RESUMEN
Sensing and responding to environmental water deficiency and osmotic stresses are essential for the growth, development, and survival of plants. Recently, an osmolality-sensing ion channel called OSCA1 was discovered that functions in sensing hyperosmolality in Arabidopsis Here, we report the cryo-electron microscopy (cryo-EM) structure and function of an OSCA1 homolog from rice (Oryza sativa; OsOSCA1.2), leading to a model of how it could mediate hyperosmolality sensing and transport pathway gating. The structure reveals a dimer; the molecular architecture of each subunit consists of 11 transmembrane (TM) helices and a cytosolic soluble domain that has homology to RNA recognition proteins. The TM domain is structurally related to the TMEM16 family of calcium-dependent ion channels and lipid scramblases. The cytosolic soluble domain possesses a distinct structural feature in the form of extended intracellular helical arms that are parallel to the plasma membrane. These helical arms are well positioned to potentially sense lateral tension on the inner leaflet of the lipid bilayer caused by changes in turgor pressure. Computational dynamic analysis suggests how this domain couples to the TM portion of the molecule to open a transport pathway. Hydrogen/deuterium exchange mass spectrometry (HDXMS) experimentally confirms the conformational dynamics of these coupled domains. These studies provide a framework to understand the structural basis of proposed hyperosmolality sensing in a staple crop plant, extend our knowledge of the anoctamin superfamily important for plants and fungi, and provide a structural mechanism for potentially translating membrane stress to transport regulation.
Asunto(s)
Anoctaminas/ultraestructura , Proteínas de Arabidopsis/ultraestructura , Canales de Calcio/ultraestructura , Oryza/ultraestructura , Conformación Proteica , Secuencia de Aminoácidos/genética , Anoctaminas/química , Anoctaminas/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Microscopía por Crioelectrón , Citoplasma/genética , Espectrometría de Masas , Potenciales de la Membrana/genética , Oryza/genética , Oryza/crecimiento & desarrollo , Presión Osmótica/fisiología , Agua/químicaRESUMEN
Cytokinesis is the final stage of cell division and produces two independent daughter cells. Vesicles derived from internal membrane stores, such as the Golgi, lysosomes, and early and recycling endosomes accumulate at the intracellular bridge (ICB) during cytokinesis. Here, we use electron tomography to show that many ICB vesicles are not independent but connected, forming a newly described ICB vesicular structure - narrow tubules that are often branched. These 'midbody tubules' labelled with horseradish peroxidase (HRP) within 10 min after addition to the surrounding medium demonstrating that they are derived from endocytosis. HRP-labelled vesicles and tubules were observed at the rim of the ICB after only 1 min, suggesting that midbody tubules are likely to be generated by local endocytosis occurring at the ICB rim. Indeed, at least one tubule was open to the extracellular space, indicative of a local origin within the ICB. Inhibition of cholesterol-dependent endocytosis by exposure to methyl-ß-cyclodextrin and filipin reduced formation of HRP-labelled midbody tubules, and induced multinucleation following ICB formation. In contrast, dynamin inhibitors, which block clathrin-mediated endocytosis, induced multinucleation but had no effect on the formation of HRP-labelled midbody tubules. Therefore, our data reveal the existence of a cholesterol-dependent endocytic pathway occurring locally at the ICB, which contributes to the accumulation of vesicles and tubules that contribute to the completion of cytokinesis.
