Morphological and biochemical characteristics associated with autophagy in gastrointestinal diseases.

Autophagy is a cellular catabolic process characterized by the formation of double-membrane autophagosomes. Transmission electron microscopy is the most rigorous method to clearly visualize autophagic engulfment and degradation. A large number of studies have shown that autophagy is closely related to the digestion, secretion, and regeneration of gastrointestinal (GI) cells. However, the role of autophagy in GI diseases remains controversial. This article focuses on the morphological and biochemical characteristics of autophagy in GI diseases, in order to provide new ideas for their diagnosis and treatment.

[Automatic classification of immune-mediated glomerular diseases based on multi-modal multi-instance learning].

OBJECTIVE: To develop a multi-modal deep learning method for automatic classification of immune-mediated glomerular diseases based on images of optical microscopy (OM), immunofluorescence microscopy (IM), and transmission electron microscopy (TEM). METHODS: We retrospectively collected the pathological images from 273 patients and constructed a multi-modal multi- instance model for classification of 3 immune-mediated glomerular diseases, namely immunoglobulin A nephropathy (IgAN), membranous nephropathy (MN), and lupus nephritis (LN). This model adopts an instance-level multi-instance learning (I-MIL) method to select the TEM images for multi-modal feature fusion with the OM images and IM images of the same patient. By comparing this model with unimodal and bimodal models, we explored different combinations of the 3 modalities and the optimal methods for modal feature fusion. RESULTS: The multi-modal multi-instance model combining OM, IM, and TEM images had a disease classification accuracy of (88.34±2.12)%, superior to that of the optimal unimodal model [(87.08±4.25)%] and that of the optimal bimodal model [(87.92±3.06)%]. CONCLUSION: This multi- modal multi- instance model based on OM, IM, and TEM images can achieve automatic classification of immune-mediated glomerular diseases with a good classification accuracy.

Square condenser apertures for square cameras in low-dose transmission electron microscopy.

In transmission electron microscopy (TEM), cameras are square or rectangular but beams are round so the circular lobes irradiate adjacent areas, precluding further neighboring acquisition for beam-sensitive samples. We present condenser aperture plates with square and rectangular shapes that improve the efficiency of area usage by 70% and enhance montage imaging for beam-sensitive specimens. We demonstrate the compatibility of these condenser aperture plates with high-resolution cryogenic TEM by reconstructing a 1.8-Å map of equine apo-ferritin.

Construction Strategy and Mechanism of a Novel Wood Preservative with Excellent Antifungal Effects.

Wood is a naturally porous material prone to microbial erosion and degradation in outdoor environments. Therefore, the development of an environmentally friendly wood preservative with excellent antibacterial effects and low toxicity is urgently needed. In this study, nitrogen-doped carbon quantum dots (N-CQDs) with excellent antifungal performance and fluorescent properties were synthesized using a one-step hydrothermal method with chitosan quaternary ammonium salt (HACC) as the raw material. The fluorescence characteristics of N-CQD preservatives can help track their position and distribution in wood. The minimum inhibitory concentration (MIC) of N-CQDs is 1.8 mg/mL, which was nearly 22 times lower than that of HACC (40.0 mg/mL) in the PDA medium. The decay resistance test demonstrated that wood treated with N-CQDs showed a considerably reduced decay degree and its mass loss rate decreased from 46 ± 0.5% to 3.8 ± 0.5%. Biological transmission electron microscopy revealed that N-CQDs effectively destroyed fungal cell structures, thereby hindering the growth of Coriolus versicolor. N-CQDs synthesized using the one-step hydrothermal method can be used as an efficient wood preservative that can effectively improve the utilization and service life of wood.

In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants.

Correlative light and electron microscopy (CLEM) is an important tool for the localisation of target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometre scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of sections by both, single-molecule localization microscopy and transmission electron microscopy using consecutive CLEM and same-section CLEM correlative workflow. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows improvements in the fluorophore blinking behavior relative to the whole-mount approaches. Here, we use a commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as fasciata1 and nucleolin1 plants and apply our in-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.

Sedimentation and Laser Light Scattering Methods for Quantifying Synthetic Tau Aggregation Propensity.

