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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

Highly efficient scarless knock-in of reporter genes into human and mouse pluripotent stem cells via transient antibiotic selection

Pluripotent stem cells (PSCs) edited with genetic reporters are useful tools for differentiation analysis and for isolation of specific cell populations for study. Reporter integration into the genome is now commonly achieved by targeted DNA nuclease-enhanced homology directed repair (HDR). However, human PSCs are known to have a low frequency of gene knock-in (KI) by HDR, making reporter line generation an arduous process. Here, we report a methodology for scarless KI of large fluorescent reporter genes into PSCs by transient selection with puromycin or zeocin. With this method, we can perform targeted KI of a single reporter gene with up to 65% efficiency, as well as simultaneous KI of two reporter genes into different loci with up to 11% efficiency. Additionally, we demonstrate that this method also works in mouse PSCs.

cell biology↗

bioRxiv2018

Context-explorer: Analysis of spatially organized protein expression in high-throughput screens

A growing body of evidence highlights the importance of the cellular microenvironment as a regulator of phenotypic and functional cellular responses to perturbations. We have previously developed cell patterning techniques to control population context parameters, and here we demonstrate Context-explorer (CE), a software tool to improve investigation of microenvironmental variables through colony level analyses. We demonstrate the capabilities of CE in the analysis of human and mouse pluripotent stem cells (hPSCs, mPSCs) patterned in colonies of defined size and shape in multi-well plates.\n\nCE employs a density-based clustering algorithm to identify cell colonies within micropatterned wells. Using this automatic colony classification methodology, we obtain accuracies comparable to manual colony counts in a fraction of the time. Classifying cells according to their relative position within a colony enables statistical analysis of radial spatial trends in protein expression within multiple colonies in the same treatment group. When applied to colonies of hPSCs, our analysis reveals a radial gradient in the expression of the pluripotency inducing transcription factors SOX2 and OCT4, and a similar trend in the intra-colony location of different cellular phenotypes. We extend these analyses to colonies of different sizes and shapes and demonstrate how the metrics derived by CE can be used to asses the patterning fidelity of micropatterned plates.\n\nWe have incorporated a number of features to enhance the usability and utility of CE. To appeal to a broad scientific community, all of the softwares functionality is accessible from a graphical user interface, and convenience functions for several common data operations are included. CE is compatible with existing image analysis programs such as CellProfiler and extends the analytical capabilities already provided by these tools. Taken together, CE facilitates investigation of spatially heterogeneous cell populations in fundamental research and drug development validation programs.

cell biology↗

bioRxiv2018

Heat-stress triggers MAPK crosstalk to turn on the hyper-osmotic response pathway

Cells make decisions based on a combination of external and internal signals. In yeast, the high osmolarity response (HOG) is a mitogen-activated protein kinase (MAPK) pathway that responds to a variety of stimuli, and it is central to the general stress response. Here we studied the effect of heat-stress (HS) on HOG. Using live-cell reporters and genetics, we show that HS promotes Hog1 phosphorylation and gene expression, exclusively via the Sln1 phosphorelay branch, and that the strength of the activation is larger in yeast adapted to high external osmolarity. HS stimulation of HOG is indirect. First, we found that it depends on the operation of a second MAPK pathway, the cell-wall integrity (CWI), a well-known mediator of HS. Second, we show that HS causes glycerol loss via the channel Fps1, and that strictly requires the CWI MAPK Slt2. Third, blocking glycerol efflux also blocks HOG activation, strongly suggesting that it is the resulting loss of turgor by the loss of the accompanying water what causes HOG stimulation. Thus, taken together, our findings highlight a central role for Fps1, and the metabolism of glycerol, in the communication between the yeast MAPK pathways, essential for survival and reproduction in changing environments.

cell biology↗

bioRxiv2018

F1FO ATP synthase responds to glycolysis inhibition by localization into the inner boundary membrane