Asunto(s)
Colesterol/metabolismo , Citocinesis/fisiología , Endocitosis/fisiología , Endosomas/metabolismo , Lisosomas/metabolismo , Aparato de Golgi/metabolismo , Peroxidasa de Rábano Silvestre/metabolismo , Humanos , Microscopía Electrónica/métodos , beta-Ciclodextrinas/metabolismoRESUMEN
We have shown previously that Rab6, a small, trans-Golgi-localized GTPase, acts upstream of the conserved oligomeric Golgi complex (COG) and ZW10/RINT1 retrograde tether complexes to maintain Golgi homeostasis. In this article, we present evidence from the unbiased and high-resolution approach of electron microscopy and electron tomography that Rab6 is essential to the trans-Golgi trafficking of two morphological classes of coated vesicles; the larger corresponds to clathrin-coated vesicles and the smaller to coat protein I (COPI)-coated vesicles. On the basis of the site of coated vesicle accumulation, cisternal dilation and the normal kinetics of cargo transport from the endoplasmic reticulum (ER) to Golgi followed by delayed Golgi to cell surface transport, we suggest that Golgi function in cargo transport is preferentially inhibited at the trans-Golgi/trans-Golgi network (TGN). The >50% increase in Golgi cisternae number in Rab6-depleted HeLa cells that we observed may well be coupled to the trans-Golgi accumulation of COPI-coated vesicles; depletion of the individual Rab6 effector, myosin IIA, produced an accumulation of uncoated vesicles with if anything a decrease in cisternal number. These results are the first evidence for a Rab6-dependent protein machine affecting Golgi-proximal, coated vesicle accumulation and probably transport at the trans-Golgi and the first example of concomitant cisternal proliferation and increased Golgi stack organization under inhibited transport conditions.
Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Tomografía con Microscopio Electrónico/métodos , Aparato de Golgi/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Transporte Biológico , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Homeostasis , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía Electrónica/métodos , Miosina Tipo II/metabolismo , Fenotipo , Isoformas de Proteínas , Transporte de Proteínas , ARN Interferente Pequeño/metabolismo , Red trans-Golgi/metabolismoRESUMEN
A subsarcolemmal tubular system network (SSTN) has been detected in skeletal muscle fibers by confocal imaging after the removal of the sarcolemma. Here we confirm the existence and resolve the fine architecture and the localization of the SSTN at an unprecedented level of detail by examining extracellularly applied tubular system markers in skeletal muscle fiber preparations with a combination of three imaging modalities: confocal fluorescence microscopy, direct stochastic optical reconstruction microscopy, and tomographic electron microscopy. Three-dimensional reconstructions showed that the SSTN was a dense two-dimensional network within the subsarcolemmal space around the fiber, running ~500-600 nm underneath and parallel to the sarcolemma. The SSTN is composed of tubules ~95 nm in width with ~60% of the tubules directed transversely and >30% directed longitudinally. The deeper regular transverse tubules located at each A-I boundary of the sarcomeres branched from the SSTN, indicating individual transverse tubules that form triads are continuous with, but do not directly contact the sarcolemma. This suggests that the SSTN plays an important role in affecting the exchange of deeper tubule lumina with the extracellular space.
Asunto(s)
Imagen Molecular , Fibras Musculares Esqueléticas/citología , Sarcolema/metabolismo , Animales , Imagenología Tridimensional , Microscopía Confocal , RatasRESUMEN
Electron tomography of the chemical synapse provides important architectural information regarding the organization of synaptic organelles including synaptic vesicles, Nissl bodies, and early endosomes. Here, we describe methods for the preparation of select murine brain regions for high-pressure freezing, freeze substitution, and EM tomographic analysis of synaptic structures. The method uses fresh brain slices prepared using a vibratome and biopsy punches to collect specific brain regions of interest suitable for subsequent preservation and EM tomographic imaging.
Asunto(s)
Tomografía con Microscopio Electrónico , Electrones , Animales , Tomografía con Microscopio Electrónico/métodos , Substitución por Congelación , Ratones , Orgánulos , SinapsisRESUMEN
Compromised protein homeostasis underlies accumulation of plaques and tangles in Alzheimer's disease (AD). To observe protein turnover at early stages of amyloid beta (Aß) proteotoxicity, we performed pulse-chase proteomics on mouse brains in three genetic models of AD that knock in alleles of amyloid precursor protein (APP) prior to the accumulation of plaques and during disease progression. At initial stages of Aß accumulation, the turnover of proteins associated with presynaptic terminals is selectively impaired. Presynaptic proteins with impaired turnover, particularly synaptic vesicle (SV)-associated proteins, have elevated levels, misfold in both a plaque-dependent and -independent manner, and interact with APP and Aß. Concurrent with elevated levels of SV-associated proteins, we found an enlargement of the SV pool as well as enhancement of presynaptic potentiation. Together, our findings reveal that the presynaptic terminal is particularly vulnerable and represents a critical site for manifestation of initial AD etiology. A record of this paper's transparent peer review process is included in the Supplemental Information.
