scholarly journals 3D structure and in situ arrangements of CatSper channel in the sperm flagellum

2021 ◽  
Author(s):  
Yanhe Zhao ◽  
Huafeng Wang ◽  
Caroline Wiesehoefer ◽  
Naman B Shah ◽  
Evan Reetz ◽  
...  
Keyword(s):  
Electron Tomography ◽  
3D Structure ◽  
Human Sperm ◽  
Structural Basis ◽  
Sperm Flagellum ◽  
Mammalian Sperm ◽  
Zigzag Pattern ◽  
Associated Proteins

The sperm calcium channel CatSper plays a central role in successful fertilization as a primary Ca2+ gateway into the sperm flagellum. However, the complex subunit composition of CatSper has impeded its reconstitution in vitro and structural elucidation. Here, we applied cryo-electron tomography to visualize the macromolecular organization of the native CatSper channel complex in intact mammalian sperm, as well as identified three additional CatSper-associated proteins. The repeating CatSper units form long zigzag-rows in four nanodomains along the flagella. In both mouse and human sperm, each CatSper repeat consists of a tetrameric pore complex. Murine CatSper contains an additional outwardly directed wing-structure connected to the tetrameric channel. The majority of the extracellular domains form a canopy above each pore-forming channel that interconnects to a zigzag-shaped roof. The intracellular domains link two neighboring channel complexes to a diagonal array. The loss of this intracellular link in Efcab9-/- sperm distorts the longitudinally aligned zigzag pattern and compromises flagellar movement. This work offers unique insights into the mechanisms underlying the assembly and transport of the CatSper complex to generate the nanodomains and provides a long-sought structural basis for understanding CatSper function in the regulation of sperm motility.

scholarly journals The structure of the COPI coat determined within the cell

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yury S Bykov ◽  
Miroslava Schaffer ◽  
Svetlana O Dodonova ◽  
Sahradha Albert ◽  
Jürgen M Plitzko ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Structural Model ◽  
Electron Tomography ◽  
Ion Beam ◽  
Membrane Thickness ◽  
In Vitro Reconstitution ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. Previously we have determined a complete structural model of the in vitro reconstituted COPI coat (Dodonova et al., 2017). Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath β’–COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from cis to trans, but the structure of the coat machinery remains constant.

scholarly journals In situ architecture of neuronal α-Synuclein inclusions

2020 ◽  
Author(s):  
Victoria A. Trinkaus ◽  
Irene Riera-Tur ◽  
Antonio Martínez-Sánchez ◽  
Felix J.B. Bäuerlein ◽  
Qiang Guo ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Electron Tomography ◽  
Structural Flexibility ◽  
Multiple Systems ◽  
Cryo Electron Tomography ◽  
Multiple Systems Atrophy ◽  
The Brain ◽  
Cellular Organelles

Summaryα-Synuclein (α-Syn) aggregation is a hallmark of devastating neurodegenerative disorders including Parkinson’s disease (PD) and multiple systems atrophy (MSA)1,2. α-Syn aggregates spread throughout the brain during disease progression2, suggesting mechanisms of intercellular seeding. Formation of α-Syn amyloid fibrils is observed in vitro3,4 and fibrillar α-Syn has been purified from patient brains5,6, but recent reports questioned whether disease-relevant α-Syn aggregates are fibrillar in structure7-9. Here we use cryo-electron tomography (cryo-ET) to image neuronal Lewy body-like α-Syn inclusions in situ at molecular resolution. We show that the inclusions consist of α-Syn fibrils crisscrossing a variety of cellular organelles such as the endoplasmic reticulum (ER), mitochondria and autophagic structures, without interacting with membranes directly. Neuronal inclusions seeded by recombinant or MSA patient-derived α-Syn aggregates have overall similar architecture, although MSA-seeded fibrils show higher structural flexibility. Using gold-labeled seeds we find that aggregate nucleation is predominantly mediated by α-Syn oligomers, with fibrils growing unidirectionally from the seed. Our results conclusively demonstrate that neuronal α-Syn inclusions contain α-Syn fibrils intermixed with cellular membranes, and illuminate the mechanism of aggregate nucleation.

scholarly journals In situ structure of virus capsids within cell nuclei by correlative light and cryo-electron tomography

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Swetha Vijayakrishnan ◽  
Marion McElwee ◽  
Colin Loney ◽  
Frazer Rixon ◽  
David Bhella
Keyword(s):  
Electron Microscopy ◽  
Structure Determination ◽  
Electron Tomography ◽  
3D Structure ◽  
Three Dimensional ◽  
Cell Nuclei ◽  
Nuclear Egress ◽  
Virus Capsids ◽  
Cryo Electron Tomography