Mitochondrial F1F0 ATP synthase is the key enzyme to fuel the cell with essential ATP. Strong indications exist that the respiratory chain and the ATP synthase are physically separated within cristae. How static this organization is, is largely unknown. Here, we investigated the effect of substrate restriction on mitochondrial respiration and the spatio-temporal organization of ATP synthase. By superresolution microscopy, the localization and mobility of single labelled mitochondrial ATP synthase was determined in live cells. We found, that the ATP synthase under oxidative respiration displayed a clear localization and confined mobility in cristae. Trajectories of individual ATP synthase proteins show a perpendicular course to the longitudinal axis of the respective mitochondrion, exactly following the ultrastructure of cristae. When substrate for TCA cycle and respiration was limited, a significant proportion of ATP synthase localized from cristae to the inner boundary membrane, and only less mobile ATP synthase remained in cristae. These observations showing the plasticity of the spatio-temporal organisation of ATP synthase can explain why ATP synthase show interactions with proteins in distinct mitochondrial subcompartments such as inner boundary membrane, cristae junctions and cristae.

cell biology↗

bioRxiv2018

NAD consumption by PARP1 in response to DNA damage triggers metabolic shift critical for damaged cell survival

DNA damage signaling is critical for the maintenance of genome integrity and cell fate decision. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor rapidly activated in a damage dose and complexity-dependent manner playing a critical role in the initial chromatin organization and DNA repair pathway choice at damage sites. However, the cell-wide consequence of its activation in damaged cells is not well delineated. Using the phasor approach to fluorescence lifetime imaging microscopy (FLIM) and fluorescence-based biosensors in combination with laser microirradiation, we found a rapid cell-wide increase of the bound/free NADH ratio in response to nuclear DNA damage, which is triggered by NAD+ depletion by PARP activation. This change is linked to the metabolic balance shift to oxidative phosphorylation (oxphos) over glycolysis. Inhibition of the respiratory chain resulted in rapid PARP-dependent ATP reduction and intracellular acidification, and eventually, PARP-dependent, AIF-independent, apoptosis indicating that oxphos becomes critical for damaged cell survival. The results reveal the novel pro-survival effect of PARP activation through a change in cellular metabolism, and demonstrate how unique applications of advanced fluorescence imaging technologies in combination with laser microirradiation can be a powerful tool to interrogate damage-induced metabolic changes at high spatiotemporal resolution in a live cell.

cell biology↗

bioRxiv2018

Annexin A1 drives macrophage skewing towards a resolving phenotype to accelerate the regeneration of muscle injury through AMPK activation

Understanding the circuits that promote an efficient resolution of inflammation is crucial to deciphering the molecular and cellular processes required to promote tissue repair. Macrophages play a central role in the regulation of inflammation, resolution and repair/regeneration. Using a model of skeletal muscle injury and repair, herein we identify Annexin A1 (AnxA1) as the extracellular trigger of macrophage skewing towards a pro-reparative phenotype. Brought into the injured tissue initially by migrated neutrophils, and then over-expressed in infiltrating macrophages, AnxA1 activates FPR2/ALX receptors and the downstream AMPK signalling cascade leading to macrophage skewing, dampening of inflammation and regeneration of muscle fibres. Mice lacking AnxA1 in all cells or in myeloid cells only display a defect in this reparative process. In vitro experiments recapitulated these properties, with AMPK null macrophages lacking AnxA1-mediated polarization. Collectively, these data identify the AnxA1/FPR2/AMPK axis as a novel pathway in skeletal muscle injury regeneration.