Asunto(s)
Enfermedad de Alzheimer/genética , Terminales Presinápticos/metabolismo , Proteómica/métodos , Animales , Modelos Animales de Enfermedad , Ratones , Ratones TransgénicosRESUMEN
Caveolae are characteristic invaginations of the mammalian plasma membrane (PM) implicated in lipid regulation, signal transduction and endocytosis. We have employed electron microscope tomography (ET) to quantify caveolae structure-function relationships in three-dimension (3D) at high resolution both in conventionally fixed and in fast-frozen/freeze-substituted (intact) cells as well as immunolabelled PM lawns. Our findings provide a detailed quantitative comparison of the average caveola dimensions for different cell types including tissue endothelial cells and cultured 3T3-L1 adipocytes. These studies revealed the presence of a spiked caveolar coat and a wide caveolar neck open to the extracellular milieu that is sensitive to conventional fixation; the neck region appeared to form a specialized microdomain with associated cytoplasmic material. In endothelial cells in situ in pancreatic islets of Langerhans, the diaphragm spanning the caveolar opening was clearly resolved by ET, and the involuted 3D topology of the cell surface mapped to measure the contribution of caveolar membranes to local increases in the surface area of the PM. The complexity of connections among caveolae and to the actin cytoskeleton and microtubules suggests that individual caveolae may be interconnected through a complex filamentous network to form a single functional unit.
Asunto(s)
Caveolas/ultraestructura , Citoesqueleto/ultraestructura , Imagenología Tridimensional , Células 3T3-L1 , Adipocitos/metabolismo , Adipocitos/ultraestructura , Animales , Caveolas/metabolismo , Caveolinas/metabolismo , Células Cultivadas , Citoesqueleto/metabolismo , Células Endoteliales/metabolismo , Feto/citología , Fibroblastos/ultraestructura , Substitución por Congelación , Islotes Pancreáticos/ultraestructura , RatonesRESUMEN
While many approaches to reduce fibrillation of amyloid-ß (Aß) have been aimed at slowing fibril formation, the degradation of fibrils remains challenging. We provide insight into fibril degradation as well as the inhibition of fiber formation by lipid vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol). In the presence of vesicles with the optimal lipid composition, fibril formation was inhibited up to 76%. Additionally, by tuning the lipid composition, mature fibril content decreased up to 74% and the ß-sheet content of Aß was significantly reduced. The reduction in fibril and ß-sheet content was consistent with a decrease in fibril diameter and could be attributed to the chaperone-like activity of the mixed vesicles. While demonstrating this remarkable activity, our findings present new evidence that lipid composition has a significant effect on the strength of the interaction between lipid bilayers and Aß peptides/fibrils. This understanding has intriguing therapeutic implications in treating protein misfolding diseases.
Asunto(s)
Amiloide/química , Fosfolípidos/química , Fosfolípidos/farmacología , Agregado de Proteínas/efectos de los fármacos , Catálisis , Glicerol/química , Modelos Moleculares , Fosforilcolina/química , Conformación Proteica en Lámina betaRESUMEN
Multi-ciliary arrays promote fluid flow and cellular motility using the polarized and coordinated beating of hundreds of motile cilia. Tetrahymena basal bodies (BBs) nucleate and position cilia, whereby BB-associated striated fibers (SFs) promote BB anchorage and orientation into ciliary rows. Mutants that shorten SFs cause disoriented BBs. In contrast to the cytotaxis model, we show that disoriented BBs with short SFs can regain normal orientation if SF length is restored. In addition, SFs adopt unique lengths by their shrinkage and growth to establish and maintain BB connections and cortical interactions in a ciliary force-dependent mechanism. Tetrahymena SFs comprise at least eight uniquely localizing proteins belonging to the SF-assemblin family. Loss of different proteins that localize to the SF base disrupts either SF steady-state length or ciliary force-induced SF elongation. Thus, the dynamic regulation of SFs promotes BB connections and cortical interactions to organize ciliary arrays.
