Biology subjects

Sun, M.

Publications and source records attributed to Sun, M..

11 recordsLinked to original sources

bioRxiv2018

Real-time structural dynamics of late steps in bacterial translation initiation visualized using time-resolved cryogenic electron microscopy

Bacterial translation initiation entails the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNAfMet)-containing 30S ribosomal initiation complex (IC) to form a 70S IC that subsequently matures into a 70S elongation-competent complex (70S EC). Rapid and accurate 70S IC formation is promoted by 30S IC-bound initiation factor (IF) 1 and the guanosine triphosphatase (GTPase) IF2, both of which must ultimately dissociate from the 70S IC before the resulting 70S EC can begin translation elongation1. Although comparison of 30S2-6 and 70S5,7-9 IC structures have revealed that the ribosome, IFs, and fMet-tRNAfMet can acquire different conformations in these complexes, the timing of conformational changes during 70S IC formation, structures of any intermediates formed during these rearrangements, and contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Moreover, lack of an authentic 70S EC structure has precluded an understanding of ribosome, IF, and fMet-tRNAfMet rearrangements that occur upon maturation of a 70S IC into a 70S EC. Using time-resolved cryogenic electron microscopy (TR cryo-EM)10 we report the first, near-atomic-resolution view of how a time-ordered series of conformational changes drive and regulate subunit joining, IF dissociation, and fMet-tRNAfMet positioning during 70S EC formation. We have found that, within ~20-80 ms, rearrangements of the 30S subunit and IF2, uniquely captured in its GDP*Pi-bound state, stabilize fMet-tRNAfMet in its intermediate, 70S P/I, configuration7 and trigger dissociation of IF1 from the 70S IC. Within the next several hundreds of ms, dissociation of IF2 from the 70S IC is coupled to further remodeling of the ribosome that positions fMet-tRNAfMet into its final, P/P, configuration within the 70S EC. Our results demonstrate the power of TR cryo-EM to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology.

molecular biology↗

bioRxiv2018

Condensin controls mitotic chromosome stiffness and stability without forming a structurally contiguous scaffold

During cell division, chromosomes must be folded into their compact mitotic form to ensure their segregation. This process is thought to be largely controlled by the action of condensin SMC protein complexes on chromatin fibers. However, how condensins organize metaphase chromosomes is not understood. We have combined micromanipulation of single human mitotic chromosomes, sub-nanonewton force measurement, siRNA interference of condensin subunit expression, and fluorescence microscopy, to analyze the role of condensin in large-scale chromosome organization. Condensin depletion leads to a dramatic (~10 fold) reduction in chromosome elastic stiffness relative to the native, non-depleted case. We also find that prolonged metaphase stalling of cells leads to overloading of chromosomes with condensin, with abnormally high chromosome stiffness. These results demonstrate that condensin is a main element controlling the stiffness of mitotic chromosomes. Isolated, slightly stretched chromosomes display a discontinuous condensing staining pattern, suggesting that condensins organize mitotic chromosomes by forming isolated compaction centers that do not form a continuous scaffold.

cell biology↗

bioRxiv2018

Stabilizing Obligatory Non-native Intermediates Along Co-transcriptional Folding Trajectories of SRP RNA Affects Cell Viability

Signal recognition particle (SRP) in Escherichia coli comprises protein Ffh and SRP RNA. Its essential functionality--co-translational protein-targeting/delivery to cellular membranes-- hinges on the RNA attaining a native long-hairpin fold that facilitates protein conformational rearrangements within the SRP complex. Since RNA folds co-transcriptionally on RNA polymerase, we use high-resolution optical tweezers to first characterize the mechanical unfolding/refolding of incrementally lengthened RNAs from stalled transcription complexes until reaching the full-length transcript. This analysis allows identification of folding intermediates adopted during the real-time co-transcriptional folding of SRP RNA. The co-transcriptional folding trajectories are surprisingly invariant to transcription rates, and involve formation of an obligatory non-native hairpin intermediate that eventually resolves into the native fold. SRP RNA variants designed to stabilize this non-native intermediate--likely sequestering the SRP ribonucleoprotein complex in an inactive form--greatly reduce cell viability, indicating that the same co-transcriptional folding mechanism operates in vivo and possible alternative antibiotic strategies.\n\nHighlightsO_LIFolding pathway of an essential functional RNA has been resolved co-transcriptionally.\nC_LIO_LIThe co-transcriptional folding pathway of SRP RNA is invariant to transcription rates.\nC_LIO_LINascent SRP RNA obligatorily forms a non-native intermediate before adopting the native fold.\nC_LIO_LIModulating transitions from the non-native to native SRP RNA hairpin fold alters cell viability.\nC_LI