Tau aggregation assays detect and quantify the conversion of soluble tau monomers into species having filamentous or oligomeric structure. Assays for filamentous aggregates in cross-ß-sheet conformation leverage optical, biochemical, or biophysical methods, each with their own advantages and throughput capacity. Here we provide protocols for two medium-throughput assays based on sedimentation and laser light scattering and compare their performance, their utility for characterizing tau aggregation dynamics, and their limitations relative to other approaches. Additionally, a protocol for transmission electron microscopy analysis is updated so as to be compatible with the truncated tau variants that have emerged as powerful tools for interrogating the structural basis of tau polymorphism. Together these methods contribute to a rich tool kit for interrogating tau aggregation kinetics and propensity over a wide range of experimental conditions.

Visualization of Engineered M13 Phages Bound to Bacterial Targets by Transmission Electron Microscopy.

The filamentous phage M13 is one of the most well-studied and characterized phages, particularly since it was introduced as a scaffold for phage display, a technique to express and evolve fusion proteins on the M13 phage's coat to study protein or peptide binding interactions. Since phages can be engineered or evolved to specifically bind to a variety of targets, engineered M13 phages have been explored for applications such as drug delivery, biosensing, and cancer therapy, among others. Specifically, with the rising challenge of antimicrobial resistance among bacteria, chimeric M13 phages have been explored both as detection and therapeutic agents due to the flexibility in tuning target specificity. Transmission electron microscopy (TEM) is a powerful tool enabling researchers to directly visualize and characterize binding of phages to bacterial surfaces. However, the filamentous phage structure poses a challenge for this technique, as the phages have similar morphology to bacterial structures such as pili. In order to differentiate between bacterial structures and the filamentous phages, here we describe a protocol to prepare TEM samples of engineered M13 phages bound to bacterial cells, in which the phage virions have been specifically labeled by decoration of the major capsid proteins with gold nanoparticles. This protocol enables clear visualization and unambiguous identification of attached filamentous phages within the context of bacterial cells expressing numerous pili.

Ultrastructure of human brain tissue vitrified from autopsy revealed by cryo-ET with cryo-plasma FIB milling.

Ultrastructure of human brain tissue has traditionally been examined using electron microscopy (EM) following fixation, staining, and sectioning, which limit resolution and introduce artifacts. Alternatively, cryo-electron tomography (cryo-ET) allows higher resolution imaging of unfixed cellular samples while preserving architecture, but it requires samples to be vitreous and thin enough for transmission EM. Due to these requirements, cryo-ET has yet to be employed to investigate unfixed, never previously frozen human brain tissue. Here we present a method for generating lamellae in human brain tissue obtained at time of autopsy that can be imaged via cryo-ET. We vitrify the tissue via plunge-freezing and use xenon plasma focused ion beam (FIB) milling to generate lamellae directly on-grid at variable depth inside the tissue. Lamellae generated in Alzheimer's disease brain tissue reveal intact subcellular structures including components of autophagy and potential pathologic tau fibrils. Furthermore, we reveal intact compact myelin and functional cytoplasmic expansions. These images indicate that plasma FIB milling with cryo-ET may be used to elucidate nanoscale structures within the human brain.

Genetically Fused Resilin-like Polypeptide-Coiled Coil Bundlemer Conjugates Exhibit Tunable Multistimuli-Responsiveness and Undergo Nanofibrillar Assembly.

Peptide-based materials are diverse candidates for self-assembly into modularly designed and stimuli-responsive nanostructures with precisely tunable compositions. Here, we genetically fused computationally designed coiled coil-forming peptides to the N- and C-termini of compositionally distinct multistimuli-responsive resilin-like polypeptides (RLPs) of various lengths. The successful expression of these hybrid polypeptides in bacterial hosts was confirmed through techniques such as gel electrophoresis, mass spectrometry, and amino acid analysis. Circular dichroism spectroscopy and ultraviolet-visible turbidimetry demonstrated that despite the fusion of disparate structural and responsive units, the coiled coils remained stable in the hybrid polypeptides, and the sequence-encoded differences in thermoresponsive phase separation of the RLPs were preserved. Cryogenic transmission electron microscopy and coarse-grained modeling showed that after thermal annealing in solution, the hybrid polypeptides adopted a closed loop conformation and assembled into nanofibrils capable of further hierarchically organizing into cluster structures and ribbon-like structures mediated by the self-association tendency of the RLPs.