Abstract Cryo electron microscopy (cryo-EM), a key method for structure determination involves imaging purified material embedded in vitreous ice. Images are then computationally processed to obtain three-dimensional structures approaching atomic resolution. There is increasing interest in extending structural studies by cryo-EM into the cell, where biological structures and processes may be imaged in context. The limited penetrating power of electrons prevents imaging of thick specimens (> 500 nm) however. Cryo-sectioning methods employed to overcome this are technically challenging, subject to artefacts or involve specialised and costly equipment. Here we describe the first structure of herpesvirus capsids determined by sub-tomogram averaging from nuclei of eukaryotic cells, achieved by cryo-electron tomography (cryo-ET) of re-vitrified cell sections prepared using the Tokuyasu method. Our reconstructions confirm that the capsid associated tegument complex is present on capsids prior to nuclear egress. We demonstrate that this method is suited to both 3D structure determination and correlative light/electron microscopy, thus expanding the scope of cryogenic cellular imaging.

scholarly journals Three-dimensional ultrastructure of the septin filament network inSaccharomyces cerevisiae

2012 ◽  
Vol 23 (3) ◽  
pp. 423-432 ◽  
Author(s):  
Aurélie Bertin ◽  
Michael A. McMurray ◽  
Jason Pierson ◽  
Luong Thai ◽  
Kent L. McDonald ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Three Dimensional ◽  
Yeast Cells ◽  
Lipid Monolayers ◽  
Morphological Description ◽  
Section Electron ◽  
Subunit Organization

Septins are conserved GTP-binding proteins involved in membrane compartmentalization and remodeling. In budding yeast, five mitotic septins localize at the bud neck, where the plasma membrane is enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2). We previously established the subunit organization within purified yeast septin complexes and how these hetero-octamers polymerize into filaments in solution and on PtdIns4,5P2-containing lipid monolayers. How septin ultrastructure in vitro relates to the septin-containing filaments observed at the neck in fixed cells by thin-section electron microscopy was unclear. A morphological description of these filaments in the crowded space of the cell is challenging, given their small cross section. To examine septin organization in situ, sections of dividing yeast cells were analyzed by electron tomography of freeze-substituted cells, as well as by cryo–electron tomography. We found networks of filaments both perpendicular and parallel to the mother–bud axis that resemble septin arrays on lipid monolayers, displaying a repeat pattern that mirrors the molecular dimensions of the corresponding septin preparations in vitro. Thus these in situ structures most likely represent septin filaments. In viable mutants lacking a single septin, in situ filaments are still present, although more disordered, consistent with other evidence that the in vivo function of septins requires filament formation.

scholarly journals Domain-swapped Dimerization of the Second PDZ Domain of ZO2 May Provide a Structural Basis for the Polymerization of Claudins

2007 ◽  
Vol 282 (49) ◽  
pp. 35988-35999 ◽  
Author(s):  
Jiawen Wu ◽  
Yinshan Yang ◽  
Jiahai Zhang ◽  
Peng Ji ◽  
Wenjing Du ◽  
...  
Keyword(s):  
Solution Structure ◽  
Pdz Domain ◽  
Three Dimensional ◽  
Structural Basis ◽  
Pdz Domains ◽  
Zonula Occludens ◽  
Associated Proteins ◽  
High Conservation

Zonula occludens proteins (ZOs), including ZO1/2/3, are tight junction-associated proteins. Each of them contains three PDZ domains. It has been demonstrated that ZO1 can form either homodimers or heterodimers with ZO2 or ZO3 through the second PDZ domain. However, the underlying structural basis is not well understood. In this study, the solution structure of the second PDZ domain of ZO2 (ZO2-PDZ2) was determined using NMR spectroscopy. The results revealed a novel dimerization mode for PDZ domains via three-dimensional domain swapping, which can be generalized to homodimers of ZO1-PDZ2 or ZO3-PDZ2 and heterodimers of ZO1-PDZ2/ZO2-PDZ2 or ZO1-PDZ2/ZO3-PDZ2 due to high conservation between PDZ2 domains in ZO proteins. Furthermore, GST pulldown experiments and immunoprecipitation studies demonstrated that interactions between ZO1-PDZ2 and ZO2-PDZ2 and their self-associations indeed exist both in vitro and in vivo. Chemical cross-linking and dynamic laser light scattering experiments revealed that both ZO1-PDZ2 and ZO2-PDZ2 can form oligomers in solution. This PDZ domain-mediated oligomerization of ZOs may provide a structural basis for the polymerization of claudins, namely the formation of tight junctions.

