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Single-molecule (SM) fluorescence microscopy allows the imaging of biomolecules in cultured cells with a precision of a few nanometres but has yet to be implemented in living adult animals. Here we used split-GFP (green fluorescent protein) fusions and complementation-activated light microscopy (CALM) for subresolution imaging of individual membrane proteins in live Caenorhabditis elegans (C. elegans). In vivo tissue-specific SM tracking of transmembrane CD4 and voltage-dependent Ca(2+) channels (VDCC) was achieved with a precision of 30 nm within neuromuscular synapses and at the surface of muscle cells in normal and dystrophin-mutant worms. Through diffusion analyses, we reveal that dystrophin is involved in modulating the confinement of VDCC within sarcolemmal membrane nanodomains in response to varying tonus of C. elegans body-wall muscles. CALM expands the applications of SM imaging techniques beyond the petri dish and opens the possibility to explore the molecular basis of homeostatic and pathological cellular processes with subresolution precision, directly in live animals.
Background
Autophagy participates in innate immunity by eliminating intracellular pathogens. Consequently, numerous microorganisms have developed strategies to impair the autophagic machinery in phagocytes. In the current study, interactions between Leishmania major (L. m.) and the autophagic machinery of bone marrow-derived macrophages (BMDM) were analyzed.
Methods
BMDM were generated from BALB/c mice, and the cells were infected with L. m. promastigotes. Transmission electron microscopy (TEM) and electron tomography were used to investigate the ultrastructure of BMDM and the intracellular parasites. Affymetrix® chip analyses were conducted to identify autophagy-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The protein expression levels of autophagy related 5 (ATG5), BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), cathepsin E (CTSE), mechanistic target of rapamycin (MTOR), microtubule-associated proteins 1A/1B light chain 3B (LC3B), and ubiquitin (UB) were investigated through western blot analyses. BMDM were transfected with specific small interfering RNAs (siRNAs) against autophagy-related genes and with mimics or inhibitors of autophagy-associated miRNAs. The infection rates of BMDM were determined by light microscopy after a parasite-specific staining.
Results
The experiments demonstrated autophagy induction in BMDM after in vitro infection with L. m.. The results suggested a putative MTOR phosphorylation-dependent counteracting mechanism in the early infection phase and indicated that intracellular amastigotes were cleared by autophagy in BMDM in the late infection phase. Transcriptomic analyses and specific downregulation of protein expression with siRNAs suggested there is an association between the infection-specific over expression of BNIP3, as well as CTSE, and the autophagic activity of BMDM. Transfection with mimics of mmu-miR-101c and mmu-miR-129-5p, as well as with an inhibitor of mmu-miR-210-5p, demonstrated direct effects of the respective miRNAs on parasite clearance in L. m.-infected BMDM. Furthermore, Affymetrix® chip analyses revealed a complex autophagy-related RNA network consisting of differentially expressed mRNAs and miRNAs in BMDM, which indicates high glycolytic and inflammatory activity in the host macrophages.
Conclusions
Autophagy in L. m.-infected host macrophages is a highly regulated cellular process at both the RNA level and the protein level. Autophagy has the potential to clear parasites from the host. The results obtained from experiments with murine host macrophages could be translated in the future to develop innovative and therapeutic antileishmanial strategies for human patients.
Fish otoliths, biominerals composed of calcium carbonate with a small amount of organic matrix, are involved in the functioning of the inner ear. Starmaker (Stm) from zebrafish (Danio rerio) was the first protein found to be capable of controlling the formation of otoliths. Recently, a gene was identified encoding the Starmaker-like (Stm-l) protein from medaka (Oryzias latipes), a putative homologue of Stm and human dentine sialophosphoprotein. Although there is no sequence similarity between Stm-l and Stm, Stm-l was suggested to be involved in the biomineralization of otoliths, as had been observed for Stm even before. The molecular properties and functioning of Stm-l as a putative regulatory protein in otolith formation have not been characterized yet. A comprehensive biochemical and biophysical analysis of recombinant Stm-l, along with in silico examinations, indicated that Stm-l exhibits properties of a coil-like intrinsically disordered protein. Stm-l possesses an elongated and pliable structure that is able to adopt a more ordered and rigid conformation under the influence of different factors. An in vitro assay of the biomineralization activity of Stm-l indicated that Stm-l affected the size, shape and number of calcium carbonate crystals. The functional significance of intrinsically disordered properties of Stm-l and the possible role of this protein in controlling the formation of calcium carbonate crystals is discussed.
