Thiorhodovibrio frisius and Trv. litoralis spp. nov., Two Novel Members from a Clade of Fastidious Purple Sulfur Bacteria That Exhibit Unique Red-Shifted Light-Harvesting Capabilities.

In the pursuit of cultivating anaerobic anoxygenic phototrophs with unusual absorbance spectra, a purple sulfur bacterium was isolated from the shoreline of Baltrum, a North Sea island of Germany. It was designated strain 970, due to a predominant light harvesting complex (LH) absorption maximum at 963-966 nm, which represents the furthest infrared-shift documented for such complexes containing bacteriochlorophyll a. A polyphasic approach to bacterial systematics was performed, comparing genomic, biochemical, and physiological properties. Strain 970 is related to Thiorhodovibrio winogradskyi DSM 6702T by 26.5, 81.9, and 98.0% similarity via dDDH, ANI, and 16S rRNA gene comparisons, respectively. The photosynthetic properties of strain 970 were unlike other Thiorhodovibrio spp., which contained typical LH absorbing characteristics of 800-870 nm, as well as a newly discovered absorption band at 908 nm. Strain 970 also had a different photosynthetic operon composition. Upon genomic comparisons with the original Thiorhodovibrio strains DSM 6702T and strain 06511, the latter was found to be divergent, with 25.3, 79.1, and 97.5% similarity via dDDH, ANI, and 16S rRNA gene homology to Trv. winogradskyi, respectively. Strain 06511 (=DSM 116345T) is thereby described as Thiorhodovibrio litoralis sp. nov., and the unique strain 970 (=DSM 111777T) as Thiorhodovibrio frisius sp. nov.

Evidence of a role for cAMP in mitochondrial regulation in ovarian granulosa cells.

In the ovary, proliferation and differentiation of granulosa cells (GCs) drive follicular growth. Our immunohistochemical study in a non-human primate, the Rhesus monkey, showed that the mitochondrial activity marker protein cytochrome c oxidase subunit 4 (COX4) increases in GCs in parallel to follicle size, and furthermore, its intracellular localization changes. This suggested that there is mitochondrial biogenesis and trafficking, and implicates the actions of gonadotropins, which regulate follicular growth and ovulation. Human KGN cells, i.e. granulosa tumour cells, were therefore used to study these possibilities. To robustly elevate cAMP, and thereby mimic the actions of gonadotropins, we used forskolin (FSK). FSK increased the cell size and the amount of mitochondrial DNA of KGN cells within 24 h. As revealed by MitoTracker™ experiments and ultrastructural 3D reconstruction, FSK treatment induced the formation of elaborate mitochondrial networks. H89, a protein kinase A (PKA) inhibitor, reduced the network formation. A proteomic analysis indicated that FSK elevated the levels of regulators of the cytoskeleton, among others (data available via ProteomeXchange with identifier PXD032160). The steroidogenic enzyme CYP11A1 (Cytochrome P450 Family 11 Subfamily A Member 1), located in mitochondria, was more than 3-fold increased by FSK, implying that the cAMP/PKA-associated structural changes occur in parallel with the acquisition of steroidogenic competence of mitochondria in KGN cells. In summary, the observations show increases in mitochondria and suggest intracellular trafficking of mitochondria in GCs during follicular growth, and indicate that they may partially be under the control of gonadotropins and cAMP. In line with this, increased cAMP in KGN cells profoundly affected mitochondrial dynamics in a PKA-dependent manner and implicated cytoskeletal changes.

Capillimicrobium parvum gen. nov., sp. nov., a novel representative of Capillimicrobiaceae fam. nov. within the order Solirubrobacterales, isolated from a grassland soil.