A systematic study of the effect of lipid architecture on cytotoxicity and cellular uptake of cationic cubosomes.

HYPOTHESIS: Lipid nanoparticles containing a cationic lipid are increasingly used in drug and gene delivery as they can display improved cellular uptake, enhanced loading for anionic cargo such as siRNA and mRNA or exhibit additional functionality such as cytotoxicity against cancer cells. This research study tests the hypothesis that the molecular structure of the cationic lipid influences the structure of the lipid nanoparticle, the cellular uptake, and the resultant cytotoxicity. EXPERIMENTS: Three potentially cytotoxic cationic lipids, with systematic variations to the hydrophobic moiety, were designed and synthesised. All the three cationic lipids synthesised contain pharmacophores such as the bicyclic coumarin group (CCA12), the tricyclic etodolac moiety (ETD12), or the large pentacyclic triterpenoid "ursolic" group (U12) conjugated to a quaternary ammonium cationic lipid containing twin C12 chains. The cationic lipids were doped into monoolein cubosomes at a range of concentrations from 0.1 mol% to 5 mol% and the effect of the lipid molecular architecture on the cubosome phase behaviour was assessed using a combination of Small Angle X-Ray Scattering (SAXS), Dynamic Light Scattering (DLS), zeta-potential and cryo-Transmission Electron Microscopy (Cryo-TEM). The resulting cytotoxicity of these particles against a range of cancerous and non-cancerous cell-lines was assessed, along with their cellular uptake. FINDINGS: The molecular architecture of the cationic lipid was linked to the internal nanostructure of the resulting cationic cubosomes with a transition to more curved cubic and hexagonal phases generally observed. Cubosomes formed from the cationic lipid CCA12 were found to have improved cellular uptake and significantly higher cytotoxicity than the cationic lipids ETD12 and U12 against the gastric cancer cell-line (AGS) at lipid concentrations ≥ 75 µg/mL. CCA12 cationic cubosomes also displayed reasonable cytotoxicity against the prostate cancer PC-3 cell-line at lipid concentrations ≥ 100 µg/mL. In contrast, 2.5 mol% ETD12 and 2.5 mol% U12 cubosomes were generally non-toxic against both cancerous and non-cancerous cell lines over the entire concentration range tested. The molecular architecture of the cationic lipid was found to influence the cubosome phase behaviour, the cellular uptake and the toxicity although further studies are necessary to determine the exact relationship between structure and cellular uptake across a range of cell lines.

Localization and quantitative distribution of a chromatin structural protein Topoisomerase II on plant chromosome using HVTEM and UHVTEM.

Topoisomerase II (TopoII) is an essential structural protein of the metaphase chromosome. It maintains the axial compaction of chromosomes during metaphase. It is localized at the axial region of chromosomes and accumulates at the centromeric region in metaphase chromosomes. However, little is known about TopoII localization and distribution in plant chromosomes, except for several publications. We used high voltage transmission electron microscopy (HVTEM) and ultra-high voltage transmission electron microscopy (UHVTEM) in conjunction with immunogold labeling and visualization techniques to detect TopoII and investigate its localization, alignment, and density on the barley chromosome at 1.4 nm scale. We found that HVTEM and UHVTEM combined with immunogold labeling is suitable for the detection of structural proteins, including a single molecule of TopoII. This is because the average size of the gold particles for TopoII visualization after silver enhancement is 8.9 ± 3.9 nm, which is well detected. We found that 31,005 TopoII molecules are distributed along the barley chromosomes in an unspecific pattern at the chromosome arms and accumulate specifically at the nucleolus organizer regions (NORs) and centromeric region. The TopoII density were 1.32-fold, 1.58-fold, and 1.36-fold at the terminal region, at the NORs, and the centromeric region, respectively. The findings of TopoII localization in this study support the multiple reported functions of TopoII in the barley metaphase chromosome.

Synthesis and characterization of TiO2-based supported materials for industrial application and recovery in a pilot photocatalytic plant using chemometric approach.