cell biology↗

bioRxiv2018

Lysosome exocytosis is required for mitosis

Mitosis, the accurate segregation of duplicated genetic material into what will become two new daughter cells, is accompanied by extensive membrane remodelling and membrane trafficking activities. Early in mitosis, adherent cells partially detach from the substratum, round up and their surface area decreases. This likely results from an endocytic uptake of plasma membrane material. As cells enter cytokinesis they re-adhere, flatten and exhibit an associated increase in surface area. The identity of the membrane donor for this phase of mitosis remains unclear. Here we show by biochemical and imaging approaches that lysosomes undergo exocytosis at telophase and that this requires the activity of phosphatidylinositol 4-kinase-III{beta}. Inhibition of lysosome exocytosis resulted in mitotic failure in a significant proportion of cells suggesting that this facet of lysosome physiology is essential and represents a new regulatory mechanism in mitosis.

cell biology↗

bioRxiv2018

Glucose intake hampers PKA-regulated HSP90 chaperone activity

Aging is an intricate phenomenon associated with the gradual loss of physiological functions, and both nutrient sensing and proteostasis control lifespan. Although multiple approaches have facilitated the identification of candidate genes that govern longevity, the molecular mechanisms that link aging pathways are still elusive. Here, we conducted a quantitative mass spectrometry screen and identified all phosphorylation/dephosphorylation sites on yeast proteins that significantly responded to calorie restriction, a well-established approach to extend lifespan. Functional screening of 135 potential regulators uncovered that Ids2 is activated by PP2C under CR and inactivated by PKA under glucose intake. ids2{Delta} or ids2 phosphomimetic cells displayed heat sensitivity and lifespan shortening. Ids2 serves as a co-chaperone to form a complex with Hsc82 or the redundant Hsp82, and phosphorylation of Ids2 impedes its association with chaperone HSP90. Thus, PP2C and PKA orchestrate glucose sensing and protein folding to enable cells to maintain protein quality for sustained longevity.

cell biology↗

bioRxiv2018

Developmental Regulation of an Organelle Tether Coordinates Mitochondrial Remodeling in Meiosis

Cellular differentiation involves remodeling cellular architecture to transform one cell type to another. By investigating mitochondrial dynamics during meiotic differentiation in budding yeast, we sought to understand how organelle morphogenesis is developmentally controlled in a system where regulators of differentiation as well as organelle architecture are known, but the interface between them remains unexplored. We found that mitochondria abruptly detach from the cell cortex shortly before segregating into gametes. Mitochondrial detachment is enabled by the programmed destruction of the mitochondria-endoplasmic reticulum-cortex anchor (MECA), an organelle tether that forms contact sites between mitochondria and the plasma membrane. MECA regulation is governed by a meiotic transcription factor, Ndt80, which promotes the activation of a conserved kinase, Ime2. We found that MECA undergoes Ime2-dependent phosphorylation. Furthermore, Ime2 promotes MECA degradation in a temporally controlled manner. Our study defines a key mechanism that coordinates mitochondrial morphogenesis with the landmark events of meiosis and demonstrates that cells can developmentally regulate tethering to induce organelle remodeling.

cell biology↗

bioRxiv2018

Cell morphological motif detector for high-resolution 3D microscopy images

Recent advances in light-sheet microscopy enable imaging of cell morphology and signaling with unprecedented detail. However, the analytical tools to systematically measure and visualize the intricate relations between cell morphodynamics, intracellular signaling, and cytoskeletal dynamics have been largely missing. Here, we introduce a set of computer vision and graphics methods to dissect molecular mechanisms underlying 3D cell morphogenesis and to test whether morphogenesis itself affects intracellular signaling. We demonstrate a machine learning based generic morphological motif detector that automatically finds lamellipodia, filopodia, and blebs on various cell types. Combining motif detection with molecular localization, we measure the differential association of PIP2 and KrasV12 with blebs. Both signals associate with bleb edges, as expected for membrane-localized proteins, but only PIP2 is enhanced on blebs. This suggests that local morphological cues differentially organize and activate sub-cellular signaling processes. Overall, our computational workflow enables the objective, automated analysis of the 3D coupling of morphodynamics with cytoskeletal dynamics and intracellular signaling.

cell biology↗

bioRxiv2018

The Drosophila Cortactin Binding Protein 2 homolog, Nausicaa, regulates lamellipodial actin dynamics in a Cortactin-dependent manner.

