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bioRxiv2018

Difference in accumulation pattern of the allergens within the same PR10 family in transgenic rice

Apple food allergen Mal d 1 and birch pollen allergen Bet v 1 belong to the same pathogen related protein 10 (PR10) family. When each of these allergens was expressed as a secretory protein by fusion with the GFP reporter in transgenic rice by ligating an N terminal signal peptide and a C terminal KDEL ER retention signal under the control of the maize ubiquitin constitutive promoter, the GFP:Mald1 highly accumulated in various tissues, whereas the accumulation level of GFP:Betv1 was remarkably reduced in vegetative tissues except for seed. Analysis by RT-PCR exhibited that there was little difference in transcript levels between them, indicating the involvement of post-transcriptional regulation. To investigate the cause of such difference in accumulation levels, deletion analysis of the Mal d 1 and domain swapping between them were carried out in transgenic rice. These results showed that the region between positions 41-90 in the Mal d 1 is predominantly implicated in higher level accumulation in vegetative tissues as well as seed compared with the Bet v 1. It is notable that GFP:Mald1 directed by the ubiquitin promoter is deposited in huge PBs in aleurone layer rather than starchy endosperm.\n\nHighlightSpecific region of PR10 proteins is mainly implicated in their stability in vegetative tissues when expressed in transgenic rice.

cell biology↗

bioRxiv2018

Tropomyosin 3.5 protects F-actin networks required for tissue biomechanical properties

Tropomyosins (Tpms) stabilize F-actin and regulate interactions with other actin-binding proteins. The eye lens changes shape in order to fine focus light to transmit a clear image, and thus lens organ function is tied to its biomechanical properties, presenting an opportunity to study Tpm functions in tissue mechanics. The major mouse lens Tpm is Tpm3.5 (TM5NM5), a previously unstudied isoform. Decreased levels of Tpm3.5 lead to softer and less mechanically resilient lenses that are unable to resume their original shape after compression. While cell organization and morphology appear unaffected, Tmod1 dissociates from the membrane in Tpm3.5-deficient lens fiber cells resulting in reorganization of the spectrin-F-actin and -actinin-F-actin networks at the membrane. These rearranged F-actin networks appear to be less able to support mechanical load and resilience leading to an overall change in tissue mechanical properties. This is the first in vivo evidence that Tpm is essential for cell biomechanical stability in a load-bearing non-muscle tissue and indicates that Tpm3.5 protects mechanically stable, load-bearing F-actin in vivo.\n\nSummaryTropomyosin 3.5 stabilizes F-actin in eye lens fiber cells and promotes normal tissue biomechanical properties. Tpm3.5 deficiency leads to F-actin network rearrangements and decreased lens stiffness and resilience.

cell biology↗

bioRxiv2018

Nuclear actin is required for transcription during Drosophila oogenesis

Actin influences gene expression at multiple levels. It regulates the activity of specific transcription factors, such as myocardin-related transcription factor (MRTF), is a component of many chromatin remodelers and linked to transcription by all three RNA polymerases (Pol). However, the molecular mechanisms by which actin participates in the gene-specific vs. general transcription have remained unclear. Here we use chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) in Drosophila ovaries to demonstrate that binding of actin to the Act5C gene is not dependent on the Mrtf transcription cofactor. At the genome-wide level, actin interacts with essentially all transcribed genes and co-occupies most gene promoters together with Pol II. On highly expressed genes, actin and Pol II can be found also on the gene bodies. Manipulation of nuclear transport factors for actin leads to decreased expression of egg shell genes, demonstrating the in vivo relevance of balanced nucleo-cytoplasmic shuttling of actin for transcription.

cell biology↗

bioRxiv2018

MEIOB and SPATA22 resemble RPA subunits and interact with the RPA complex to promote meiotic recombination