In this contribution, the performance of powdered titanium dioxide (TiO2)-based photocatalysts was evaluated in a pilot photocatalytic plant for the degradation of different dyes, with an investigated volume of 1 L and solar simulated light as irradiation source. Five different samples, synthesized in our laboratories, were tested in the pilot plant, each consisting of TiO2 nanoparticles (NPs) coupled with a different material (persistent luminescent material and semiconductor material) and treated in different thermal conditions. All synthesized samples have been subjected to X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Brunauer-Emmett-Teller analysis (BET), and transmission electron microscopy (TEM) characterization, to shed light on the influence of introducing other materials on titania characteristics. To study and evaluate the significance of the parameters affecting the process in the pilot plant, a chemometric approach was applied, by selecting a mathematical model (D-Optimal) to simultaneously monitor a large number of variables (i.e., 7), both qualitative and quantitative, over a wide range of levels. At the same time, the recovery of the synthesized photocatalysts was studied following a novel promising recuperation method, i.e., annulling the surface charge of the suspended samples by reaching the isoelectric point (pHPZC) of each sample, for the quantitative precipitation of TiO2 nanoparticles.

Protocol for synthesis of spherical silver nanoparticles with stable optical properties and characterization by transmission electron microscopy.

The synthesis of metallic plasmonic nanoparticles (NPs) faces challenges in stability and reproducibility, especially with silver. Here, we present a protocol for tunable synthesis of spherical silver NPs (AgNPs) with stable optical properties. We describe steps for preparing solutions, morphological characterization of AgNPs by transmission electron microscopy, and testing stability. AgNPs exhibit enduring stability and compatibility with various pH values. Moreover, they can be functionalized for optical biosensing applications, offering versatility in nanomaterial applications.

Characterization of cleavage patterns and assembly of N-terminally modified GII.6 norovirus VP1 proteins.

When expressed in vitro, the major capsid protein VP1 of a norovirus (NoV) can self-assemble into virus-like particles (VLPs), and its N-terminus can tolerate foreign sequences without the assembly being affected. We explored the effects of adding an N-terminal sequence to the VP1 of a GII.6 NoV strain on its cleavage and assembly. Sequences of varying lengths derived from the minor capsid protein VP2 were added to the VP1 N-terminus. Using a recombinant baculovirus expression system, the fusion proteins were expressed, and their cleavage patterns and assembly were analyzed using mass spectrometry and transmission electron microscopy, respectively. All of the fusion proteins were successfully expressed and exhibited varying degrees of enzyme cleavage, most probably at the N-terminus. LC-MS results revealed that similar fragments were obtained for wild-type VP1 and fusion proteins, indicating that the cleavage sites were conserved. EM analysis indicated that VLPs of different sizes were successfully assembled for certain fusion proteins. The study data demonstrate that NoV VP1 can tolerate foreign sequences of a certain length at its N-terminus and that a conserved cleavage pattern exists, which might facilitate further investigation of the assembly and cleavage mechanisms of NoV.

Stimulation of microvesicle secretion in Trichomonas vaginalis.

Trichomonas vaginalis is an extracellular flagellate protozoan and the etiological agent of human trichomoniasis, a sexually transmitted infection (STI) with a high incidence. Several reports have shown that this protozoan releases microvesicles into the culture medium, which show high potential in modulating cell-to-cell communication and the host response to infections. However, the biogenesis of these vesicles has not been analyzed in detail. In the present study, high-resolution ion scanning microscopy (SEM) and transmission electron microscopy (TEM) were used to analyze the surface of control cells and cells incubated in the presence of Ca2+ alone or with A 23187 calcium ionophore. Two different strains of T. vaginalis were analyzed. Most control cells displayed relatively smooth surfaces, whereas cells incubated with Ca2+ had many surface projections of variable shape and size (from 40 nm to around 1 µm). Quantitative analyses were performed directly in the scanning electron microscope and showed a significant increase in the number of cells with surface projections after incubation in the presence of calcium. TEM showed that treated cells presented several cytoplasmic multivesicular structures, suggesting membrane fusion and exosomes in the extracellular medium. The amount and size of the released vesicles were quantitatively analyzed using light scattering and TEM on negatively stained samples. The observations show that incubation of both parasite strains in the presence of Ca2+ significantly increased the release of microvesicles into the extracellular medium in a time-dependent process. Sequential incubation in the presence of Ca2+ and the calcium ionophore A23187 increases the presence of vesicles on the parasite surface only at a short incubation time (5 min). Transmission electron microscopy showed that at least part of the vesicles are originated from cytoplasmic multivesicular structures. This information contributes to a better understanding of the biogenesis of extracellular vesicles secreted by T. vaginalis.

Lighting lantern above Psalteriomonadidae: Unveiling novel diversity within the genus Psalteriomonas (Discoba: Heterolobosea).

Psalteriomonadidae are a small family of anaerobic free-living protists belonging to Heterolobosea, Discoba. We cultured 74 new strains of mostly amoeboid Psalteriomonadidae obtained from mainly freshwater habitats and sequenced their 18S rRNA gene. Based on the phylogenetic analysis and genetic distances, we report multiple novel species, four of which we formally describe based on the light-microscopic morphology (Psalteriomonas minuta, P. australis, P. fimbriata, and P. parva). We also examined the ultrastructure of two Psalteriomonas species using transmission electron microscopy. We transfer Sawyeria marylandensis into the genus Psalteriomonas and synonymize Sawyeria with Psalteriomonas. In addition, we studied the flagellate stage of P. marylandensis comb. nov. for the first time, using light and scanning electron microscopy.

Improvement of Na+-K+ ATPase Activity and Gene Expression Associated with Glomerular Ultrastructural Protection in Diabetic Nephropathy by Acacia senegal: Role of Oxidative Stress Disturbances / Mejora de la actividad Na+-K+ ATPasa y la Expresión Génica Asociada con la Protección Ultraestructural Glomerular en la Nefropatía Diabética por Acacia senegal: Papel de las Alteraciones del Estrés Oxidativo

SUMMARY: This study assessed the effects of Acacia Senegal (AS) combined with insulin on Na+/K+-ATPase (NKA) activity and mRNA expression, serum glucose, renal function, and oxidative stress in a rat model of diabetic nephropathy (DN). Sixty rats were equally divided into six groups: normal control, normal+AS, diabetic (DM), DM+insulin, DM+AS, and DM+insulin+AS groups. Diabetes mellitus (type 1) was induced by a single injection of streptozotocin (65 mg/kg), and insulin and AS treatments were carried until rats were culled at the end of week 12. Serum glucose and creatinine levels, hemoglobin A1c (HbA1c) were measured. Renal homogenate levels of NKA activity and gene expression, malondialdehyde, superoxide dismutase (SOD), catalase and reduced glutathione (GSH) were evaluated as well as kidney tissue histology and ultrastructure. Diabetes caused glomerular damage and modulation of blood and tissue levels of creatinine, glucose, HbA1c, malondialdehyde, NKA activity and gene expression, SOD, catalase and GSH, which were significantly (p<0.05) treated with AS, insulin, and insulin plus AS. However, AS+insulin treatments were more effective. In conclusion, combined administration of AS with insulin to rats with DN decreased NKA activity and gene expression as well as oxidative stress, and improved glycemic state and renal structure and function.

Este estudio evaluó los efectos de Acacia senegal (AS) combinada con insulina sobre la actividad Na+/K+- ATPasa (NKA) y la expresión de ARNm, la glucosa sérica, la función renal y el estrés oxidativo en un modelo de nefropatía diabética (ND) en ratas. Sesenta ratas se dividieron equitativamente en seis grupos: control normal, normal+AS, diabética (DM), DM+insulina, DM+AS y DM+insulina+AS. La diabetes mellitus (tipo 1) se indujo mediante una única inyección de estreptozotocina (65 mg/kg), y los tratamientos con insulina y AS se llevaron a cabo hasta que las ratas fueron sacrificadas al final de la semana 12. Se midieron niveles séricos de glucosa y creatinina, hemoglobina A1c (HbA1c). Se evaluaron los niveles de homogeneizado renal de actividad NKA y expresión génica, malondialdehído, superóxido dismutasa (SOD), catalasa y glutatión reducido (GSH), así como la histología y ultraestructura del tejido renal. La diabetes causó daño glomerular y modulación de los niveles sanguíneos y tisulares de creatinina, glucosa, HbA1c, malondialdehído, actividad y expresión génica de NKA, SOD, catalasa y GSH, los cuales fueron tratados significativamente (p<0,05) con AS, insulina e insulina más AS. Sin embargo, los tratamientos con AS+insulina fueron más efectivos. En conclusión, la administración combinada de AS con insulina a ratas con DN disminuyó la actividad de NKA y la expresión genética, así como el estrés oxidativo, y mejoró el estado glucémico y la estructura y función renal.