Sex preselection by flow cytometric separation of X and Y chromosome-bearing sperm based on DNA difference: a review

1995 ◽  
Vol 7 (4) ◽  
pp. 893 ◽  
Author(s):  
LA Johnson
Keyword(s):  
Y Chromosome ◽  
Sex Chromosome ◽  
Human Sperm ◽  
Specific Marker ◽  
Current Form ◽  
Vitro Fertilization ◽  
Flow Cytometric ◽  
Flow Cytometric Sorting

Recent research on the flow cytometry of sperm for the purpose of predetermining gender of offspring has led to a validated method to separate X from Y chromosome-bearing sperm for use with in vitro fertilization and embryo transfer, intratubal insemination or intracytoplasmic sperm injection. The basis for the method is the sex chromosome-specific marker, DNA, which is present in greater amounts in X-bearing sperm than in Y-bearing sperm of mammals. Sperm are exposed to the vital dye Hoechst 33342 which binds to the minor groove of the DNA helix. Flow cytometric sorting of the sperm using a laser as the excitation source results in populations of Y- or X-bearing sperm that are 85-90% pure. Several hundred offspring have been produced from swine, rabbits, sheep and cattle that confirm the predicted sex. The method is currently being applied to the commercial embryo market. The method is not likely to be used in conjunction with standard cattle or swine artificial insemination practice in its current form since only about 4 x 10(5) sorted sperm can be produced per hour of sorting. The technology has also been applied to human sperm for use by couples that are at risk to sex-linked disease expression in their offspring. Populations of human sperm have been sorted with X and Y purities of about 80% as confirmed by DNA probe technology and fluorescence in situ hybridization.

Cryo-Electron Tomography of Dynein in vitro and in situ

2007 ◽  
Vol 13 (S02) ◽  
Author(s):  
V Tang ◽  
JR McIntosh ◽  
ME Porter ◽  
RD Vale ◽  
D Nicastro
Keyword(s):  
Electron Tomography ◽  
Cryo Electron Tomography

Biomineralization of the Hydroxyapatite with 3D-Structure for Enamel Reconstruction

2011 ◽  
Vol 391-392 ◽  
pp. 633-637
Author(s):  
Kun Tian ◽  
Min Peng ◽  
Ping Wu ◽  
Chu Hang Liao ◽  
Fa Yin Huang
Keyword(s):  
3D Structure ◽  
Oral Epithelium ◽  
Dentinal Tubule ◽  
Dental Tissue ◽  
Reciprocal Interactions ◽  
Mineralized Tissues ◽  
Parallel Direction ◽  
Dental Tissue Engineering

Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin. Based on these basic theory, we design a organic molecules model to induced the crystallization of hydroxyapatite to synthesized tooth-like calcium phosphate/hydroxyapatite with 3D-structure in a controllable way in vitro. We observed that hydroxyapatite nanorods can be controlled followed by in situ phosphorylation process and triggered by conditions of pH and ionic strength. The results showed that he dentinal tubule were blocked by neonatal hydroxyapatite layer and this composite a continuous structure of columns crystal with size of 30-80nm. At the same time, XRD showed that the precipitation was calcium fluoride phosphate and Ca:P was 1.6. Furthermore, there were column crystal with parallel direction inside, as same as the crystal array in the top of enamel rod. The results suggest that collagen monolayer may be useful in the modulation of mineral behavior during in situ dental tissue engineering.

scholarly journals A lamin A/C variant causing striated muscle disease provides insights into filament organization

2021 ◽  
Vol 134 (6) ◽  
pp. jcs256156
Author(s):  
Rafael Kronenberg-Tenga ◽  
Meltem Tatli ◽  
Matthias Eibauer ◽  
Wei Wu ◽  
Ji-Yeon Shin ◽  
...  
Keyword(s):  
Striated Muscle ◽  
Electron Tomography ◽  
Nuclear Lamina ◽  
Muscle Disease ◽  
Relative Reduction ◽  
Embryonic Fibroblasts ◽  
Thick Filaments ◽  
Cryo Electron Tomography ◽  
Associated Proteins

ABSTRACTThe LMNA gene encodes the A-type lamins, which polymerize into ∼3.5-nm-thick filaments and, together with B-type lamins and associated proteins, form the nuclear lamina. Mutations in LMNA cause a wide variety of pathologies. In this study, we analyzed the nuclear lamina of embryonic fibroblasts from LmnaH222P/H222P mice, which develop cardiomyopathy and muscular dystrophy. Although the organization of the lamina appeared unaltered, there were changes in chromatin and B-type lamin expression. An increase in nuclear size and consequently a relative reduction in heterochromatin near the lamina allowed for a higher resolution structural analysis of lamin filaments using cryo-electron tomography. This was most apparent when visualizing lamin filaments in situ and using a nuclear extraction protocol. Averaging of individual segments of filaments in LmnaH222P/H222P mouse fibroblasts resolved two polymers that constitute the mature filaments. Our findings provide better views of the organization of lamin filaments and the effect of a striated muscle disease-causing mutation on nuclear structure.

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