biophysics↗

bioRxiv2018

For comparing phylogenetic diversity among communities, go ahead and use synthesis phylogenies

Should we build our own phylogenetic trees based on gene sequence data, or can we simply use available synthesis phylogenies? This is a fundamental question that any study involving a phylogenetic framework must face at the beginning of the project. Building a phylogeny from gene sequence data (purpose-built phylogeny) requires more effort, expertise, and cost than subsetting an already available phylogeny (synthesis-based phylogeny). However, we still lack a comparison of how these two approaches to building phylogenetic trees influence common community phylogenetic analyses such as comparing community phylogenetic diversity and estimating trait phylogenetic signal. Here, we generated three purpose-built phylogenies and their corresponding synthesis-based trees (two from Phylomatic and one from the Open Tree of Life [OTL]). We simulated 1,000 communities and 12,000 continuous traits along each purpose-built phylogeny. We then compared the effects of different trees on estimates of phylogenetic diversity (alpha and beta) and phylogenetic signal (Pagels {lambda} and Blombergs K). Synthesis-based phylogenies generally yielded higher estimates of phylogenetic diversity when compared to purpose-built phylogenies. However, resulting measures of phylogenetic diversity from both types of phylogenies were highly correlated (Spearmans{rho} > 0.8 in most cases). Mean pairwise distance (both alpha and beta) is the index that is most robust to the differences in tree construction that we tested. Measures of phylogenetic diversity based on the OTL showed the highest correlation with measures based on the purpose-built phylogenies. Trait phylogenetic signal estimated with synthesis-based phylogenies, especially from the OTL, were also highly correlated with estimates of Blombergs K or close to Pagels {lambda} from purpose-built phylogenies when traits were simulated under Brownian Motion. For commonly employed community phylogenetic analyses, our results justify taking advantage of recently developed and continuously improving synthesis trees, especially the Open Tree of Life.

ecology↗

bioRxiv2018

Study of Sterilization Effects on Marine Vibrio sp. using Interaction of Cavitation with Shock Wave in a Narrow Water Chamber

When underwater shock waves are generated by an electric discharge in a narrow water chamber, the instant release of a great amount of energy causes the propagation of a shear wave in wall material with the deformation of the chamber wall. The shear waves produce decompression in water and result in the growth of bubble nuclei. Subsequently, those oscillating cavitation bubbles are exposed to the shock pressures, and thus free radicals and rebound shock waves are generated due to their violent collapses. Eventually, marine bacteria around them are inactivated by these productions. In the present study, we investigate the sterilization effects of these oscillating bubbles and cavitation-shock interaction by bio-experiments, respectively. Furthermore, the chemical action of free radicals on marine bacteria is discussed. The generation of the OH radicals is clarified by measuring the concentration of the H2O2. To estimate the generation condition of the OH radicals, a bubble dynamic model consisting of an oscillation model for the growth of bubble nuclei and an impact model to describe the cavitation-shock interaction is developed. Finally, the theoretical estimation by the bubble dynamic model is discussed under the conditions of the present experiments.

biophysics↗

bioRxiv2018

Experimental infection characteristics of Bordetella pertussis via aerosol challenge on rhesus macaques

