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bioRxiv2018

Change in sexual signaling traits outruns morphological divergence in a recent avian radiation across an ecological gradient

The relative roles of natural and sexual selection in promoting evolutionary lineage divergence remains controversial and difficult to assess in natural systems. Local adaptation through natural selection is known to play a central role in adaptive radiations, yet secondary sexual traits can vary widely among species in recent radiations, suggesting that sexual selection may also be important in the early stages of speciation. Here we compare rates of divergence in ecologically relevant traits (morphology) and sexually selected signaling traits (coloration) relative to neutral structure in genome-wide molecular markers, and examine patterns of variation in sexual dichromatism to understand the roles of natural and sexual selection in the diversification of the songbird genus Junco (Aves: Passerellidae). Juncos include divergent lineages in Central America and several dark-eyed junco (J. hyemalis) lineages that diversified recently as the group recolonized North America following the last glacial maximum (c.a. 18,000 years ago). We found an accelerated rate of divergence in sexually selected characters relative to ecologically relevant traits. Moreover, a synthetic index of sexual dichromatism comparable across lineages revealed a positive relationship between the degree of color divergence and the strength of sexual selection, especially when controlling for neutral genetic distance. We also found a positive correlation between dichromatism and latitude, which coincides with the latitudinal pattern of decreasing lineage age but also with a steep ecological gradient. Finally, we detected an association between outlier loci potentially under selection and both sexual dichromatism and latitude of breeding range. These results suggest that the joint effects of sexual and ecological selection have played a role in the junco radiation and can be important in the early stages of lineage formation.

evolutionary biology↗

bioRxiv2018

Plant odour and sex pheromone are integral elements of specific mate recognition in an insect herbivore

Specific mate recognition strongly relies on the chemical senses in many animals, and especially in nocturnal insects. Two signal types lend to premating olfactory communication in terrestrial habitats: sex signals blend into an atmosphere of habitat odorants, where plant volatiles prevail. We show for the first time that males of the African cotton leafworm Spodoptera littoralis perceive female sex pheromone and volatiles of its plant host cotton as a unit, rather than as independent messages. In clean air, S. littoralis males are attracted to flawed pheromone signals, such as single synthetic pheromone components or even the pheromone of a sibling species, Oriental leafworm S. litura. Presence of host plant volatiles, however, strongly reduces the male response to deficient or heterospecific pheromone signals. That plant cues enhance discrimination of sex pheromone quality confirms the idea that specific mate recognition in noctuid moths has evolved in concert with host plant adaptation. A participation of host plant odour in sexual communication suggests that mate recognition is under natural and sexual selection. Moreover, shifts in either female host preference or sex pheromone biosynthesis give rise to new communication channels that have the potential to initiate or contribute to reproductive isolation.

evolutionary biology↗

bioRxiv2015

Apoptosis in snowflake yeast: novel trait, or side effect of toxic waste?

Recent experiments evolving de novo multicellularity in yeast have found that large-cluster forming genotypes also exhibit higher rates of programmed cell death (apoptosis). This was previously interpreted as the evolution of a simple form of cellular division of labor: apoptosis results in the scission of cell-cell connections, allowing snowflake yeast to produce proportionally smaller, faster-growing propagules. Through spatial simulations, Duran-Nebreda and Sole (2015) develop the novel null hypothesis that apoptosis is not an adaptation, per se, but is instead caused by the accumulation of toxic metabolites in large clusters. Here we test this hypothesis by synthetically creating unicellular derivatives of snowflake yeast through functional complementation with the ancestral ACE2 allele. We find that multicellular snowflake yeast with elevated apoptosis exhibit a similar rate of apoptosis when cultured as single cells. We also show that larger snowflake yeast clusters tend to contain a greater fraction of older, senescent cells, which may explain why larger clusters of a given genotype are more apoptotic. Our results show that apoptosis is not caused by side effects of spatial structure, such as starvation or waste product accumulation, and are consistent with the hypothesis that elevated apoptosis is a trait which co-evolves with large cluster size.