Drosophila CG10915 is an uncharacterized protein coding gene with sequence similarity to human Cortactin Binding Protein 2 (CTTNBP2) and Cortactin Binding Protein 2 N-terminal like (CTTNBP2NL). We have named this gene Nausicaa (naus) and characterize it through a combination of quantitative live-cell total internal reflection fluorescence (TIRF) microscopy, electron microscopy, RNAi depletion, and genetics. We found that Naus co-localizes with F-actin and Cortactin in the lamellipodia of Drosophila S2R+ and D25c2 cells and this localization is lost following Cortactin or Arp2/3 depletion or by mutations that disrupt a conserved proline patch found in its mammalian homologs. Using Permeabilization Activated Reduction in Fluorescence (PARF) and Fluorescence Recovery after Photo-bleaching (FRAP), we find that depletion of Cortactin alters Naus dynamics leading to a decrease in its half-life. Furthermore, we discovered that Naus depletion in S2R+ cells led to a decrease in actin retrograde flow and a lamellipodia characterized by long, unbranched filaments. We demonstrate that these alterations to the dynamics and underlying actin architecture also affect D25c2 cell migration and decrease arborization in Drosophila neurons. We present the novel hypothesis that Naus functions to slow Cortactins disassociation from Arp2/3 nucleated branch junctions, thereby increasing both branch nucleation and junction stability.

cell biology↗

bioRxiv2018

Platypus Induced Pluripotent Stem Cells: the Unique Pluripotency Signature of a Monotreme

Summary StatementThe generation and transcriptome analysis of the first induced pluripotent stem cells from the platypus reveals SOX2 has been a key driver of the expanded pluripotency regulatory network in placental mammals.\n\nABSTRACTThe mechanisms by which pluripotency has evolved remain unclear. To gain insight into the evolution of mammalian pluripotency we have generated induced pluripotent stem cells (piPSCs) from the platypus. Deep sequencing of the piPSC transcriptome revealed that piPSCs robustly express the core eutherian pluripotency factors OCT4, SOX2 and NANOG. Given the more extensive role of SOX3 over SOX2 in avian pluripotency, our data indicate that between 315 million years and 166 million years ago primitive mammals replaced the role of SOX3 in the vertebrate pluripotency network with SOX2. DAX1/NR0B1 is not expressed in piPSCs and an analysis of the platypus DAX1 promoter revealed the absence of a proximal SOX2-binding DNA motif known to be critical for DAX1 expression in eutherian pluripotent stem cells, suggesting that the acquisition of SOX2 responsiveness by DAX1 has facilitated its recruitment into the pluripotency network of eutherians. We further show that the expression ratio of X chromosomes to autosomes (X1-5 X1-5:AA) is approximately equal to 1 indicating that there is no upregulation of X-linked genes and that there is no preference for silencing of maternal or paternal alleles (ie imprinting).

cell biology↗

bioRxiv2018

PDEδ inhibition impedes the proliferation and survival of human colorectal cancer cell lines harboring oncogenic KRas

