Plasmodesmal connectivity in C4 Gynandropsis gynandra is induced by light and dependent on photosynthesis.

In leaves of C4 plants, the reactions of photosynthesis become restricted between two compartments. Typically, this allows accumulation of C4 acids in mesophyll (M) cells and subsequent decarboxylation in the bundle sheath (BS). In C4 grasses, proliferation of plasmodesmata between these cell types is thought to increase cell-to-cell connectivity to allow efficient metabolite movement. However, it is not known whether C4 dicotyledons also show this enhanced plasmodesmal connectivity and so whether this is a general requirement for C4 photosynthesis is not clear. How M and BS cells in C4 leaves become highly connected is also not known. We investigated these questions using 3D- and 2D-electron microscopy on the C4 dicotyledon Gynandropsis gynandra as well as phylogenetically close C3 relatives. The M-BS interface of C4 G. gynandra showed higher plasmodesmal frequency compared with closely related C3 species. Formation of these plasmodesmata was induced by light. Pharmacological agents that perturbed photosynthesis reduced the number of plasmodesmata, but this inhibitory effect could be reversed by the provision of exogenous sucrose. We conclude that enhanced formation of plasmodesmata between M and BS cells is wired to the induction of photosynthesis in C4 G. gynandra.

Ectopic callose deposition into woody biomass modulates the nano-architecture of macrofibrils.

Plant biomass plays an increasingly important role in the circular bioeconomy, replacing non-renewable fossil resources. Genetic engineering of this lignocellulosic biomass could benefit biorefinery transformation chains by lowering economic and technological barriers to industrial processing. However, previous efforts have mostly targeted the major constituents of woody biomass: cellulose, hemicellulose and lignin. Here we report the engineering of wood structure through the introduction of callose, a polysaccharide novel to most secondary cell walls. Our multiscale analysis of genetically engineered poplar trees shows that callose deposition modulates cell wall porosity, water and lignin contents and increases the lignin-cellulose distance, ultimately resulting in substantially decreased biomass recalcitrance. We provide a model of the wood cell wall nano-architecture engineered to accommodate the hydrated callose inclusions. Ectopic polymer introduction into biomass manifests in new physico-chemical properties and offers new avenues when considering lignocellulose engineering.

Electron counting detectors in scanning transmission electron microscopy via hardware signal processing.

Transmission electron microscopy is a pivotal instrument in materials and biological sciences due to its ability to provide local structural and spectroscopic information on a wide range of materials. However, the electron detectors used in scanning transmission electron microscopy are often unable to provide quantified information, that is the number of electrons impacting the detector, without exhaustive calibration and processing. This results in arbitrary signal values with slow response times that cannot be used for quantification or comparison to simulations. Here we demonstrate and optimise a hardware signal processing approach to augment electron detectors to perform single electron counting.

Downregulation of extraembryonic tension controls body axis formation in avian embryos.

Embryonic tissues undergoing shape change draw mechanical input from extraembryonic substrates. In avian eggs, the early blastoderm disk is under the tension of the vitelline membrane (VM). Here we report that the chicken VM characteristically downregulates tension and stiffness to facilitate stage-specific embryo morphogenesis. Experimental relaxation of the VM early in development impairs blastoderm expansion, while maintaining VM tension in later stages resists the convergence of the posterior body causing stalled elongation, failure of neural tube closure, and axis rupture. Biochemical and structural analysis shows that VM weakening is associated with the reduction of outer-layer glycoprotein fibers, which is caused by an increasing albumen pH due to CO2 release from the egg. Our results identify a previously unrecognized potential cause of body axis defects through mis-regulation of extraembryonic tissue tension.

Multiple gene co-options underlie the rapid evolution of sexually deceptive flowers in Gorteria diffusa.

Gene co-option, the redeployment of an existing gene in an unrelated developmental context, is an important mechanism underlying the evolution of morphological novelty. In most cases described to date, novel traits emerged by co-option of a single gene or genetic network. Here, we show that the integration of multiple co-opted genetic elements facilitated the rapid evolution of complex petal spots that mimic female bee-fly pollinators in the sexually deceptive South African daisy Gorteria diffusa. First, co-option of iron homeostasis genes altered petal spot pigmentation, producing a color similar to that of female pollinators. Second, co-option of the root hair gene GdEXPA7 enabled the formation of enlarged papillate petal epidermal cells, eliciting copulation responses from male flies. Third, co-option of the miR156-GdSPL1 transcription factor module altered petal spot placement, resulting in better mimicry of female flies resting on the flower. The three genetic elements were likely co-opted sequentially, and strength of sexual deception in different G. diffusa floral forms strongly correlates with the presence of the three corresponding morphological alterations. Our findings suggest that gene co-options can combine in a modular fashion, enabling rapid evolution of novel complex traits.