Ultrastructural analysis of wild-type and RIM1α knockout active zones in a large cortical synapse
(2022)
Rab3A-interacting molecule (RIM) is crucial for fast Ca\(^{2+}\)-triggered synaptic vesicle (SV) release in presynaptic active zones (AZs). We investigated hippocampal giant mossy fiber bouton (MFB) AZ architecture in 3D using electron tomography of rapid cryo-immobilized acute brain slices in RIM1α\(^{−/−}\) and wild-type mice. In RIM1α\(^{−/−}\), AZs are larger with increased synaptic cleft widths and a 3-fold reduced number of tightly docked SVs (0–2 nm). The distance of tightly docked SVs to the AZ center is increased from 110 to 195 nm, and the width of their electron-dense material between outer SV membrane and AZ membrane is reduced. Furthermore, the SV pool in RIM1α\(^{−/−}\) is more heterogeneous. Thus, RIM1α, besides its role in tight SV docking, is crucial for synaptic architecture and vesicle pool organization in MFBs.
Stalk cell polar ion transport provide for bladder‐based salinity tolerance in Chenopodium quinoa
(2022)
Chenopodium quinoa uses epidermal bladder cells (EBCs) to sequester excess salt. Each EBC complex consists of a leaf epidermal cell, a stalk cell, and the bladder.
Under salt stress, sodium (Na\(^{+}\)), chloride (Cl\(^{−}\)), potassium (K\(^{+}\)) and various metabolites are shuttled from the leaf lamina to the bladders. Stalk cells operate as both a selectivity filter and a flux controller.
In line with the nature of a transfer cell, advanced transmission electron tomography, electrophysiology, and fluorescent tracer flux studies revealed the stalk cell’s polar organization and bladder‐directed solute flow.
RNA sequencing and cluster analysis revealed the gene expression profiles of the stalk cells. Among the stalk cell enriched genes, ion channels and carriers as well as sugar transporters were most pronounced. Based on their electrophysiological fingerprint and thermodynamic considerations, a model for stalk cell transcellular transport was derived.
Automatic image reconstruction is critical to cope with steadily increasing data from advanced microscopy. We describe here the Fiji macro 3D ART VeSElecT which we developed to study synaptic vesicles in electron tomograms. We apply this tool to quantify vesicle properties (i) in embryonic Danio rerio 4 and 8 days past fertilization (dpf) and (ii) to compare Caenorhabditis elegans N2 neuromuscular junctions (NMJ) wild-type and its septin mutant (unc-59(e261)). We demonstrate development-specific and mutant-specific changes in synaptic vesicle pools in both models. We confirm the functionality of our macro by applying our 3D ART VeSElecT on zebrafish NMJ showing smaller vesicles in 8 dpf embryos then 4 dpf, which was validated by manual reconstruction of the vesicle pool. Furthermore, we analyze the impact of C. elegans septin mutant unc-59(e261) on vesicle pool formation and vesicle size. Automated vesicle registration and characterization was implemented in Fiji as two macros (registration and measurement). This flexible arrangement allows in particular reducing false positives by an optional manual revision step. Preprocessing and contrast enhancement work on image-stacks of 1nm/pixel in x and y direction. Semi-automated cell selection was integrated. 3D ART VeSElecT removes interfering components, detects vesicles by 3D segmentation and calculates vesicle volume and diameter (spherical approximation, inner/outer diameter). Results are collected in color using the RoiManager plugin including the possibility of manual removal of non-matching confounder vesicles. Detailed evaluation considered performance (detected vesicles) and specificity (true vesicles) as well as precision and recall. We furthermore show gain in segmentation and morphological filtering compared to learning based methods and a large time gain compared to manual segmentation. 3D ART VeSElecT shows small error rates and its speed gain can be up to 68 times faster in comparison to manual annotation. Both automatic and semi-automatic modes are explained including a tutorial.
The diffraction limit of light confines fluorescence imaging of subcellular structures in fungi. Different super-resolution methods are available for the analysis of fungi that we briefly discuss. We exploit the filamentous fungus Fusarium fujikuroi expressing a YFP-labeled membrane protein showing the benefit of correlative light- and electron microscopy (CLEM), that combines structured illumination microscopy (SIM) and scanning election microscopy (SEM).
The cell wall synthesis pathway producing peptidoglycan is a highly coordinated and tightly regulated process. Although the major components of bacterial cell walls have been known for decades, the complex regulatory network controlling peptidoglycan synthesis and many details of the cell division machinery are not well understood. The eukaryotic-like serine/threonine kinase Stk and the cognate phosphatase Stp play an important role in cell wall biosynthesis and drug resistance in S. aureus. We show that stp deletion has a pronounced impact on cell wall synthesis. Deletion of stp leads to a thicker cell wall and decreases susceptibility to lysostaphin. Stationary phase Δstp cells accumulate peptidoglycan precursors and incorporate higher amounts of incomplete muropeptides with non-glycine, monoglycine and monoalanine interpeptide bridges into the cell wall. In line with this cell wall phenotype, we demonstrate that the lipid II:glycine glycyltransferase FemX can be phosphorylated by the Ser/Thr kinase Stk in vitro. Mass spectrometric analyses identify Thr32, Thr36 and Ser415 as phosphoacceptors. The cognate phosphatase Stp dephosphorylates these phosphorylation sites. Moreover, Stk interacts with FemA and FemB, but is unable to phosphorylate them. Our data indicate that Stk and Stp modulate cell wall synthesis and cell division at several levels.
Assigning functions to uncultivated environmental microorganisms continues to be a challenging endeavour. Here, we present a new microscopy protocol for fluorescence in situ hybridisation-correlative light and electron microscopy (FISH-CLEM) that enabled, to our knowledge for the first time, the identification of single cells within their complex microenvironment at electron microscopy resolution. Members of the candidate phylum Poribacteria, common and uncultivated symbionts of marine sponges, were used towards this goal. Cellular 3D reconstructions revealed bipolar, spherical granules of low electron density, which likely represent carbon reserves. Poribacterial activity profiles were retrieved from prokaryotic enriched sponge metatranscriptomes using simulation-based optimised mapping. We observed high transcriptional activity for proteins related to bacterial microcompartments (BMC) and we resolved their subcellular localisation by combining FISH-CLEM with immunohistochemistry (IHC) on ultra-thin sponge tissue sections. In terms of functional relevance, we propose that the BMC-A region may be involved in 1,2-propanediol degradation. The FISH-IHC-CLEM approach was proven an effective toolkit to combine -omics approaches with functional studies and it should be widely applicable in environmental microbiology.
Highlights
• Dopamine receptor-1 activation induces TrkB cell-surface expression in striatal neurons
• Dopaminergic deficits cause TrkB accumulation and clustering in the ER
• TrkB clusters colocalize with cargo receptor SORCS-2 in direct pathway striatal neurons
• Intracellular TrkB clusters fail to fuse with lysosomes after dopamine depletion
Summary
Disturbed motor control is a hallmark of Parkinson’s disease (PD). Cortico-striatal synapses play a central role in motor learning and adaption, and brain-derived neurotrophic factor (BDNF) from cortico-striatal afferents modulates their plasticity via TrkB in striatal medium spiny projection neurons (SPNs). We studied the role of dopamine in modulating the sensitivity of direct pathway SPNs (dSPNs) to BDNF in cultures of fluorescence-activated cell sorting (FACS)-enriched D1-expressing SPNs and 6-hydroxydopamine (6-OHDA)-treated rats. DRD1 activation causes enhanced TrkB translocation to the cell surface and increased sensitivity for BDNF. In contrast, dopamine depletion in cultured dSPN neurons, 6-OHDA-treated rats, and postmortem brain of patients with PD reduces BDNF responsiveness and causes formation of intracellular TrkB clusters. These clusters associate with sortilin related VPS10 domain containing receptor 2 (SORCS-2) in multivesicular-like structures, which apparently protects them from lysosomal degradation. Thus, impaired TrkB processing might contribute to disturbed motor function in PD.
A fine balance of regulatory (T\(_{reg}\)) and conventional CD4\(^+\) T cells (T\(_{conv}\)) is required to prevent harmful immune responses, while at the same time ensuring the development of protective immunity against pathogens. As for many cellular processes, sphingolipid metabolism also crucially modulates the T\(_{reg}\)/T\(_{conv}\) balance. However, our understanding of how sphingolipid metabolism is involved in T cell biology is still evolving and a better characterization of the tools at hand is required to advance the field. Therefore, we established a reductionist liposomal membrane model system to imitate the plasma membrane of mouse T\(_{reg}\) and T\(_{conv}\) with regards to their ceramide content. We found that the capacity of membranes to incorporate externally added azide-functionalized ceramide positively correlated with the ceramide content of the liposomes. Moreover, we studied the impact of the different liposomal preparations on primary mouse splenocytes in vitro. The addition of liposomes to resting, but not activated, splenocytes maintained viability with liposomes containing high amounts of C\(_{16}\)-ceramide being most efficient. Our data thus suggest that differences in ceramide post-incorporation into T\(_{reg}\) and T\(_{conv}\) reflect differences in the ceramide content of cellular membranes.
Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridization (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs. To overcome these limitations, we have hybridized slices of high pressure frozen, freeze-substituted and LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localization that can be complemented with immunofluorescence and electron microscopy, as well as array tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA structures, and we show that the method can be employed to determine mRNA compactness. We apply the method to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing in single-cellular trypanosomes and we show an example of differential gene expression in the metazoan Caenorhabditis elegans.
The amyotrophic lateral sclerosis (ALS) neurodegenerative disorder has been associated with multiple genetic lesions, including mutations in the gene for fused in sarcoma (FUS), a nuclear-localized RNA/DNA-binding protein. Neuronal expression of the pathological form of FUS proteins in Caenorhabditis elegans results in mislocalization and aggregation of FUS in the cytoplasm, and leads to impairment of motility. However, the mechanisms by which the mutant FUS disrupts neuronal health and function remain unclear. Here we investigated the impact of ALS-associated FUS on motor neuron health using correlative light and electron microscopy, electron tomography, and electrophysiology. We show that ectopic expression of wild-type or ALS-associated human FUS impairs synaptic vesicle docking at neuromuscular junctions. ALS-associated FUS led to the emergence of a population of large, electron-dense, and filament-filled endosomes. Electrophysiological recording revealed reduced transmission from motor neurons to muscles. Together, these results suggest a pathological effect of ALS-causing FUS at synaptic structure and function organization.
The Johnston's organ (JO) in the insect antenna is a multisensory organ involved in several navigational tasks including wind‐compass orientation, flight control, graviception, and, possibly, magnetoreception. Here we investigate the three dimensional anatomy of the JO and its neuronal projections into the brain of the desert ant Cataglyphis, a marvelous long‐distance navigator. The JO of C. nodus workers consists of 40 scolopidia comprising three sensory neurons each. The numbers of scolopidia slightly vary between different sexes (female/male) and castes (worker/queen). Individual scolopidia attach to the intersegmental membrane between pedicel and flagellum of the antenna and line up in a ring‐like organization. Three JO nerves project along the two antennal nerve branches into the brain. Anterograde double staining of the antennal afferents revealed that JO receptor neurons project to several distinct neuropils in the central brain. The T5 tract projects into the antennal mechanosensory and motor center (AMMC), while the T6 tract bypasses the AMMC via the saddle and forms collaterals terminating in the posterior slope (PS) (T6I), the ventral complex (T6II), and the ventrolateral protocerebrum (T6III). Double labeling of JO and ocellar afferents revealed that input from the JO and visual information from the ocelli converge in tight apposition in the PS. The general JO anatomy and its central projection patterns resemble situations in honeybees and Drosophila. The multisensory nature of the JO together with its projections to multisensory neuropils in the ant brain likely serves synchronization and calibration of different sensory modalities during the ontogeny of navigation in Cataglyphis.
