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bioRxiv2018

Single-cell analysis identifies EpCAM+/CDH6+/TROP-2- cells as human liver progenitors.

The liver is largely composed of hepatocytes and bile duct epithelial cells (BECs). Controversy exists as to whether a liver stem/progenitor cell capable of renewing both hepatocytes and BECs exists. Single cell RNA sequencing of freshly isolated human foetal and healthy adult liver identified hepatocyte, hepatoblast and liver progenitor cell (hLPC) populations. hLPCs, found at the interface between hepatocytes and bile ducts in both foetal and adult tissue, were distinguishable from BECs by their negative expression of TROP-2. Prospective isolation followed by in vitro culture demonstrated their potential for expansion and bi-lineage differentiation. The hLPC expression signature was also conserved within expanded cell populations specific to certain cases of liver injury and cancer. These data support the idea of a true progenitor existing within healthy adult liver that can be activated upon injury. Further work to define the mechanisms regulating hLPC behaviour could advance understanding of human development and disease.

cell biology↗

bioRxiv2018

Off-target inhibition by active site-targeting SHP2 inhibitors

Due to the involvement of SHP2 (SH2 domain-containing protein tyrosine phosphatase) in human disease, including Noonan syndrome and cancer, several inhibitors targeting SHP2 have been developed. Here, we report that the commonly used SHP2 inhibitor NSC-78788 does not exhibit robust inhibitory effects on growth factor-dependent MAPK (mitogen-activated protein kinase) pathway activation, and that the recently developed active site-targeting SHP2 inhibitors IIB-08, 11a-1, and GS-493 show off-target effects on ligand-evoked activation/trans-phosphorylation of the PDGFR{beta} (platelet-derived growth factor receptor {beta}). GS-493 also inhibits purified human PDGFR{beta} and SRC in vitro, whereas PDGFR{beta} inhibition by IIB-08 and 11a-1 occurs only in the cellular context. Our results argue for extreme caution in inferring specific functions for SHP2 based on studies using these inhibitors.

cell biology↗

bioRxiv2018

Timely double-strand break repair and pathway choice in pericentromeric heterochromatin depend on the histone demethylase dKDM4A

Introduction Introduction Results Discussion Materials Methods References One of the most harmful DNA lesions is a double-strand break (DSB), whose improper repair can lead to formation of aberrant chromosomes linked to cancer and developmental diseases 1. At DSBs, the severed strands of the DNA helix are repaired by a variety of mechanisms. The two major DSB repair pathways are Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). NHEJ repairs DNA by ligating both ends of the DSB together, often resulting in small insertions and deletions at the break site. HR repair involves more extensive processing of the DSB site, in which 5 to 3 end-resection of the DSB ends results in a single-stranded DNA sequence that invades and perfectly copies homologous sequences to repair the DS ...

cell biology↗

bioRxiv2018

Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence

Lysosomes receive and degrade cargo from endocytosis, phagocytosis and autophagy. They also play an important role in sensing and instructing cells on their metabolic state. The lipid kinase PIKfyve generates phosphatidylinositol-3,5-bisphosphate to modulate lysosome function. PIKfyve inhibition leads to impaired degradative capacity, ion dysregulation, abated autophagic flux, and a massive enlargement of lysosomes. Collectively, this leads to various physiological defects including embryonic lethality, neurodegeneration and overt inflammation. While being the most dramatic phenotype, the reasons for lysosome enlargement remain unclear. Here, we examined whether biosynthesis and/or fusion-fission dynamics contribute to swelling. First, we show that PIKfyve inhibition activates TFEB, TFE3 and MITF enhancing lysosome gene expression. However, this did not augment lysosomal protein levels during acute PIKfyve inhibition and deletion of TFEB and/or related proteins did not impair lysosome swelling. Instead, PIKfyve inhibition led to fewer but enlarged lysosomes, suggesting that an imbalance favouring lysosome fusion over fission causes lysosome enlargement. Indeed, conditions that abated fusion curtailed lysosome swelling in PIKfyve-inhibited cells.\n\nSummary statementPIKfyve inhibition causes lysosomes to coalesce, resulting in fewer, enlarged lysosomes. We also show that TFEB-mediated lysosome biosynthesis does not contribute to swelling.

cell biology↗

bioRxiv2018

Active presynaptic ribosomes in mammalian brain nerve terminals, and increased transmitter release after protein synthesis inhibition