Asunto(s)
Cuerpos Basales/fisiología , Cilios/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Protozoarias/metabolismo , Tetrahymena thermophila/crecimiento & desarrollo , Tetrahymena thermophila/metabolismo , Fenómenos Mecánicos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Protozoarias/genética , Tetrahymena thermophila/genéticaRESUMEN
We have developed a simplified, efficient approach for the 3D reconstruction and analysis of mammalian cells in toto by electron microscope tomography (ET), to provide quantitative information regarding 'global' cellular organization at approximately 15-20 nm resolution. Two insulin-secreting beta cells-deemed 'functionally equivalent' by virtue of their location at the periphery of the same pancreatic islet-were reconstructed in their entirety in 3D after fast-freezing/freeze-substitution/plastic embedment in situ within a glucose-stimulated islet of Langerhans isolated intact from mouse pancreata. These cellular reconstructions have afforded several unique insights into fundamental structure-function relationships among key organelles involved in the biosynthesis and release of the crucial metabolic hormone, insulin, that could not be provided by other methods. The Golgi ribbon, mitochondria and insulin secretory granules in each cell were segmented for comparative analysis. We propose that relative differences between the two cells in terms of the number, dimensions and spatial distribution (and for mitochondria, also the extent of branching) of these organelles per cubic micron of cellular volume reflects differences in the two cells' individual capacity (and/or readiness) to respond to secretagogue stimulation, reflected by an apparent inverse relationship between the number/size of insulin secretory granules versus the number/size of mitochondria and the Golgi ribbon. We discuss the advantages of this approach for quantitative cellular ET of mammalian cells, briefly discuss its application relevant to other complementary techniques, and summarize future strategies for overcoming some of its current limitations.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Islotes Pancreáticos/ultraestructura , Orgánulos/ultraestructura , Tomografía/métodos , Animales , Crioultramicrotomía , RatonesRESUMEN
Endocrine cells are continually regulating the balance between hormone biosynthesis, secretion, and intracellular degradation to ensure that cellular hormone stores are maintained at optimal levels. In pancreatic beta-cells, intracellular insulin stores in beta-granules are mostly upheld by efficiently up-regulating proinsulin biosynthesis at the translational level to rapidly replenish the insulin lost via exocytosis. Under normal circumstances, intracellular degradation of insulin plays a relatively minor janitorial role in retiring aged beta-granules, apparently via crinophagy. However, this mechanism alone is not sufficient to maintain optimal insulin storage in beta-cells when insulin secretion is dysfunctional. Here, we show that despite an abnormal imbalance of glucose/glucagon-like peptide 1 regulated insulin production over secretion in Rab3A(-/-) mice compared with control animals, insulin storage levels were maintained due to increased intracellular beta-granule degradation. Electron microscopy analysis indicated that this was mediated by a significant 12-fold up-regulation of multigranular degradation vacuoles in Rab3A(-/-) mouse islet beta-cells (P Asunto(s)
Autofagia/fisiología
, Células Secretoras de Insulina/metabolismo
, Insulina/metabolismo
, Animales
, Secreción de Insulina
, Células Secretoras de Insulina/patología
, Ratones
, Ratones Endogámicos C57BL
, Ratones Noqueados
, Proinsulina/biosíntesis
, Proteína de Unión al GTP rab3A/deficiencia
, Proteína de Unión al GTP rab3A/genética
RESUMEN
Meiotic chromosome segregation leads to the production of haploid germ cells. During meiosis I (MI), the paired homologous chromosomes are separated. Meiosis II (MII) segregation leads to the separation of paired sister chromatids. In the budding yeast Saccharomyces cerevisiae, both of these divisions take place in a single nucleus, giving rise to the four-spored ascus. We have modeled the microtubules in 20 MI and 15 MII spindles by using reconstruction from electron micrographs of serially sectioned meiotic cells. Meiotic spindles contain more microtubules than their mitotic counterparts, with the highest number in MI spindles. It is possible to differentiate between MI versus MII spindles based on microtubule numbers and organization. Similar to mitotic spindles, kinetochores in either MI or MII are attached by a single microtubule. The models indicate that the kinetochores of paired homologous chromosomes in MI or sister chromatids in MII are separated at metaphase, similar to mitotic cells. Examination of both MI and MII spindles reveals that anaphase A likely occurs in addition to anaphase B and that these movements are concurrent. This analysis offers a structural basis for considering meiotic segregation in yeast and for the analysis of mutants defective in this process.
Asunto(s)
Cromosomas Fúngicos/ultraestructura , Saccharomyces cerevisiae/metabolismo , Huso Acromático/química , Huso Acromático/ultraestructura , Anafase , Núcleo Celular/metabolismo , Cromátides/ultraestructura , Segregación Cromosómica , Proteínas Fúngicas/metabolismo , Proteínas Fluorescentes Verdes/química , Haploidia , Procesamiento de Imagen Asistido por Computador , Cinetocoros/metabolismo , Meiosis , Microscopía Electrónica , Microtúbulos/metabolismo , Modelos Teóricos , Mutación , FenotipoRESUMEN
Centromere protein (CENP) A, a histone H3 variant, is a key epigenetic determinant of chromosome domains known as centromeres. Centromeres nucleate kinetochores, multi-subunit complexes that capture spindle microtubules to promote chromosome segregation during mitosis. Two kinetochore proteins, CENP-C and CENP-N, recognize CENP-A in the context of a rare CENP-A nucleosome. Here, we reveal the structural basis for the exquisite selectivity of CENP-N for centromeres. CENP-N uses charge and space complementarity to decode the L1 loop that is unique to CENP-A. It also engages in extensive interactions with a 15-base pair segment of the distorted nucleosomal DNA double helix, in a position predicted to exclude chromatin remodelling enzymes. Besides CENP-A, stable centromere recruitment of CENP-N requires a coincident interaction with a newly identified binding motif on nucleosome-bound CENP-C. Collectively, our studies clarify how CENP-N and CENP-C decode and stabilize the non-canonical CENP-A nucleosome to enforce epigenetic centromere specification and kinetochore assembly.
Asunto(s)
Proteína A Centromérica/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN/metabolismo , Línea Celular , Centrómero/química , Proteína A Centromérica/química , Proteínas Cromosómicas no Histona/química , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN/química , Humanos , Cinetocoros/química , Cinetocoros/metabolismo , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
Toll-like receptor (TLR) signaling is a key innate immunity response to pathogens. Recruitment of signaling adapters such as MAL (TIRAP) and MyD88 to the TLRs requires Toll/interleukin-1 receptor (TIR)-domain interactions, which remain structurally elusive. Here we show that MAL TIR domains spontaneously and reversibly form filaments in vitro. They also form cofilaments with TLR4 TIR domains and induce formation of MyD88 assemblies. A 7-Å-resolution cryo-EM structure reveals a stable MAL protofilament consisting of two parallel strands of TIR-domain subunits in a BB-loop-mediated head-to-tail arrangement. Interface residues that are important for the interaction are conserved among different TIR domains. Although large filaments of TLR4, MAL or MyD88 are unlikely to form during cellular signaling, structure-guided mutagenesis, combined with in vivo interaction assays, demonstrated that the MAL interactions defined within the filament represent a template for a conserved mode of TIR-domain interaction involved in both TLR and interleukin-1 receptor signaling.