Novelty and ImpactThe undruggable KRas is a prevalent oncogene in CRC with poor prognosis. In hPDAC cells pharmacological targeting of PDE{delta} affects oncogenic KRas signaling, but it remained unclear whether this approach is transferable to other cancer cells. Here, we show that genetic and pharmacologic PDE{delta} inhibition also impedes the proliferation of oncogenic, but not wild-type KRas bearing CRC cells indicating that PDE{delta} inhibition is a specific tool for targeting growth of oncogenic KRas bearing CRC.\n\nAbstractRas proteins, most notably KRas, are prevalent oncogenes in human cancer. Plasma membrane localization and thereby signaling of KRas is regulated by the prenyl-binding protein PDE{delta}. Recently, we have reported the specific anti-proliferative effects of PDE{delta} inhibition in KRas-dependent human pancreatic ductal adenocarcinoma cell lines. Here, we investigated the proliferative dependence on the solubilizing activity of PDE{delta} of human colorectal cancer (CRC) cell lines with or without oncogenic KRas mutations. Our results show that genetic and pharmacologic interference with PDE{delta} specifically inhibits proliferation and survival of CRC cell lines harboring oncogenic KRas mutations whereas isogenic cell lines in which the KRas oncogene has been removed, or cell lines with oncogenic BRaf mutations or EGFR overexpression are not dependent on PDE{delta}. Pharmacological PDE{delta} inhibition is therefore a possible new avenue to target oncogenic KRas bearing CRC.

cell biology↗

bioRxiv2018

Enforced expression of phosphatidylinositol 4-phosphate 5-kinase homolog (PIPKH) alters phosphatidylinositol 4,5-bisphosphate distribution and the localization of small G-proteins

The generation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) is essential for many of the functions including the control of cytoskeleton, signal transduction and endocytosis. Additionally, due to its presence in the plasma membrane and its anionic charge PtdIns(4,5)P2, together with phosphatidylserine, imbue the inner leaflet of the plasma membrane with a negative surface charge. This negative charge helps to define the identity of the plasma membrane as serves to recruit or regulate a multitude of proteins that contain polybasic domains or patches. Here we determine that the phosphatidylinositol 4-phosphate 5-kinase homolog (PIPKH) alters the subcellular distribution of PtdIns(4,5)P2 by re-localizing the PIP5Ks to endomembranes. Consistently, we find a redistribution of the PIP5K family members to endomembrane structures upon PIPKH overexpression that is accompanied by an accumulation of PtdIns(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), which further influences the distribution of endosomes and lysosomes. Additionally, we demonstrate that the accumulation of polyphosphoinositides increases their negative surface charge that in turn leads to the relocalization of surface charge probes as well as the polycationic proteins K-Ras and Rac1.

cell biology↗

bioRxiv2018

Filopodome mapping identifies p130Cas as a mechanosensitive regulator of filopodia stability

Filopodia are adhesive cellular protrusions specialised in the detection of extracellular matrix (ECM)-derived cues. While ECM engagement at focal adhesions is known to trigger the recruitment of hundreds of proteins (\"adhesome\") to fine-tune cellular behaviour, the components of the filopodia adhesions remain undefined. Here, we performed a structured illumination microscopy-based screen to map the localisation of 80 target proteins, linked to cell adhesion and migration, within filopodia. We demonstrate preferential enrichment of several adhesion proteins to either filopodia tips, filopodia shafts, or shaft subdomains suggesting divergent, spatially restricted functions for these proteins. Moreover, proteins with phospho-inositide (PI) binding sites are particularly enriched in filopodia. This, together with the strong localisation of PI(3,4)P2 in filopodia tips, predicts critical roles for PIs in regulating filopodia ultra-structure and function. Our mapping further reveals that filopodia adhesions consist of a unique set of proteins, the filopodome, that are distinct from classical nascent adhesions, focal adhesions and fibrillar adhesions. Using live imaging, we observe that filopodia adhesions can give rise to nascent adhesions, which, in turn, form focal adhesions. Finally, we demonstrate that p130Cas (BCAR1) is recruited to filopodia tips via its CCHD domain and acts as a mechanosensitive regulator of filopodia stability.

cell biology↗

bioRxiv2018

Curcumin inhibits cystogenesis in autosomal dominant polycystic kidney disease cells via altering JAK2/STAT3 activity.

