intermediate states in viral entry are transient and thermodynamically unstable, making it impossible to understand these transitions using standard methods, yet understanding these transition states is important for expanding our knowledge of the viral entry process. In this study, we use cryo-electron tomography (cryo-ET) to dissect the stepwise process by which the receptor-binding protein triggers F-mediated fusion, when forming a complex with receptor-bearing membranes. Using an on-grid antibody capture method that facilitates examination of fresh, biologically active strains of virus directly from supernatant fluids and a series of biological tools that permit the capture of intermediate states in the fusion process, we visualize the series of events that occur when a pristine, authentic viral particle interacts with target receptors and proceeds from the viral entry steps of receptor engagement to membrane fusion.


Assuntos
Membrana Celular/metabolismo , Proteína HN/metabolismo , Vírus da Parainfluenza 3 Humana/metabolismo , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus , Animais , Membrana Celular/ultraestrutura , Chlorocebus aethiops , Humanos , Vírus da Parainfluenza 3 Humana/ultraestrutura , Células Vero
3.
Proc Natl Acad Sci U S A ; 117(33): 19713-19719, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32759217

RESUMO

Images of micrometer-scale domains in lipid bilayers have provided the gold standard of model-free evidence to understand the domains' shapes, sizes, and distributions. Corresponding techniques to directly and quantitatively assess smaller (nanoscale and submicron) liquid domains have been limited. Researchers commonly seek to correlate activities of membrane proteins with attributes of the domains in which they reside; doing so hinges on identification and characterization of membrane domains. Although some features of membrane domains can be probed by indirect methods, these methods are often constrained by the limitation that data must be analyzed in the context of models that require multiple assumptions or parameters. Here, we address this challenge by developing and testing two methods of identifying submicron domains in biomimetic membranes. Both methods leverage cryo-electron tomograms of ternary membranes under vitrified, hydrated conditions. The first method is optimized for probe-free applications: Domains are directly distinguished from the surrounding membrane by their thickness. This technique quantitatively and accurately measures area fractions of domains, in excellent agreement with known phase diagrams. The second method is optimized for applications in which a single label is deployed for imaging membranes by both high-resolution cryo-electron tomography and diffraction-limited optical microscopy. For this method, we test a panel of probes, find that a trimeric mCherry label performs best, and specify criteria for developing future high-performance, dual-use probes. These developments have led to direct and quantitative imaging of submicron membrane domains in vitrified, hydrated vesicles.


Assuntos
Membrana Celular/ultraestrutura , Tomografia com Microscopia Eletrônica/métodos , Bicamadas Lipídicas/química , Membrana Celular/química , Microdomínios da Membrana/química , Microdomínios da Membrana/ultraestrutura
4.
Proc Natl Acad Sci U S A ; 117(31): 18497-18503, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32680969

RESUMO

Membrane proteins (MPs) used to be the most difficult targets for structural biology when X-ray crystallography was the mainstream approach. With the resolution revolution of single-particle electron cryo-microscopy (cryo-EM), rapid progress has been made for structural elucidation of isolated MPs. The next challenge is to preserve the electrochemical gradients and membrane curvature for a comprehensive structural elucidation of MPs that rely on these chemical and physical properties for their biological functions. Toward this goal, here we present a convenient workflow for cryo-EM structural analysis of MPs embedded in liposomes, using the well-characterized AcrB as a prototype. Combining optimized proteoliposome isolation, cryo-sample preparation on graphene grids, and an efficient particle selection strategy, the three-dimensional (3D) reconstruction of AcrB embedded in liposomes was obtained at 3.9 Å resolution. The conformation of the homotrimeric AcrB remains the same when the surrounding membranes display different curvatures. Our approach, which can be widely applied to cryo-EM analysis of MPs with distinctive soluble domains, lays out the foundation for cryo-EM analysis of integral or peripheral MPs whose functions are affected by transmembrane electrochemical gradients or/and membrane curvatures.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lipossomos/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Lipossomos/metabolismo , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Proteínas Associadas à Resistência a Múltiplos Medicamentos/ultraestrutura , Conformação Proteica
5.
PLoS One ; 15(7): e0236373, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32702063