Evolutionary Biology↗

bioRxiv2018

Polygonal motion and adaptable phototaxis via flagellar beat switching in Euglena gracilis

Biological microswimmers exhibit versatile strategies for sensing and navigating their environment 1-7, e.g., run-and-tumble 2 and curvature modulation 3. Here we report a striking behavior of Euglena gracilis, where Euglena cells swim in polygonal trajectories due to exposure to increasing light intensities. While smoothly curved trajectories are common for microswimmers 3, 8, such quantized ones have not been reported previously. This polygonal behavior emerges from periodic switching between the flagellar beating patterns of helical swimming 6, 9 and spinning 10 behaviors. We develop and experimentally validate a biophysical model that describes the phase relationship between the eyespot, cell orientation, light detection, and cellular reorientation, that accounts for all three behavioral states. Coordinated switching between these behaviors allows ballistic, superdiffusive, diffusive, or subdiffusive motion 11,12 (i.e., the tuning of the diffusion constant over 3 orders of magnitude) and enables navigation in structured light fields, e.g., edge avoidance and gradient descent. This feedback-control links multiple system scales (flagellar beats, cellular behaviors, phototaxis strategies) with implications for other natural and synthetic microswimmers 13.

biophysics↗

bioRxiv2018

Imaging Striatal Dopamine Release Using a Non-Genetically Encoded Near-Infrared Fluorescent Catecholamine Nanosensor

Neuromodulation plays a critical role in brain function in both health and disease. New optical tools, and their validation in biological tissues, are needed that can image neuromodulation with high spatial and temporal resolution, which will add an important new dimension of information to neuroscience research. Here, we demonstrate the use of a catecholamine nanosensor with fluorescent emission in the 1000-1300 nm near-infrared window to measure dopamine transmission in ex vivo brain slices. These near-infrared catecholamine nanosensors (nIRCats) represent a broader class of nanosensors that can be synthesized from non-covalent conjugation of single wall carbon nanotubes (SWNT) with single strand oligonucleotides. We show that nIRCats can be used to detect catecholamine efflux in brain tissue driven by both electrical stimulation or optogenetic stimulation. Spatial analysis of electrically-evoked signals revealed dynamic regions of interest approximately 2 microns in size in which transients scaled with simulation intensity. Optogenetic stimulation of dopaminergic terminals produced similar transients, whereas optogenetic stimulation of glutamatergic terminals showed no effect on nIRCat signal. Bath application of nomifensine prolonged nIRCat fluorescence signal, consistent with reuptake blockade of dopamine. We further show that the chemically synthetic molecular recognition elements of nIRCats permit measurement of dopamine dynamics in the presence of dopamine receptor agonists and antagonists. These nIRCat nanosensors may be advantageous for future use because i) they do not require virus delivery, gene delivery, or protein expression, ii) their near-infrared fluorescence facilitates imaging in optically scattering brain tissue and is compatible for use in conjunction with other optical neuroscience tool sets, iii) the broad availability of unique near-infrared colors have the potential for simultaneous detection of multiple neurochemical signals, and iv) they are compatible with pharmacology. Together, these data suggest nIRCats and other nanosensors of this class can serve as versatile new optical tools to report dynamics of extracellular neuromodulation in the brain.

neuroscience↗

bioRxiv2018

Gamete fusion rapidly reconstitutes a bi-partite transcription factor to block re-fertilization

