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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

Strategies to improve photosynthetic nitrogen-use efficiency with no yield penalty: lessons from late-sown winter wheat

HighlightOptimal N allocation at several integration levels accounts for improved canopy PNUE while maintaining high grain yield in winter wheat\n\nAbstractImproving canopy photosynthetic nitrogen-use efficiency (PNUE) may maintain or even increase yield with reduced N input. In this study, later-sown winter wheat was studied to reveal the mechanism underlying improved canopy PNUE while maintaining high yield. N allocation at several levels was optimised in late-sown wheat plants. N content per plant increased. Increased N was allocated to the flag leaf and second leaf, and to ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) in upper leaves. Constant or reduced N was allocated to leaf 3, leaf 4, and Rubisco in lower leaves. The specific green leaf area nitrogen (SLN) of upper leaves increased, while that of lower leaves remained unchanged or decreased. N allocation to the cell wall decreased in all leaves. As a result, the maximum carboxylation rate of upper leaves increased, and that of lower leaves remained constant or decreased. CO2 diffusion capacity was enhanced in all leaves. Outperformance by light-saturated net photosynthetic rate (Pmax) over SLN led to improved PNUE in upper leaves. Enhanced Pmax coupled with unchanged or decreased SLN resulted in improved PNUE in lower leaves. High yield was maintained because enhanced photosynthetic capacity at the leaf and whole plant levels compensated for reduced canopy leaf area.

cell biology↗

bioRxiv2018

An amphipathic helix enables septins to sense micron-scale membrane curvature

The geometry of cells is well described by membrane curvature. Septins are filament forming, GTP-binding proteins that assemble on positive, micrometer-scale curvatures. Here, we examine the molecular basis of curvature sensing by septins. We show that differences in affinity and the number of binding sites drive curvature-specific adsorption of septins. Moreover, we find septin assembly onto curved membranes is cooperative and show that geometry influences higher-order arrangement of septin filaments. Although septins must form polymers to stay associated with membranes, septin filaments do not have to span micrometers in length to sense curvature, as we find that single septin complexes have curvature-dependent association rates. We trace this ability to an amphipathic helix (AH) located on the C-terminus of Cdc12. The AH domain is necessary and sufficient for curvature sensing both in vitro and in vivo. These data show that curvature sensing by septins operates at much smaller length scales than the micrometer curvatures being detected.

cell biology↗

bioRxiv2018

Studying the fate of tumor extracellular vesicles at high spatio-temporal resolution using the zebrafish embryo

Tumor extracellular vesicles (tumor EVs) mediate the communication between tumor and stromal cells mostly to the benefit of tumor progression. Notably, tumor EVs have been reported to travel in the blood circulation, reach specific distant organs and locally modify the microenvironment. However, visualizing these events in vivo still faces major hurdles. Here, we show a new method for tracking individual circulating tumor EVs in a living organism: we combine novel, bright and specific fluorescent membrane probes, MemBright, with the transparent zebrafish embryo as an animal model. We provide the first description of tumor EVs hemodynamic behavior and document their arrest before internalization. Using transgenic lines, we show that circulating tumor EVs are uptaken by endothelial cells and blood patrolling macrophages, but not by leukocytes, and subsequently stored in acidic degradative compartments. Finally, we prove that the MemBright can be used to follow naturally released tumor EVs in vivo. Overall, our study demonstrates the usefulness and prospects of zebrafish embryo to track tumor EVs in vivo.\n\nHighlightsO_LIMemBright, a new family of membrane probes, allows for bright and specific staining of EVs\nC_LIO_LIZebrafish melanoma EVs are very similar to human and mouse melanoma EVs in morphology and protein content\nC_LIO_LIThe zebrafish embryo is an adapted model to precisely track tumor EVs dynamics and fate in a living organism from light to electron microscopy\nC_LIO_LICirculating tumor EVs are rapidly uptaken by endothelial cells and patrolling macrophages\nC_LIO_LICorrelated light and electron microscopy can be used in zebrafish to identify cells and compartments uptaking tumor EVs\nC_LI\n\nBlurbDispersion of tumor extracellular vesicles (EVs) throughout the body promotes tumor progression. However the behavior of tumor EVs in body fluids remains mysterious due to their small size and the absence of adapted animal model. Here we show that the zebrafish embryo can be used to track circulating tumor EVs in vivo and provide the first high-resolution description of their dissemination and uptake.