RESUMO

The diagnosis of patients with malignancies relies on the results of a clinical cytological examination. To enhance the diagnostic qualities of cytological examinations, it is important to have a detailed analysis of the cell's characteristics. There is, therefore, a need for developing a new auxiliary method for cytological diagnosis. In this study, we focused on studying the charge of the cell membrane surface of fixed cells, which is one of important cell's characteristics. Although fixed cells lose membrane potential which is observed in living cells owing to ion dynamics, we hypothesized that fixed cells still have a cell membrane surface charge due to cell membrane components and structure. We used 5 cell lines in this study (ARO, C32TG, RT4, TK, UM-UC-14). After fixation with CytoRich Red, we measured the cell membrane surface charge of fixed cells in solution using zeta potential measurements and fixed cells on glass slides, visualizing it using antibody-labeled beads and positively-charged beads. Furthermore, we measured the cell membrane surface charge of fixed cells under different conditions, such as different solution of fixative, ion concentration, pH, and pepsin treatments. The zeta potential measurements and visualization using the beads indicated that the cell membrane surface of fixed cells was negatively charged, and also that the charge varied among fixed cells. The charge state was affected by the different treatments. Moreover, the number of cell-bound beads was small in interphase, anaphase, and apoptotic cells. We concluded that the negative cell membrane surface charge was influenced by the three-dimensional structure of proteins as well as the different types of amino acids and lipids on the cell membrane. Thus, cell surface charge visualization can be applied as a new auxiliary method for clinical cytological diagnosis. This is the first systematic report of the cell membrane surface charge of fixed cells.


Assuntos
Linhagem Celular/ultraestrutura , Membrana Celular/ultraestrutura , Células Cultivadas/ultraestrutura , Citodiagnóstico , Anáfase/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Fixadores/farmacologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Pepsina A/farmacologia , Propriedades de Superfície
6.
PLoS One ; 15(7): e0236842, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730363

RESUMO

Cyanobacteria can form biofilms in nature, which have ecological roles and high potential for practical applications. In order to study them we need biofilm models that contain healthy cells and can withstand physical manipulations needed for structural studies. At present, combined studies on the structural and physiological features of axenic cyanobacterial biofilms are limited, mostly due to the shortage of suitable model systems. Here, we present a simple method to establish biofilms using the cyanobacterium Synechocystis PCC6803 under standard laboratory conditions to be directly used for photosynthetic activity measurements and scanning electron microscopy (SEM). We found that glass microfiber filters (GMF) with somewhat coarse surface features provided a suitable skeleton to form Synechocystis PCC6803 biofilms. Being very fragile, untreated GMFs were unable to withstand the processing steps needed for SEM. Therefore, we used polyhydroxybutyrate coating to stabilize the filters. We found that up to five coats resulted in GMF stabilization and made possible to obtain high resolution SEM images of the structure of the surface-attached cells and the extensive exopolysaccharide and pili network, which are essential features of biofilm formation. By using pulse-amplitude modulated variable chlorophyll fluorescence imaging, it was also demonstrated that the biofilms contain photosynthetically active cells. Therefore, the Synechocystis PCC6803 biofilms formed on coated GMFs can be used for both structural and functional investigations. The model presented here is easy to replicate and has a potential for high-throughput studies.


Assuntos
Biofilmes/crescimento & desenvolvimento , Membrana Celular/metabolismo , Microscopia Eletrônica de Varredura/métodos , Polissacarídeos Bacterianos/metabolismo , Synechocystis/crescimento & desenvolvimento , Synechocystis/ultraestrutura , Membrana Celular/ultraestrutura , Polissacarídeos Bacterianos/ultraestrutura , Synechocystis/metabolismo
7.
Circ Arrhythm Electrophysiol ; 13(7): e008241, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32536203