Presynaptic neuronal activity requires the localization of thousands of proteins that are typically synthesized in the soma and transported to nerve terminals. Local translation for some dendritic proteins occurs, but local translation in mammalian presynaptic nerve terminals is difficult to demonstrate. Here, we present evidence for local presynaptic protein synthesis in the mammalian brain at a glutamatergic nerve terminal. We show an essential ribosomal component, 5.8s rRNA, in terminals. We also show active translation in nerve terminals, in situ, in brain slices demonstrating ongoing presynaptic protein synthesis. After inhibiting translation for ~1 hour, the presynaptic terminal exhibits increased spontaneous release, and increased evoked release with an increase in vesicle recycling during stimulation trains. Postsynaptic response, shape and amplitude were not affected. We conclude that ongoing protein synthesis limits vesicle release at the nerve terminal which reduces the need for presynaptic vesicle replenishment, thus conserving energy required for maintaining synaptic transmission.

cell biology↗

bioRxiv2018

Identification of Miro as a mitochondrial receptor for myosin XIX

Mitochondrial distribution in cells is critical for cellular function and proper inheritance during cell division. In mammalian cells, mitochondria are transported predominantly along microtubules by kinesin and dynein and along actin filaments by myosin. Myosin XIX (Myo19) associates with the outer mitochondrial membrane, but no specific receptor has been identified. Using proximity BioID labeling, we identified Miro-1 and Miro-2 as potential binding partners of Myo19. Interaction studies show that Miro-1 binds to a C-terminal fragment of the Myo19 tail region and that Miro recruits the Myo19 tail in vivo. This recruitment is regulated by the nucleotide-state of the N-terminal Rho-like GTPase domain of Miro. Notably, Myo19 protein stability in cells depends on its association with Miro. Finally, Myo19 regulates the subcellular distribution of mitochondria. Downregulation, as well as overexpression, of Myo19 induces perinuclear collapse of mitochondria, phenocopying the loss of kinesin KIF5 or its mitochondrial receptor Miro. These results suggest that Miro coordinates microtubule- and actin-based mitochondrial movement.

cell biology↗

bioRxiv2018

Golgi Renaissance: the pivotal role of the largest Golgi protein giantin

Golgi undergoes disorganization in response to the drugs or alcohol, but it is able to restore compact structure under recovery. This self-organization mechanism remains mostly elusive, as does the role of giantin, the largest Golgi matrix dimeric protein. Here, we found that in cells treated with Brefeldin A (BFA) or ethanol (EtOH), Golgi disassembly is associated with giantin de-dimerization, which was restored to the dimer form after BFA or EtOH washout. Cells lacking giantin are disabled for the restoration of the classical ribbon Golgi, and they demonstrate altered trafficking of proteins to the cell surface. The fusion of the nascent Golgi membranes is mediated by the cross-membrane interaction of Rab6a GTPase and giantin. Giantin is involved in the formation of long intercisternal connections, which in giantin-depleted cells was replaced by the short bridges that formed via oligomerization of GRASP65. This phenomenon occurs in advanced prostate cancer cells, in which a fragmented Golgi phenotype is maintained by the dimerization of GRASP65. Thus, we provide a model of Golgi Renaissance, which is impaired in aggressive prostate cancer.

cell biology↗

bioRxiv2018

Organizing neuronal ER-PM junctions is a conserved nonconducting function of Kv2 plasma membrane ion channels

Endoplasmic reticulum (ER) and plasma membrane (PM) form junctions crucial to ion and lipid signaling and homeostasis. The Kv2.1 ion channel is unique among PM proteins in organizing ER-PM junctions. Here, we show that this organizing function is conserved between Kv2 family members that differ in their biophysical properties, modulation and cellular expression. Manipulation of actin cytoskeleton surrounding Kv2 ER-PM junctions affects their spatial organization. Kv2-containing ER-PM junctions overlap with those formed by canonical ER-PM tethers. ER-PM junction organization by Kv2 channels is unchanged by point mutations that eliminate ion conduction, but abolished by those that eliminate PM clustering without impacting ion channel function. Kv2.2 is distinct in lacking the reversible modulation of junction organization present in Kv2.1. Brain neurons in Kv2 double knockout mice have altered ER-PM junctions, demonstrating a conserved in vivo function for Kv2 family members distinct from their canonical role as ion-conducting channels shaping neuronal excitability.

