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bioRxiv2018

Mutations generated by repair of Cas9-induced double strand breaks are predictable from surrounding sequence

The exact DNA mutation produced by cellular repair of a CRISPR/Cas9-generated double strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, and depend on DNA sequence at the targeted location. Here, we present a systematic study of this link. We created a high throughput assay to directly measure the edits generated by over 40,000 guide RNAs, and applied it in a range of genetic backgrounds and for alternative CRISPR/Cas9 reagents. In total, we gathered data for over 1,000,000,000 mutational outcomes in synthetic constructs, which mirror those at endogenous loci. The majority of reproducible mutations are insertions of a single base, short deletions, or long microhomology-mediated deletions. gRNAs have a cell-line dependent preference for particular outcomes, especially favouring single base insertions and microhomology-mediated deletions. We uncover sequence determinants of the produced mutations at individual loci, and use these to derive a predictor of Cas9 editing outcomes with accuracy close to the theoretical maximum. This improved understanding of sequence repair allows better design of editing experiments, and may lead to future therapeutic applications.

cell biology↗

bioRxiv2018

Dual role for fungal-specific outer kinetochore proteins during cell cycle and development in Magnaporthe oryzae

The outer kinetochore DASH/DAM complex ensures proper spindle structure and chromosome segregation. While DASH complex protein requirement diverges among different yeasts, its role in filamentous fungi has not been investigated so far. We studied the dynamics and role of middle (Mis12) and outer (Dam1 and Ask1) kinetochore proteins in the filamentous fungal pathogen, Magnaporthe oryzae, which undergoes multiple cell cycle linked developmental transitions. Both Dam1 and Ask1, unlike Mis12, were recruited to the nucleus specifically during mitosis. While Dam1 was not required for viability, loss of its function (dam1{Delta} mutant) delayed mitotic progression, resulting in impaired conidial and hyphal development in Magnaporthe. Intriguingly, both Dam1 and Ask1 also localised to the hyphal tips, in the form of punctae oscillating back and forth from the growing ends, suggesting that Magnaporthe DASH complex proteins may play a non-canonical role in polarised growth during interphase, in addition to their function in nuclear segregation during mitosis. Impaired appressorial (infection structure) development and function in the dam1{Delta} mutant suggest that fungus-specific Dam1 complex proteins could be an attractive target for a novel anti-fungal strategy.\n\nSummary StatementDASH complex proteins are differentially recruited to the nucleus during cell division and are intriguingly involved in polarised growth during development and differentiation in the rice blast fungus.

cell biology↗

bioRxiv2018

Pirfenidone attenuates lung fibrotic fibroblast-mediated fibrotic responses to transforming growth factor-β1

Pirfenidone, an antifibrotic agent used for treatment of idiopathic pulmonary fibrosis (IPF), functions by inhibiting myofibroblast differentiation, which is involved in transforming growth factor (TGF)-{beta}1-induced IPF pathogenesis. However, unlike normal lung fibroblasts, the relationship between pirfenidone responses of TGF-{beta}1-induced human fibrotic lung fibroblasts and lung fibrosis is unknown. Here, we investigated the effect of pirfenidone on the functions of two new targets, collagen triple helix repeat containing protein 1 (CTHRC1) and four-and-a-half LIM domain protein 2 (FHL2), which included fibroblast activity, collagen gel contraction, and migration toward fibronectin. Compared to control lung fibroblasts, pirfenidone restored TGF-{beta}1-stimulated fibroblast-mediated collagen gel contraction, migration, and CTHRC1 release in lung fibrotic fibroblasts. Furthermore, pirfenidone attenuated TGF-{beta}1- and CTHRC1-induced fibroblast activity, bone morphogenic protein-4/Gremlin1 upregulation, and -smooth muscle actin, fibronectin, and FHL2 downregulation, similar to that observed post-CTHRC1 inhibition. In contrast, FHL2 inhibition suppressed migration and fibronectin expression but did not downregulate CTHRC1. Overall, pirfenidone suppressed fibrotic fibroblast-mediated fibrotic processes via inverse regulation of CTHRC1-induced lung fibroblast activity. Thus, CTHRC1 can be used for predicting pirfenidone response and developing new therapeutic target for lung fibrosis.\n\nSummary statementPirfenidone suppressed TGF-{beta}1-mediated fibrotic processes in fibrotic lung fibroblasts by attenuating CTHRC1 expression, suggesting that CTHRC1 may be a novel therapeutic target for treating patients with lung fibrosis.