Dysregulated JAK/STAT signalling is implicated in polycystic kidney disease, which is a common genetic disease leading to renal failure. However, the mechanisms underlying JAK/STAT-mediated cystogenesis arepoorlyunderstood.TheroleofJAK2wasinvestigated immunohistochemically in a murine model of cystic disease (Pkd1nl/nl). In the normal kidney, JAK2 is restricted to tubular epithelial and vascular cells with lesser staining in bowmans capsule and is undetectable in the interstitium. By contrast, in the diseased kidney JAK2 appears stronger in cyst-lining cells when compared to normal tubules and appears mislocalised in the interstitium. Given that JAK2 is a major tyrosine kinase activating JAK/STAT, we considered whether its inhibition can attenuate cystic growth in vitro. To assess this we used curcumin, a natural phytochemical, which significantly reduced JAK2 levels and STAT3 activity. Consistently, reduced JAK2/STAT3 activity was correlated with reduced cystic growth of cystic cells in three-dimensional cyst assays. Taken together, our results suggest that JAK2 is a key signalling molecule that functions to inhibit cystic growth in cystic tubular epithelial cells, thus providing the foundation for its development as a novel therapeutic in polycystic kidney disease.

cell biology↗

bioRxiv2018

Migration and Differentiation of Osteoclast Precursors under Gradient Fluid Shear Stress

The skeleton is able to adapt to mechanical loading through bone remodeling, i.e. bone resorption followed by bone formation. The osteoclasts close to microdamages are believed to initiate bone resorption, but whether local mechanical loading such as fluid flow regulates recruitment and differentiation of osteoclast precursors at the site of bone resorption has yet to be investigated. In the present study, finite element analysis first revealed that there exists low fluid shear stress (FSS) field inside microdamage. Basing on a custom-made device of cone-and-plate fluid chamber, finite element analysis and particle image velocimetry measurement were performed to verify the formation of gradient FSS flow field. Furthermore, the effects of gradient FSS on the migration, aggregation, and fusion of osteoclast precursors were observed. Results showed that osteoclast precursor RAW264.7 cells migrate along radial direction toward the region with lower FSS during exposure to gradient FSS stimulation for 40 min, obviously deviating from the direction of actual fluid flow indicated by fluorescent particles. When inhibiting calcium signaling pathway with gadolinium and thapsigargin, cell migration toward low-FSS region was significantly reduced. For other cell lines, MC3T3-E1, PDLF, rMSC and MDCK, gradient FSS stimulation did not lead to the low-FSS-inclined migration. After being cultured under gradient FSS stimulation for 6 days, the density of RAW264.7 cells and the ratio of TRAP-positive multinucleated osteoclasts in low-FSS region were significantly higher than those in high-FSS region. Therefore, osteoclast precursor cells may have special ability to sense FSS gradient and tend to actively migrate toward low-FSS region, which is regulated by calcium signaling pathway.

cell biology↗

bioRxiv2018

A Cytoplasmic GOLD Protein Controls Cell Polarity

Phosphoinositide lipids provide spatial landmarks during polarized secretion. Here, we elucidate a role for phosphatidylinositol 4-phosphate (PI4P) metabolism in the control of cell polarity. In budding yeast, PI4P is enriched at the plasma membrane of growing daughter cells. Upon heat shock however, PI4P rapidly increases at the plasma membrane in mother cells resulting in a more uniform PI4P distribution. Rather than phosphoinositide kinase activation, PI4P hydrolysis is impaired to generate the heat-induced PI4P signal in mother cells. This fine tune control of PI4P metabolism is mediated through attenuation of the Osh3 protein that binds and presents PI4P to a phosphoinositide phosphatase. Importantly, Osh3 undergoes phase transitions upon environmental stress conditions, resulting in intracellular aggregates and reduced cortical localization. The chaperone Hsp104 co-assembles with intracellular Osh3 granules, but is not required for their formation. Interestingly, the Osh3 GOLD domain, also present in the ER-localized p24 cargo adaptor family, is sufficient to form stress granules. Accordingly, GOLD-mediated phase transitions may provide a general mechanism to modulate secretion and growth upon transient changes in physiological and environmental conditions.

cell biology↗