cell biology↗

bioRxiv2018

Nucleolar stress triggers the irreversible cell cycle slow down leading to cell death during replicative aging in Saccharomyces cerevisiae

The accumulation of Extrachromosomal rDNA Circles (ERCs) and their asymmetric segregation upon division have been hypothesized to be responsible for replicative senescence in mother yeasts and rejuvenation in daughter cells. However, it remains unclear by which molecular mechanisms ERCs would trigger the irreversible cell cycle slow-down leading to cell death. We show that ERCs accumulation is concomitant with a nucleolar stress, characterized by a massive accumulation of pre-rRNAs in the nucleolus, leading to a loss of nucleus-to-cytoplasm ratio, decreased growth rate and cell-cycle slow-down. This nucleolar stress, observed in old mothers, is not inherited by rejuvenated daughters. Unlike WT, in the long-lived mutant fob1{triangleup}, a majority of cells is devoid of nucleolar stress and does not experience replicative senescence before death. Our study provides a unique framework to order the successive steps that govern the transition to replicative senescence and highlights the causal role of nucleolar stress in cellular aging.

cell biology↗

bioRxiv2018

TOXOPLASMA ACTIVATES HOST HYPOXIA INDUCIBLE FACTOR-1 BY CYTOPLASMIC TRAPPING AND LAMP1-DEPENDENT LYSOSOMAL DEGRADATION OF PROLYL-HYDROXYLASE 2

Hypoxia Inducible Factor-1 is a metazoan heterodimeric transcription factor that senses changes in O2 levels. HIF-1 subunit abundance is post-translationally regulated by prolyl-hydroxylase domain enzymes (PHDs), which use molecular O2 and -ketoglutarate to hydroxylate two prolyl-residues in HIF-1. Three PHDs have been identified and PHD2 is the most critical regulator of HIF-1. HIF-1 can also be activated independently of hypoxia and in some cases this is due to changes in PHD2 abundance through poorly understood mechanisms. Previously, we reported that under O2-replete conditions that the intracellular parasite Toxoplasma gondii activates HIF-1 by reducing PHD2 protein abundance. Here, we demonstrate that Toxoplasma regulates PHD2 through a multistep process. First, PHD2 is a nucleocytoplasmic protein and Toxoplasma induces PHD2 cytoplasmic accumulation to separate it from nuclear HIF-1. PHD2 is then degraded by lysosomes independently of the major autophagic processes, macroautophagy or chaperone-mediated autophagy. Rather, PHD2 interacts with the major lysosomal membrane protein, LAMP1, which is required for HIF-1 activation. These data therefore highlight for the first time that cytoplasmic trapping and subsequent lysosomal degradation of a host nucleocytoplasmic protein is a mechanism used by a microbial pathogen to regulate host gene expression.

cell biology↗

bioRxiv2018

The RZZ complex integrates spindle checkpoint maintenance with dynamic expansion of unattached kinetochores

The Mad1-Mad2 heterodimer is the catalytic hub of the spindle assembly checkpoint (SAC), which controls mitosis through assembly of a multi-subunit anaphase inhibitor, the mitotic checkpoint complex (MCC) [1, 2]. Mad1-Mad2 first catalyzes MCC assembly at interphase nuclear pores [3], then migrates to kinetochores at nuclear envelope breakdown (NEBD) and resumes MCC assembly until bipolar spindle attachment is complete [1, 2]. There is significant debate about the factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes [4-9]. Through gene editing and live-cell imaging, we found that the human Rod-Zw10-Zwilch (RZZ) complex is dispensable for cell viability and initial recruitment of Mad1-Mad2 to kinetochores at NEBD, but then becomes necessary to tether Mad1-Mad2 at kinetochores and sustain SAC arrest in cells challenged with spindle poisons. We also show that RZZ forms the mesh-like fibrous corona, a structural expansion of the outer kinetochore important for timely chromosome congression [10-13] once Mps1 phosphorylates the N-terminus of Rod. Artificially tethering Mad1-Mad2 to kinetochores enabled long-term mitotic arrest in the absence of RZZ. Conversely, blocking early RZZ-independent recruitment of Mad1-Mad2 eliminated the transient SAC response in RZZ-null cells. We conclude that RZZ drives structural changes in the outer kinetochore that facilitate chromosome bi-orientation and chronic SAC transduction, a key determinant of cytotoxicity during anti-mitotic drug therapy [14-16].