cell biology↗

bioRxiv2018

Label-free digital holographic microscopy reveals virus-induced cytopathic effects in live cells

Cytopathic effects (CPEs) are a hallmark of infections. CPEs can be observed by phase contrast or fluorescence light microscopy, albeit at the cost of phototoxicity. We report that digital holo-tomographic microscopy (DHTM) reveals distinct patterns of virus infections in live cells with minimal perturbation. DHTM is label-free, and records the phase shift of low energy light passing through the specimen on a transparent surface. DHTM infers a 3-dimensional (3D) tomogram based on the refractive index (RI). By measuring RI and computing the refractive index gradient (RIG) values DHTM unveils on optical heterogeneity in cells upon virus infection. We find that vaccinia virus (VACV), herpes simplex virus (HSV) and rhinovirus (RV) infections progressively and distinctly increased RIG. VACV, but not HSV and RV infection induced oscillations of cell volume, while all three viruses altered cytoplasmic membrane dynamics, and induced apoptotic features akin to the chemical compound staurosporin, but with virus-specific signatures. In sum, we introduce DHTM for quantitative label-free microscopy in infection research, and uncover virus-type specific changes and CPE in living cells at minimal interference.

cell biology↗

bioRxiv2018

Cyclodextrin triggers MCOLN1-dependent endo-lysosome secretion in Niemann-Pick type C cells

In specialized cell types, lysosome-related organelles support regulated secretory pathways, while in non-specialized cells, lysosomes can undergo fusion with the plasma membrane in response to a transient rise in cytosolic calcium. Recent evidence also indicates that lysosome secretion can be controlled transcriptionally and promote clearance in lysosome storage diseases. In addition, evidence is also accumulating that low concentrations of cyclodextrins reduce the cholesterol storage phenotype in cells and animals with the cholesterol storage disease Niemann-Pick type C, via an unknown mechanism. Here, we report that cyclodextrin triggers the secretion of the endo/lysosomal content in non-specialized cells, and that this mechanism is responsible for the decreased cholesterol overload in Niemann-Pick type C cells. We also find that that the secretion of the endo/lysosome content occurs via a mechanism dependent on the endosomal calcium channel MCOLN1, as well as FYCO1, the AP1 adaptor and its partner Gadkin. We conclude that endolysosomes in non-specialized cells can acquire secretory functions elicited by cyclodextrin, and that this pathway is responsible for the decrease in cholesterol storage in Niemann-Pick C cells.

cell biology↗

bioRxiv2018

Centromere repositioning induced by inner kinetochore impairment generates a meiosis barrier

Centromeres dictate the sites for kinetochore assembly on chromosomes, while their own position on each chromosome is determined epigenetically by a specific histone H3 variant CENP-A. For all eukaryotic species, the chromosomal position of each centromere is distinct and inherited with high fidelity, although the mechanisms underlying the epigenetic stability and its functional significance remain largely unknown. Here in the fission yeast Schizosaccharomyces pombe, we show that mutations in inner kinetochore components influence centromeric chromatin organization to various levels. In extreme cases, a single deletion of wip1, mhf1 and mhf2 (the conserved CENP-T-W-S-X complex subunits) or double deletions of cnp3 (a homologue of mammalian CENP-C) and fta6 (a pombe specific component) induce centromere repositioning - inactivation of the original centromere and formation of a neocentromere - in one of the three chromosomes at random. Neocentromeres tend to locate in pericentromeric heterochromatin regions, although heterochromatin is not required for centromere inactivation. Cells carrying a neocentromere are competent in mitosis and in meiosis of homozygotes. However, when these cells are crossed to cells carrying the original centromere, the progeny suffers severe lethality due to defects in meiotic chromosome segregation. These results recapitulate a meiosis barrier that could initiate genetic divergence between two populations with mismatched centromeres, documenting a potential role of the Evolutionary New Centromeres (ENCs) in speciation.\n\nSignificance StatementIn eukaryotes, centromeres are chromosomal regions where kinetochores are assembled and the positions of centromeres are accurately inherited. While the centromere and kinetochore assembly are extensively studied, the mechanisms that each centromere maintain its identity on chromosomes are still not well understood. In this study, we demonstrated that the inner kinetochore is required for the normal centromere identity as single depletion of the inner kinetochore CENP-T-W-S-X complex or double deletions of cnp3/CENP-C and fta6 induce centromere repositioning. We further showed cells carrying a neocentromere are reproductively isolated from the wildtype population carrying the original centromere. Taken together, these results suggest that induced centromere repositioning mimics the evolutionary new centromeres and is sufficient to cause reproductive isolation.

