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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.
Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses.
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.
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.