Synaptic vesicles (SVs) are a key component of neuronal signaling and fulfil different roles depending on their composition. In electron micrograms of neurites, two types of vesicles can be distinguished by morphological criteria, the classical “clear core” vesicles (CCV) and the typically larger “dense core” vesicles (DCV), with differences in electron density due to their diverse cargos. Compared to CCVs, the precise function of DCVs is less defined. DCVs are known to store neuropeptides, which function as neuronal messengers and modulators [1]. In C. elegans, they play a role in locomotion, dauer formation, egg-laying, and mechano- and chemosensation [2]. Another type of DCVs, also referred to as granulated vesicles, are known to transport Bassoon, Piccolo and further constituents of the presynaptic density in the center of the active zone (AZ), and therefore are important for synaptogenesis [3].
To better understand the role of different types of SVs, we present here a new automated approach to classify vesicles. We combine machine learning with an extension of our previously developed vesicle segmentation workflow, the ImageJ macro 3D ART VeSElecT. With that we reliably distinguish CCVs and DCVs in electron tomograms of C. elegans NMJs using image-based features. Analysis of the underlying ground truth data shows an increased fraction of DCVs as well as a higher mean distance between DCVs and AZs in dauer larvae compared to young adult hermaphrodites. Our machine learning based tools are adaptable and can be applied to study properties of different synaptic vesicle pools in electron tomograms of diverse model organisms.
Many (poly‐)phenolic natural products, for example, curcumin and taxifolin, have been studied for their activity against specific hallmarks of neurodegeneration, such as amyloid‐β 42 (Aβ42) aggregation and neuroinflammation. Due to their drawbacks, arising from poor pharmacokinetics, rapid metabolism, and even instability in aqueous medium, the biological activity of azobenzene compounds carrying a pharmacophoric catechol group, which have been designed as bioisoteres of curcumin has been examined. Molecular simulations reveal the ability of these compounds to form a hydrophobic cluster with Aβ42, which adopts different folds, affecting the propensity to populate fibril‐like conformations. Furthermore, the curcumin bioisosteres exceeded the parent compound in activity against Aβ42 aggregation inhibition, glutamate‐induced intracellular oxidative stress in HT22 cells, and neuroinflammation in microglial BV‐2 cells. The most active compound prevented apoptosis of HT22 cells at a concentration of 2.5 μm (83 % cell survival), whereas curcumin only showed very low protection at 10 μm (21 % cell survival).
The bacterial endosymbiont Blochmannia floridanus of the carpenter ant Camponotus floridanus contributes to its hosts' ontogeny via nutritional upgrading during metamorphosis. This primary endosymbiosis is essential for both partners and vertical transmission of the endosymbionts is guaranteed by bacterial infestation of oocytes. Here we present a detailed analysis of the presence and localisation of B. floridanus in the ants' ovaries obtained by FISH and TEM analyses. The most apical part of the germarium harbouring germ-line stem cells (GSCs) is not infected by the bacteria. The bacteria are detectable for the first time in lower parts of the germarium when cystocytes undergo the 4th and 5th division and B. floridanus infects somatic cells lying under the basal lamina surrounding the ovarioles. With the beginning of cystocyte differentiation, the endosymbionts are exclusively transported from follicle cells into the growing oocytes. This infestation of the oocytes by bacteria very likely involves exocytosis endocytosis processes between follicle cells and the oocytes. Nurse cells were never found to harbour the endosymbionts. Furthermore we present first gene expression data in C floridanus ovaries. These data indicate a modulation of immune gene expression which may facilitate tolerance towards the endosymbionts and thus may contribute to their transovarial transmission.