The order Solirubrobacterales is a deep-branching lineage within the phylum Actinomycetota. Most representatives have been isolated from terrestrial environments. A strain isolated from a grassland soil was found to be affiliated with this order and therefore characterized by a polyphasic approach. Cells of strain 0166_1T are Gram-positive, short rods, non-motile, non-spore-forming and divide by binary fission. A surface layer with protrusions covers the majority of the cells. Strain 0166_1T grows optimally around neutral to slightly alkaline pH (pH 7.1-7.9) and at temperatures between 24-36 °C in SSE/HD 1 : 10 medium. It grows optimally with 0-0.5% NaCl (w/v) but can withstand concentrations up to 5 %. The major fatty acids are C18 : 1 ω9c, C16 : 1 ω7c, C17 : 0 cyclo ω7c, C18 : 1 ω7c methyl and C19 : 0 cyclo ω9c. The major polar lipids are diphosphatidylglycerol, two unidentified phospholipids and one unidentified glycolipid. MK-7(H4) and MK-7(H2) are the predominant respiratory quinones. meso-2,6-Diaminopimelic acid is the diagnostic diamino acid in the cell-wall peptidoglycan. The G+C content for strain 0166_1T is 72.8 mol%. 16S rRNA gene sequence analysis indicated that this bacterium was related to Conexibacter arvalis KV-962T and Conexibacter stalactiti YC2-25T with 95.5 and 95.2 % sequence similarity, respectively. Based on the phenotypic, genomic and phylogenetic data, we propose the novel species Capillimicrobium parvum sp. nov. (type strain 0166_1T=DSM 104329T=LMG 29999T=CECT 9240T) of the novel genus Capillimicrobium gen. nov. within the novel family Capillimicrobiaceae fam. nov.

Changes in Envelope Structure and Cell-Cell Communication during Akinete Differentiation and Germination in Filamentous Cyanobacterium Trichormus variabilis ATCC 29413.

Planktonic freshwater filamentous cyanobacterium Trichormus variabilis ATCC 29413 (previously known as Anabaena variabilis) can differentiate heterocysts and akinetes to survive under different stress conditions. Whilst heterocysts enable diazotrophic growth, akinetes are spore-like resting cells that make the survival of the species possible under adverse growth conditions. Under suitable environmental conditions, they germinate to produce new vegetative filaments. Several morphological and physiological changes occur during akinete formation and germination. Here, using scanning electron microscopy (SEM), we found that the mature akinetes had a wrinkled envelope, and the surface of the envelope smoothened as the cell size increased during germination. Thereupon, the akinete envelope ruptured to release the short emerging filament. Focused ion beam-scanning electron microscopy (FIB/SEM) tomography of immature akinetes revealed the presence of cytoplasmic granules, presumably consisting of cyanophycin or glycogen. In addition, the akinete envelope architecture of different layers, the exopolysaccharide and glycolipid layers, could be visualized. We found that this multilayered envelope helped to withstand osmotic stress and to maintain the structural integrity. Furthermore, by fluorescence recovery after photobleaching (FRAP) measurements, using the fluorescent tracer calcein, we found that intercellular communication decreased during akinete formation as compared with the vegetative cells. In contrast, freshly germinating filaments restored cell communication.

In situ visualization of a simple bipartite kinetochore with a single microtubule attachment in Giardia intestinalis (Metamonada).

To understand general features in evolution of kinetochore organization, investigating a wide range of mitotic mechanisms in various non-model eukaryotes is necessary. A binucleate flagellate Giardia intestinalis is a representative of highly divergent eukaryotic lineage of Metamonads. FIB/SEM tomography was used to investigate ultrastructural details of its mitotic architecture, including kinetochores. Giardia undergoes semi-open mitosis, with the nuclear envelope remaining intact except for polar fenestrae, allowing microtubules to enter the nucleoplasm. At the onset of mitosis, the nuclear envelope bends inward, forming a concave depression at the spindle poles. Spindle microtubules emanate from a cytoplasmic fuzzy microtubule organizing center near the flagellar basal bodies. Kinetochoral microtubules enter the nucleoplasm and bind to kinetochores. A small bipartite kinetochore composed of a dense inner disk, approximately 46 nm in diameter, and a two-armed outer fork, is attached to just one microtubule. To our knowledge, this is the first in situ evidence of a one-microtubule attachment to a kinetochore, which could represent a basic eukaryotic situation.