RESUMO

BACKGROUND: Mutations in the gene encoding the cardiac voltage-gated sodium channel Nav1.5 cause various cardiac arrhythmias. This variety may arise from different determinants of Nav1.5 expression between cardiomyocyte domains. At the lateral membrane and T-tubules, Nav1.5 localization and function remain insufficiently characterized. METHODS: We used novel single-molecule localization microscopy and computational modeling to define nanoscale features of Nav1.5 localization and distribution at the lateral membrane, the lateral membrane groove, and T-tubules in cardiomyocytes from wild-type (N=3), dystrophin-deficient (mdx; N=3) mice, and mice expressing C-terminally truncated Nav1.5 (ΔSIV; N=3). We moreover assessed T-tubules sodium current by recording whole-cell sodium currents in control (N=5) and detubulated (N=5) wild-type cardiomyocytes. RESULTS: We show that Nav1.5 organizes as distinct clusters in the groove and T-tubules which density, distribution, and organization partially depend on SIV and dystrophin. We found that overall reduction in Nav1.5 expression in mdx and ΔSIV cells results in a nonuniform redistribution with Nav1.5 being specifically reduced at the groove of ΔSIV and increased in T-tubules of mdx cardiomyocytes. A T-tubules sodium current could, however, not be demonstrated. CONCLUSIONS: Nav1.5 mutations may site-specifically affect Nav1.5 localization and distribution at the lateral membrane and T-tubules, depending on site-specific interacting proteins. Future research efforts should elucidate the functional consequences of this redistribution.


Assuntos
Membrana Celular/metabolismo , Ativação do Canal Iônico , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Imagem Individual de Molécula , Animais , Membrana Celular/ultraestrutura , Simulação por Computador , Distrofina/genética , Distrofina/metabolismo , Potenciais da Membrana , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Modelos Cardiovasculares , Miócitos Cardíacos/ultraestrutura , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Transporte Proteico
8.
PLoS Genet ; 16(6): e1008890, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32579558

RESUMO

The Drosophila apical photoreceptor membrane is defined by the presence of two distinct morphological regions, the microvilli-based rhabdomere and the stalk membrane. The subdivision of the apical membrane contributes to the geometrical positioning and the stereotypical morphology of the rhabdomeres in compound eyes with open rhabdoms and neural superposition. Here we describe the characterization of the photoreceptor specific protein PIP82. We found that PIP82's subcellular localization demarcates the rhabdomeric portion of the apical membrane. We further demonstrate that PIP82 is a phosphorylation target of aPKC. PIP82 localization is modulated by phosphorylation, and in vivo, the loss of the aPKC/Crumbs complex results in an expansion of the PIP82 localization domain. The absence of PIP82 in photoreceptors leads to misshapped rhabdomeres as a result of misdirected cellular trafficking of rhabdomere proteins. Comparative analyses reveal that PIP82 originated de novo in the lineage leading to brachyceran Diptera, which is also characterized by the transition from fused to open rhabdoms. Taken together, these findings define a novel factor that delineates and maintains a specific apical membrane domain, and offers new insights into the functional organization and evolutionary history of the Drosophila retina.


Assuntos
Membrana Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Fotorreceptoras de Invertebrados/metabolismo , Retina/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Evolução Biológica , Diferenciação Celular/genética , Membrana Celular/genética , Membrana Celular/ultraestrutura , Polaridade Celular/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação com Perda de Função , Masculino , Microscopia Eletrônica de Transmissão , Fosforilação , Células Fotorreceptoras de Invertebrados/citologia , Células Fotorreceptoras de Invertebrados/ultraestrutura , Filogenia , Proteína Quinase C/metabolismo , Retina/citologia , Retina/ultraestrutura , Transcrição Genética
9.
Proc Natl Acad Sci U S A ; 117(27): 15684-15693, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571921

RESUMO

Mitochondria are known to play an essential role in photoreceptor function and survival that enables normal vision. Within photoreceptors, mitochondria are elongated and extend most of the inner-segment length, where they supply energy for protein synthesis and the phototransduction machinery in the outer segment, as well as acting as a calcium store. Here, we examined the arrangement of the mitochondria within the inner segment in detail using three-dimensional (3D) electron microscopy techniques and show they are tethered to the plasma membrane in a highly specialized arrangement. Remarkably, mitochondria and their cristae openings align with those of neighboring inner segments. The pathway by which photoreceptors meet their high energy demands is not fully understood. We propose this to be a mechanism to share metabolites and assist in maintaining homeostasis across the photoreceptor cell layer. In the extracellular space between photoreceptors, Müller glial processes were identified. Due to the often close proximity to the inner-segment mitochondria, they may, too, play a role in the inner-segment mitochondrial arrangement as well as metabolite shuttling. OPA1 is an important factor in mitochondrial homeostasis, including cristae remodeling; therefore, we examined the photoreceptors of a heterozygous Opa1 knockout mouse model. The cristae structure in the Opa1 +/- photoreceptors was not greatly affected, but the mitochondria were enlarged and had reduced alignment to neighboring inner-segment mitochondria. This indicates the importance of key regulators in maintaining this specialized photoreceptor mitochondrial arrangement.