Homologous recombination is a conserved DNA repair process mandatory for chromosome segregation during meiosis. RPA, a ubiquitous complex essential to recombination, is thought to play a similar role during mitotic and meiotic recombination. MEIOB, a meiosis-specific factor with unknown molecular function, ressembles a RPA subunit. Here we use in vivo approaches to show that in mouse spermatocytes, DMC1 and RAD51 appear to be normally loaded in the absence of MEIOB but are prematurely lost from unrepaired recombination sites. This loss correlates with an accumulation of the BLM helicase on meiotic chromosomes. We also show that MEIOB alters the immunodetection of RPA subunits at meiotic recombination sites. Using electron microscopy and purified proteins, we demonstrate that the MEIOB-SPATA22 complex associates with and modifies the conformation of RPA-coated ssDNA. Finally, we identify structural homology between MEIOB, SPATA22 and RPA subunits, and show that MEIOB and SPATA22 interact through C-terminal OB-fold containing domains (OBCDs) like RPA subunits. Moreover, MEIOB and SPATA22 cooperate to interact with RPA through their OBCDs. Our results suggest that MEIOB, SPATA22 and RPA work together to ensure proper processing of meiotic recombination intermediates.

cell biology↗

bioRxiv2018

Defining Hepatic Modifiers Of ATTR Amyloidosis Through Therapeutic Gene Editing In Patient iPSCs

Systemic amyloidosis represents a class of disorders in which misfolded proteins are secreted by effector organs and deposited as proteotoxic aggregates at downstream target tissues. Despite being well-described clinically, the contribution of effector organs such as the liver to the pathogenesis of these diseases is poorly understood. Here, we utilize a patient-specific induced pluripotent stem cell (iPSC)-based model of hereditary transthyretin (TTR) amyloid disease (ATTR amyloidosis) in order to define the contributions of hepatic cells to the distal proteotoxicity of secreted TTR. To this end, we employ a gene correction strategy to generate isogenic, ATTR amyloidosis patient-specific iPSCs expressing either amyloidogenic or wild-type TTR. We further utilize this gene editing strategy in combination with single cell RNAseq to identify multiple hepatic proteostasis factors, including many components of adaptive unfolded protein response (UPR) signaling pathways, whose expression correlates with the production of destabilized TTR variants in iPSC-derived hepatic cells. We further demonstrate that enhancing ER proteostasis within ATTR amyloidosis iPSC-derived hepatic lineages via stress-independent activation of aforementioned adaptive UPR signaling preferentially reduces the secretion of destabilized amyloidogenic TTR. Together, these results suggest the potential of the liver to chaperone-at-a-distance and impact pathogenesis at downstream target cells in the context of systemic amyloid disease, and further highlight the promise of UPR modulating therapeutics for the treatment of TTR-mediated and other amyloid diseases.

cell biology↗

bioRxiv2018

APC/CCdh1-mediated degradation of Cdh1 during meiosis in S. cerevisiae

The Anaphase-Promoting Complex/Cyclosome (APC/C) is a ubiquitin ligase that promotes the ubiquitination and subsequent degradation of numerous cell cycle regulators during mitosis and in G1. Proteins are recruited to the APC/C by activator proteins such as Cdh1. During the cell cycle, Cdh1 is subject to precise regulation so that substrates are not degraded prematurely. We have explored the regulation of Cdh1 during the developmental transition into meiosis and sporulation in the budding yeast S. cerevisiae. Transition to sporulation medium triggers the degradation of Cdh1. Degradation requires that cells be of the a/a mating type and be starved for glucose, but they do not actually need to enter into the meiotic program. Degradation requires an intact SNF1 protein kinase complex (orthologous to the mammalian AMPK nutritional sensor), which is activated by the absence of glucose. Cdh1 degradation is mediated by the APC/C itself in a trans mechanism in which one molecule of Cdh1 recruits a second molecule of Cdh1 to the APC/C for ubiquitination. However, Cdh1-Cdh1 recognition does not depend on the degradation motifs or binding sites involved in the recognition of typical APC/C substrates. We hypothesize that Cdh1 degradation is necessary for the preservation of cell cycle regulators and chromosome cohesion proteins between the reductional and equational meiotic divisions, which occur without the intervening Gap or S phases found in mitotic cell cycles.

