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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

β-actin mRNA interactome mapping by proximity biotinylation

The molecular function and fate of mRNAs are controlled by RNA binding proteins (RBPs). However, identification of the interacting proteome of a specific mRNA in vivo is still very challenging. Based on the widely-used RNA tagging with MS2 aptamers for RNA visualization, we developed a novel RNA proximity biotinylation (RNA-BioID) method by tethering biotin ligase (BirA*) via MS2 coat protein (MCP) at the 3UTR of endogenously MS2 tagged {beta}-actin mRNA in mouse embryonic fibroblasts (MEFs). We demonstrate the dynamics of the {beta}-actin mRNA interactome by characterizing its changes upon serum-induced localization of the mRNA. Apart from the previously known interactors, we identified over 60 additional {beta}-actin associated RBPs by RNA-BioID, among them the KH-domain containing protein FUBP3/MARTA2 has shown to be required for {beta}-actin mRNA localization. This protein binds to the 3-untranslated region of {beta}-actin mRNA, is essential for {beta}-actin mRNA localization but does not interact with the characterized {beta}-actin zipcode element. RNA-BioID provides a tool to identify new mRNA interactors and to study the dynamic view of the interacting proteome of endogenous mRNAs in space and time.

cell biology↗

bioRxiv2018

Icariin induces MC3T3-E1 cell proliferation and differentiation via the BMP-2/Smads/Runx2 signal pathway

Icariin, the main active ingredient of Epimedium, has played an important role in bone anabolism. However, the molecular mechanism for this effect was not convincingly reported yet. In this paper, the role of icariin on cell morphology, viability, cell cycling and the activity of alkaline phosphatase (ALP) were studied, and the molecular mechanism of icariin induced osteogenic effect was also investigated. Icariin with different concentrations (10, 20 and 40 ng/ml) was used to modify the pre-osteoblastic MC3T3-E1 cells for 48, 72 and 96 h, and the proliferation, morphology, and the cell cycle of the cells were evaluated by Cell Counting Kit-8 (CCK-8), microscopy and flow cytometry, respectively. Bone morphogenic protein-2 (BMP-2), bone morphogenic protein receptor-2 (BMPR-2), Smad4, Smadl/5/8 proteins expression levels were obtained by Western blotting and the expression levels of runt-related transcription factor 2 (Runx2) mRNA was examined by reverse transcription-polymerase chain reaction (RT-PCR). In this study, we found that icariin could promote the proliferation and differentiation of MC3T3-E1 cells in a dose - and time-dependent manner. Icariin could stimulate the expression of the BMP-2, BMPR-2, Smad4 and Smadl/5/8 proteins. Furthermore, icariin could upregulate the expression of Runx2 mRNA. These results showed that icariin played an important role in upregulating BMP-2 expression to activate the BMP-2/Smads/Runx2 signal pathway for increasing both the proliferation and differentiation of the MC3T3-E1 cells. However, the osteogenic effects of icariin can be suppressed by the BMP-2 antagonist (Noggin). In conclusion, we demonstrate that icariin is an osteoinductive factor that exerts its osteogenic effect by regulating the BMP-2/Smads/Runx2 signal pathway in MC3T3-E1 cells.

cell biology↗

bioRxiv2018

The ER chaperone PfGRP170 is essential for asexual development and is linked to stress response in malaria parasites.

The vast majority of malaria mortality is attributed to one parasite species: Plasmodium falciparum. Asexual replication of the parasite within the red blood cell is responsible for the pathology of the disease. In Plasmodium, the endoplasmic reticulum (ER) is a central hub for protein folding and trafficking as well as stress response pathways. In this study, we tested the role of an uncharacterized ER protein, PfGRP170, in regulating these key functions by generating conditional mutants. Our data show that PfGRP170 localizes to the ER and is essential for asexual growth, specifically required for proper development of schizonts. PfGRP170 is essential for surviving heat shock, suggesting a critical role in cellular stress response. The data demonstrate that PfGRP170 interacts with the Plasmodium orthologue of the ER chaperone, BiP. Finally, we found that loss of PfGRP170 function leads to the activation of the Plasmodium eIF2 kinase, PK4, suggesting a specific role for this protein in this parasite stress response pathway.

cell biology↗

bioRxiv2018

Yes-associated protein (YAP) is required in maintaining normal ovarian follicle development and function