Asunto(s)
Proteínas Proteolipídicas Asociadas a Mielina y Linfocito/metabolismo , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito/ultraestructura , Factor 88 de Diferenciación Mieloide/metabolismo , Factor 88 de Diferenciación Mieloide/ultraestructura , Multimerización de Proteína , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/ultraestructura , Línea Celular , Microscopía por Crioelectrón , Análisis Mutacional de ADN , Humanos , Modelos Moleculares , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito/genética , Conformación Proteica , Dominios Proteicos , Transducción de SeñalRESUMEN
Neuronal communication relies on synaptic vesicles undergoing regulated exocytosis and recycling for multiple rounds of fusion. Whether all synaptic vesicles have identical protein content has been challenged, suggesting that their recycling ability may differ greatly. Botulinum neurotoxin type-A (BoNT/A) is a highly potent neurotoxin that is internalized in synaptic vesicles at motor nerve terminals and induces flaccid paralysis. Recently, BoNT/A was also shown to undergo retrograde transport, suggesting it might enter a specific pool of synaptic vesicles with a retrograde trafficking fate. Using high-resolution microscopy techniques including electron microscopy and single molecule imaging, we found that the BoNT/A binding domain is internalized within a subset of vesicles that only partially co-localize with cholera toxin B-subunit and have markedly reduced VAMP2 immunoreactivity. Synaptic vesicles loaded with pHrodo-BoNT/A-Hc exhibited a significantly reduced ability to fuse with the plasma membrane in mouse hippocampal nerve terminals when compared with pHrodo-dextran-containing synaptic vesicles and pHrodo-labeled anti-GFP nanobodies bound to VAMP2-pHluorin or vGlut-pHluorin. Similar results were also obtained at the amphibian neuromuscular junction. These results reveal that BoNT/A is internalized in a subpopulation of synaptic vesicles that are not destined to recycle, highlighting the existence of significant molecular and functional heterogeneity between synaptic vesicles.
Asunto(s)
Toxinas Botulínicas Tipo A/farmacología , Neuronas Motoras/metabolismo , Neurotoxinas/farmacología , Vesículas Sinápticas/metabolismo , Animales , Exocitosis/efectos de los fármacos , Exocitosis/genética , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Ratones , Neuronas Motoras/efectos de los fármacos , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/metabolismo , Transporte de Proteínas/genética , Vesículas Sinápticas/efectos de los fármacosRESUMEN
The triacylglycerol (TAG) crystal structures and morphologies of fractionated milk lipids in nanoemulsions were investigated at 4°C. Droplet size (0.17 versus 1.20 µm), lipid composition (stearin versus olein) and cooling rate (1 versus 10°C min(-1)) had an influence on the structural properties. Five crystal polymorphs (α, ß'1, ß'2, ß1, and ß2) were formed with either triple and/or double chain length structures in the solid phases of the emulsified systems. X-ray scattering peak intensities were reduced with the nanoemulsion particles. The internal structure of TAG exhibited stacking of individual lamellar layers (3.8-4.2 nm). Various anisometric shapes of fat nanoparticles were formed due to a highly sharp curvature of the nano-size droplets. The shape of olein nanoparticles was more polyhedral compared to the stearin. TAG crystals arranged in a planar-layered organisation at the slower cooling rate. These differences imply that the nanometric confinement of oil droplets modifies the fat crystal habit.
Asunto(s)
Emulsiones/química , Proteínas de la Leche/química , Leche/química , Animales , Anisotropía , Rastreo Diferencial de Calorimetría , Microscopía por Crioelectrón , Cristalización , Cristalografía por Rayos X , Lípidos/química , Microscopía Electrónica de Transmisión , Nanopartículas/química , Nanotecnología , Tamaño de la Partícula , Dispersión de Radiación , Temperatura , Triglicéridos/química , Rayos XRESUMEN
Dysfunction of caveolae is involved in human muscle disease, although the underlying molecular mechanisms remain unclear. In this paper, we have functionally characterized mouse and zebrafish models of caveolae-associated muscle disease. Using electron tomography, we quantitatively defined the unique three-dimensional membrane architecture of the mature muscle surface. Caveolae occupied around 50% of the sarcolemmal area predominantly assembled into multilobed rosettes. These rosettes were preferentially disassembled in response to increased membrane tension. Caveola-deficient cavin-1(-/-) muscle fibers showed a striking loss of sarcolemmal organization, aberrant T-tubule structures, and increased sensitivity to membrane tension, which was rescued by muscle-specific Cavin-1 reexpression. In vivo imaging of live zebrafish embryos revealed that loss of muscle-specific Cavin-1 or expression of a dystrophy-associated Caveolin-3 mutant both led to sarcolemmal damage but only in response to vigorous muscle activity. Our findings define a conserved and critical role in mechanoprotection for the unique membrane architecture generated by the caveolin-cavin system.