cell biology↗

bioRxiv2018

Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

Idiopathic pulmonary fibrosis is a fatal, incurable lung disease in which the intricate alveolar network of the human lung is progressively replaced by fibrotic scars, eventually leading to respiratory failure. Myofibroblasts are the effector cells that lead to abnormal deposition of extracellular matrix proteins and therefore mediate fibrotic disease not only in the lung but also in other organs. Emerging literature suggests a correlation between fibrosis and metabolic alterations in IPF. In this study, we show that the first-line antidiabetic drug, metformin, exerts potent antifibrotic effects in the lung by modulating metabolic pathways, inhibiting TGF{beta}1 action, suppressing collagen formation, activating PPAR{gamma} signaling and inducing lipogenic differentiation in lung myofibroblasts derived from human patients. Using genetic lineage tracing in a murine model of lung fibrosis, we show that metformin alters the fate of myofibroblasts and accelerates fibrosis resolution by inducing myofibroblast-tolipofibroblast transdifferentiation. Detailed pathway analysis showed that the reduction of collagen synthesis was largely AMPK-dependent, whereas the transdifferentiation of myo- to lipofibroblasts occurred in a BMP2-PPAR{gamma}-dependent fashion and was largely AMPK-independent. Our data report an unprecedented role for metformin in lung fibrosis, thus warranting further therapeutic evaluation.

cell biology↗

bioRxiv2018

Protect TUDCA stimulated CKD-derived hMSCs against the CKD-Ischemic disease via upregulation of PrPC

Although autologous human mesenchymal stem cells (hMSCs) are a promising source for regenerative stem cell therapy, the barriers associated with pathophysiological conditions in this disease limit therapeutic applicability to patients. We proved treatment of CKD-hMSCs with TUDCA enhanced the mitochondrial function of these cells and increased complex I & IV enzymatic activity, increasing PINK1 expression and decreasing mitochondrial O2*- and mitochondrial fusion in a PrPC-dependent pathway. Moreover, TH-1 cells enhanced viability when co-cultured in vitro with TUDCA-treated CKD-hMSC. In vivo, tail vein injection of TUDCA-treated CKD-hMSCs into the mouse model of CKD associated with hindlimb ischemia enhanced kidney recovery, the blood perfusion ratio, vessel formation, and prevented limb loss, and foot necrosis along with restored expression of PrPC in the blood serum of the mice. These data suggest that TUDCA-treated CKD-hMSCs are a promising new autologous stem cell therapeutic intervention that dually treats cardiovascular problems and CKD in patients.

cell biology↗

bioRxiv2018

A unified model for microtubule rescue

How microtubules transition from depolymerization to polymerization, known as rescue, is poorly understood. Here we examine two models for rescue: 1) an end-driven model in which the depolymerizing end stochastically switches to a stable state; and 2) a lattice-driven model in which rescue-sites are integrated into the microtubule prior to depolymerization. We test these models using a combination of computational simulations and in vitro experiments with purified tubulin. Our findings support the lattice-driven model by identifying repeated rescue sites in microtubules. In addition, we discover an important role for divalent cations in determining the frequency and location of rescue sites. We use wash-in experiments to show that divalent cations inhibit rescue during depolymerization, but not during the polymerization. We propose a unified model in which rescues are driven by embedded rescue sites in microtubules, but the activity of these sites is influenced by changes in the depolymerizing ends.