Assuntos
GTP Fosfo-Hidrolases/genética , Mitocôndrias/genética , Membranas Mitocondriais/ultraestrutura , Visão Ocular/genética , Animais , Membrana Celular/genética , Membrana Celular/ultraestrutura , Células Ependimogliais/metabolismo , Células Ependimogliais/ultraestrutura , Humanos , Camundongos , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Células Fotorreceptoras/ultraestrutura , Visão Ocular/fisiologia
10.
PLoS One ; 15(6): e0234430, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32511274

RESUMO

Excess presence of the human epidermal growth factor receptor 2 (HER2) as well as of the focal adhesion protein complexes are associated with increased proliferation, migratory, and invasive behavior of cancer cells. A cross-regulation between HER2 and integrin signaling pathways has been found, but the exact mechanism remains elusive. Here, we investigated whether HER2 colocalizes with focal adhesion complexes on breast cancer cells overexpressing HER2. For this purpose, vinculin or talin green fluorescent protein (GFP) fusion proteins, both key constituents of focal adhesions, were expressed in breast cancer cells. HER2 was either extracellularly or intracellularly labeled with fluorescent quantum dots nanoparticles (QDs). The cell-substrate interface was analyzed at the location of the focal adhesions by means of total internal reflection fluorescent microscopy or correlative fluorescence- and scanning transmission electron microscopy. Expression of HER2 at the cell-substrate interface was only observed upon intracellular labeling, and was heterogeneous with both HER2-enriched and -low regions. In contrast to an expected enrichment of HER2 at focal adhesions, an anti-correlated expression pattern was observed for talin and HER2. Our findings suggest a spatial anti-correlation between HER2 and focal adhesion complexes for adherent cells.


Assuntos
Membrana Celular/metabolismo , Adesões Focais/metabolismo , Receptor ErbB-2/metabolismo , Análise Espacial , Adesão Celular , Linhagem Celular Tumoral , Membrana Celular/ultraestrutura , Adesões Focais/ultraestrutura , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia Eletrônica de Transmissão e Varredura , Microscopia de Fluorescência , Receptor ErbB-2/análise , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Talina/análise , Talina/genética , Talina/metabolismo , Vinculina/análise , Vinculina/genética , Vinculina/metabolismo
11.
Proc Natl Acad Sci U S A ; 117(25): 14209-14219, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513731

RESUMO

The physical dimensions of proteins and glycans on cell surfaces can critically affect cell function, for example, by preventing close contact between cells and limiting receptor accessibility. However, high-resolution measurements of molecular heights on native cell membranes have been difficult to obtain. Here we present a simple and rapid method that achieves nanometer height resolution by localizing fluorophores at the tip and base of cell surface molecules and determining their separation by radially averaging across many molecules. We use this method, which we call cell surface optical profilometry (CSOP), to quantify the height of key multidomain proteins on a model cell, as well as to capture average protein and glycan heights on native cell membranes. We show that average height of a protein is significantly smaller than its contour length, due to thermally driven bending and rotation on the membrane, and that height strongly depends on local surface and solution conditions. We find that average height increases with cell surface molecular crowding but decreases with solution crowding by solutes, both of which we confirm with molecular dynamics simulations. We also use experiments and simulations to determine the height of an epitope, based on the location of an antibody, which allows CSOP to profile various proteins and glycans on a native cell surface using antibodies and lectins. This versatile method for profiling cell surfaces has the potential to advance understanding of the molecular landscape of cells and the role of the molecular landscape in cell function.