cell biology↗

bioRxiv2018

Asymmetric Distribution of Glucose Transporter mRNA Provides Growth Advantage

Asymmetric localization of mRNA is important for cell fate decisions in eukaryotes and provides the means for localized protein synthesis in a variety of cell types. Here we show that hexose transporter mRNAs are retained in the mother cell of S. cerevisiae until metaphase-anaphase transition (MAT) and then are released into the bud. The retained mRNA was translationally inactive but bound to ribosomes before MAT. Importantly, when cells were shifted from starvation to glucose-rich conditions, HXT2 mRNA, but none of the other HXT mRNAs, was enriched in the bud after MAT. This enrichment was dependent on the Ras/cAMP/PKA pathway, the APC ortholog Kar9 and nuclear segregation into the bud. Competition experiments between strains that only expressed one hexose transporter at a time revealed that HXT2 only cells grow faster than their counterparts when released from starvation. Therefore, asymmetric distribution of HXT2 mRNA provides a growth advantage for young daughters, who are better prepared for nutritional changes in the environment. Our data provide evidence that asymmetric mRNA localization is an important factor in determining cellular fitness.

cell biology↗

bioRxiv2018

Osteoblastic PLEKHO1 contributes to joint inflammation in rheumatoid arthritis

Osteoblasts participating in the inflammation regulation gradually obtain concerns. However, its role in joint inflammation of rheumatoid arthritis (RA) is largely unknown. Pleckstrin homology domain-containing family O member 1 (PLEKHO1) was previously identified as a negative regulator of osteogenic lineage activity. Here we demonstrated that PLEKHO1 was highly expressed in osteoblasts of articular specimens from RA patients and inflammatory arthritis mice. Genetic deletion of osteoblastic Plekho1 ameliorated joint inflammation in mice with collagen-induced arthritis (CIA) and K/BxN serum-transfer arthritis (STA), whereas overexpressing Plekho1 only within osteoblasts in CIA and STA mice demonstrated exacerbated local inflammation. Further in vitro studies indicated that PLEKHO1 was required for TRAF2-mediated RIP1 ubiquitination to activate NF-kB for inducing inflammatory cytokines production in osteoblasts. Moreover, osteoblastic PLEKHO1 inhibition improved joint inflammation and attenuated bone formation reduction in CIA mice and non-human primate arthritis model. These data strongly suggest that highly expressed PLEKHO1 in osteoblast mediates joint inflammation in RA. Targeting osteoblastic PLEKHO1 may exert dual therapeutic action of alleviating joint inflammation and promoting bone formation in RA.

cell biology↗

bioRxiv2018

Live tracking of inter-organ communication by endogenous exosomes in vivo

Extracellular vesicles (EVs) are released by most cell types but the definitive demonstration of their functional relevance remains challenging due to the lack of appropriate model organisms. Here we developed an in vivo model to study EV physiology by expressing CD63-pHluorin in zebrafish embryos. A combination of microscopy techniques and proteomic analysis allowed us to study the biogenesis, composition, transfer, uptake and fate of individual endogenous EVs in vivo. We identified an exosome population released in a syntenin-dependent manner from the Yolk Syncytial Layer into the blood circulation. These exosomes were specifically captured, endocytosed and degraded by patrolling macrophages and endothelial cells in the Caudal Vein Plexus (CVP) in a scavenger receptor and dynamin-dependent manner. Interference with exosome secretion affected CVP growth, supporting their trophic role. Altogether, our work provides a unique model to track in vivo inter-organ communication by endogenous exosomes at individual vesicle level and high spatio-temporal accuracy.\n\nHighlights- Single endogenous EVs can be live-visualized in the whole embryo with CD63-pHluorin\n- In the YSL, syntenin regulates exosome release into the blood for their propagation\n- YSL exosomes reach the tail to be taken up by macrophages and endothelial cells\n- Uptake is scavenger receptor and dynamin-dependent and provides trophic support\n\n\nBlurbWe propose zebrafish embryos expressing a fluorescent reporter for exosomes as a relevant model organism to live-track production, journey and fate of individual extracellular vesicles in vivo. Our model allows investigation of the composition of EVs and the molecular mechanisms controlling their biogenesis and fate and functions in receiving cells.