cell biology↗

bioRxiv2018

Increased Adhesion Of CML Cells By ABL1 Tyrosine Kinase Inhibitors Induce Tunneling Nanotubes

Summary statementThis study describes the effects of tyrosine kinase inhibitors on tunneling nanotube formation via increased adhesion through {beta}-integrin in chronic myeloid leukemia cells.\n\nAbstractThe actin-containing cell-to-cell communicator tunneling nanotube (TNT) is involved in regulation of cell death threshold of leukemic cells, while the mechanism of TNT regulation is mostly unknown. We have investigated TNT formation and its response to treatment in chronic myeloid leukemia (CML) cells with the pathognomonic chimeric fusion kinase BCR-ABL1 after treatment with the tyrosine kinase inhibitor nilotinib and interferon-. Bone marrow cells of chronic phase CML patients and the CML cell line Kcl-22 formed few or no TNTs. Nilotinib and interferon- treatment induced TNT formation in Kcl-22 cells and were found to be linked to increased adherence to fibronectin coated surfaces by restoration of {beta}1-integrin function. This suggests modulation of TNT cell-cell communication in CML as a novel mechanism in kinase inhibitor therapy of CML.

cell biology↗

bioRxiv2018

Increased green autofluorescence is a marker for non-invasive prediction of H2O2-induced cell death and decreases in the intracellular ATP of HaCaT cells

Since oxidative stress plays important pathological roles in numerous diseases, it is of both critical theoretical and clinical significance to search for the approaches for predicting oxidative damage. Cellular models have great value for studying oxidative damage, which would be significantly promoted if non-invasive approaches for predicting oxidative damage can be established without the need of exogenous probes. In our current study, we tested our hypothesis that changes of the autofluorescence (AF) of cells may be used for predicting oxidative cellular damage. Our study found that H2O2 dose-dependently increased the green AF of HaCaT keratinocyte cell line at non-nuclear regions assessed at 1 hr or 3 hrs after the H2O2 exposures, while H2O2 did not affect the green AF of other cell types tested in our study, including PC 12 cells and BV2 microglia. We further found that the increases in the AF of HaCaT cells are highly correlated with the H2O2-induced increases in early-stage apoptosis, late-stage apoptosis and necrosis assessed at 18 hrs after the H2O2 exposures, which are also negatively correlated with the intracellular ATP levels of the H2O2-treated cells assessed at 18 hrs after the H2O2 exposures. Collectively, our study has suggested that increased AF may become the first endogenous marker for non-invasive prediction of oxidative damage selectively for such cell types as HaCaT cells. Compared with traditional approaches, our method may have significant value for studying oxidative damage of keratinocytes with significantly higher efficiency and lower cost.

cell biology↗

bioRxiv2018

Conditional control of fluorescent protein degradation by an auxin-dependent nanobody

The conditional and reversible depletion of proteins by auxin-mediated degradation is a powerful tool to investigate protein functions in cells and whole organisms. However, its wider applications require fusing the auxin-inducible degron (AID) to individual target proteins. Thus, establishing the auxin system for multiple proteins can be challenging. Another approach for directed protein degradation are anti-GFP nanobodies, which can be applied to GFP stock collections that are readily available in different experimental models. Here, we combine the advantages of auxin and nanobody-based degradation technologies creating an AID-nanobody to degrade GFP-tagged proteins at different cellular structures in a conditional and reversible manner in human cells. We demonstrate efficient and reversible inactivation of the anaphase promoting complex/cyclosome (APC/C) and thus provide new means to study the functions of this essential ubiquitin E3 ligase. Further, we establish auxin degradation in a vertebrate model organism by employing AID-nanobodies in zebrafish.

cell biology↗

bioRxiv2018

Peptides derived from plant virus VPg protein inhibit eIF4E oncogene

Viruses of the Potyviridae family have VPg protein covalently attached to the 5 end of their linear RNA genome. The protein interacts with the host translation initiation factor eIF4E that occurs in plant cells in two isoforms, one being the preferable target of a given VPg, the remaining one still acting in host protein synthesis. In animal cells only one form of eIF4E is directly involved in protein synthesis. The human eIF4E is known to be an oncogene; elevated expression of eIF4E leads to oncogenic transformation, cancers in animal models and poor prognosis in human cancers, while reduction of the eIF4E level can reverse the transformed phenotype. We show that VPg protein delivery to cells containing only one eIF4E isoform involved in protein synthesis resulted in immobilization of eIF4E in the cytoplasm. The region of VPg involved in the interaction with eIF4E has been partially identified. Peptides derived from this region interacted better with eIF4E than complete VPg protein. Here we characterized one of VPg peptides, VPg5 and we show that VPg5 delivered to colon carcinoma HCT116 cells is able to inhibit cell growth, which is accompanied by reduction in eIF4E level.\n\nList of abbreviations

cell biology↗

bioRxiv2018

Validation of a library of cGMP-compliant human pluripotent stem cell lines for use in liver therapy