cell biology↗

bioRxiv2018

NudCL2 is an Hsp90 cochaperone to regulate sister chromatid cohesion by stabilizing cohesin subunits

Sister chromatid cohesion plays a key role in ensuring precise chromosome segregation during mitosis, which is mediated by the multisubunit complex cohesin. However, the molecular regulation of cohesin subunits stability remains unclear. Here, we show that NudCL2 (NudC-like protein 2) is essential for the stability of cohesin subunits by regulating Hsp90 ATPase activity in mammalian cells. Depletion of NudCL2 induces mitotic defects and premature sister chromatid separation and destabilizes cohesin subunits that interact with NudCL2. Similar defects are also observed upon inhibition of Hsp90 ATPase activity. Interestingly, ectopic expression of Hsp90 efficiently rescues the protein instability and functional deficiency of cohesin induced by NudCL2 depletion, but not vice versa. Moreover, NudCL2 not only binds to Hsp90, but also significantly modulates Hsp90 ATPase activity and promotes the chaperone function of Hsp90. Taken together, these data suggest that NudCL2 is a previously undescribed Hsp90 cochaperone to modulate sister chromatid cohesion by stabilizing cohesin subunits, providing a hitherto unrecognized mechanism that is crucial for faithful chromosome segregation during mitosis.

cell biology↗

bioRxiv2018

Three-dimensional culture of chicken primordial germ cells in chemically defined media containing the functional polymer FP003

Scalable production of avian suspension cell exhibits a valuable potential on therapeutic application by producing recombinant protein and as the substrate for virus growth. This study sought to establish a system with chemically defined components for three-dimensional (3D) culture of chicken primordial germ cells (cPGCs), a pluripotent avian cell type. cPGCs were cultured in medium supplemented with the functional polymer FP003. Viscoelasticity was low in this medium, and cPGCs did not sediment,and consequently their expansion was improved. The total number of cPGCs increased by 17-fold after 1week of culture in 3D-FAot medium, an aseric chemically defined medium containing FP003, indicating that this medium enhances the expansion of cPGCs. Moreover, cPGC cell lines stably expressed the germline-specific reporter VASA:tdTOMATO, as well as other markers of cPGCs, for more than 1 month upon culture in 3D-FAot medium, indicating that the characteristics of these cells are maintained. cPGCs harboring both PGK:EGFP and VASA:tdTOMATO robustly expressed both fluorescent proteins upon culture in 3D-FAot, suggesting that this approach is perspective for recombinant protein production. In summary,this novel 3D culture system can be used to efficiently expand cPGCs in suspension without mechanical stirring or loss of cellular properties. This system provides a platform for large-scale culture ofcPGCs in industry.

cell biology↗

bioRxiv2018

OTUB1 non-catalytically regulates the stability of the E2 ubiquitin conjugating enzyme UBE2E1

OTUB1 is a deubiquitinating enzyme that cleaves K48-linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, non-catalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin conjugating enzymes and inhibits their activity by trapping the E2~ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to members of the UBE2D (UBCH5) and UBE2E families, as well as to UBC13 (UBE2N). Cellular roles have been identified for the interaction of OTUB1 with UBC13 and members of the UBE2D family, but not for UBE2E E2 enzymes. We report here a novel role for OTUB1-E2 interactions in modulating E2 protein ubiquitination. We find that depletion of OTUB1 dramatically destabilizes the E2 conjugating enzyme UBE2E1 (UBE2E1) in cells and that this effect is independent of the catalytic activity of OTUB1 but depends on the ability of OTUB1 to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitination in vitro and in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we have found a new role for OTUB1 in rescuing specific E2s from degradation in vivo.