Assuntos
Membrana Celular/química , Proteínas de Membrana/química , Polissacarídeos/química , Anticorpos , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Epitopos , Imunofluorescência , Células HEK293 , Humanos , Lectinas , Bicamadas Lipídicas , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Polissacarídeos/metabolismo , Domínios Proteicos
12.
PLoS One ; 15(6): e0233856, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32492073

RESUMO

The enzyme ß-glucosidase 2 (GBA2) is clinically relevant because it is targeted by the drug miglustat (Zavesca®) and because it is involved in inherited diseases. Mutations in the GBA2 gene are associated with two neurological diseases on the ataxia-spasticity spectrum, hereditary spastic paraplegia 46 (SPG46) and Marinesco-Sjögren-like syndrome (MSS). To establish how GBA2 mutations give rise to neurological pathology, we have begun to investigate mutant forms of GBA2 encoded by disease-associated GBA2 alleles. Previously, we found that five GBA2 missense mutants and five C-terminally truncated mutants lacked enzyme activity. Here we have examined the cellular locations of wild-type (WT) and mutant forms of GBA2 by confocal and electron microscopy, using transfected cells. Similar to GBA2-WT, the D594H and M510Vfs*17 GBA2 mutants were located at the plasma membrane, whereas the C-terminally truncated mutants terminating after amino acids 233 and 339 (GBA2-233 and -339) were present in the mitochondrial matrix, induced mitochondrial fragmentation and loss of mitochondrial transmembrane potential. Deletional mutagenesis indicated that residues 161-200 are critical for the mitochondrial fragmentation of GBA2-233 and -339. Considering that the mitochondrial fragmentation induced by GBA2-233 and -339 is consistently accompanied by their localization to the mitochondrial matrix, our deletional analysis raises the possibility that that GBA2 residues 161-200 harbor an internal targeting sequence for transport to the mitochondrial matrix. Altogether, our work provides new insights into the behaviour of GBA2-WT and disease-associated forms of GBA2.


Assuntos
Glucosilceramidase/metabolismo , Mitocôndrias/patologia , Alelos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Glucosilceramidase/genética , Humanos , Potencial da Membrana Mitocondrial , Microscopia Eletrônica , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Mutação , Paraplegia Espástica Hereditária/genética , Degenerações Espinocerebelares/genética
13.
Nat Commun ; 11(1): 2848, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503964

RESUMO

The natural antibiotic teixobactin kills pathogenic bacteria without detectable resistance. The difficult synthesis and unfavourable solubility of teixobactin require modifications, yet insufficient knowledge on its binding mode impedes the hunt for superior analogues. Thus far, teixobactins are assumed to kill bacteria by binding to cognate cell wall precursors (Lipid II and III). Here we present the binding mode of teixobactins in cellular membranes using solid-state NMR, microscopy, and affinity assays. We solve the structure of the complex formed by an improved teixobactin-analogue and Lipid II and reveal how teixobactins recognize a broad spectrum of targets. Unexpectedly, we find that teixobactins only weakly bind to Lipid II in cellular membranes, implying the direct interaction with cell wall precursors is not the sole killing mechanism. Our data suggest an additional mechanism affords the excellent activity of teixobactins, which can block the cell wall biosynthesis by capturing precursors in massive clusters on membranes.


Assuntos
Antibacterianos/farmacologia , Membrana Celular/metabolismo , Depsipeptídeos/farmacologia , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Membrana Celular/ultraestrutura , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Depsipeptídeos/química , Lipossomos/metabolismo , Espectroscopia de Ressonância Magnética , Microscopia de Fluorescência , Estrutura Molecular , Relação Estrutura-Atividade , Uridina Difosfato Ácido N-Acetilmurâmico/química , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismo
14.
Nat Commun ; 11(1): 2270, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385301

RESUMO

Mycobacterium tuberculosis is a global health problem in part as a result of extensive cytotoxicity caused by the infection. Here, we show how M. tuberculosis causes caspase-1/NLRP3/gasdermin D-mediated pyroptosis of human monocytes and macrophages. A type VII secretion system (ESX-1) mediated, contact-induced plasma membrane damage response occurs during phagocytosis of bacteria. Alternatively, this can occur from the cytosolic side of the plasma membrane after phagosomal rupture in infected macrophages. This damage causes K+ efflux and activation of NLRP3-dependent IL-1ß release and pyroptosis, facilitating the spread of bacteria to neighbouring cells. A dynamic interplay of pyroptosis with ESCRT-mediated plasma membrane repair also occurs. This dual plasma membrane damage seems to be a common mechanism for NLRP3 activators that function through lysosomal damage.