The effect of aerosol challenge of rhesus macaques with Bordetella pertussis and the feasibility of using rhesus monkeys as an animal model for pertussis infection were evaluated in this study. Four 1-year old rhesus macaques were aerosol challenged with B. pertussis at the concentration of 105 CFU/mL for 30 min (group 1) or 60 min (group 2). Rectal temperature was found slightly increased at days 3 and 5 and returned to baseline levels at day 21 after challenge. White blood cell counts peaked at day 7, with a 4.7~6.1-fold increase and returned to baseline levels at day 45. Bacteria colonization of nasopharyngeal swabs was observed, and the number of colonies was gradually increased and peaked at day 14, reaching 5.4-8.1 x 106/mL. The seroconversion rate of anti-pertussis toxin (PT), pertactin (PRN), and filamentous hemagglutinin(FHA) antibodies was 100%, with an increase in geometric mean titers after challenge. Analysis of cytokines revealed that the levels of cytokines including IL-2, IL-6, IL-8, IL-10, IL-17A, IL-13, IL-12, and IL-18 were significantly increased at days 5 to 14 in group 2. These results demonstrate that the characteristic of pertussis infection in infant rhesus macaque was similar as in human beings, which provide a clue to using infant rhesus macaque as a candidate model for pertussis infection in the future studies

microbiology↗

bioRxiv2018

Identification and characterization of cherry (Cerasus pseudocerasus G. Don) genes in response to parthenocarpy induced by GA3 through transcriptome analysis

Fruit set after successful pollination is a key process in the production of sweet cherries, but low fruit rate is the main problem for crop production in sweet cherries. Gibberellin treatment can directly induce parthenogenesis without pollination; therefore, gibberellin treatment is a very important method to improve the fruit setting rate of sweet cherries. Exogenous gibberellin can satisfy the hormone requirement during fruit growth and development. Some related studies have mainly focused on physiological aspects, such as breeding, branching, fertilization, etc., and the molecular mechanism is not clear. In this study, we analyzed the transcriptome of Meizao sweet cherry fruit treated with gibberellin during the anthesis and hard-core period to determine the genes associated with parthenocarpic fruit set. A total of 765 and 186 differentially expressed genes (DEGs) were found at anthesis and the hard-core stage after gibberellin 3 (GA3) treatment, respectively. The differentially expressed genes between the control and GA3 treatment showed that the GA3 response mainly included parthenocarpic fruit set and cell division. Exogenous gibberellin stimulated sweet cherry parthenocarpy and enlargement, which were verified by qRT-PCR results of related genes and the parthenocarpic fruit set and fruit size. Based on our research and previous studies on Arabidopsis thaliana, we identified the key genes associated with parthenocarpic fruit set and cell division. Briefly, we found patterns in the sweet cherry fruit setting-related DEGs, especially those associated with hormone balance, cytoskeleton formation and cell wall modification. Overall, the result provides a possible molecular mechanism regulating parthenocarpic fruit set, which is important for basic research and industrial development of sweet cherries.\n\nHighlightcherry genes in response to parthenocarpy and promote to fruit setting induced by GA3.

molecular biology↗

bioRxiv2018

The N-cadherin interactome in primary cardiomyocytes as defined by quantitative proximity proteomics.

The junctional complexes that couple cardiomyocytes must transmit the mechanical forces of contraction while maintaining adhesive homeostasis. The adherens junction (AJ) connects the actomyosin networks of neighboring cardiomyocytes and is required for proper heart function. Yet little is known about the molecular composition of the cardiomyocyte AJ or how it is organized to function under mechanical load. Here we define the architecture, dynamics and proteome of the cardiomyocyte AJ. Mouse neonatal cardiomyocytes assemble stable AJs along intercellular contacts with organizational and structural hallmarks similar to mature contacts. We combine quantitative mass spectrometry with proximity labeling to identify the N-cadherin (CDH2) interactome. We define over 350 proteins in this interactome, nearly 200 of which are unique to CDH2 and not part of the E-cadherin (CDH1) interactome. CDH2-specific interactors are comprised primarily of adaptor and adhesion proteins that promote junction specialization. Finally, we find evidence of dynamic interplay between AJ and Z-disc proteins. Together, our results provide novel insight into the cardiomyocyte AJ and provide a proteomic atlas for defining the molecular complexes that regulate cardiomyocyte intercellular adhesion.\n\nSummary StatementProximity proteomics reveals a specific and specialized N-cadherin (CDH2) interactome along the cell-cell contacts of primary cardiomyocytes.