cell biology↗

bioRxiv2018

A positive feedback loop drives centrosome maturation in flies

Centrosomes are formed when mother centrioles recruit pericentriolar material (PCM) around themselves. The PCM expands dramatically as cells prepare to enter mitosis (a process termed centrosome maturation), but it is unclear how this expansion is achieved. In flies, Spd-2 and Cnn form an extensive scaffold around the mother centriole that recruits other components of the mitotic PCM, and the Polo-dependent phosphorylation of Cnn at the centrosome is crucial for scaffold assembly. Here we show that, like Cnn, Spd-2 is specifically phosphorylated at centrosomes. This phosphorylation appears to create multiple phosphorylated S-S/T(p) motifs that allow Spd-2 to recruit Polo to the expanding scaffold. If Spd-2 cannot recruit Polo to the expanding scaffold, the scaffold is initially assembled around the mother centriole, but it cannot expand outwards, and centrosome maturation fails. We conclude that Spd-2, Polo and Cnn cooperate to form a positive feedback loop that drives the dramatic expansion of the mitotic centrosome in flies.

cell biology↗

bioRxiv2018

In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics

The long-term expansion of keratinocytes under serum- and feeder free conditions generally results in diminished proliferation and an increased commitment to terminal differentiation. Here we present a serum and xenogeneic feeder free culture system that retains the self-renewal capacity of primary human keratinocytes. In vivo, the tissue microenvironment is a major contributor to determining cell fate and a key component of the microenvironment is the extracellular matrix (ECM). Accordingly, acellular ECMs derived from human dermal fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, were used as the basis for a xenogeneic-free keratinocyte expansion protocol. A phospholipase A2 decellularisation procedure produced matrices which, by proteomics analysis, resembled in composition the core matrix proteins of skin dermis. On these ECMs keratinocytes proliferated rapidly, retained their small size, expressed p63, did not express keratin 10 and rarely expressed keratin 16. Moreover, the colony forming efficiency of keratinocytes cultured on these acellular matrices was markedly enhanced. Collectively these data indicate that the dermal fibroblast-derived matrices support the in vitro expansion of keratinocytes that maintained stem-like characteristics under serum free conditions.

cell biology↗

bioRxiv2018

Mitochondrial biogenesis is transcriptionally repressed in lysosomal lipid storage diseases

Perturbations in mitochondrial function and homeostasis are pervasive in lysosomal storage diseases, but the underlying mechanisms remain unknown. Here, we report a transcriptional program that represses mitochondrial biogenesis and function in lysosomal storage diseases Niemann-Pick type C (NPC) and acid sphingomyelinase deficiency (ASM), in patient cells and mouse tissues. This mechanism is mediated by the transcription factors KLF2 and ETV1, which are both induced in NPC and ASM patient cells. Mitochondrial biogenesis and function defects in these cells are rescued by the silencing of KLF2 or ETV1. Increased ETV1 expression is regulated by KLF2, while the increase of KLF2 protein levels in NPC and ASM stems from impaired signaling downstream sphingosine-1-phosphate receptor 1 (S1PR1), which normally represses KLF2. In patient cells, S1PR1 is undetectable at the plasma membrane and thus unable to repress KLF2. This manuscript provides a mechanistic pathway for the prevalent mitochondrial defects in lysosomal storage diseases.