Assuntos
Membrana Celular/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose , Tuberculose/metabolismo , Tuberculose/patologia , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Catepsinas/metabolismo , Membrana Celular/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Inflamassomos/metabolismo , Inflamassomos/ultraestrutura , Mitocôndrias/metabolismo , Mycobacterium tuberculosis/metabolismo , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Células THP-1
15.
Nat Commun ; 11(1): 1516, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32471995

RESUMO

ESCRT-III proteins assemble into ubiquitous membrane-remodeling polymers during many cellular processes. Here we describe the structure of helical membrane tubes that are scaffolded by bundled ESCRT-III filaments. Cryo-ET reveals how the shape of the helical membrane tube arises from the assembly of two distinct bundles of helical filaments that have the same helical path but bind the membrane with different interfaces. Higher-resolution cryo-EM of filaments bound to helical bicelles confirms that ESCRT-III filaments can interact with the membrane through a previously undescribed interface. Mathematical modeling demonstrates that the interface described above is key to the mechanical stability of helical membrane tubes and helps infer the rigidity of the described protein filaments. Altogether, our results suggest that the interactions between ESCRT-III filaments and the membrane could proceed through multiple interfaces, to provide assembly on membranes with various shapes, or adapt the orientation of the filaments towards the membrane during membrane remodeling.


Assuntos
Membrana Celular/química , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Anisotropia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Lipossomos/ultraestrutura , Modelos Biológicos , Polímeros/química , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo
17.
Nat Struct Mol Biol ; 27(4): 373-381, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32231289

RESUMO

The plasma membrane adenosine triphosphate (ATP) release channel pannexin 1 (PANX1) has been implicated in many physiological and pathophysiological processes associated with purinergic signaling, including cancer progression, apoptotic cell clearance, inflammation, blood pressure regulation, oocyte development, epilepsy and neuropathic pain. Here we present near-atomic-resolution structures of human and frog PANX1 determined by cryo-electron microscopy that revealed a heptameric channel architecture. Compatible with ATP permeation, the transmembrane pore and cytoplasmic vestibule were exceptionally wide. An extracellular tryptophan ring located at the outer pore created a constriction site, potentially functioning as a molecular sieve that restricts the size of permeable substrates. The amino and carboxyl termini, not resolved in the density map, appeared to be structurally dynamic and might contribute to narrowing of the pore during channel gating. In combination with functional characterization, this work elucidates the previously unknown architecture of pannexin channels and establishes a foundation for understanding their unique channel properties.


Assuntos
Trifosfato de Adenosina/química , Membrana Celular/ultraestrutura , Conexinas/ultraestrutura , Microscopia Crioeletrônica , Proteínas do Tecido Nervoso/ultraestrutura , Trifosfato de Adenosina/genética , Animais , Anuros/genética , Membrana Celular/química , Membrana Celular/genética , Conexinas/química , Conexinas/genética , Humanos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Conformação Proteica , Transdução de Sinais/genética
18.
Nat Struct Mol Biol ; 27(4): 392-399, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251413

RESUMO

The endosomal sorting complexes required for transport (ESCRTs) mediate diverse membrane remodeling events. These typically require ESCRT-III proteins to stabilize negatively curved membranes; however, recent work has indicated that certain ESCRT-IIIs also participate in positive-curvature membrane-shaping reactions. ESCRT-IIIs polymerize into membrane-binding filaments, but the structural basis for negative versus positive membrane remodeling by these proteins remains poorly understood. To learn how certain ESCRT-IIIs shape positively curved membranes, we determined structures of human membrane-bound CHMP1B-only, membrane-bound CHMP1B + IST1, and IST1-only filaments by cryo-EM. Our structures show how CHMP1B first polymerizes into a single-stranded helical filament, shaping membranes into moderate-curvature tubules. Subsequently, IST1 assembles a second strand on CHMP1B, further constricting the membrane tube and reducing its diameter nearly to the fission point. Each step of constriction thins the underlying bilayer, lowering the barrier to membrane fission. Our structures reveal how a two-component, sequential polymerization mechanism drives membrane tubulation, constriction and bilayer thinning.