cell biology↗

bioRxiv2018

Bacillus thuringiensis targets the host intestinal epithelial junctions for successful infection of Caenorhabditis elegans

Pathogenic bacteria use different strategies to infect their hosts including the simultaneous production of pore forming toxins and several virulence factors that help to synergize their pathogenic effects. However, how the pathogenic bacteria are able to complete their life cycle and break out the host intestinal barrier is poorly understood. The infectious cycle of Bacillus thuringiensis (Bt) bacterium in Caenorhabditis elegans is a powerful model system to study the early stages of the infection process. Bt produces Cry pore-forming toxins during the sporulation phase that are key virulence factors involved in Bt pathogenesis. Here we show that during the early stages of infection, the Cry toxins disrupt the midgut epithelial tissue allowing the germination of spores. The vegetative Bt cells then trigger a quorum sensing response that is activated by PlcR regulator resulting in production of different virulence factors, such as the metalloproteinases ColB and Bmp1, that besides Cry toxins are necessary to disrupt the nematode epithelial junctions causing efficient bacterial host infection and dead of the nematode. Overall our work describes a novel mechanism for Bt infection, targeting the epithelial junctions of its host midgut cells.\n\nAuthor summaryThe entomopathogenic bacteria Bacillus thuringiensis (Bt) are used worldwide as biopesticides due to their insecticidal properties. Crystal proteins (Cry) produced by Bt during the sporulation phase of growth are mainly responsible for their insecticidal properties. The infection process of Bt includes three successive steps, virulence, necrotrophic, and sporulation processes. During the virulence process, after ingestion by the susceptible hosts, the Cry toxins form pores in the apical membrane of intestinal cells, inducing favorable conditions for bacterial spore germination. Vegetative bacteria multiply in the host and coordinate their behavior by using the quorum sensor regulator PlcR, which leads to the production of virulence factors allowing the bacteria to kill the host. However, how the bacteria are able to disrupt the host intestinal barrier during the early stages of infection remains unknown. Here we show that Bt employs the nematicidal Cry toxins and additional virulence factors controlled by the PlcR regulon to disrupt the intestinal epithelial junctions of C. elegans at the early stages of infection allowing that Bt bacteria complete its life cycle in the worms. Our work provides new insights into the pathogenesis of Bt, and highlights the importance of breaking down host epithelial junctions for a successful infection, a similar mechanism could be used by other pathogens-host interactions since epithelial junctions are conserved structures from insects to mammals.

microbiology↗

bioRxiv2018

An unconventional myosin, myosin 1d regulates Kupffer’s vesicle morphogenesis and laterality

INTRODUCTION INTRODUCTION RESULTS MATERIALS AND METHODS AUTHOR CONTRIBUTIONS REFERENCES Establishing left-right (LR) asymmetry is a fundamental process essential for arrangement of visceral organs during development. In vertebrates, motile cilia driven fluid flow in the left-right organizer (LRO) is essential for initiating symmetry breaking event1-3. Without a definite LRO structure in invertebrates, LR asymmetry is initiated at a cellular level by actin-myosin driven chirality4, 5. In Drosophila, myosin1D drives tissue-specific chirality in hind-gut looping6, 7. Here, we show that myosin 1d (myo1d) is essential for establishing LR asymmetry in zebrafish. Using super-r ...

developmental biology↗

bioRxiv2017

Trabecular Meshwork Failure In A Model Of Pigmentary Glaucoma

Pigment dispersion syndrome can lead to pigmentary glaucoma (PG), a poorly understood condition of younger, myopic eyes with fluctuating, high intraocular pressure (IOP). The absence of a model similar in size and behavior to human eyes has made it difficult to investigate its pathogenesis. Here, we present a porcine ex vivo model that recreates the features of PG including intraocular hypertension, pigment accumulation in the trabecular meshwork and relative failure of phagocytosis. In in vitro monolayer cultures as well as in ex vivo eye perfusion cultures, we found that the trabecular meshwork (TM) cells that regulate outflow, form actin stress fibers and have a decreased phagocytosis. Gene expression microarray and pathway analysis indicated key roles of RhoA in regulating the TM cytoskeleton, motility, and phagocytosis thereby providing new targets for PG therapy.

cell biology↗