Recent advancements in the production of hepatocytes from human pluripotent stem cells (hPSC-Heps) afford tremendous possibilities for treatment of patients with liver disease. Validated current good manufacturing practice (cGMP) lines are an essential prerequisite for such applications but have only recently been established. Whether such cGMP lines are capable of hepatic differentiation is not known. To address this knowledge gap, we examined the proficiency of three recently derived cGMP lines (two hiPSC and one hESC) to differentiate into hepatocytes and their suitability for therapy. hPSC-Heps generated using a chemically defined four-step hepatic differentiation protocol uniformly demonstrated highly reproducible phenotypes and functionality. Seeding into a 3D PEG-DA fabricated inverted colloid crystal (ICC) scaffold converted these immature progenitors into more advanced hepatic tissue structures. Hepatic constructs could also be successfully encapsulated into the immune-privileged material alginate. This is the first report we are aware of demonstrating cGMP-compliant hPSCs can generate cells with advanced hepatic function potentially suitable for future therapeutic applications.

cell biology↗

bioRxiv2018

MECHANOSENSATION DYNAMICALLY COORDINATES POLAR GROWTH AND CELL WALL ASSEMBLY TO PROMOTE CELL SURVIVAL

How growing cells cope with size expansion while ensuring mechanical integrity is not known. In walled cells, such as those of microbes and plants, growth and viability are both supported by a thin and rigid encasing cell wall (CW). We deciphered the dynamic mechanisms controlling wall surface assembly during cell growth, using a novel sub-resolution microscopy approach to monitor CW thickness in live rod-shaped fission yeast cells. We found that polar cell growth yielded wall thinning, and that thickness negatively influenced growth. Thickness at growing tips exhibited oscillating behavior with thickening phases followed by thinning phases, indicative of a delayed feedback promoting thickness homeostasis. This feedback was mediated by mechanosensing through the cell wall integrity pathway, which probes strain in the wall to adjust synthase localization and activity to surface growth. Mutants defective in thickness homeostasis lysed by rupturing the wall demonstrating its essential role for walled cell survival.

cell biology↗

bioRxiv2018

Myricetin Attenuates LPS-induced Inflammation in RAW 264.7 Macrophages and Mouse Models

BackgroundMyricetin has been demonstrated to inhibit inflammation in a variety of diseases, but little is known about its characters in acute lung injury (ALI). In this study, we aimed to investigate the protective effects of myricetin on inflammation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and a LPS-induced lung injury model.\n\nMethodsSpecifically, we investigated its effects on lung edema and histological damage by lung W/D weight ratio, HE staining and Evans Blue dye. Then macrophage activation was detected by evaluating the TNF-, IL-6 and IL-1{beta} mRNA and protein iNOS and COX-2. Myricetin was used to detect the impact on the inflammatory responses in LPS-induced RAW264.7 cells with the same manners in mouse model. Finally, NF-{kappa}B and MAPK signaling pathways were investigated with Western blot assay in LPS-induced RAW264.7 cells.\n\nResultsMyricetin significantly inhibited the production of the pro-inflammatory cytokines in vitro and in vivo. The in vivo experiments showed that pretreatment with Myricetin markedly attenuated the development of pulmonary edema, histological severities and macrophage activation in mice with ALI. The underlying mechanisms were further demonstrated in vitro that myricetin exerted an anti-inflammatory effect through suppressing the NF-{kappa}B p65 and AKT activation in NF-{kappa}B pathway and JNK, p-ERK and p38 in mitogen-activated protein kinases signaling pathway.\n\nConclusionMyricetin alleviated ALI by inhibiting macrophage activation, and inhibited inflammation in vitro and in vivo. It may be a potential therapeutic candidate for the prevention of inflammatory diseases.

cell biology↗