Assuntos
Membrana Celular/ultraestrutura , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Proteínas Oncogênicas/ultraestrutura , Membrana Celular/química , Membrana Celular/genética , Citocinese/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/química , Endossomos/genética , Endossomos/ultraestrutura , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Polimerização , Conformação Proteica
19.
Biochim Biophys Acta Biomembr ; 1862(8): 183291, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32234322

RESUMO

Antimicrobial peptides (AMPs) selectively kill bacteria by disrupting their cell membranes, and are promising compounds to fight drug-resistant microbes. Biophysical studies on model membranes have characterized AMP/membrane interactions and the mechanism of bilayer perturbation, showing that accumulation of cationic peptide molecules in the external leaflet leads to the formation of pores ("carpet" mechanism). However, similar quantitative studies on real cells are extremely limited. Here, we investigated the interaction of the dansylated PMAP23 peptide (DNS-PMAP23) with a Gram-positive bacterium, showing that 107 bound peptide molecules per cell are needed to kill it. This result is consistent with our previous finding for Gram-negative strains, where a similar high threshold for killing was determined, demonstrating the general relevance of the carpet model for real bacteria. However, in the case of the Gram-positive strain, this number of molecules even exceeds the total surface available on the bacterial membrane. The high affinity of DNS-PMAP23 for the anionic teichoic acids of the Gram-positive cell wall, but not for the lipopolysaccharides of Gram-negative bacteria, provides a rationale for this finding. To better define the role of anionic lipids in peptide/cell association, we studied DNS-PMAP23 interaction with E. coli mutant strains lacking phosphatidylglycerol and/or cardiolipin. Surprisingly, these strains showed a peptide affinity similar to that of the wild type. This finding was rationalized by observing that these bacteria have an increased content of other anionic lipids, thus maintaining the total membrane charge essentially constant. Finally, studies of DNS-PMAP23 association to dead bacteria showed an affinity an order of magnitude higher compared to that of live cells, suggesting strong peptide binding to intracellular components that become accessible after membrane perturbation. This effect could play a role in population resistance to AMP action, since dead bacteria could protect the surviving cells by sequestering significant amounts of peptide molecules. Overall, our data indicate that quantitative studies of peptide association to bacteria can lead to a better understanding of the mechanism of action of AMPs.


Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Parede Celular/efeitos dos fármacos , Relação Estrutura-Atividade , Sequência de Aminoácidos/genética , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Parede Celular/química , Parede Celular/ultraestrutura , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/patogenicidade , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/patogenicidade , Humanos , Lipopolissacarídeos/química , Testes de Sensibilidade Microbiana
20.
Biochim Biophys Acta Biomembr ; 1862(4): 183242, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32135146

RESUMO

Antimicrobial Peptides (AMPs) are host defense molecules that initiate microbial death by binding to the membrane. On membrane binding, AMPs undergo changes in conformation and aggregation state to enable killing action. Depending on the AMP and cell membrane characteristics, the nature of binding can be aggregating or non-aggregating, with high/low cooperativity, at single or multiple sites with high/low affinity leading to a unique killing action that needs to be studied individually. In the present study, a steady-state model that simulates AMP-membrane interaction was developed and was used to predict the mechanism of AMP binding. The predictions obtained from the model were validated with experimentally deciphered values available in literature. The model was further used to predict the mechanism for a set of designed AMPs with high sequence similarity to Myeloid Antimicrobial Peptide (MAP) family. Depending on the predicted mechanism, a unique half saturation constant and steepness of response (Hill coefficient) was obtained which was further validated with available data from literature. The model could reliably predict the mechanism, the half saturation constant and the Hill coefficient values. Further based on the analysis, it was observed that aggregation and oligomerization result in drastic killing action in a short range of peptide concentration owing to high Hill coefficient values. Mechanisms such as monomers binding at multiple sites with/without cooperativity result in antimicrobial activity at low half saturation constant though the killing action may not be steep. Thus, the methodology developed here can be used to develop hypothesis for studying AMP-membrane interaction mechanisms.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/química , Proteínas de Membrana/química , Modelos Moleculares , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Membrana Celular/genética , Membrana Celular/ultraestrutura , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Testes de Sensibilidade Microbiana , Ligação Proteica/genética , Biologia de Sistemas
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA