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1.
iScience ; 27(1): 108700, 2024 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-38213623

RESUMO

Mitochondria are key organelles to provide ATP for synaptic transmission. This study aims to unravel the structural adaptation of mitochondria to an increase in presynaptic energy demand and upon the functional impairment of the auditory system. We use the anteroventral cochlear nucleus (AVCN) of wild-type and congenital deaf mice before and after hearing onset as a model system for presynaptic states of lower and higher energy demands. We combine focused ion beam scanning electron microscopy and electron tomography to investigate mitochondrial morphology. We found a larger volume of synaptic boutons and mitochondria after hearing onset with a higher crista membrane density. In deaf animals lacking otoferlin, we observed a shallow increase of mitochondrial volumes toward adulthood in endbulbs, while in wild-type animals mitochondria further enlarged. We propose that in the AVCN, presynaptic mitochondria undergo major structural changes likely to serve higher energy demands upon the onset of hearing and further maturation.

2.
Nat Struct Mol Biol ; 30(12): 1902-1912, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37857822

RESUMO

Glutaminase (GLS), which deaminates glutamine to form glutamate, is a mitochondrial tetrameric protein complex. Although inorganic phosphate (Pi) is known to promote GLS filamentation and activation, the molecular basis of this mechanism is unknown. Here we aimed to determine the molecular mechanism of Pi-induced mouse GLS filamentation and its impact on mitochondrial physiology. Single-particle cryogenic electron microscopy revealed an allosteric mechanism in which Pi binding at the tetramer interface and the activation loop is coupled to direct nucleophile activation at the active site. The active conformation is prone to enzyme filamentation. Notably, human GLS filaments form inside tubulated mitochondria following glutamine withdrawal, as shown by in situ cryo-electron tomography of cells thinned by cryo-focused ion beam milling. Mitochondria with GLS filaments exhibit increased protection from mitophagy. We reveal roles of filamentous GLS in mitochondrial morphology and recycling.


Assuntos
Glutaminase , Mitofagia , Camundongos , Humanos , Animais , Glutaminase/química , Glutaminase/metabolismo , Glutamina/metabolismo , Mitocôndrias/metabolismo
3.
Front Cell Dev Biol ; 11: 1178992, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37635868

RESUMO

In mammals, spatial orientation is synaptically-encoded by sensory hair cells of the vestibular labyrinth. Vestibular hair cells (VHCs) harbor synaptic ribbons at their presynaptic active zones (AZs), which play a critical role in molecular scaffolding and facilitate synaptic release and vesicular replenishment. With advancing age, the prevalence of vestibular deficits increases; yet, the underlying mechanisms are not well understood and the possible accompanying morphological changes in the VHC synapses have not yet been systematically examined. We investigated the effects of maturation and aging on the ultrastructure of the ribbon-type AZs in murine utricles using various electron microscopic techniques and combined them with confocal and super-resolution light microscopy as well as metabolic imaging up to 1 year of age. In older animals, we detected predominantly in type I VHCs the formation of floating ribbon clusters, mostly consisting of newly synthesized ribbon material. Our findings suggest that VHC ribbon-type AZs undergo dramatic structural alterations upon aging.

4.
EMBO Rep ; 24(9): e56702, 2023 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-37477166

RESUMO

Cochlear inner hair cells (IHCs) form specialized ribbon synapses with spiral ganglion neurons that tirelessly transmit sound information at high rates over long time periods with extreme temporal precision. This functional specialization is essential for sound encoding and is attributed to a distinct molecular machinery with unique players or splice variants compared to conventional neuronal synapses. Among these is the active zone (AZ) scaffold protein piccolo/aczonin, which is represented by its short splice variant piccolino at cochlear and retinal ribbon synapses. While the function of piccolo at synapses of the central nervous system has been intensively investigated, the role of piccolino at IHC synapses remains unclear. In this study, we characterize the structure and function of IHC synapses in piccolo gene-trap mutant rats (Pclogt/gt ). We find a mild hearing deficit with elevated thresholds and reduced amplitudes of auditory brainstem responses. Ca2+ channel distribution and ribbon morphology are altered in apical IHCs, while their presynaptic function seems to be unchanged. We conclude that piccolino contributes to the AZ organization in IHCs and is essential for normal hearing.


Assuntos
Células Ciliadas Auditivas Internas , Neuropeptídeos , Ratos , Animais , Audição/fisiologia , Sinapses/fisiologia , Cóclea , Gânglio Espiral da Cóclea/metabolismo , Proteínas do Citoesqueleto/metabolismo
5.
J Cell Biol ; 222(3)2023 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-36562752

RESUMO

In recent years, Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) has emerged as a flexible method that enables semi-automated volume ultrastructural imaging. We present a toolset for adherent cells that enables tracking and finding cells, previously identified in light microscopy (LM), in the FIB-SEM, along with the automatic acquisition of high-resolution volume datasets. We detect the underlying grid pattern in both modalities (LM and EM), to identify common reference points. A combination of computer vision techniques enables complete automation of the workflow. This includes setting the coincidence point of both ion and electron beams, automated evaluation of the image quality and constantly tracking the sample position with the microscope's field of view reducing or even eliminating operator supervision. We show the ability to target the regions of interest in EM within 5 µm accuracy while iterating between different targets and implementing unattended data acquisition. Our results demonstrate that executing volume acquisition in multiple locations autonomously is possible in EM.


Assuntos
Imageamento Tridimensional , Microscopia Eletrônica de Volume , Microscopia Eletrônica de Varredura , Imageamento Tridimensional/métodos , Software
6.
Glia ; 71(3): 509-523, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36354016

RESUMO

Healthy myelin sheaths consist of multiple compacted membrane layers closely encasing the underlying axon. The ultrastructure of CNS myelin requires specialized structural myelin proteins, including the transmembrane-tetraspan proteolipid protein (PLP) and the Ig-CAM myelin-associated glycoprotein (MAG). To better understand their functional relevance, we asked to what extent the axon/myelin-units display similar morphological changes if PLP or MAG are lacking. We thus used focused ion beam-scanning electron microscopy (FIB-SEM) to re-investigate axon/myelin-units side-by-side in Plp- and Mag-null mutant mice. By three-dimensional reconstruction and morphometric analyses, pathological myelin outfoldings extend up to 10 µm longitudinally along myelinated axons in both models. More than half of all assessed outfoldings emerge from internodal myelin. Unexpectedly, three-dimensional reconstructions demonstrated that both models displayed complex axonal pathology underneath the myelin outfoldings, including axonal sprouting. Axonal anastomosing was additionally observed in Plp-null mutant mice. Importantly, normal-appearing axon/myelin-units displayed significantly increased axonal diameters in both models according to quantitative assessment of electron micrographs. These results imply that healthy CNS myelin sheaths facilitate normal axonal diameters and shape, a function that is impaired when structural myelin proteins PLP or MAG are lacking.


Assuntos
Sistema Nervoso Central , Proteína Proteolipídica de Mielina , Bainha de Mielina , Glicoproteína Associada a Mielina , Animais , Camundongos , Axônios/metabolismo , Sistema Nervoso Central/metabolismo , Camundongos Knockout , Microscopia Eletrônica de Varredura , Proteínas da Mielina/metabolismo , Bainha de Mielina/metabolismo , Glicoproteína Associada a Mielina/genética , Proteína Proteolipídica de Mielina/genética
7.
Elife ; 112022 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-35274615

RESUMO

Oligodendrocytes facilitate rapid impulse propagation along the axons they myelinate and support their long-term integrity. However, the functional relevance of many myelin proteins has remained unknown. Here, we find that expression of the tetraspan-transmembrane protein CMTM5 (chemokine-like factor-like MARVEL-transmembrane domain containing protein 5) is highly enriched in oligodendrocytes and central nervous system (CNS) myelin. Genetic disruption of the Cmtm5 gene in oligodendrocytes of mice does not impair the development or ultrastructure of CNS myelin. However, oligodendroglial Cmtm5 deficiency causes an early-onset progressive axonopathy, which we also observe in global and tamoxifen-induced oligodendroglial Cmtm5 mutants. Presence of the WldS mutation ameliorates the axonopathy, implying a Wallerian degeneration-like pathomechanism. These results indicate that CMTM5 is involved in the function of oligodendrocytes to maintain axonal integrity rather than myelin biogenesis.


Assuntos
Bainha de Mielina , Oligodendroglia , Animais , Axônios/fisiologia , Sistema Nervoso Central/metabolismo , Camundongos , Proteínas da Mielina/genética , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo
8.
Nat Commun ; 13(1): 1163, 2022 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-35246535

RESUMO

Myelin, the electrically insulating sheath on axons, undergoes dynamic changes over time. However, it is composed of proteins with long lifetimes. This raises the question how such a stable structure is renewed. Here, we study the integrity of myelinated tracts after experimentally preventing the formation of new myelin in the CNS of adult mice, using an inducible Mbp null allele. Oligodendrocytes survive recombination, continue to express myelin genes, but they fail to maintain compacted myelin sheaths. Using 3D electron microscopy and mass spectrometry imaging we visualize myelin-like membranes failing to incorporate adaxonally, most prominently at juxta-paranodes. Myelinoid body formation indicates degradation of existing myelin at the abaxonal side and the inner tongue of the sheath. Thinning of compact myelin and shortening of internodes result in the loss of about 50% of myelin and axonal pathology within 20 weeks post recombination. In summary, our data suggest that functional axon-myelin units require the continuous incorporation of new myelin membranes.


Assuntos
Bainha de Mielina , Substância Branca , Animais , Axônios/metabolismo , Camundongos , Bainha de Mielina/metabolismo , Oligodendroglia
9.
Science ; 375(6581): eabj3944, 2022 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-35143306

RESUMO

Human oocytes are prone to assembling meiotic spindles with unstable poles, which can favor aneuploidy in human eggs. The underlying causes of spindle instability are unknown. We found that NUMA (nuclear mitotic apparatus protein)-mediated clustering of microtubule minus ends focused the spindle poles in human, bovine, and porcine oocytes and in mouse oocytes depleted of acentriolar microtubule-organizing centers (aMTOCs). However, unlike human oocytes, bovine, porcine, and aMTOC-free mouse oocytes have stable spindles. We identified the molecular motor KIFC1 (kinesin superfamily protein C1) as a spindle-stabilizing protein that is deficient in human oocytes. Depletion of KIFC1 recapitulated spindle instability in bovine and aMTOC-free mouse oocytes, and the introduction of exogenous KIFC1 rescued spindle instability in human oocytes. Thus, the deficiency of KIFC1 contributes to spindle instability in human oocytes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinesinas/deficiência , Oócitos/fisiologia , Oócitos/ultraestrutura , Fuso Acromático/fisiologia , Polos do Fuso/fisiologia , 1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Animais , Bovinos , Complexo Dinactina/metabolismo , Dineínas/metabolismo , Feminino , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/fisiologia , Centro Organizador dos Microtúbulos/ultraestrutura , Microtúbulos/metabolismo , Proteínas Recombinantes/metabolismo , Fuso Acromático/ultraestrutura , Polos do Fuso/ultraestrutura , Suínos
10.
PLoS Biol ; 18(11): e3000943, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33196637

RESUMO

In several neurodegenerative disorders, axonal pathology may originate from impaired oligodendrocyte-to-axon support of energy substrates. We previously established transgenic mice that allow measuring axonal ATP levels in electrically active optic nerves. Here, we utilize this technique to explore axonal ATP dynamics in the Plpnull/y mouse model of spastic paraplegia. Optic nerves from Plpnull/y mice exhibited lower and more variable basal axonal ATP levels and reduced compound action potential (CAP) amplitudes, providing a missing link between axonal pathology and a role of oligodendrocytes in brain energy metabolism. Surprisingly, when Plpnull/y optic nerves are challenged with transient glucose deprivation, both ATP levels and CAP decline slower, but recover faster upon reperfusion of glucose. Structurally, myelin sheaths display an increased frequency of cytosolic channels comprising glucose and monocarboxylate transporters, possibly facilitating accessibility of energy substrates to the axon. These data imply that complex metabolic alterations of the axon-myelin unit contribute to the phenotype of Plpnull/y mice.


Assuntos
Trifosfato de Adenosina/metabolismo , Bainha de Mielina/metabolismo , Paraplegia/metabolismo , Potenciais de Ação , Animais , Axônios/metabolismo , Modelos Animais de Doenças , Metabolismo Energético , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Proteína Proteolipídica de Mielina/deficiência , Proteína Proteolipídica de Mielina/genética , Bainha de Mielina/patologia , Nervo Óptico/metabolismo , Nervo Óptico/patologia , Paraplegia/genética , Paraplegia/patologia , Fenótipo
11.
Nat Commun ; 11(1): 3208, 2020 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-32587250

RESUMO

Inner hair cells (IHCs) are the primary receptors for hearing. They are housed in the cochlea and convey sound information to the brain via synapses with the auditory nerve. IHCs have been thought to be electrically and metabolically independent from each other. We report that, upon developmental maturation, in mice 30% of the IHCs are electrochemically coupled in 'mini-syncytia'. This coupling permits transfer of fluorescently-labeled metabolites and macromolecular tracers. The membrane capacitance, Ca2+-current, and resting current increase with the number of dye-coupled IHCs. Dual voltage-clamp experiments substantiate low resistance electrical coupling. Pharmacology and tracer permeability rule out coupling by gap junctions and purinoceptors. 3D electron microscopy indicates instead that IHCs are coupled by membrane fusion sites. Consequently, depolarization of one IHC triggers presynaptic Ca2+-influx at active zones in the entire mini-syncytium. Based on our findings and modeling, we propose that IHC-mini-syncytia enhance sensitivity and reliability of cochlear sound encoding.


Assuntos
Cóclea , Células Ciliadas Auditivas Internas , Audição/fisiologia , Animais , Sinalização do Cálcio , Cóclea/citologia , Cóclea/inervação , Nervo Coclear/metabolismo , Tomografia com Microscopia Eletrônica , Células Gigantes , Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/fisiologia , Camundongos , Técnicas de Patch-Clamp , Roedores/fisiologia , Sinapses/metabolismo
12.
EMBO J ; 39(14): e104105, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32567732

RESUMO

Mitochondrial function is critically dependent on the folding of the mitochondrial inner membrane into cristae; indeed, numerous human diseases are associated with aberrant crista morphologies. With the MICOS complex, OPA1 and the F1 Fo -ATP synthase, key players of cristae biogenesis have been identified, yet their interplay is poorly understood. Harnessing super-resolution light and 3D electron microscopy, we dissect the roles of these proteins in the formation of cristae in human mitochondria. We individually disrupted the genes of all seven MICOS subunits in human cells and re-expressed Mic10 or Mic60 in the respective knockout cell line. We demonstrate that assembly of the MICOS complex triggers remodeling of pre-existing unstructured cristae and de novo formation of crista junctions (CJs) on existing cristae. We show that the Mic60-subcomplex is sufficient for CJ formation, whereas the Mic10-subcomplex controls lamellar cristae biogenesis. OPA1 stabilizes tubular CJs and, along with the F1 Fo -ATP synthase, fine-tunes the positioning of the MICOS complex and CJs. We propose a new model of cristae formation, involving the coordinated remodeling of an unstructured crista precursor into multiple lamellar cristae.


Assuntos
Proteínas de Membrana/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Complexos Multiproteicos/metabolismo , Células HeLa , Humanos , Proteína Cofatora de Membrana/genética , Proteína Cofatora de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Complexos Multiproteicos/genética
13.
J Struct Biol ; 211(1): 107528, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32387573

RESUMO

Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) is an invaluable tool to visualize the 3D architecture of cell constituents and map cell networks. Recently, amorphous ice embedding techniques have been associated with FIB-SEM to ensure that the biological material remains as close as possible to its native state. Here we have vitrified human HeLa cells and directly imaged them by cryo-FIB-SEM with the secondary electron InLens detector at cryogenic temperature and without any staining. Image stacks were aligned and processed by denoising, removal of ion beam milling artefacts and local charge imbalance. Images were assembled into a 3D volume and the major cell constituents were modelled. The data illustrate the power of the workflow to provide a detailed view of the internal architecture of the fully hydrated, close-to-native, entire HeLa cell. In addition, we have studied the feasibility of combining cryo-FIB-SEM imaging with live-cell protein detection. We demonstrate that internalized gold particles can be visualized by detecting back scattered primary electrons at low kV while simultaneously acquiring signals from the secondary electron detector to image major cell features. Furthermore, gold-conjugated antibodies directed against RNA polymerase II could be observed in the endo-lysosomal pathway while labelling of the enzyme in the nucleus was not detected, a shortcoming likely due to the inadequacy between the size of the gold particles and the voxel size. With further refinements, this method promises to have a variety of applications where the goal is to localize cellular antigens while visualizing the entire native cell in three dimensions.


Assuntos
Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Microscopia Eletrônica de Varredura , Proteínas/ultraestrutura , Células HeLa , Humanos , Proteínas/isolamento & purificação , Coloração e Rotulagem
14.
J Struct Biol ; 210(2): 107492, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32156581

RESUMO

Advances in electron microscopy including improved imaging techniques and state-of-the-art detectors facilitate imaging of larger tissue volumes with electron microscopic resolution. In combination with genetic tools for the generation of mouse mutants this allows assessing the three-dimensional (3D) characteristics of pathological features in disease models. Here we revisited the axonal pathology in the central nervous system of a mouse model of spastic paraplegia type 2, the Plp-/Y mouse. Although PLP is a bona fide myelin protein, the major hallmark of the disease in both SPG2 patients and mouse models are axonal swellings comprising accumulations of numerous organelles including mitochondria, gradually leading to irreversible axonal loss. To assess the number and morphology of axonal mitochondria and the overall myelin preservation we evaluated two sample preparation techniques, chemical fixation or high-pressure freezing and freeze substitution, with respect to the objective of 3D visualization. Both methods allowed visualizing distribution and morphological details of axonal mitochondria. In Plp-/Y mice the number of mitochondria is 2-fold increased along the entire axonal length. Mitochondria are also found in the excessive organelle accumulations within axonal swellings. In addition, organelle accumulations were detected within the myelin sheath and the inner tongue. We find that 3D electron microscopy is required for a comprehensive understanding of the size, content and frequency of axonal swellings, the hallmarks of axonal pathology.


Assuntos
Axônios/patologia , Microscopia Eletrônica de Transmissão/métodos , Animais , Sistema Nervoso Central/patologia , Camundongos , Microscopia Eletrônica de Varredura , Bainha de Mielina/patologia
15.
Sci Rep ; 10(1): 2004, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32029771

RESUMO

Alignment of stacks of serial images generated by Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) is generally performed using translations only, either through slice-by-slice alignments with SIFT or alignment by template matching. However, limitations of these methods are two-fold: the introduction of a bias along the dataset in the z-direction which seriously alters the morphology of observed organelles and a missing compensation for pixel size variations inherent to the image acquisition itself. These pixel size variations result in local misalignments and jumps of a few nanometers in the image data that can compromise downstream image analysis. We introduce a novel approach which enables affine transformations to overcome local misalignments while avoiding the danger of introducing a scaling, rotation or shearing trend along the dataset. Our method first computes a template dataset with an alignment method restricted to translations only. This pre-aligned dataset is then smoothed selectively along the z-axis with a median filter, creating a template to which the raw data is aligned using affine transformations. Our method was applied to FIB-SEM datasets and showed clear improvement of the alignment along the z-axis resulting in a significantly more accurate automatic boundary segmentation using a convolutional neural network.

16.
Methods Cell Biol ; 152: 1-21, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31326016

RESUMO

Focused ion beam-scanning electron microscopy (FIB-SEM) has become a widely used technique in life sciences. To achieve the best data quality, sample preparation is important and has to be adapted to the specimen and the specific application. Here we illustrate three preparation procedures for mouse nervous tissue: First, the use of high-pressure freezing followed by direct imaging of vitrified tissue without any staining in the FIB-SEM under cryo-conditions as direct and fast procedure. Second, a slow procedure involving freeze substitution of frozen samples combined with additional staining for enhanced contrast and plastic embedding. Third, a fast preparation applying microwave-assisted chemical fixation and processing for resin embedding. All three methods of sample preparation are suitable for obtaining data stacks by FIB-SEM acquisition and 3D reconstruction.


Assuntos
Microscopia Eletrônica de Varredura/métodos , Sistema Nervoso/citologia , Animais , Microscopia Crioeletrônica/métodos , Substituição ao Congelamento/métodos , Imageamento Tridimensional/métodos , Camundongos , Inclusão em Plástico/métodos , Coloração e Rotulagem/métodos
17.
Science ; 364(6447)2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31249032

RESUMO

Mammalian oocytes segregate chromosomes with a microtubule spindle that lacks centrosomes, but the mechanisms by which acentrosomal spindles are organized and function are largely unclear. In this study, we identify a conserved subcellular structure in mammalian oocytes that forms by phase separation. This structure, which we term the liquid-like meiotic spindle domain (LISD), permeates the spindle poles and forms dynamic protrusions that extend well beyond the spindle. The LISD selectively concentrates multiple microtubule regulatory factors and allows them to diffuse rapidly within the spindle volume. Disruption of the LISD via different means disperses these factors and leads to severe spindle assembly defects. Our data suggest a model whereby the LISD promotes meiotic spindle assembly by serving as a reservoir that sequesters and mobilizes microtubule regulatory factors in proximity to spindle microtubules.


Assuntos
Centrossomo/fisiologia , Meiose , Microtúbulos/fisiologia , Oócitos/fisiologia , Fuso Acromático/fisiologia , Animais , Aurora Quinase A/metabolismo , Cadeias Pesadas de Clatrina/metabolismo , Feminino , Proteínas Fetais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Células NIH 3T3
18.
J Vis Exp ; (145)2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30958469

RESUMO

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.


Assuntos
Congelamento , Imageamento Tridimensional , Micro-Ondas , Pressão , Animais , Caenorhabditis elegans/fisiologia , Substituição ao Congelamento , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura
19.
Elife ; 82019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30672734

RESUMO

Myelin serves as an axonal insulator that facilitates rapid nerve conduction along axons. By transmission electron microscopy, a healthy myelin sheath comprises compacted membrane layers spiraling around the cross-sectioned axon. Previously we identified the assembly of septin filaments in the innermost non-compacted myelin layer as one of the latest steps of myelin maturation in the central nervous system (CNS) (Patzig et al., 2016). Here we show that loss of the cytoskeletal adaptor protein anillin (ANLN) from oligodendrocytes disrupts myelin septin assembly, thereby causing the emergence of pathological myelin outfoldings. Since myelin outfoldings are a poorly understood hallmark of myelin disease and brain aging we assessed axon/myelin-units in Anln-mutant mice by focused ion beam-scanning electron microscopy (FIB-SEM); myelin outfoldings were three-dimensionally reconstructed as large sheets of multiple compact membrane layers. We suggest that anillin-dependent assembly of septin filaments scaffolds mature myelin sheaths, facilitating rapid nerve conduction in the healthy CNS.


Assuntos
Sistema Nervoso Central/metabolismo , Proteínas Contráteis/fisiologia , Bainha de Mielina/metabolismo , Septinas/metabolismo , Animais , Sistema Nervoso Central/patologia , Proteínas Contráteis/genética , Camundongos , Dobramento de Proteína
20.
Nat Struct Mol Biol ; 25(1): 21-28, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29323269

RESUMO

The nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and the structures of assembling NPCs in a correlative 3D EM time course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets whose holes progressively shrink. NPC precursors are found in small membrane holes and dilate radially during assembly of the inner ring complex, forming thousands of transport channels within minutes. This mechanism is fundamentally different from that of interphase NPC assembly and explains how mitotic cells can rapidly establish a closed nuclear compartment while making it transport competent.


Assuntos
Núcleo Celular/metabolismo , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/metabolismo , Animais , Membrana Celular/metabolismo , Cromossomos , Citoplasma/metabolismo , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático/metabolismo , Edição de Genes , Células HeLa , Humanos , Interfase , Cinética , Microscopia Eletrônica de Varredura , Mitose , Xenopus
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