cell biology↗

bioRxiv2018

The C-type lectin Schlaff ensures epidermal barrier compactness in Drosophila

The stability of extracellular matrices is in general ensured by cross-linking of its components. Previously, we had shown that the integrity of the layered Drosophila cuticle relies on the presence of a covalent cuticular dityrosine network. Production and composition of this structure remained unstudied. In this work, we present our analyses of the schlaff (slf) gene coding for a C-type lectin that is needed for the adhesion between the horizontal cuticle layers. The Slf protein mainly localizes between the two layers called epicuticle and procuticle that separate from each other when the function of Slf is reduced or eliminated paralleling the phenotype of a cuticle with reduced extracellular dityrosine. Localisation of the dityrosinylated protein Resilin to the epicuticle-procuticle interface suggests that the dityrosine network mediates the adhesion of the epicuticle to the procuticle. Ultimately, compromised Slf function is associated with massive water loss. In summary, we propose that Slf is implied in the stabilisation of a dityrosine layer especially between the epicuticle and the procuticle that in turn constitutes an outward barrier against uncontrolled water flow.\n\nSummary statementExtracellular matrices adopt a stereotypic organisation for function during development. The lectin Schlaff assists adhesion reactions to ensure compactness of the epidermal cuticle in Drosophila.

cell biology↗

bioRxiv2018

Quantitative Interactome Proteomics Reveals a Molecular Basis for ATF6-Dependent Regulation of a Destabilized Amyloidogenic Protein

Activation of the unfolded protein response (UPR)-associated transcription factor ATF6 has emerged as a promising strategy to selectively reduce the secretion and subsequent toxic aggregation of destabilized, amyloidogenic proteins implicated in diverse systemic amyloid diseases. However, the molecular mechanism by which ATF6 activation reduces the secretion of amyloidogenic proteins remains poorly defined. Here, we establish a quantitative interactomics platform with improved throughput and sensitivity to define how ATF6 activation selectively reduces secretion of a destabilized, amyloidogenic immunoglobulin light chain (LC) associated with Light Chain Amyloidosis (AL). We show that ATF6 activation increases the targeting of this destabilized LC to a select subset of pro-folding ER proteostasis factors that retains the amyloidogenic LC within the ER, preventing its secretion to downstream secretory environments. Our results define a molecular basis for the selective, ATF6-dependent reduction in destabilized LC secretion and highlight the advantage for targeting this endogenous UPR-associated transcription factor to reduce secretion of destabilized, amyloidogenic proteins implicated in AL and related systemic amyloid diseases.

cell biology↗

bioRxiv2018

Triple A patient cells suffering from mitotic defects fail to localize PGRMC1 to mitotic kinetochore fibers

HIGHLIGHT SUMMARYO_LIInvestigating cell division in human adrenal cells we show that proliferation is decreased upon overexpression of ALADIN, PGRMC1 or PGRMC2.\nC_LIO_LIIn immunofluorescence experiments using human adrenal cells and triple A patient fibroblasts we observed that during cell division PGRMC1 localizes to the microtubule kinetochore-fibers in metaphase and to the mid-body in telophase.\nC_LIO_LIDepletion of ALADIN results in mis-localization of Aurora A and PGRMC1 in metaphase cells of the human adrenal cell line and fibroblasts derived from patients with triple A syndrome.\nC_LIO_LIIn real time PCR using RNA of fibroblasts of triple A syndrome patients and healthy controls we measured an increased expression of PGRMC2 in cells with ALADIN mis-function compared to the control cells.\nC_LIO_LIWe hypothesize that a loss of the regulatory interaction between ALADIN and PGRMC2 leads to an over-regulation and over-expression of PGRMC2 and displaces PGRMC1 at the metaphase spindle. This diminishing of PGRMC1 concentration at kinetochore fibers may lead to mitotic errors and pro - liferation arrest.\nC_LI\n\nABSTRACTMembrane-associated progesterone receptors are restricted to the endoplasmic reticulum and are shown to regulate the activity of cytochrome P450 enzymes which are involved in steroidogenesis or drug detoxification. PGRMC1 and PGRMC2 belong to this group of microsomal receptors and are of interest due to their suspected role during cell cycle. PGRMC1 and PGRMC2 are thought to bind to each other thereby suppressing entry into mitosis. We could previously report that PGRMC2 interacts with the nucleoporin ALADIN which when mutated results in the autosomal recessive disorder triple A syndrome. ALADIN is a novel regulator of mitotic controller Aurora kinase A and depletion of this nucleoporin leads to microtubule instability. In the current study, we present that proliferation is decreased when ALADIN, PGRMC1 or PGRMC2 are over-expressed. Furthermore, we find that depletion of ALADIN results in mis-localization of Aurora kinase A and PGRMC1 in metaphase cells. Additionally, PGRMC2 is over-expressed in triple A patient fibroblasts. Our results emphasize the possibility that loss of the regulatory interaction between ALADIN and PGRMC2 gives rise to a depletion of PGRMC1 at kinetochore fibers and to mitotic errors. This observation may explain part of the symptoms seen in triple A syndrome patients.

cell biology↗

bioRxiv2018

The Cdc42 GEF, Gef1, promotes uniform protein distribution along the actomyosin ring to enable concentric furrowing

During cytokinesis, fission yeast coordinates actomyosin ring constriction with septum ingression, resulting in concentric furrow formation. Mechanisms coordinating septum ingression with the actomyosin ring remain unclear. We report that cells lacking the Cdc42 activator Gef1, combined with an activated allele of the formin, Cdc12, display non-concentric furrowing. Although cells that furrow non-concentrically display normal actomyosin rings, the scaffold Cdc15 is unevenly distributed along the ring. This suggests that after ring assembly, uniform Cdc15 distribution along the ring drives proper furrow formation. We find that Cdc15 levels at the ring are reduced in the activated cdc12 mutant, or upon disruption of Arp2/3 complex-dependent endocytic patches. Furthermore, Cdc15 levels in endocytic patches increase in gef1 mutants. We hypothesize that assembled rings recruit Cdc15 from endocytic patches. Patches with higher Cdc15 levels and slower ring-association rate lead to uneven Cdc15 distribution. Based on this hypothesis we developed a mathematical model that captures experimentally observed Cdc15 distributions along the ring. We propose that, at the ring, Gef1 and endocytic events promote uniform Cdc15 distribution to enable proper septum ingression and concentric furrow formation.\n\nSummary StatementGef1 and endocytic events at the assembled actomyosin ring facilitate uniform Cdc15 distribution along the ring thus enabling concentric furrow formation.

cell biology↗

bioRxiv2018

RAB6 and microtubules restrict secretion to focal adhesions

To ensure their homeostasis and sustain differentiated functions, cells continuously transport diverse cargos to various cell compartments and in particular to the cell surface. Secreted proteins are transported along intracellular routes from the endoplasmic reticulum through the Golgi complex before reaching the plasma membrane along microtubule tracks. Using a synchronized secretion assay, we report here that exocytosis does not occur randomly at the cell surface but on localized hotspots juxtaposed to focal adhesions. Although microtubules are involved, the RAB6-dependent machinery plays an essential role. We observed that, irrespective of the transported cargos, most post-Golgi carriers are positive for RAB6 and that its inactivation leads to a broad reduction of protein secretion. RAB6 may thus be a general regulator of post-Golgi secretion.

cell biology↗

bioRxiv2018

Niacin fine-tunes energy homeostasis through canonical GPR109A signaling

Niacin has long been considered as a high-potency drug for beneficially treating lipid abnormalities, however, its anti-atherosclerotic effects have been challenged by recent studies. Here, we demonstrated that oral supplementation of niacin resulted in a significant reduction in body weight and fat mass without affecting food intake in high-fat diet-fed wild-type mice, but not in GPR109A-defeicient mice. Further investigation showed that niacin challenge led to a remarkable inhibition of hepatic lipogenesis via a GPR109A-dependent ERK1/2/AMPK pathway. Additionally, we demonstrated that niacin treatment stimulated thermogenesis in brown adipose tissue by induction of thermogenic genes via GPR109A. Moreover, we observed that mice exposed to niacin exhibited a dramatic decrease in intestinal absorption of fatty acids. Together, our data demonstrate that acting on GPR109A, niacin shows the potential to maintain energy homeostasis by fine-tuning hepatic lipogenesis, BAT/beige thermogenesis and intestinal fat absorption, representing a potential approach to the treatment of lipid abnormalities.