Yes-associated protein (YAP) is one of the major components of the Hippo signaling pathway, also known as the Salvador/Warts/Hippo (SWH) pathway. Although the exact extracellular signal that controls the Hippo pathway is currently unknown, increasing evidence supports a critical role of the Hippo pathway in embryonic development, regulation of organ size, and carcinogenesis. The ovary is one of few adult tissues that exhibit cyclical changes. Ovarian follicles, the basic units of ovary, are composed of a single oocyte surrounded by expanding layers of granulosa and theca cells. Granulosa cells (GCs) produce sex steroids and growth factors, which facilitate the development of the follicle and maturation of the oocyte. It has been reported that YAP is highly expressed in human GC tumors, but the role of YAP in normal ovarian follicle development is largely unknown. In current study, we examined YAP expression in bovine ovaries. We demonstrate that downstream hippo signaling effector protein, YAP and transcription co-activator, TAZ, are present and localization of both YAP and TAZ are density-dependent. Likewise, YAP and TAZ are critically involved in granulosa cell proliferation. Furthermore, reducing YAP in granulosa cells inhibits FSH-induced aromatase expression and estradiol biosynthesis. The data suggest that YAP plays an important role in the development of ovarian follicles and estradiol synthesis, which are necessary for maintaining normal ovarian function.

cell biology↗

bioRxiv2018

TANGO1 and SEC12 are co-packaged with procollagen I to facilitate the generation of large COPII carriers

Large COPII-coated vesicles serve to convey the large cargo procollagen I (PC1) from the endoplasmic reticulum (ER). The link between large cargo in the lumen of the ER and modulation of the COPII machinery remains unresolved. TANGO1 is required for procollagen (PC) secretion and interacts with PC and COPII on opposite sides of the ER membrane, but evidence suggests that TANGO1 is retained in the ER, and not included in normal size (<100nm) COPII vesicles. Here we show that TANGO1 is exported out of the ER in large COPII-coated PC1 carriers, and retrieved back to the ER by the retrograde coat, COPI, mediated by the C-terminal RDEL retrieval sequence of HSP47. TANGO1 is known to target the COPII initiation factor SEC12 to ER exit sites through an interacting protein, cTAGE5. SEC12 is important for the growth of COPII vesicles, but it is not sorted into small budded vesicles. We found both cTAGE5 and SEC12 were exported with TANGO1 in large COPII carriers. In contrast to its exclusion from small transport vesicles, SEC12 was particularly enriched around ER membranes and large COPII carriers that contained PC1. We constructed a split GFP system to recapitulate the targeting of SEC12 to PC1 via the luminal domain of TANGO1. The minimal targeting system enriched SEC12 around PC1 and generated large PC1 carriers. We conclude that TANGO1, cTAGE5, and SEC12 are co-packed with PC1 into COPII carriers to increase the size of COPII thus ensuring the capture of large cargo.

cell biology↗

bioRxiv2018

Live-cell single-molecule analysis of β2-adrenergic receptor diffusion dynamics and confinement

Signal transduction mechanisms and successive regulatory processes alter the lateral mobility of {beta}2-adrenergic receptors ({beta}2AR). In this work we combined modern single particle tracking methods in order to analyze the diffusion dynamics of SNAP-tagged {beta}2AR in HEK wild-type cells and HEK {beta}-arrestin knockout cells before and after agonist stimulation. For analysis of trajectories we first used mean squared displacement (MSD) analysis. Secondly, we applied an advanced variational Bayesian treatment of hidden Markov models (vbSPT) in combination with the recently introduced packing coefficient (Pc), which together provided a detailed model of three discrete diffusive states, state transitioning and spatial confinement. Interesting to note, state switching between S3 (fast-diffusing) and S1 (slow-diffusing) occurred sequentially over an intermediate state S2. After ligand stimulation more SNAP-tagged {beta}2AR in HEK wild-type cells switched occupancy into the slow-diffusing state, whereas less receptors were found in the fast diffusive state. Unexpectedly, all three states showed a fraction of confined receptor mobility that increased under stimulation, but confinement sizes were unaffected. Receptor diffusion characteristics were comparable in HEK {beta}-arrestin knockout cells under basal conditions and only minor but non-significant changes occurred upon stimulation, as expected from the depletion of {beta}-arrestin, an important regulatory protein. The data presented here on the occurrence of different diffusion states, their transitioning and variable spatial confinements clearly indicate that lateral mobility of {beta}2AR is much more complex than previously thought.

cell biology↗

bioRxiv2018

In vivo, regulated reconstitution of spindle checkpoint arrest and silencing through chemical-induced dimerisation

Chemical-induced dimerisation (CID) uses small molecules to control specific protein-protein interactions. Here, we employ CID dependent on the plant hormone abscisic acid (ABA) to reconstitute spindle checkpoint signalling in fission yeast. The spindle checkpoint signal usually originates at unattached or inappropriately attached kinetochores. These are complex, multi-protein structures with several important functions. To bypass kinetochore complexity, we take a reductionist approach to study checkpoint signalling. We generate a synthetic checkpoint arrest ectopically by inducing hetero-dimerisation of the checkpoint proteins Mph1Mps1 and Spc7KNL1. These proteins are engineered such that they cant localise to kinetochores, and only form a complex in the presence of ABA. Using this novel assay we are able to checkpoint arrest a synchronous population of cells within 30 minutes of ABA addition. This assay allows for detailed genetic dissection of checkpoint activation and importantly it also provides a valuable tool for studying checkpoint silencing. To analyse silencing of the checkpoint and the ensuing mitotic exit, we simply wash-out the ABA from arrested cells. We show here that silencing is critically-dependent on PP1Dis2 recruitment to Mph1Mps1-Spc7KNL1 signalling platforms.

cell biology↗

bioRxiv2018

Peroxisomes control mitochondrial dynamics and the mitochondrion-dependent pathway of apoptosis

Summary StatementsWe unveil a previously unrecognized role of peroxisomes in the regulation of mitochondrial fission-fusion dynamics, mitochondrion-dependent caspase activation, and cellular apoptosis.\n\nAbstractPeroxisomes cooperate with mitochondria in the performance of cellular metabolic functions such as fatty acid oxidation and maintenance of redox homeostasis. Whether peroxisomes also regulate mitochondrial fission-fusion dynamics or mitochondrion-dependent apoptosis has remained unclear, however. We now show that genetic ablation of the peroxins Pex3 or Pex5, which are essential for peroxisome biogenesis, resulted in mitochondrial fragmentation in mouse embryonic fibroblasts (MEFs) in a manner dependent on dynamin-related protein 1 (Drp1). Conversely, treatment with 4-phenylbutyric acid, an inducer of peroxisome proliferation, resulted in mitochondrial elongation in wild-type MEFs, but not in Pex3-deficient MEFs. We further found that peroxisome deficiency increased the levels of cytosolic cytochrome c and caspase activity under basal conditions without inducing apoptosis. It also greatly enhanced etoposide-induced caspase activation and apoptosis, indicative of an enhanced cellular sensitivity to death signals. Together, our data unveil a previously unrecognized role of peroxisomes in the regulation of mitochondrial dynamics and mitochondrion-dependent apoptosis. Given that mutations of peroxin genes are responsible for lethal disorders such as Zellweger syndrome, effects of such mutations on mitochondrion-dependent apoptosis may contribute to disease pathogenesis.

cell biology↗

bioRxiv2018

Label free 3D analysis of organelles in living cells by refractive index shows pre-mitotic organelle spinning in mammalian stem cells

Holo-tomographic microscopy (HTM) is a label-free non-phototoxic microscopy method reporting the fine changes of a cells refractive indexes (RI) in 3D. By combining HTM with epifluorescence, we demonstrate that cellular organelles such as Lipid droplets and mitochondria show a specific RI signature that distinguishes them with high resolution and contrast. We further show that HTM allows to follow in unprecedented ways the dynamics of mitochondria, lipid droplets as well as that of endocytic structures in live cells over long period of time, which led us to observe to our knowledge for the first time a global organelle spinning occurring before mitosis.

cell biology↗

bioRxiv2018

A role for Gic1 and Gic2 in Cdc42 polarization

The conserved Rho-family GTPase Cdc42 is a master regulator of polarity establishment in many cell types. Cdc42 becomes activated and concentrated in a region of the cell cortex, and recruits a variety of effector proteins to that site. In turn, many effectors participate in regulation of cytoskeletal elements in order to remodel the cytoskeleton in a polarized manner. The budding yeast Saccharomyces cerevisiae has served as a tractable model system for studies of cell polarity. In yeast cells, Cdc42 polarization involves a positive feedback loop in which effectors called p21-activated kinases (PAKs) act to recruit a Cdc42-directed guanine nucleotide exchange factor (GEF), generating more GTP-Cdc42 in areas that already have GTP-Cdc42. The GTPase-interacting components (GICs) Gic1 and Gic2 are also Cdc42 effectors, and have been implicated in regulation of the actin and septin cytoskeleton. However, we report that cells lacking GICs are primarily defective in polarizing Cdc42 itself, suggesting that they act upstream as well as downstream of Cdc42 in yeast. Our findings suggest that feedback pathways involving GTPase effectors may be more prevalent than had been appreciated.

cell biology↗

bioRxiv2018

Gp78 E3 ubiquitin ligase mediates both basal and damage-induced mitophagy

Mitophagy, the elimination of mitochondria by the autophagy machinery, evolved to monitor mitochondrial health and maintain mitochondrial integrity. PINK1 is a sensor of mitochondrial health that recruits Parkin and other mitophagy-inducing ubiquitin ligases to depolarized mitochondria. However, mechanisms underlying mitophagic control of mitochondrial homeostasis, basal mitophagy, remain poorly understood. The Gp78 E3 ubiquitin ligase, an endoplasmic reticulum membrane protein, induces mitochondrial fission, endoplasmic reticulum-mitochondria contacts and mitophagy of depolarized mitochondria. CRISPR/Cas9 knockout of Gp78 in HT-1080 fibrosarcoma cells results in reduced ER-mitochondria contacts, increased mitochondrial volume and resistance to CCCP-induced mitophagy. Knockdown (KD) of the essential autophagy protein ATG5 increased mitochondrial volume of wild-type cells but did not impact mitochondrial volume of Gp78 knockout cells. This suggests that endogenous Gp78 actively eliminates mitochondria by autophagy in wild-type HT-1080 cells. Damage-induced mitophagy of depolarized mitochondria, in the presence of CCCP, but not basal mitophagy was prevented by knockdown of PINK1. This suggests that endogenous Gp78 plays dual roles in mitophagy induction: 1) control of mitochondrial homeostasis through mitophagy of undamaged mitochondria; and 2) elimination of damaged mitochondria through PINK1.

cell biology↗

bioRxiv2018

Budding yeast complete DNA replication after chromosome segregation begins

To faithfully transmit genetic information, cells must replicate their entire genome before division. This is thought to be ensured by the temporal separation of replication and chromosome segregation. Here we show that in a substantial fraction of unperturbed yeast cells, DNA replication finishes during anaphase, late in mitosis. High cyclin-Cdk activity inhibits replication in metaphase, and the decrease in cyclin-Cdk activity during mitotic exit allows DNA replication to finish at difficult-to-replicate regions. Replication during late mitosis correlates with elevated mutation rates, including copy number variation. Thus, yeast cells temporally overlap replication and chromosome segregation during normal growth, possibly allowing cells to maximize population-level growth rate while simultaneously exploring greater genetic space.\n\nOne Sentence SummaryCompletion of DNA replication is coupled to downregulation of Cyclin-Dependent Kinase during mitotic exit.

cell biology↗

bioRxiv2018

Iron and Heme Coordinate Erythropoiesis through HRI-Mediated Regulation of Protein Translation and Gene Expression

Iron and heme play central roles in red blood cell production. However, the mechanisms by which iron and heme levels coordinate erythropoiesis remain incompletely understood. HRI is a heme-regulated kinase that controls translation by phosphorylating eIF2. Here, we investigate the global impact of iron, heme and HRI on protein translation in vivo in murine primary erythroblasts using ribosome profiling. By defining the underlying changes in translation during iron and HRI deficiencies, we validate known regulators of this process, including Atf4, and identify novel pathways such as co-regulation of ribosomal protein mRNA translation. Surprisingly, we found that heme and HRI pathways, but not iron-regulated pathways, mediate the major protein translational and transcriptional responses to iron deficiency in erythroblasts in vivo and thereby identify previously unappreciated regulators of erythropoiesis. Our genome-wide study uncovers the major impact of the HRI-mediated integrated stress response for the adaptation to iron deficiency anemia.

cell biology↗

bioRxiv2018

A contraction stress model of hypertrophic cardiomyopathy due to thick filament sarcomere mutations

Thick filament sarcomere mutations are the most common cause of hypertrophic cardiomyopathy (HCM), a disorder of heart muscle thickening associated with sudden cardiac death and heart failure, with unclear mechanisms. We engineered an isogenic panel of four human HCM induced pluripotent stem cell (iPSc) models using CRISPR/Cas9, and studied iPSc-derived cardiomyocytes (iCMs) in 3-dimensional cardiac microtissue (CMT) assays that resemble in vivo cardiac architecture and biomechanics. HCM mutations result in hypercontractility in association with prolonged relaxation kinetics in proportion to mutation pathogenicity but not calcium dysregulation. RNA sequencing and protein expression studies identified that HCM mutations result in p53 activation secondary to increased oxidative stress, which results in increased cytotoxicity that can be reversed by p53 genetic ablation. Our findings implicate hypercontractility as an early consequence of thick filament mutations, and the p53 pathway as a molecular marker and candidate therapeutic target for thick filament HCM.