cell biology↗

bioRxiv2018

The Desmosome is a Mesoscale Lipid Raft-Like Membrane Domain

Desmogleins are cadherin family adhesion molecules essential for epidermal integrity. Previous studies have shown that desmogleins associate with lipid rafts, but the significance of this association was not clear. Here, we report that the desmoglein transmembrane domain (TMD) is the primary determinant of raft association. Further, we identify a novel mutation in the DSG1 TMD (G562R) that causes severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome. Molecular modeling predicts that this G to R mutation shortens the DSG1 TMD, and experiments directly demonstrate that this mutation compromises both lipid raft association and desmosome incorporation. Finally, cryo-electron tomography (cryo-ET) indicates that the lipid bilayer within the desmosome is ~10% thicker than adjacent regions of the plasma membrane. These findings suggest that differences in bilayer thickness influence the organization of adhesion molecules within the epithelial plasma membrane, with cadherin TMDs recruited to the desmosome via establishment of a specialized mesoscale lipid raft-like membrane domain.

cell biology↗

bioRxiv2018

Topokaryotyping demonstrates single cell variability and stress dependent variations in nuclear envelope associated domains

Analysis of large-scale interphase genome positioning with reference to a nuclear landmark has recently been studied using sequencing-based single cell approaches. However, these approaches are dependent upon technically challenging, time consuming and costly high throughput sequencing technologies, requiring specialized bioinformatics tools and expertise. Here, we propose a novel, affordable and robust microscopy-based single cell approach, termed Topokaryotyping, to analyze and reconstruct the interphase positioning of genomic loci relative to a given nuclear landmark, detectable as banding pattern on mitotic chromosomes. This is accomplished by proximity-dependent histone labeling, where biotin ligase BirA fused to nuclear envelope marker Emerin was coexpressed together with Biotin Acceptor Peptide (BAP)-histone fusion followed by (i) biotin labeling, (ii) generation of mitotic spreads, (iii) detection of the biotin label on mitotic chromosomes and (iv) their identification by karyotyping. Using Topokaryotyping, we identified both cooperativity and stochasticity in the positioning of emerin-associated chromatin domains in individual cells. Furthermore, the chromosome-banding pattern showed dynamic changes in emerin-associated domains upon physical and radiological stress. In summary, Topokaryotyping is a sensitive and reliable technique to quantitatively analyze spatial positioning of genomic regions interacting with a given nuclear landmark at the single cell level in various experimental conditions.

cell biology↗

bioRxiv2018

Polarity signaling ensures epidermal homeostasis by coupling cellular mechanics and genomic integrity

Epithelial homeostasis requires balanced progenitor cell proliferation and differentiation, whereas disrupting this equilibrium fosters degeneration or cancer. Here we studied how cell polarity signaling orchestrates epidermal self-renewal and differentiation. Using genetic ablation, quantitative imaging, mechanochemical reconstitution and atomic force microscopy, we find that mammalian Par3 couples genome integrity and epidermal fate through shaping keratinocyte mechanics, rather than mitotic spindle orientation. Par3 inactivation impairs actomyosin contractility and viscoelasticity, and elicits mitotic failures that trigger aneuploidy, mitosis-dependent DNA damage responses, p53 stabilization and premature differentiation. Importantly, reconstituting myosin activity is sufficient to restore mitotic fidelity, genome integrity, and balanced differentiation and stratification. Collectively, this study deciphers a mechanical signaling network in which Par3 acts upstream of Rho/ROCK-mediated actomyosin contractility to promote intrinsic force generation, thereby maintaining mitotic accuracy and cellular fitness at the genomic level. Disturbing this network may compromise not only epidermal homeostasis but potentially also that of other self-renewing epithelia.