The ploidy cycle, integral to sexual reproduction, requires not only meiosis to halve the number of chromosomes, but also mechanisms that ensure zygotes are formed by exactly two partners1-5. During sexual reproduction of the fungal model organism Schizosaccharomyces pombe, haploid P- and M-cells normally fuse to form a diploid zygote that immediately enters meiosis6. Here, we reveal that fast post-fusion reconstitution of a bi-partite transcription factor actively blocks re-fertilization. We first identify mutants that undergo transient cell fusion involving cytosol exchange but not karyogamy, and show this drives distinct cell fates in the two gametes: The P-partner undergoes lethal, haploid meiosis while the M-cell persists in mating. Consistently, we find that the zygotic transcription that drives meiosis is initiated rapidly only from the P-parental genome, even in wild type cells. This asymmetric gene expression depends on a bi-partite complex formed post-fusion between the nuclear P-cell-specific homeobox protein Pi and a cytosolic M-specific peptide Mi7,8, which is captured by Pi in the P-nucleus. Zygotic transcription is thus poised to initiate in the P-nucleus as fast as Mi reaches it. The asymmetric nuclear accumulation is inherent to the transcription factor design, and is reconstituted by a pair of synthetic interactors, one localized to the nucleus of one gamete and the other in the cytosol of its partner. Strikingly, imposing a delay in zygotic transcription, by postponing Mi expression or deleting its transcriptional target in the P-genome, leads to zygotes fusing with additional gametes, thus forming polyploids and eventually aneuploid progeny. We further show that the signaling cascade to block re-fertilization shares components with, but bifurcates from, meiotic induction9-11. Thus, cytoplasmic connection upon gamete fusion leads to rapid reconstitution of a bi-partite transcription factor in one partner to block re-fertilization and induce meiosis, thus ensuring genome maintenance during sexual reproduction.

cell biology↗

bioRxiv2018

Dissecting PCNA function with a systematically designed mutation library in yeast

Proliferating cell nuclear antigen (PCNA), encoded by POL30 in Saccharomyces cerevisiae, is a key component of DNA metabolism. Here a library consisting of 308 PCNA mutants was designed and synthesized to probe the contribution of each residue to its biological function. Five regions were identified with elevated sensitivity to DNA damaging reagents using high-throughput phenotype screening. Using a series of genetic and biochemical analyses, we demonstrated that one particular mutant, K168A, which displayed severe DNA damage sensitivity, abolished the DNA damage tolerance (DDT) pathway by disrupting interactions between PCNA and Rad5p. Subsequent domain analysis showed that the PCNA/Rad5p interaction is prerequisite for the function of Rad5p in DDT. Our study not only provides a resource in the form of a library of versatile mutants to study PCNA functions, but also reveals a key regulatory function of Rad5p, which highlights the importance of the PCNA-Rad5p interaction.\n\nAuthor summaryPCNA is regarded as the maestro of DNA replication fork because of the astonishing ability to interact with lots of partner proteins that participate in various DNA metabolism processes. However, it has remained elusive as to how does PCNA orchestrate these functions in harmony. Here, we constructed a systematic mutation library of PCNA, which covers every amino acid to map the functional sites of it. This carefully designed synthetic mutant pool could be generally useful and serve as a flexible resource, such as dissecting the functional mechanism of PCNA by genetic relationship analysis with key proteins through Synthetic genetic array. We further dissected the intrinsic mechanism for damage sensitivity of PCNAK168A, the most severe DNA damage sensitive mutant in our alanine scanning mutation library, this helps us to get better understanding of how PCNA participates in DNA damage tolerance (DDT) pathways. Our findings indicate that K168 site is vital for the interaction between DDT related partner proteins and PCNA, and also highlight the importance of the PCNA-Rad5p interaction.

genetics↗

bioRxiv2018

Mapping the human kinome in response to DNA damage

We provide a catalog for the effects of the human kinome on cell survival in response to DNA damaging agents, selected to cover all major DNA repair pathways. By treating 313 kinase-deficient cell lines with ten diverse DNA damaging agents, including seven commonly used chemotherapeutics, we were able to identify kinase specific vulnerabilities and resistances. In order to identify novel synthetic lethal interactions, we investigate the cellular response to carmustine for 25 cell lines, by establishing a phenotypic FACS assay designed to mechanistically investigate and validate gene-drug interactions. We show apoptosis, cell cycle, DNA damage and proliferation after alkylation or crosslink-induced damage for selected cell lines and rescue the cellular sensitivity of DYRK4, EPHB6, MARK3, PNCK as a proof of principle for our study. Our data suggest that some cancers with inactivated DYRK4, EPHB6, MARK3 or PNCK gene could be particularly vulnerable to treatment by alkylating chemotherapeutic agents carmustine or temozolomide.