Drosophila SPG12 ortholog, reticulon-like 1, governs presynaptic ER organization and Ca2+ dynamics.

Neuronal endoplasmic reticulum (ER) appears continuous throughout the cell. Its shape and continuity are influenced by ER-shaping proteins, mutations in which can cause distal axon degeneration in Hereditary Spastic Paraplegia (HSP). We therefore asked how loss of Rtnl1, a Drosophila ortholog of the human HSP gene RTN2 (SPG12), which encodes an ER-shaping protein, affects ER organization and the function of presynaptic terminals. Loss of Rtnl1 depleted ER membrane markers at Drosophila presynaptic motor terminals and appeared to deplete narrow tubular ER while leaving cisternae largely unaffected, thus suggesting little change in resting Ca2+ storage capacity. Nevertheless, these changes were accompanied by major reductions in activity-evoked Ca2+ fluxes in the cytosol, ER lumen, and mitochondria, as well as reduced evoked and spontaneous neurotransmission. We found that reduced STIM-mediated ER-plasma membrane contacts underlie presynaptic Ca2+ defects in Rtnl1 mutants. Our results show the importance of ER architecture in presynaptic physiology and function, which are therefore potential factors in the pathology of HSP.

Polysaccharide metabolism regulates structural colour in bacterial colonies.

The brightest colours in nature often originate from the interaction of light with materials structured at the nanoscale. Different organisms produce such coloration with a wide variety of materials and architectures. In the case of bacterial colonies, structural colours stem for the periodic organization of the cells within the colony, and while considerable efforts have been spent on elucidating the mechanisms responsible for such coloration, the biochemical processes determining the development of this effect have not been explored. Here, we study the influence of nutrients on the organization of cells from the structurally coloured bacteria Flavobacterium strain IR1. By analysing the optical properties of the colonies grown with and without specific polysaccharides, we found that the highly ordered organization of the cells can be altered by the presence of fucoidans. Additionally, by comparing the organization of the wild-type strain with mutants grown in different nutrient conditions, we deduced that this regulation of cell ordering is linked to a specific region of the IR1 chromosome. This region encodes a mechanism for the uptake and metabolism of polysaccharides, including a polysaccharide utilization locus (PUL operon) that appears specific to fucoidan, providing new insight into the biochemical pathways regulating structural colour in bacteria.

Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins.

Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that represent a target for insecticides. Peptide neurotoxins are known to block nAChRs by binding to their target subunits, however, a better understanding of this mechanism is needed for effective insecticide design. To facilitate the analysis of nAChRs we used a CRISPR/Cas9 strategy to generate null alleles for all ten nAChR subunit genes in a common genetic background. We studied interactions of nAChR subunits with peptide neurotoxins by larval injections and styrene maleic acid lipid particles (SMALPs) pull-down assays. For the null alleles, we determined the effects of α-Bungarotoxin (α-Btx) and ω-Hexatoxin-Hv1a (Hv1a) administration, identifying potential receptor subunits implicated in the binding of these toxins. We employed pull-down assays to confirm α-Btx interactions with the Drosophila α5 (Dα5), Dα6, Dα7 subunits. Finally, we report the localisation of fluorescent tagged endogenous Dα6 during Drosophila CNS development. Taken together, this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins and provides a resource for the in vivo analysis of insect nAChRs.

Microscopy and chemical analyses reveal flavone-based woolly fibres extrude from micron-sized holes in glandular trichomes of Dionysia tapetodes.