Inheritance of the reduced mitochondria of Giardia intestinalis is coupled to the flagellar maturation cycle.

BACKGROUND: The presence of mitochondria is a distinguishing feature between prokaryotic and eukaryotic cells. It is currently accepted that the evolutionary origin of mitochondria coincided with the formation of eukaryotes and from that point control of mitochondrial inheritance was required. Yet, the way the mitochondrial presence has been maintained throughout the eukaryotic cell cycle remains a matter of study. Eukaryotes control mitochondrial inheritance mainly due to the presence of the genetic component; still only little is known about the segregation of mitochondria to daughter cells during cell division. Additionally, anaerobic eukaryotic microbes evolved a variety of genomeless mitochondria-related organelles (MROs), which could be theoretically assembled de novo, providing a distinct mechanistic basis for maintenance of stable mitochondrial numbers. Here, we approach this problem by studying the structure and inheritance of the protist Giardia intestinalis MROs known as mitosomes. RESULTS: We combined 2D stimulated emission depletion (STED) microscopy and focused ion beam scanning electron microscopy (FIB/SEM) to show that mitosomes exhibit internal segmentation and conserved asymmetric structure. From a total of about forty mitosomes, a small, privileged population is harnessed to the flagellar apparatus, and their life cycle is coordinated with the maturation cycle of G. intestinalis flagella. The orchestration of mitosomal inheritance with the flagellar maturation cycle is mediated by a microtubular connecting fiber, which physically links the privileged mitosomes to both axonemes of the oldest flagella pair and guarantees faithful segregation of the mitosomes into the daughter cells. CONCLUSION: Inheritance of privileged Giardia mitosomes is coupled to the flagellar maturation cycle. We propose that the flagellar system controls segregation of mitochondrial organelles also in other members of this supergroup (Metamonada) of eukaryotes and perhaps reflects the original strategy of early eukaryotic cells to maintain this key organelle before mitochondrial fusion-fission dynamics cycle as observed in Metazoa was established.

Usitatibacter rugosus gen. nov., sp. nov. and Usitatibacter palustris sp. nov., novel members of Usitatibacteraceae fam. nov. within the order Nitrosomonadales isolated from soil.

Members of the metabolically diverse order Nitrosomonadales inhabit a wide range of environments. Two strains affiliated with this order were isolated from soils in Germany and characterized by a polyphasic approach. Cells of strains 0125_3T and Swamp67T are Gram-negative rods, non-motile, non-spore-forming, non-capsulated and divide by binary fission. They tested catalase-negative, but positive for cytochrome c-oxidase. Both strains form small white colonies on agar plates and grow aerobically and chemoorganotrophically on SSE/HD 1 : 10 medium, preferably utilizing organic acids and proteinaceous substrates. Strains 0125_3T and Swamp67T are mesophilic and grow optimally without NaCl addition at slightly alkaline conditions. Major fatty acids are C16 : 1 ω7c, C16 : 0 and C14 : 0. The major polar lipids are diphosphatidylglycerol, phosphatidylethanolamine and phosphatidyglycerol. The predominant respiratory quinone is Q-8. The G+C content for 0125_3T and Swamp67T was 67 and 66.1 %, respectively. The 16S rRNA gene analysis indicated that the closest relatives (<91 % sequence similarity) of strain 0125_3T were Nitrosospira multiformis ATCC 25196T, Methyloversatilis universalis FAM5T and Denitratisoma oestradiolicum AcBE2-1T, while Nitrosospira multiformis ATCC 25196T, Nitrosospira tenuis Nv1T and Nitrosospira lacus APG3T were closest to strain Swamp67T. The two novel strains shared 97.4 % 16S rRNA gene sequence similarity with one another and show low average nucleotide identity of their genomes (83.8 %). Based on the phenotypic, chemotaxonomic, genomic and phylogenetic analysis, we propose the two novel species Usitatibacter rugosus sp. nov (type strain 0125_3T=DSM 104443T=LMG 29998T=CECT 9241T) and Usitatibacter palustris sp. nov. (type strain Swamp67T=DSM 104440T=LMG 29997T=CECT 9242T) of the novel genus Usitatibacter gen. nov., within the novel family Usitatibacteraceae fam. nov.