systems biology↗

bioRxiv2017

Improved de novo Genome Assembly: Synthetic long read sequencing combined with optical mapping produce a high quality mammalian genome at relatively low cost

Current short-read methods have come to dominate genome sequencing because they are cost-effective, rapid, and accurate. However, short reads are most applicable when data can be aligned to a known reference. Two new methods for de novo assembly are linked-reads and restriction-site labeled optical maps. We combined commercial applications of these technologies for genome assembly of an endangered mammal, the Hawaiian Monk seal.\n\nWe show that the linked-reads produced with 10X Genomics Chromium chemistry and assembled with Supernova v1.1 software produced scaffolds with an N50 of 22.23 Mbp with the longest individual scaffold of 84.06 Mbp. When combined with Bionano Genomics optical maps using Bionano RefAligner, the scaffold N50 increased to 29.65 Mbp for a total of 170 hybrid scaffolds, the longest of which was 84.78 Mbp. These results were 161X and 215X, respectively, improved over DISCOVAR de novo assemblies. The quality of the scaffolds was assessed using conserved synteny analysis of both the DNA sequence and predicted seal proteins relative to the genomes of humans and other species. We found large blocks of conserved synteny suggesting that the hybrid scaffolds were high quality. An inversion in one scaffold complementary to human chromosome 6 was found and confirmed by optical maps.\n\nThe complementarity of linked-reads and optical maps is likely to make the production of high quality genomes more routine and economical and, by doing so, significantly improve our understanding of comparative genome biology.

genomics↗

bioRxiv2017

The Peach RGF/GLV Signalling Peptide pCTG134 Is Involved In A Regulatory Circuit That Sustains Auxin And Ethylene Actions

Peach is a climacteric species whose ripening is regulated by the plant hormone ethylene. A crosstalk mechanism with auxin is necessary to support climacteric ethylene synthesis. The homeostasis control of auxin is regulated also by the activity of peptide hormones (PHs), acting both as short and long distant ligands. In this work, we investigated the role of CTG134, a peach gene encoding a GOLVEN-like PH isolated in mesocarp at the onset of ripening.\n\nIn peach fruit, CTG134 was expressed during the climacteric transition and its mRNA level was induced by auxin and 1-methylcyclopropene (1-MCP) treatments, whereas it was minimally affected by ethylene. To better elucidate its function, CTG134 was overexpressed in Arabidopsis and tobacco, which showed abnormal root hair growth, similar to wild-type plants treated with a synthetic form of the peptide. Molecular surveys demonstrated an impaired hormonal crosstalk, resulting in a re-modulated expression of a set of genes involved in both ethylene and auxin domains. In addition, the promoter of pCTG134 fused with GUS reporter highlighted gene activity in plant organs in which the auxin-ethylene interplay is known to occur. These data support the role of pCTG134 as mediator in an auxin-ethylene regulatory circuit.\n\nHighlightThe role of the peach RGF/GLV peptide during root hair formation in Arabidopsis and tobacco supports its involvement in a cross-hormonal auxin-ethylene regulatory circuit.

plant biology↗

bioRxiv2017

Mechanistic causes of constrained phenotypic variation revealed by synthetic gene regulatory networks

Phenotypic variation is the raw material of adaptive Darwinian evolution. The phenotypic variation found in organismal development is biased towards certain phenotypes, but the molecular mechanisms behind such restrictions are still poorly understood. Gene regulatory networks have been proposed as one cause of constrained phenotypic variation. However, most of the evidence for this is theoretical rather than experimental. Here, we study evolutionary biases in two synthetic gene regulatory circuits expressed in E. coli that produce a gene expression stripe - a pivotal pattern in embryonic development. The two parental circuits produce the same phenotype, but create it through different regulatory mechanisms. We show that mutations cause distinct novel phenotypes in the two networks and use a combination of experimental measurements, mathematical modelling and DNA sequencing to understand why mutations bring forth only some but not other novel gene expression phenotypes. Our results reveal that the regulatory mechanisms of networks restrict the possible phenotypic variation upon mutation. Consequently, seemingly equivalent networks can indeed be distinct in how they constrain the outcome of further evolution.