Septins are a highly conserved family of small GTPases that form cytoskeletal filaments. Their cellular functions, especially in the nervous system, still remain largely enigmatic, but there are accumulating lines of evidence that septins play important roles in neuronal physiology and pathology. In order to further dissect septin function in the nervous system a detailed temporal resolved analysis in the genetically well tractable model vertebrate zebrafish (Danio rerio) is crucially necessary. To close this knowledge gap we here provide a reference dataset describing the expression of selected septins (sept3, sept5a and sept5b) in the zebrafish central nervous system. Strikingly, proliferation zones are devoid of expression of all three septins investigated, suggesting that they have a role in post-mitotic neural cells. Our finding that three septins are mainly expressed in non-proliferative regions was further confirmed by double-stainings with a proliferative marker. Our RNA in situ hybridization (ISH) study, detecting sept3, sept5a and sept5b mRNAs, shows that all three septins are expressed in largely overlapping regions of the developing brain. However, the expression of sept5a is much more confined compared to sept3 and sept5b. In contrast, the expression of all the three analyzed septins is largely similar in the adult brain.
Background
Bacterial meningitis is a life-threatening disease that occurs when pathogens such as Neisseria meningitidis cross the meningeal blood cerebrospinal fluid barrier (mBCSFB) and infect the meninges. Due to the human-specific nature of N. meningitidis, previous research investigating this complex host–pathogen interaction has mostly been done in vitro using immortalized brain endothelial cells (BECs) alone, which often do not retain relevant barrier properties in culture. Here, we developed physiologically relevant mBCSFB models using BECs in co-culture with leptomeningeal cells (LMCs) to examine N. meningitidis interaction.
Methods
We used BEC-like cells derived from induced pluripotent stem cells (iBECs) or hCMEC/D3 cells in co-culture with LMCs derived from tumor biopsies. We employed TEM and structured illumination microscopy to characterize the models as well as bacterial interaction. We measured TEER and sodium fluorescein (NaF) permeability to determine barrier tightness and integrity. We then analyzed bacterial adherence and penetration of the cell barrier and examined changes in host gene expression of tight junctions as well as chemokines and cytokines in response to infection.
Results
Both cell types remained distinct in co-culture and iBECs showed characteristic expression of BEC markers including tight junction proteins and endothelial markers. iBEC barrier function as determined by TEER and NaF permeability was improved by LMC co-culture and remained stable for seven days. BEC response to N. meningitidis infection was not affected by LMC co-culture. We detected considerable amounts of BEC-adherent meningococci and a relatively small number of intracellular bacteria. Interestingly, we discovered bacteria traversing the BEC-LMC barrier within the first 24 h post-infection, when barrier integrity was still high, suggesting a transcellular route for N. meningitidis into the CNS. Finally, we observed deterioration of barrier properties including loss of TEER and reduced expression of cell-junction components at late time points of infection.
Conclusions
Here, we report, for the first time, on co-culture of human iPSC derived BECs or hCMEC/D3 with meningioma derived LMCs and find that LMC co-culture improves barrier properties of iBECs. These novel models allow for a better understanding of N. meningitidis interaction at the mBCSFB in a physiologically relevant setting.
Sphingolipids, including ceramides, are a diverse group of structurally related lipids composed of a sphingoid base backbone coupled to a fatty acid side chain and modified terminal hydroxyl group. Recently, it has been shown that sphingolipids show antimicrobial activity against a broad range of pathogenic microorganisms. The antimicrobial mechanism, however, remains so far elusive. Here, we introduce 'click-AT-CLEM', a labeling technique for correlated light and electron microscopy (CLEM) based on the super-resolution array tomography (srAT) approach and bio-orthogonal click chemistry for imaging of azido-tagged sphingolipids to directly visualize their interaction with the model Gram-negative bacterium Neisseria meningitidis at subcellular level. We observed ultrastructural damage of bacteria and disruption of the bacterial outer membrane induced by two azido-modified sphingolipids by scanning electron microscopy and transmission electron microscopy. Click-AT-CLEM imaging and mass spectrometry clearly revealed efficient incorporation of azido-tagged sphingolipids into the outer membrane of Gram-negative bacteria as underlying cause of their antimicrobial activity.