FIB-SEM-based analysis of Borrelia intracellular processing by human macrophages.

Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic disorder affecting primarily skin, joints and nervous system. Successful internalization and intracellular processing of borreliae by immune cells, like macrophages, is decisive for the outcome of a respective infection. Here, we use, for the first time, focused ion beam scanning electron microscopy tomography (FIB-SEM tomography) to visualize the interaction of borreliae with primary human macrophages with high resolution. We report that interaction between macrophages and the elongated and highly motile borreliae can lead to formation of membrane tunnels that extend deeper into the host cytoplasm than the actual phagosome, most probably as a result of partial extrication of captured borreliae. We also show that membrane tubulation at borreliae-containing phagosomes, a process suggested earlier as a mechanism leading to phagosome compaction but hard to visualize in live-cell imaging, is apparently a frequent phenomenon. Finally, we demonstrate that the endoplasmic reticulum (ER) forms multiple STIM1-positive contact sites with both membrane tunnels and phagosome tubulations, confirming the important role of the ER during uptake and intracellular processing of borreliae.

Fusion of Mitochondria to 3-D Networks, Autophagy and Increased Organelle Contacts are Important Subcellular Hallmarks during Cold Stress in Plants.

Low temperature stress has a severe impact on the distribution, physiology, and survival of plants in their natural habitats. While numerous studies have focused on the physiological and molecular adjustments to low temperatures, this study provides evidence that cold induced physiological responses coincide with distinct ultrastructural alterations. Three plants from different evolutionary levels and habitats were investigated: The freshwater alga Micrasterias denticulata, the aquatic plant Lemna sp., and the nival plant Ranunculus glacialis. Ultrastructural alterations during low temperature stress were determined by the employment of 2-D transmission electron microscopy and 3-D reconstructions from focused ion beam-scanning electron microscopic series. With decreasing temperatures, increasing numbers of organelle contacts and particularly the fusion of mitochondria to 3-dimensional networks were observed. We assume that the increase or at least maintenance of respiration during low temperature stress is likely to be based on these mitochondrial interconnections. Moreover, it is shown that autophagy and degeneration processes accompany freezing stress in Lemna and R. glacialis. This might be an essential mechanism to recycle damaged cytoplasmic constituents to maintain the cellular metabolism during freezing stress.

Cell Wall Reinforcements Accompany Chilling and Freezing Stress in the Streptophyte Green Alga Klebsormidium crenulatum.

Adaptation strategies in freezing resistance were investigated in Klebsormidium crenulatum, an early branching streptophyte green alga related to higher plants. Klebsormidium grows naturally in unfavorable environments like alpine biological soil crusts, exposed to desiccation, high irradiation and cold stress. Here, chilling and freezing induced alterations of the ultrastructure were investigated. Control samples (kept at 20°C) were compared to chilled (4°C) as well as extracellularly frozen algae (-2 and -4°C). A software-controlled laboratory freezer (AFU, automatic freezing unit) was used for algal exposure to various temperatures and freezing was manually induced. Samples were then high pressure frozen and cryo-substituted for electron microscopy. Control cells had a similar appearance in size and ultrastructure as previously reported. While chilling stressed algae only showed minor ultrastructural alterations, such as small inward facing cell wall plugs and minor alterations of organelles, drastic changes of the cell wall and in organelle distribution were found in extracellularly frozen samples (-2°C and -4°C). In frozen samples, the cytoplasm was not retracted from the cell wall, but extensive three-dimensional cell wall layers were formed, most prominently in the corners of the cells, as determined by FIB-SEM and TEM tomography. Similar alterations/adaptations of the cell wall were not reported or visualized in Klebsormidium before, neither in controls, nor during other stress scenarios. This indicates that the cell wall is reinforced by these additional wall layers during freezing stress. Cells allowed to recover from freezing stress (-2°C) for 5 h at 20°C lost these additional cell wall layers, suggesting their dynamic formation. The composition of these cell wall reinforcement areas was investigated by immuno-TEM. In addition, alterations of structure and distribution of mitochondria, dictyosomes and a drastically increased endoplasmic reticulum were observed in frozen cells by TEM and TEM tomography. Measurements of the photosynthetic oxygen production showed an acclimation of Klebsormidium to chilling stress, which correlates with our findings on ultrastructural alterations of morphology and distribution of organelles. The cell wall reinforcement areas, together with the observed changes in organelle structure and distribution, are likely to contribute to maintenance of an undisturbed cell physiology and to adaptation to chilling and freezing stress.

A rapid workflow for the characterization of small numbers of unicellular eukaryotes by using correlative light and electron microscopy.

The limited availability of biological samples hinders phylogenetic efforts to define structural differences among various biological groups. A novel workflow enabling the analysis of protists in low cell numbers by electron microscopy (EM) is described with cysts of Giardia intestinalis, a single-celled eukaryotic parasite. Correlative light and electron microscopy (CLEM) allows for the selection of individual cells and is economical in terms of time and cost. We describe a cyst purification protocol in combination with an adhesive coating for fixation and ultrathin embedding that results in excellent preservation of cell morphology. The application of advanced structural and analytical EM methods, such as high-resolution field emission scanning electron microscopy (FESEM), focused ion beam tomography (FIB/SEM), and energy-dispersive X-ray spectroscopy (EDX) analysis, is demonstrated. The workflow represents a new approach for studying the cellular and organelle architecture of rare and "difficult to culture" microorganisms.

Lack of FIBRILLIN6 in Arabidopsis thaliana affects light acclimation and sulfate metabolism.

Arabidopsis thaliana contains 13 fibrillins (FBNs), which are all localized to chloroplasts. FBN1 and FBN2 are involved in photoprotection of photosystem II, and FBN4 and FBN5 are thought to be involved in plastoquinone transport and biosynthesis, respectively. The functions of the other FBNs remain largely unknown. To gain insight into the function of FBN6, we performed coexpression and Western analyses, conducted fluorescence and transmission electron microscopy, stained reactive oxygen species (ROS), measured photosynthetic parameters and glutathione levels, and applied transcriptomics and metabolomics. Using coexpression analyses, FBN6 was identified as a photosynthesis-associated gene. FBN6 is localized to thylakoid and envelope membranes, and its knockout results in stunted plants. The delayed-growth phenotype cannot be attributed to altered basic photosynthesis parameters or a reduced CO2 assimilation rate. Under moderate light stress, primary leaves of fbn6 plants begin to bleach and contain enlarged plastoglobules. RNA sequencing and metabolomics analyses point to an alteration in sulfate reduction in fbn6. Indeed, glutathione content is higher in fbn6, which in turn confers cadmium tolerance of fbn6 seedlings. We conclude that loss of FBN6 leads to perturbation of ROS homeostasis. FBN6 enables plants to cope with moderate light stress and affects cadmium tolerance.

Insights into replicative senescence of human testicular peritubular cells.

There is evidence for an age-related decline in male reproductive functions, yet how the human testis may age is not understood. Human testicular peritubular cells (HTPCs) transport sperm, contribute to the spermatogonial stem cell (SSC) niche and immune surveillance, and can be isolated and studied in vitro. Consequences of replicative senescence of HTPCs were evaluated to gain partial insights into human testicular aging. To this end, early and advanced HTPC passages, in which replicative senescence was indicated by increased cell size, altered nuclear morphology, enhanced ß-galactosidase activity, telomere attrition and reduced mitochondrial DNA (mtDNA), were compared. These alterations are typical for senescent cells, in general. To examine HTPC-specific changes, focused ion beam scanning electron microscopy (FIB/SEM) tomography was employed, which revealed a reduced mitochondrial network and an increased lysosome population. The results coincide with the data of a parallel proteomic analysis and indicate deranged proteostasis. The mRNA levels of typical contractility markers and growth factors, important for the SSC niche, were not significantly altered. A secretome analysis identified, however, elevated levels of macrophage migration inhibitory factor (MIF) and dipeptidyl peptidase 4 (DPP4), which may play a role in spermatogenesis. Testicular DPP4 may further represent a possible drug target.

Halobacterium salinarum virus ChaoS9, a Novel Halovirus Related to PhiH1 and PhiCh1.

The unexpected lysis of a large culture of Halobacterium salinarum strain S9 was found to be caused by a novel myovirus, designated ChaoS9. Virus purification from the culture lysate revealed a homogeneous population of caudovirus-like particles. The viral genome is linear, dsDNA that is partially redundant and circularly permuted, has a unit length of 55,145 nt, a G + C% of 65.3, and has 85 predicted coding sequences (CDS) and one tRNA (Arg) gene. The left arm of the genome (0⁻28 kbp) encodes proteins similar in sequence to those from known caudoviruses and was most similar to myohaloviruses phiCh1 (host: Natrialbamagadii) and phiH1 (host: Hbt. salinarum). It carries a tail-fiber gene module similar to the invertible modules present in phiH1 and phiCh1. However, while the tail genes of ChaoS9 were similar to those of phiCh1 and phiH1, the Mcp of ChaoS9 was most similar (36% aa identity) to that of Haloarcula hispanica tailed virus 1 (HHTV-1). Provirus elements related to ChaoS9 showed most similarity to tail/assembly proteins but varied in their similarity with head/assembly proteins. The right arm (29⁻55 kbp) of ChaoS9 encoded proteins involved in DNA replication (ParA, RepH, and Orc1) but the other proteins showed little similarity to those from phiH1, phiCh1, or provirus elements, and most of them could not be assigned a function. ChaoS9 is probably best classified within the genus Myohalovirus, as it shares many characteristics with phiH1 (and phiCh1), including many similar proteins. However, the head/assembly gene region appears to have undergone a recombination event, and the inferred proteins are different to those of phiH1 and phiCh1, including the major capsid protein. This makes the taxonomic classification of ChaoS9 more ambiguous. We also report a revised genome sequence and annotation of Natrialba virus phiCh1.

Engineering extracellular vesicles as novel treatment options: exploiting herpesviral immunity in CLL.

Extracellular vesicles (EVs) are important mediators of cell-cell communication. Intriguingly, EVs can be engineered and thus exploited for the targeted transfer of functional proteins of interest. Thus, engineered EVs may constitute attractive tools for the development of novel therapeutic interventions, like cancer immunotherapies, vaccinations or targeted drug delivery. Here, we describe a novel experimental immunotherapeutic approach for the adjuvant treatment of chronic lymphocytic leukaemia (CLL) based on engineered EVs carrying gp350, the major glycoprotein of Epstein-Barr virus (EBV), CD40L, a central immune accessory molecule and pp65, an immunodominant antigen of the human cytomegalovirus (CMV). We show that these engineered EVs specifically interact with malignant B cells from CLL patients and render these cells immunogenic to allogeneic and autologous EBV- and CMV-specific CD4+ and CD8+ T cells. Collectively, co-opting engineered EVs to re-target the strong herpesviral immunity in CLL patients to malignant cells constitutes an attractive strategy for the adjuvant treatment of a still incurable disease. Abbreviations: CLL: chronic lymphocytic leukaemia; EBV: Epstein-Barr virus; CMV: cytomegalovirus.

In vivo Ca2+ imaging of astrocytic microdomains reveals a critical role of the amyloid precursor protein for mitochondria.

The investigation of amyloid precursor protein (APP) has been mainly confined to its neuronal functions, whereas very little is known about its physiological role in astrocytes. Astrocytes exhibit a particular morphology with slender extensions protruding from somata and primary branches. Along these fine extensions, spontaneous calcium transients occur in spatially restricted microdomains. Within these microdomains mitochondria are responsible for local energy supply and Ca2+ buffering. Using two-photon in vivo Ca2+ imaging, we report a significant decrease in the density of active microdomains, frequency of spontaneous Ca2+ transients and slower Ca2+ kinetics in mice lacking APP. Mechanistically, these changes could be potentially linked to mitochondrial malfunction as our in vivo and in vitro data revealed severe, APP-dependent structural mitochondrial fragmentation in astrocytes. Functionally, such mitochondria exhibited prolonged kinetics and morphology dependent signal size of ATP-induced Ca2+ transients. Our results highlight a prominent role of APP in the modulation of Ca2+ activity in astrocytic microdomains whose precise functioning is crucial for the reinforcement and modulation of synaptic function. This study provides novel insights in APP physiological functions which are important for the understanding of the effects of drugs validated in Alzheimer's disease treatment that affect the function of APP.