cell biology↗

bioRxiv2018

PBRM1 regulates stress response in epithelial cells

Polybromo1 (PBRM1) is a chromatin remodeler subunit highly mutated in cancer, particularly renal clear cell carcinoma. PBRM1 is a member of the SWI/SNF subcomplex, PBAF (PBRM1-Brg1/Brm Associated Factors) and is characterized by six tandem bromodomains. Here we establish a role for PBRM1 in epithelial cell maintenance through the expression of genes involved in cell adhesion, metabolism, stress response, and apoptosis. In support of a general role for PBRM1 in stress response and apoptosis, we observe that loss of PBRM1 results in an increase in reactive oxygen species generation and a decrease in cellular viability under stress conditions. We find that loss of PBRM1 promotes cell growth under favorable conditions but is required for cell survival under conditions of cellular stress.

cell biology↗

bioRxiv2018

Mutant p53 triggers a dynamin-1/APPL1 endosome feedback loop that regulates β1 integrin recycling and migration

Multiple mechanisms contribute to cancer cell progression and metastatic activity, including changes in endocytic trafficking and signaling of cell surface receptors. We report that gain-of-function (GOF) mutant p53 expression enhances {beta} integrin and EGF receptor recycling and increases cell migration by triggering a positive feedback loop involving the activation of dynamin-1 (Dyn1) and accumulation of a spatially-restricted subpopulation of APPL1-positive perimeter endosomes. DNM1 is upregulated at both the mRNA and protein levels in a manner dependent on expression of GOF mutant p53. Perimeter APPL1 endosomes are required for rapid recycling of EGFR and {beta}1 integrins and modulate Akt signaling and Dyn1 activation to create the positive feedback loop that culminates in increased focal adhesion turnover and cell migration. Thus, Dyn1- and Akt-dependent perimeter APPL1 endosomes function as a nexus, integrating signaling and receptor trafficking, that can be co-opted by cancer cells for mutant p53-driven migration and invasion.

cell biology↗

bioRxiv2018

MYO1C facilitates arrival at the Golgi apparatus through stabilization of branched actin

We aim at the identification of myosin motor proteins that control trafficking at the Golgi apparatus. In addition to the known Golgi-associated myosins MYO6, MYO18A and MYH9 (myosin IIA), we identify MYO1C as a novel player at the Golgi. We demonstrate that depletion of MYO1C induces Golgi apparatus fragmentation and decompaction. MYO1C accumulates at dynamic structures around the Golgi apparatus that colocalize with Golgi-associated actin dots. Interestingly, MYO1C depletion leads to loss of cellular F-actin, and Golgi apparatus decompaction is also observed after the inhibition or loss of the Arp2/3 complex. We show that the functional consequences of MYO1C depletion is a delay in the arrival of incoming transport carriers, both from the anterograde and retrograde routes. We propose that MYO1C stabilizes branched actin at the Golgi apparatus that facilitates the arrival of incoming transport at the Golgi.

cell biology↗

bioRxiv2018

Mitochondrial network fragmentation modulates mutant mtDNA accumulation independently of absolute fission-fusion rates

Mitochondrial DNA (mtDNA) mutations cause severe congenital diseases but may also be associated with healthy aging. MtDNA is stochastically replicated and degraded, and exists within organelles which undergo dynamic fusion and fission. The role of the resulting mitochondrial networks in the time evolution of the cellular proportion of mutated mtDNA molecules (heteroplasmy), and cell-to-cell variability in heteroplasmy (heteroplasmy variance), remains incompletely understood. Heteroplasmy variance is particularly important since it modulates the number of pathological cells in a tissue. Here, we provide the first wide-reaching theoretical framework which bridges mitochondrial network and genetic states. We show that, under a range of conditions, the (genetic) rate of increase in heteroplasmy variance and de novo mutation are proportionally modulated by the (physical) fraction of unfused mitochondria, independently of the absolute fission-fusion rate. In the context of selective fusion, we show that intermediate fusion/fission ratios are optimal for the clearance of mtDNA mutants. Our findings imply that modulating network state, mitophagy rate and copy number to slow down heteroplasmy dynamics when mean heteroplasmy is low could have therapeutic advantages for mitochondrial disease and healthy aging.

cell biology↗