BACKGROUND: Dionysia tapetodes, a small cushion-forming mountainous evergreen in the Primulaceae, possesses a vast surface-covering of long silky fibres forming the characteristic "woolly" farina. This contrasts with some related Primula which instead form a fine powder. Farina is formed by specialized cellular factories, a type of glandular trichome, but the precise composition of the fibres and how it exits the cell is poorly understood. Here, using a combination of cell biology (electron and light microscopy) and analytical chemical techniques, we present the principal chemical components of the wool and its mechanism of exit from the glandular trichome. RESULTS: We show the woolly farina consists of micron-diameter fibres formed from a mixture of flavone and substituted flavone derivatives. This contrasts with the powdery farina, consisting almost entirely of flavone. The woolly farina in D. tapetodes is extruded through specific sites at the surface of the trichome's glandular head cell, characterised by a small complete gap in the plasma membrane, cell wall and cuticle and forming a tight seal between the fibre and hole. The data is consistent with formation and thread elongation occurring from within the cell. CONCLUSIONS: Our results suggest the composition of the D. tapetodes farina dictates its formation as wool rather than powder, consistent with a model of thread integrity relying on intermolecular H-bonding. Glandular trichomes produce multiple wool fibres by concentrating and maintaining their extrusion at specific sites at the cell cortex of the head cell. As the wool is extensive across the plant, there may be associated selection pressures attributed to living at high altitudes.

Neural stem cells traffic functional mitochondria via extracellular vesicles.

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.

Rubisco and carbon-concentrating mechanism co-evolution across chlorophyte and streptophyte green algae.

Green algae expressing a carbon-concentrating mechanism (CCM) are usually associated with a Rubisco-containing micro-compartment, the pyrenoid. A link between the small subunit (SSU) of Rubisco and pyrenoid formation in Chlamydomonas reinhardtii has previously suggested that specific RbcS residues could explain pyrenoid occurrence in green algae. A phylogeny of RbcS was used to compare the protein sequence and CCM distribution across the green algae and positive selection in RbcS was estimated. For six streptophyte algae, Rubisco catalytic properties, affinity for CO2 uptake (K0.5 ), carbon isotope discrimination (δ13 C) and pyrenoid morphology were compared. The length of the ßA-ßB loop in RbcS provided a phylogenetic marker discriminating chlorophyte from streptophyte green algae. Rubisco kinetic properties in streptophyte algae have responded to the extent of inducible CCM activity, as indicated by changes in inorganic carbon uptake affinity, δ13 C and pyrenoid ultrastructure between high and low CO2 conditions for growth. We conclude that the Rubisco catalytic properties found in streptophyte algae have coevolved and reflect the strength of any CCM or degree of pyrenoid leakiness, and limitations to inorganic carbon in the aquatic habitat, whereas Rubisco in extant land plants reflects more recent selective pressures associated with improved diffusive supply of the terrestrial environment.

Glycation changes molecular organization and charge distribution in type I collagen fibrils.

Collagen fibrils are central to the molecular organization of the extracellular matrix (ECM) and to defining the cellular microenvironment. Glycation of collagen fibrils is known to impact on cell adhesion and migration in the context of cancer and in model studies, glycation of collagen molecules has been shown to affect the binding of other ECM components to collagen. Here we use TEM to show that ribose-5-phosphate (R5P) glycation of collagen fibrils - potentially important in the microenvironment of actively dividing cells, such as cancer cells - disrupts the longitudinal ordering of the molecules in collagen fibrils and, using KFM and FLiM, that R5P-glycated collagen fibrils have a more negative surface charge than unglycated fibrils. Altered molecular arrangement can be expected to impact on the accessibility of cell adhesion sites and altered fibril surface charge on the integrity of the extracellular matrix structure surrounding glycated collagen fibrils. Both effects are highly relevant for cell adhesion and migration within the tumour microenvironment.

ThX - a next-generation probe for the early detection of amyloid aggregates.

Neurodegenerative diseases such as Alzheimer's and Parkinson's are associated with protein misfolding and aggregation. Recent studies suggest that the small, rare and heterogeneous oligomeric species, formed early on in the aggregation process, may be a source of cytotoxicity. Thioflavin T (ThT) is currently the gold-standard fluorescent probe for the study of amyloid proteins and aggregation processes. However, the poor photophysical and binding properties of ThT impairs the study of oligomers. To overcome this challenge, we have designed Thioflavin X, (ThX), a next-generation fluorescent probe which displays superior properties; including a 5-fold increase in brightness and 7-fold increase in binding affinity to amyloidogenic proteins. As an extrinsic dye, this can be used to study unique structural amyloid features both in bulk and on a single-aggregate level. Furthermore, ThX can be used as a super-resolution imaging probe in single-molecule localisation microscopy. Finally, the improved optical properties (extinction coefficient, quantum yield and brightness) of ThX can be used to monitor structural differences in oligomeric species, not observed via traditional ThT imaging.

Poly(ADP-Ribose) Links the DNA Damage Response and Biomineralization.