Independent yet overlapping pathways ensure the robustness and responsiveness of trans-Golgi network functions in Arabidopsis.

The trans-Golgi-network (TGN) has essential housekeeping functions in secretion, endocytosis and protein sorting, but also more specialized functions in plant development. How the robustness of basal TGN function is ensured while specialized functions are differentially regulated is poorly understood. Here, we investigate two key regulators of TGN structure and function, ECHIDNA and the Transport Protein Particle II (TRAPPII) tethering complex. An analysis of physical, network and genetic interactions suggests that two network communities are implicated in TGN function and that ECHIDNA and TRAPPII belong to distinct yet overlapping pathways. Whereas ECHIDNA and TRAPPII colocalized at the TGN in interphase cells, their localization diverged in dividing cells. Moreover, ECHIDNA and TRAPPII localization patterns were mutually independent. TGN structure, endocytosis and sorting decisions were differentially impacted in echidna and trappii mutants. Our analyses point to a partitioning of specialized TGN functions, with ECHIDNA being required for cell elongation and TRAPPII for cytokinesis. Two independent pathways able to compensate for each other might contribute to the robustness of TGN housekeeping functions and to the responsiveness and fine tuning of its specialized functions.

From Light Microscopy to Analytical Scanning Electron Microscopy (SEM) and Focused Ion Beam (FIB)/SEM in Biology: Fixed Coordinates, Flat Embedding, Absolute References.

Correlative light and electron microscopy (CLEM) has been in use for several years, however it has remained a costly method with difficult sample preparation. Here, we report a series of technical improvements developed for precise and cost-effective correlative light and scanning electron microscopy (SEM) and focused ion beam (FIB)/SEM microscopy of single cells, as well as large tissue sections. Customized coordinate systems for both slides and coverslips were established for thin and ultra-thin embedding of a wide range of biological specimens. Immobilization of biological samples was examined with a variety of adhesives. For histological sections, a filter system for flat embedding was developed. We validated ultra-thin embedding on laser marked slides for efficient, high-resolution CLEM. Target cells can be re-located within minutes in SEM without protracted searching and correlative investigations were reduced to a minimum of preparation steps, while still reaching highest resolution. The FIB/SEM milling procedure is facilitated and significantly accelerated as: (i) milling a ramp becomes needless, (ii) significant re-deposition of milled material does not occur; and (iii) charging effects are markedly reduced. By optimizing all technical parameters FIB/SEM stacks with 2 nm iso-voxels were achieved over thousands of sections, in a wide range of biological samples.

Label-free 3D-CLEM Using Endogenous Tissue Landmarks.

Emerging 3D correlative light and electron microscopy approaches enable studying neuronal structure-function relations at unprecedented depth and precision. However, established protocols for the correlation of light and electron micrographs rely on the introduction of artificial fiducial markers, such as polymer beads or near-infrared brandings, which might obscure or even damage the structure under investigation. Here, we report a general applicable "flat embedding" preparation, enabling high-precision overlay of light and scanning electron micrographs, using exclusively endogenous landmarks in the brain: blood vessels, nuclei, and myelinated axons. Furthermore, we demonstrate feasibility of the workflow by combining in vivo 2-photon microscopy and focused ion beam scanning electron microscopy to dissect the role of astrocytic coverage in the persistence of dendritic spines.

Correction to: Precise and economic FIB/SEM for CLEM: with 2 nm voxels through mitosis.

Unfortunately, part of the legend to Fig. 6 has been incorrectly published.