cell biology↗

bioRxiv2018

A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites

Endosymbiosis has driven major molecular and cellular innovations. Plasmodium spp. parasites that cause malaria contain an essential, non-photosynthetic plastid, the apicoplast, which originated from a secondary (eukaryote-eukaryote) endosymbiosis. To discover organellar pathways with evolutionary and biomedical significance, we performed a mutagenesis screen for essential genes required for apicoplast biogenesis in P. falciparum. Apicoplast-minus mutants were isolated using a chemical rescue that permits conditional disruption of the apicoplast and a new fluorescent reporter for organelle loss. Five candidate genes were validated (out of 12 identified), including a TIM-barrel protein that likely derived from a core metabolic enzyme but evolved a new activity. Our results demonstrate the first forward genetic screen to assign essential cellular functions to unannotated P. falciparum genes. A putative TIM-barrel enzyme and other newly-identified apicoplast biogenesis proteins open opportunities to discover new mechanisms of organelle biogenesis, molecular evolution underlying eukaryotic diversity, and drug targets against multiple parasitic diseases.

cell biology↗

bioRxiv2018

Dot6 is a major regulator of cell size and a transcriptional activator of ribosome biogenesis in the opportunistic yeast Candida albicans

In most species, size homeostasis appears to be exerted in late G1 phase as cells commit to division, called Start in yeast and the Restriction Point in metazoans. This size threshold couples cell growth to division and thereby establishes long-term size homeostasis. Our former investigations have shown that hundreds of genes markedly altered cell size under homeostatic growth conditions in the opportunistic yeast Candida albicans, but surprisingly only few of these overlapped with size control genes in the budding yeast Saccharomyces cerevisiae. Here, we investigated one of the divergent potent size regulators in C. albicans, the Myb-like HTH transcription factor Dot6. Our data demonstrated that Dot6 is a negative regulator of Start and also acts as a transcriptional activator of ribosome biogenesis (Ribi) genes. Genetic epistasis uncovered that Dot6 interacted with the master transcriptional regulator of the G1 machinery, SBF complex, but not with the Ribi and cell size regulators Sch9, Sfp1 and p38/Hog1. Dot6 was required for carbon-source modulation of cell size and it is regulated at the level of nuclear localization by TOR pathway. Our findings support a model where Dot6 acts as a hub that integrate directly growth cues via the TOR pathway to control the commitment to mitotic division at G1.

cell biology↗

bioRxiv2018

The RNA-Binding Protein DND1 Acts Sequentially as a Negative Regulator of Pluripotency and a Positive Regulator of Epigenetic Modifiers Required for Germ Cell Reprogramming

The adult spermatogonial stem cell population arises from pluripotent primordial germ cells (PGCs) that enter the fetal testis around embryonic day 10.5 (E10.5). These cells undergo rapid mitotic proliferation, then enter a prolonged period of cell cycle arrest (G1/G0) during which they transition to pro-spermatogonia. In mice homozygous for the Ter mutation in the RNA-binding protein DND1 (DND1Ter/Ter), many germ cells fail to enter G1/G0, and give rise to teratomas, tumors in which many embryonic cell types are represented. To investigate the origin of these tumors, we sequenced the transcriptome of male germ cells in DND1Ter/Ter mutants at E12.5, E13.5, and E14.5, just prior to the formation of teratomas, and correlated this information with direct targets of DND1 identified by DO-RIP-Seq. Consistent with previous results, we found that DND1 controls the down regulation of many genes associated with pluripotency and active cell cycle, including elements of the mTor, Hippo and Bmp/Nodal signaling pathways. However, DND1 targets also include genes associated with male differentiation including a large group of chromatin regulators activated in wild type but not mutant germ cells during the transition between E13.5 and E14.5. These results suggest multiple functions of DND1, and link DND1 to the initiation of epigenetic modifications in male germ cells.

cell biology↗

bioRxiv2018

CTP synthase regulation by miR-975 controls cell proliferation and differentiation in Drosophila melanogaster