cell biology↗

bioRxiv2018

microRNA-dependent regulation of biomechanical genes establishes tissue stiffness homeostasis

The mechanical properties of tissues, which are determined primarily by their extracellular matrix (ECM), are largely stable over time despite continual turnover of ECM constituents 1,2. These observations imply active homeostasis, where cells sense and adjust rates of matrix synthesis, assembly and degradation to keep matrix and tissue properties within the optimal range. However, the regulatory pathways that mediate this process are essentially unknown3. Genome-wide analyses of endothelial cells revealed abundant microRNA-mediated regulation of cytoskeletal, adhesive and extracellular matrix (CAM) mRNAs. High-throughput assays showed co-transcriptional regulation of microRNA and CAM genes on stiff substrates, which buffers CAM expression. Disruption of global or individual microRNA-dependent suppression of CAM genes induced hyper-adhesive, hyper-contractile phenotypes in multiple systems in vitro, and increased tissue stiffness in the zebrafish fin-fold during homeostasis and regeneration in vivo. Thus, a network of microRNAs and CAM mRNAs mediate tissue mechanical homeostasis.

cell biology↗

bioRxiv2018

Atypical septate junctions maintain the somatic enclosure around maturing spermatids and prevent premature sperm release in Drosophila testis.

Tight junctions prevent the paracellular flow and maintain cell polarity in an epithelium. These are also essential for maintaining the blood-testis-barrier involved in regulating sperm differentiation. Septate junctions are orthologous to the tight junctions in insects. In Drosophila testis, major septate junction components co-localize at the interface of germline and somatic cells initially and then condense between the two somatic cells in a cyst after germline meiosis. Their localization is extensively remodeled in subsequent stages. We find that characteristic septate junctions are formed between the somatic cyst cells at the elongated spermatid stage. Consistent with the previous reports, knockdown of essential junctional components, Discs-large-1 and Neurexin-IV, in the somatic cyst cells, during the early stages, disrupted sperm differentiation beyond the spermatocyte stage. Somatic knockdown of these proteins during the final stages of spermatid maturation caused premature release of spermatids inside the testes, resulting in partial loss of male fertility. These results indicate the importance of maintaining mechanical integrity of the somatic enclosure during spermatid coiling and release in Drosophila testis. It also highlights the functional similarity with the tight junction proteins during spermatogenesis in mammalian testes.\n\nSummary statementDubey et al., showed that septate junctions stitch the somatic enclosure around maturing spermatids in Drosophila testis. Maintaining the integrity of this junction is essential for proper release of spermatids.

cell biology↗

bioRxiv2018

Mating in wild yeast: variable interest in sex after spore germination

Studies of lab strains of Saccharomyces cerevisiae have uncovered signaling pathways involved in mating, including information processing strategies to optimize decisions to mate or to bud. However, lab strains are heterothallic (unable to self-mate) while wild yeast are homothallic. And while mating of lab strains is studied using cycling haploid cells, mating of wild yeast is thought to involve germinating spores. Thus, it was unclear whether lab strategies would be appropriate in the wild. Here, we have investigated the behaviors of several yeast strains derived from wild isolates. Following germination, these strains displayed large differences in their propensity to mate or to enter the cell cycle. The variable interest in sex following germination was correlated with differences in pheromone production, which were due to both cis- and trans-acting factors. Our findings suggest that yeast spores germinating in the wild may often enter the cell cycle and form microcolonies prior to engaging in mating.

cell biology↗

bioRxiv2018

Translational control of TFEB and autophagy via eIF5A rejuvenates B cell immunity

Failure to make adaptive immune responses is a hallmark of aging. In particular reduced B cell function leads to poor vaccination efficacy and a high prevalence of infections in the elderly. However, the molecular mechanism underlying immune senescence is largely unknown. Here we show that autophagy levels are specifically reduced in mature lymphoid cells, leading to compromised memory B cell responses in old individuals. Spermidine, a naturally occurring polyamine metabolite, induces autophagy in vivo and rejuvenates memory B cell responses in an autophagy-dependent manner. Mechanistically, spermidine post-translationally modifies the translation factor eIF5A, which assists the synthesis of TFEB, a key transcription factor of autophagy. Spermidine is depleted in the elderly, leading to reduced TFEB expression and autophagy. Replenishing spermidine restored this pathway and improved the responses of old human B cells. Taken together, our results reveal an unexpected autophagy regulatory mechanism mediated by eIF5A at the translational level, and this pathway can be harnessed to rejuvenate immune senescence in humans.

cell biology↗

bioRxiv2018

Modulation of corneal tissue mechanics influences epithelial cell phenotype

Whilst the control of stem cell differentiation using substrates of differing compliance has been extensively explored in vitro, the significance of this mechanism at a physiological level is not known. Here we set to explore the role of corneal surface biomechanics in controlling epithelial cell proliferation and differentiation. Using non-contact high-resolution Brillouin spectro-microscopy we showed that the corneal outer edge (limbus) has significantly lower bulk modulus compared to the central cornea, and that this difference is precisely delimited in the organ. Furthermore, the areas of the limbus with distinctly softer properties were shown to be associated with limbal epithelial stem cell (LESC) residence. Based on these findings, we then provided the first demonstration of the capacity to modulate LESC phenotype, both in vivo and ex vivo, solely through the recreation/restoration of suitable biomechanical niches. These results thus confirm the fundamental role of corneal biomechanics in directing epithelial stem cell behavior.

cell biology↗

bioRxiv2018

A non-coding genetic variant maximally associated with serum urate levels is functionally linked to HNF4A-dependent PDZK1 expression

Several dozen genetic variants associate with serum urate levels, but the precise molecular mechanisms by which they affect serum urate are unknown. Here we tested for functional linkage of the maximally-associated genetic variant rs1967017 at the PDZK1 locus to elevated PDZK1 expression.\n\nWe performed expression quantitative trait locus (eQTL) and likelihood analyses followed by gene expression assays. Zebrafish were used to determine the ability of rs1967017 to direct tissue-specific gene expression. Luciferase assays in HEK293 and HepG2 cells measured the effect of rs1967017 on transcription amplitude.\n\nPAINTOR analysis revealed rs1967017 as most likely to be causal and rs1967017 was an eQTL for PDZK1 in the intestine. The region harboring rs1967017 was capable of directly driving green fluorescent protein expression in the kidney, liver and intestine of zebrafish embryos, consistent with a conserved ability to confer tissue-specific expression. The urate-increasing T-allele of rs1967017 strengthens a binding site for the transcription factor HNF4A. siRNA depletion of HNF4A reduced endogenous PDZK1 expression in HepG2 cells. Luciferase assays showed that the T-allele of rs1967017 gains enhancer activity relative to the urate-decreasing C-allele, with T-allele enhancer activity abrogated by HNF4A depletion. HNF4A physically binds the rs1967017 region, suggesting direct transcriptional regulation of PDZK1 by HNF4A.\n\nWith other reports our data predict that the urate-raising T-allele of rs1967017 enhances HNF4A binding to the PDZK1 promoter, thereby increasing PDZK1 expression. As PDZK1 is a scaffold protein for many ion channel transporters, increased expression can be predicted to increase activity of urate transporters and alter excretion of urate.

cell biology↗

bioRxiv2018

The Growth Hormone Secretagogue Receptor, Ghrelin and Biochemical Signaling Molecules in Human Heart Failure

BackgroundCurrently, the early pre-clinical detection of left ventricular (LV) dysfunction is difficult as biomarkers are not specific for the cardiomyopathic process. The underlying molecular mechanisms leading to heart failure remain elusive, highlighting the need for identification of cardiac-specific markers. The growth hormone secretagogue receptor (GHSR) and its ligand ghrelin are present in cardiac tissue and are known to contribute to myocardial energetics. Here, we examined tissue ghrelin-GHSR levels as specific markers of cardiac dysfunction in patients who underwent cardiac transplantation.\n\nMethods and ResultsSamples of cardiac tissue were obtained from 10 cardiac transplant patients at the time of organ harvesting, and during serial post-transplant biopsies. Quantitative fluorescence microscopy using a novel fluorescent ghrelin analog was used to measure levels of GHSR, and immunofluorescence was used to measure levels of ghrelin, b-type natriuretic peptide (BNP) and tissue markers of cardiomyocyte contractility and growth. GHSR and ghrelin expression levels were highly variable in the explanted heart, less in the grafted heart biopsies. GHSR and ghrelin were strongly positively correlated, and both markers were negatively correlated with LV ejection fraction. Ghrelin had stronger positive correlations than BNP with the signaling markers for contractility and growth.\n\nConclusionsThese data suggest that GHSR-ghrelin have potential use as an integrated marker of cardiac dysfunction. Interestingly, tissue ghrelin appeared to be a more sensitive indicator than BNP to the biochemical processes that are characteristic of heart failure. This work allows for further use of ghrelin-GHSR to interrogate cardiac-specific biochemical mechanisms in pre-clinical stages of HF.\n\nPrecisThis study shows the relationships between GHSR, ghrelin, and signaling molecules with relation to heart function in human heart failure with tissue from diseased heart and healthy heart biopsies.

cell biology↗

bioRxiv2018

Drosophila kinesin-8 stabilises kinetochore-microtubule interaction

Kinesin-8 is required for proper chromosome alignment in a variety of animal and yeast cell types. However, how this conserved motor protein controls chromosome alignment remains unclear, as multiple biochemical activities, including inconsistent ones between studies, have been identified for this motor family. Here, we show that Drosophila kinesin-8 Klp67A possesses both microtubule (MT) plus-end-stabilising and -destabilising activities in addition to commonly observed MT plus-end-directed motility and tubulin-binding activity in vitro, and is required for stable kinetochore-MT attachment during prometaphase in S2 cells In the absence of kinesin-8Klp67A, abnormally-long MTs interact in an \"end-on\" fashion with kinetochores at normal frequency. However, the interaction was not stable and, once-attached, MTs were frequently detached. This phenotype was rescued by ectopic expression of MT plus-end-stabilising factor CLASP, but not by artificial shortening of MTs. These results suggest that MT-stabilising activity of kinesin-8Klp67A is critical for stable kinetochore-MT attachment. Finally, human kinesin-8KIF18A was also shown important to ensure proper MT attachment.

cell biology↗

bioRxiv2018

Structure of the ciliary axoneme at nanometer resolution reconstructed by TYGRESS

The resolution of subtomogram averages calculated from cryo-electron tomograms (cryo-ET) of crowded cellular environments is often limited due to signal loss in, and misalignment of the subtomograms. In contrast, single-particle cryo-electron microcopy (SP-cryo-EM) routinely reaches near-atomic resolution of isolated complexes. We developed a novel hybrid-method called \"TomographY-Guided 3D REconstruction of Subcellular Structures\" (TYGRESS) that combines cryo-ET with SP-cryo-EM to achieve close-to-nanometer resolution of complexes inside crowded environments. Using TYGRESS, we determined the native 3D structures of the intact ciliary axoneme with up to 12 [A] resolution. These results reveal many structures and details that were not visible by cryo-ET. TYGRESS is generally applicable to cellular complexes that are amenable to subtomogram averaging, bringing us a step closer to (pseudo-)atomic models of cells.\n\nOne Sentence SummaryA hybrid cryo-electron microscopy method reveals subcellular structures at unprecedented resolution.

cell biology↗