Asunto(s)
Caveolinas/metabolismo , Mecanotransducción Celular , Proteínas de la Membrana/metabolismo , Actividad Motora/fisiología , Fibras Musculares Esqueléticas/fisiología , Proteínas de Unión al ARN/metabolismo , Estrés Mecánico , Animales , Caveolinas/genética , Tomografía con Microscopio Electrónico , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Distrofias Musculares/genética , Distrofias Musculares/patología , Proteínas de Unión al ARN/genética , Sarcolema/genética , Sarcolema/patología , Pez CebraRESUMEN
Members of phylum Planctomycetes have been proposed to possess atypical cell organisation for the Bacteria, having a structure of sectioned cells consistent with internal compartments surrounded by membranes. Here via electron tomography we confirm the presence of compartments in the planctomycete Gemmata obscuriglobus cells. Resulting 3-D models for the most prominent structures, nuclear body and riboplasm, demonstrate their entirely membrane - enclosed nature. Immunogold localization of the FtsK protein also supports the internal organisation of G.obscuriglobus cells and their unique mechanism of cell division. We discuss how these new data expand our knowledge on bacterial cell biology and suggest evolutionary consequences of the findings.
Asunto(s)
Compartimento Celular , Planctomycetales/ultraestructura , Proteínas Bacterianas/metabolismo , Pared Celular/ultraestructura , Espacio Intracelular/metabolismo , Planctomycetales/metabolismo , Transporte de ProteínasRESUMEN
Caveolae are cell-surface membrane invaginations that play critical roles in cellular processes including signaling and membrane homeostasis. The cavin proteins, in cooperation with caveolins, are essential for caveola formation. Here we show that a minimal N-terminal domain of the cavins, termed HR1, is required and sufficient for their homo- and hetero-oligomerization. Crystal structures of the mouse cavin1 and zebrafish cavin4a HR1 domains reveal highly conserved trimeric coiled-coil architectures, with intersubunit interactions that determine the specificity of cavin-cavin interactions. The HR1 domain contains a basic surface patch that interacts with polyphosphoinositides and coordinates with additional membrane-binding sites within the cavin C terminus to facilitate membrane association and remodeling. Electron microscopy of purified cavins reveals the existence of large assemblies, composed of a repeating rod-like structural element, and we propose that these structures polymerize through membrane-coupled interactions to form the unique striations observed on the surface of caveolae in vivo.
Asunto(s)
Caveolas/química , Caveolas/metabolismo , Caveolinas/química , Caveolinas/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Animales , Caveolas/ultraestructura , Cristalografía por Rayos X , Citoplasma/química , Citoplasma/ultraestructura , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Transducción de Señal/fisiología , Pez Cebra/metabolismoRESUMEN
The nucleoid of the planctomycete Gemmata obscuriglobus is unique within the Bacteria in being both highly condensed and enclosed by a double-membrane nuclear envelope, seemingly analogous to the nucleus of eukaryotes. Here we have applied electron tomography to study high-pressure frozen, cryosubstituted cells of G. obscuriglobus and found multiple nested orders of DNA organization within the condensed nucleoid structure. Detailed examination of the nucleoid revealed a series of nested arcs characteristic of liquid crystalline cholesteric DNA structure. The finest fibers were arranged in parallel concentrically in a double-twist organization. At the highest order of nucleoid organization, several of these structures come together to form the core of the G. obscuriglobus nucleoid. The complex structure of DNA within this nucleoid may have implications for understanding the evolutionary significance of compartmentalized planctomycete cells.