cell biology↗

bioRxiv2018

Molecular and topological reorganizations in mitochondrial architecture interplay during Bax-mediated steps of apoptosis

During apoptosis, Bcl-2 proteins such as Bax and Bak mediate the release of pro-apoptotic proteins from the mitochondria by clustering on the outer mitochondrial membrane and thereby permeabilizing it. However, it remains unclear how outer membrane openings form. Here, we combined different correlative microscopy and electron cryo-tomography approaches to visualize the effects of Bax activity on mitochondria in human cells. Our data show that Bax clusters localize near outer membrane ruptures of highly variable size. Bax clusters contain structural elements suggesting a higher-order organization of their components. Furthermore, unfolding of inner membrane cristae is coupled to changes in the supramolecular assembly of ATP synthases, particularly pronounced at membrane segments exposed to the cytosol by ruptures. Based on our results, we propose a comprehensive model in which molecular reorganizations of the inner membrane and sequestration of outer membrane components into Bax clusters interplay in the formation of outer membrane ruptures.

cell biology↗

bioRxiv2018

SSU72 phosphatase is a telomere replication terminator

Telomeres, the protective ends of eukaryotic chromosomes, are replicated through concerted actions by conventional DNA polymerases and telomerase, though the regulation of this process is not fully understood. Telomere replication requires (C)-Stn1-Ten1, a telomere ssDNA-binding complex that is homologous to RPA. Here, we show that the evolutionarily conserved phosphatase Ssu72 is responsible for terminating the cycle of telomere replication in fission yeast. Ssu72 controls the recruitment of Stn1 to telomeres by regulating Stn1 phosphorylation at S74, a residue that lies within the conserved OB fold domain. Consequently, ssu72{Delta} mutants are defective in telomere replication and exhibit long 3 overhangs, which are indicative of defective lagging strand DNA synthesis. We also show that hSSU72 regulates telomerase activation in human cells by controlling the recruitment of hSTN1 to telomeres. Thus, in this study, we demonstrate a previously unknown yet conserved role for the phosphatase SSU72, whereby this enzyme controls telomere homeostasis by activating lagging strand DNA synthesis, thus terminating the cycle of telomere replication.

cell biology↗

bioRxiv2018

Cell type-specific structural plasticity of the ciliary transition zone in C. elegans

Background informationThe current consensus on cilia development posits that the ciliary transition zone (TZ) is formed via extension of nine centrosomal microtubules. In this model, TZ structure remains unchanged in microtubule number throughout the cilium life cycle. This model does not however explain structural variations of TZ structure seen in nature, and could also lend itself to the misinterpretation that deviations from nine-doublet microtubule ultrastructure represent an abnormal phenotype. Thus, a better understanding of events that occur at the TZ in vivo during metazoan development is required.\n\nResultsTo address this issue, we characterized ultrastructure of two types of sensory cilia in developing Caenorhabditis elegans. We discovered that, in cephalic male (CEM) and inner labial quadrant (IL2Q) sensory neurons, ciliary TZs are structurally plastic and remodel from one structure to another during animal larval development. The number of microtubules doublets forming the TZ can be increased or decreased over time, depending on cilia type. Both cases result in structural TZ intermediates different from TZ in adult cilia. In CEM cilia, axonemal extension and maturation occurs concurrently with TZ structural maturation.\n\nConclusions and SignificanceOur work extends the current model to include the structural plasticity of metazoan transition zone, which can be structurally delayed, maintained or remodeled in cell type-specific manner.

cell biology↗