Enhanced extracellular vesicles mediated uttroside B (Utt-B) delivery to Hepatocellular carcinoma cell: Pharmacokinetics based on PBPK modelling.

Our prior investigation has confirmed that the anti-hepatocellular carcinoma activity of the plant saponin, specifically Uttroside B (Utt-B), derived from the leaves of Solanum nigrum Linn. This study concentrated on formulating a novel biocompatible nanocarrier utilizing Extracellular vesicles (EVs) to enhance the delivery of plant saponin into cells. The physicochemical attributes of Extracellular Vesicles/UttrosideB (EVs/Utt-B) were comprehensively characterized through techniques such as Transmission Electron Microscopy (TEM) and Fourier-transform infrared spectroscopy (FTIR). Despite the promising therapeutic potential of this uttroside B, mechanistic know-how about its entry into cells is still in its infancy. Our research sheds light on the extracellular vesicle-mediated mechanism facilitating the entry of the saponin into cells, a phenomenon confirmed through the use of by confocal microscopy. We further analysed drug-releasing kinetics and simulated the Pharmacokinetics by PBPK modelling. The simulated pharmacokinetics revealed the bioavailability of Uttroside-B in oral administration against intravenous administration.

A transmission electron microscopy investigation suggests that telocytes, skeletal muscles, myoblasts, and stem cells in common carp (Cyprinus carpio) respond to salinity challenges.

BACKGROUND: Telocytes are modified interstitial cells that communicate with other types of cells, including stem cells. Stemness properties render them more susceptible to environmental conditions. The current morphological investigation examined the reactions of telocytes to salt stress in relation to stem cells and myoblasts. The common carp are subjected to salinity levels of 0.2, 6, and 10 ppt. The gill samples were preserved and prepared for TEM. RESULTS: The present study observed that telocytes undergo morphological change and exhibit enhanced secretory activities in response to changes in salinity. TEM can identify typical telocytes. This research gives evidence for the communication of telocytes with stem cells, myoblasts, and skeletal muscles. Telocytes surround stem cells. Telopodes made planar contact with the cell membrane of the stem cell. Telocytes and their telopodes surrounded the skeletal myoblast. These findings show that telocytes may act as nurse cells for skeletal stem cells and myoblasts, which undergo fibrillogenesis. Not only telocytes undergo morphological alternations, but also skeletal muscles become hypertrophied, which receive telocyte secretory vesicles in intercellular compartments. CONCLUSION: In conclusion, the activation of telocytes is what causes stress adaptation. They might act as important players in intercellular communication between cells. It is also possible that reciprocal interaction occurs between telocytes and other cells to adapt to changing environmental conditions.

Ultrastructure of macromolecular assemblies contributing to bacterial spore resistance revealed by in situ cryo-electron tomography.

Bacterial spores owe their incredible resistance capacities to molecular structures that protect the cell content from external aggressions. Among the determinants of resistance are the quaternary structure of the chromosome and an extracellular shell made of proteinaceous layers (the coat), the assembly of which remains poorly understood. Here, in situ cryo-electron tomography on lamellae generated by cryo-focused ion beam micromachining provides insights into the ultrastructural organization of Bacillus subtilis sporangia. The reconstructed tomograms reveal that early during sporulation, the chromosome in the forespore adopts a toroidal structure harboring 5.5-nm thick fibers. At the same stage, coat proteins at the surface of the forespore form a stack of amorphous or structured layers with distinct electron density, dimensions and organization. By analyzing mutant strains using cryo-electron tomography and transmission electron microscopy on resin sections, we distinguish seven nascent coat regions with different molecular properties, and propose a model for the contribution of coat morphogenetic proteins.