CTP synthase (CTPsyn) is an essential metabolic enzyme. As a key regulator of the nucleotide pool, the protein has been found to be elevated in cancer models. In many organisms, CTPsyn compartmentalizes into filaments termed cytoophidia. For D. melanogaster, it is only its Isoform C i.e. CTPsynIsoC which forms the structure. The fruit flys testis is home to somatic and germline stem cells. Both micro and macro-cytoophidia are normally seen in the transit amplification regions close to its apical tip, where the stem-cell niche is located and development is at its most rapid. Here, we report that CTPsynIsoC overexpression causes the lengthening of cytoophidia throughout the entirety of the testicular body. A bulging apical tip is found in approximately one-third of like-genotyped males. Immunostaining shows that the cause of this tumour-like phenotype is most likely due to increased numbers of both germline cells and spermatocytes. We also report that under conditions whereby miR-975 is overexpressed, greater incidences of the same bulged-phenotype coincides with induced upregulation of CTPsynIsoC. However, RT-qPCR assays reveal that either overexpression genotype provokes a differential response in expression of a number of genes concurrently associated with CTPsyn and cancer, showing that the pathways CTPsynIsoC affect and miR-975 regulate may be completely independent of each other. This study presents the first instance of consequences of miRNA-asserted regulation upon CTPsyn in D. melanogaster, and further reaffirms the enzymes close ties to cancer and carcinogenesis.

cell biology↗

bioRxiv2018

Superresolution architecture of pluripotency guarding adhesions

Human pluripotent stem cells (hPSC) can generate almost all adult cell lineages. While it is clear that key transcriptional programmes are important elements for maintaining pluripotency, the equally essential requirement for cell adhesion to specific extracellular matrix components remains poorly defined. Our recent observation that hPSC colonies form unusually large \"cornerstone\" focal adhesions (FA), distinct from parental somatic cells, that are lost following differentiation, emphasises the potential of these atypical FA as gatekeepers of pluripotency. Here, using nanopatterns, we further demonstrate that physical restriction of adhesion size, in hPSC colonies, is sufficient to trigger differentiation. Using superresolution two-colour interfero-metric photo-activated localization microscopy (iPALM), we examined the three-dimensional architecture of these cornerstone adhesions and report vertical lamination of FA proteins with three main structural peculiarities: 1) integrin {beta}5 and talin are present at high density, at the edges of cornerstone FA, adjacent to a vertical kank-rich protein wall. 2) Vinculin localises higher than expected with respect to the substrata and displays a head-above-tail orientation, and 3) surprisingly, actin and -actinin are present in two discrete layers, a previously undescribed localisation for these proteins. Finally, we report that depletion of kanks diminishes FA patterning, and actin organisation within the colony, indicating a key role for kanks in hPSC colony architecture.

cell biology↗

bioRxiv2018

Anti-angiogenic effects of VEGF stimulation on endothelium deficient in phosphoinositide recycling

Anti-angiogenic therapies have generated significant interest for their potential to combat tumor growth (1-6). However, the ability of tumors to overproduce pro-angiogenic ligands and overcome targeted inhibitory therapies has hampered this approach (7, 8). A novel way to circumvent this problem might be to target the resynthesis of critical substrates consumed during intracellular transduction of pro-angiogenic signals in endothelial cells, thus harnessing the tumors own production of excess stimulatory ligands to deplete adjacent host endothelial cells of the capacity to respond to these signals (9-12). Here we show using zebrafish and human endothelial cells in vitro that endothelial cells deficient in CDP-diacylglycerol synthase 2 are uniquely sensitive to increased VEGF stimulation due to a reduced capacity to re-synthesize phosphoinositides, including phosphatidylinositol 4,5-bisphosphate (PIP2) a key substrate for VEGF signal transduction, resulting in VEGF-exacerbated defects in angiogenesis and angiogenic signaling (9-22). Using murine tumor allograft models (23) we show that either systemic or endothelial cell specific suppression of phosphoinositide recycling results in reduced tumor growth and reduced tumor angiogenesis. Our results suggest that inhibition of phosphoinositide recycling may provide a useful anti-angiogenic approach, and highlights the general potential of targeting the resynthesis of rate limiting signaling substrates as a valuable therapeutic strategy.\n\nSUMMARY STATEMENTTargeting phosphoinositide recycling during tumor angiogenesis provides a potentially uniquely effective anti-cancer therapy.

cell biology↗