Biomineralization of the extracellular matrix is an essential, regulated process. Inappropriate mineralization of bone and the vasculature has devastating effects on patient health, yet an integrated understanding of the chemical and cell biological processes that lead to mineral nucleation remains elusive. Here, we report that biomineralization of bone and the vasculature is associated with extracellular poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerases in response to oxidative and/or DNA damage. We use ultrastructural methods to show poly(ADP-ribose) can form both calcified spherical particles, reminiscent of those found in vascular calcification, and biomimetically calcified collagen fibrils similar to bone. Importantly, inhibition of poly(ADP-ribose) biosynthesis in vitro and in vivo inhibits biomineralization, suggesting a therapeutic route for the treatment of vascular calcifications. We conclude that poly(ADP-ribose) plays a central chemical role in both pathological and physiological extracellular matrix calcification.

Silver Nanowire Particle Reactivity with Human Monocyte-Derived Macrophage Cells: Intracellular Availability of Silver Governs Their Cytotoxicity.

Silver nanowires (AgNWs) are increasingly being used in the production of optoelectronic devices, with manufacturing processes posing a risk for occupational exposures via inhalation. Although some studies have explored the environmental effects of AgNWs, few data exist on human health effects. Alveolar macrophages are central in the clearance of inhaled fibers from the lungs, with frustrated phagocytosis often stated as a key determinant for the onset of inflammatory reactions. However, the mechanisms through which fully ingested AgNWs interact with, degrade, and transform within primary macrophages over time, and whether the reactivity of the AgNWs arises due to ionic or particulate effects, or both, are poorly understood. Here, a combination of elemental quantification, 3D tomography, analytical transmission electron microscopy (TEM), and confocal microscopy were employed to monitor the uptake, intracellular Ag+ availability, and processing of AgNWs of two different lengths (1 and 10 µm) inside human monocyte-derived macrophages (HMMs). Using AgNO3 and spherical silver nanoparticles (AgNPs) as a comparison, the amount of total bioavailable/intracellular Ag highly correlated to the cytotoxicity of AgNWs. The 10 µm AgNWs were completely internalized in HMMs, with numerous lysosomal vesicles observed in close vicinity to the AgNWs. Following cellular uptake, AgNWs dissolved and transformed intracellularly, with precipitation of AgCl as well as Ag2S. These transformation processes were likely due to AgNW degradation in the acidic environment of lysosomes, leading to the release of Ag+ ions that rapidly react with Cl- and SH- species of the cell microenvironment. Our data suggest that, in HMMs, not only frustrated phagocytosis but also the extent of intracellular uptake and dissolution of AgNWs dictates their cytotoxicity.

In Vitro Targeting and Selective Killing of T47D Breast Cancer Cells by Purpurin and 5-Fluorouracil Anchored to Magnetic CNTs: Nitrene-Based Functionalization versus Uptake, Cytotoxicity, and Intracellular Fate.

Fe-encapsulated multiwall carbon nanotubes (Fe@MWCNTs) are candidates for magnetically targeted Drug Delivery Systems (mt-DDSs) against breast cancer. However, their full potential as versatile and biosafe vectors has yet to be developed. Key challenges that remain are relating surface functionalization to cytotoxicity and inducing selective cytotoxicity to cancer cells. We have studied quantitative uptake of pristine and functionalized Fe@MWCNTs (f-Fe@MWCNTs) in correlation to their in vitro cytotoxicity. Human monocyte macrophages (HMMs) and T47D breast cancer cells were selected as models to test selective cytotoxicity. [2+1]-Cycloaddition of nitrenes to Fe@MWCNTs yielded both effective functionalization and drug "tethering". Hydrophilization of Fe@MWCNTs was critical for efficient active cell uptake. f-Fe@MWCNTs were considerably more toxic to T47D cells than HMMs, in spite of longer exposure times of the latter. Eventually, Fe@MWCNTs loaded with 5-fluorouracil in a ß-cyclodextrin cage or with covalently linked purpurin emerged as the most cytotoxic and steerable in a magnetic field toward promising mt-DDSs.

Characterization and Visualization of Vesicles in the Endo-Lysosomal Pathway with Surface-Enhanced Raman Spectroscopy and Chemometrics.