Background
The plant endophytic fungus Serendipita indica colonizes roots of a wide range of plant species and can enhance growth and stress resistance of these plants. Due to its ease of axenic cultivation and its broad host plant range including the model plant Arabidopsis thaliana and numerous crop plants, it is widely used as a model fungus to study beneficial fungus-root interactions. In addition, it was suggested to be utilized for commercial applications, e.g. to enhance yield in barley and other species. To produce inoculum, S. indica is mostly cultivated in a complex Hill-Kafer medium (CM medium), however, growth in this medium is slow, and yield of chlamydospores, which are often used for plant root inoculation, is relatively low.
Results
We tested and optimized a simple vegetable juice-based medium for an enhanced yield of fungal inoculum. The described vegetable juice (VJ) medium is based on commercially available vegetable juice and is easy to prepare. VJ medium was superior to the currently used CM medium with respect to biomass production in liquid medium and hyphal growth on agar plates. Using solid VJ medium supplemented with sucrose (VJS), a high amount of chlamydospores developed already after 8 days of cultivation, producing significantly more spores than on CM medium. Use of VJ medium is not restricted to S. indica, as it also supported growth of two pathogenic fungi often used in plant pathology experiments: the ascomycete Fusarium graminearum, the causal agent of Fusarium head blight disease on wheat and barley, and Verticillium longisporum, the causal agent of verticillium wilt.
Conclusions
The described VJ medium is recommended for streamlined and efficient production of inoculum for the plant endophytic fungus Serendipita indica and might prove superior for the propagation of other fungi for research purposes.
At the end of the first larval stage, the nematode Caenorhabditis elegans developing in harsh environmental conditions is able to choose an alternative developmental path called the dauer diapause. Dauer larvae exhibit different physiology and behaviors from non-dauer larvae. Using focused ion beam-scanning electron microscopy (FIB-SEM), we volumetrically reconstructed the anterior sensory apparatus of C. elegans dauer larvae with unprecedented precision. We provide a detailed description of some neurons, focusing on structural details that were unknown or unresolved by previously published studies. They include the following: (1) dauer-specific branches of the IL2 sensory neurons project into the periphery of anterior sensilla and motor or putative sensory neurons at the sub-lateral cords; (2) ciliated endings of URX sensory neurons are supported by both ILso and AMso socket cells near the amphid openings; (3) variability in amphid sensory dendrites among dauers; and (4) somatic RIP interneurons maintain their projection into the pharyngeal nervous system. Our results support the notion that dauer larvae structurally expand their sensory system to facilitate searching for more favorable environments.
Meiotic chromosome movement is important for the pairwise alignment of homologous chromosomes, which is required for correct chromosome segregation. Movement is driven by cytoplasmic forces, transmitted to chromosome ends by nuclear membrane-spanning proteins. In animal cells, lamins form a prominent scaffold at the nuclear periphery, yet the role lamins play in meiotic chromosome movement is unclear. We show that chromosome movement correlates with reduced lamin association with the nuclear rim, which requires lamin phosphorylation at sites analogous to those that open lamina network crosslinks in mitosis. Failure to remodel the lamina results in delayed meiotic entry, altered chromatin organization, unpaired or interlocked chromosomes, and slowed chromosome movement. The remodeling kinases are delivered to lamins via chromosome ends coupled to the nuclear envelope, potentially enabling crosstalk between the lamina and chromosomal events. Thus, opening the lamina network plays a role in modulating contacts between chromosomes and the nuclear periphery during meiosis.