Surface-enhanced Raman spectroscopy (SERS) is an ultrasensitive vibrational fingerprinting technique widely used in analytical and biosensing applications. For intracellular sensing, typically gold nanoparticles (AuNPs) are employed as transducers to enhance the otherwise weak Raman spectroscopy signals. Thus, the signature patterns of the molecular nanoenvironment around intracellular unlabeled AuNPs can be monitored in a reporter-free manner by SERS. The challenge of selectively identifying molecular changes resulting from cellular processes in large and multidimensional data sets and the lack of simple tools for extracting this information has resulted in limited characterization of fundamental cellular processes by SERS. Here, this shortcoming in analysis of SERS data sets is tackled by developing a suitable methodology of reference-based PCA-LDA (principal component analysis-linear discriminant analysis). This method is validated and exemplarily used to extract spectral features characteristic of the endocytic compartment inside cells. The voluntary uptake through vesicular endocytosis is widely used for the internalization of AuNPs into cells, but the characterization of the individual stages of this pathway has not been carried out. Herein, we use reporter-free SERS to identify and visualize the stages of endocytosis of AuNPs in cells and map the molecular changes via the adaptation and advantageous use of chemometric methods in combination with tailored sample preparation. Thus, our study demonstrates the capabilities of reporter-free SERS for intracellular analysis and its ability to provide a way of characterizing intracellular composition. The developed analytical approach is generic and enables the application of reporter-free SERS to identify unknown components in different biological matrices and materials.

NMR spectroscopy of native and in vitro tissues implicates polyADP ribose in biomineralization.

Nuclear magnetic resonance (NMR) spectroscopy is useful to determine molecular structure in tissues grown in vitro only if their fidelity, relative to native tissue, can be established. Here, we use multidimensional NMR spectra of animal and in vitro model tissues as fingerprints of their respective molecular structures, allowing us to compare the intact tissues at atomic length scales. To obtain spectra from animal tissues, we developed a heavy mouse enriched by about 20% in the NMR-active isotopes carbon-13 and nitrogen-15. The resulting spectra allowed us to refine an in vitro model of developing bone and to probe its detailed structure. The identification of an unexpected molecule, poly(adenosine diphosphate ribose), that may be implicated in calcification of the bone matrix, illustrates the analytical power of this approach.

Citrate bridges between mineral platelets in bone.

We provide evidence that citrate anions bridge between mineral platelets in bone and hypothesize that their presence acts to maintain separate platelets with disordered regions between them rather than gradual transformations into larger, more ordered blocks of mineral. To assess this hypothesis, we take as a model for a citrate bridging between layers of calcium phosphate mineral a double salt octacalcium phosphate citrate (OCP-citrate). We use a combination of multinuclear solid-state NMR spectroscopy, powder X-ray diffraction, and first principles electronic structure calculations to propose a quantitative structure for this material, in which citrate anions reside in a hydrated layer, bridging between apatitic layers. To assess the relevance of such a structure in native bone mineral, we present for the first time, to our knowledge, (17)O NMR data on bone and compare them with (17)O NMR data for OCP-citrate and other calcium phosphate minerals relevant to bone. The proposed structural model that we deduce from this work for bone mineral is a layered structure with thin apatitic platelets sandwiched between OCP-citrate-like hydrated layers. Such a structure can explain a number of known structural features of bone mineral: the thin, plate-like morphology of mature bone mineral crystals, the presence of significant quantities of strongly bound water molecules, and the relatively high concentration of hydrogen phosphate as well as the maintenance of a disordered region between mineral platelets.

The effect of particle agglomeration on the formation of a surface-connected compartment induced by hydroxyapatite nanoparticles in human monocyte-derived macrophages.

Agglomeration dramatically affects many aspects of nanoparticle-cell interactions. Here we show that hydroxyapatite nanoparticles formed large agglomerates in biological medium resulting in extensive particle uptake and dose-dependent cytotoxicity in human macrophages. Particle citration and/or the addition of the dispersant Darvan 7 dramatically reduced mean agglomerate sizes, the amount of particle uptake and concomitantly cytotoxicity. More surprisingly, agglomeration governed the mode of particle uptake. Agglomerates were sequestered within an extensive, interconnected membrane labyrinth open to the extracellular space. In spite of not being truly intracellular, imaging studies suggest particle degradation occurred within this surface-connected compartment (SCC). Agglomerate dispersion prevented the SCC from forming, but did not completely inhibit nanoparticle uptake by other mechanisms. The results of this study could be relevant to understanding particle-cell interactions during developmental mineral deposition, in ectopic calcification in disease, and during application of hydroxyapatite nanoparticle vectors in biomedicine.