systems biology↗

bioRxiv2018

phospho-ERK is a response biomarker to a combination of sorafenib and MEK inhibition in liver cancer

Treatment of liver cancer remains challenging, due to a paucity of drugs that target critical dependencies. Sorafenib is a multikinase inhibitor that is approved as the standard therapy for advanced hepatocellular carcinoma patients, but it can only provide limited survival benefit for patients. To investigate the cause of this limited therapeutic effect, we performed a CRISPR-Cas9 based synthetic lethality screen to search for kinases whose knockout synergize with sorafenib. We find that suppression of ERK2 sensitizes several liver cancer cell lines to sorafenib. Drugs inhibiting the MEK or ERK kinases reverse unresponsiveness to sorafenib in vitro and in vivo in a subset of liver cancer cell lines characterized by high levels of active phospho-ERK levels through synergistic inhibition of ERK kinase activity. Our data provide a combination strategy for treating liver cancer and suggest that tumors with activation of p-ERK, which is seen in some 30% of liver cancers, are most likely to benefit from such combinatorial treatment.

cancer biology↗

bioRxiv2018

Molecular dissection of early defense signaling underlying volatile-mediated defense priming and herbivore resistance in rice

Herbivore-induced plant volatiles prime plant defenses and resistance. How volatiles are integrated into early defense signaling is not well understood. Furthermore, whether there is a causal relationship between volatile defense priming and herbivore resistance is unclear. Here, we investigated the impact of indole, a common herbivore-induced plant volatile and known defense priming cue, on early defense signaling and herbivore resistance in rice. We show that rice plants infested by Spodoptera frugiperda caterpillars release up to 25 ng*h-1. Exposure to equal doses of synthetic indole enhances rice resistance to S. frugiperda. Screening of early signaling components reveals that indole directly enhances the expression of the receptor like kinase OsLRR-RLK1. Furthermore, indole specifically primes the transcription, accumulation and activation of the mitogen-activated protein kinase OsMPK3 as well as the expression of the downstream WRKY transcription factor OsWRKY70 and several jasmonate biosynthesis genes, resulting in a higher accumulation of jasmonic acid (JA). Using transgenic plants defective in early signaling, we show that OsMPK3 is required, and that OsMPK6 and OsWRKY70 contribute to indole-mediated defense priming of JA-dependent herbivore resistance. We conclude that volatiles can increase herbivore resistance of plants by priming early defense signaling components.

plant biology↗

bioRxiv2017

Jellyfish Oocyte Maturation Inducing Hormone And Neuroendocrine Regulation Of Reproduction

Oocyte meiotic maturation is a critical process for sexually reproducing animals, and its core cytoplasmic regulators are highly conserved between species. In contrast, the few known Maturation Inducing Hormones (MIHs) that act on oocytes to initiate this process have highly variable molecular natures. Using the hydrozoan jellyfish species Clytia and Cladonema, which undergo oocyte maturation in response to dark-light and light-dark transitions respectively, we deduced from gonad transcriptome data amidated tetrapeptide sequences and found that synthetic peptides could induce maturation of isolated oocytes at nanomolar concentrations. Antibody preabsorption experiments conclusively demonstrated that these W/RPRPamide-related neuropeptides account for endogenous MIH activity produced by isolated gonads. We further showed that the MIH peptides are synthesised by neural-type cells in the gonad, are released following dark-light / light-dark transitions, and probably act on the oocyte surface. They are produced by male as well as female jellyfish and can trigger both sperm and egg release, suggesting a role in spawning coordination. We propose an evolutionary link between hydrozoan MIH and the neuropeptide hormones that regulate reproduction upstream of MIH in bilaterian species.

developmental biology↗

bioRxiv2017

Evidence for a role of calcium in STING signaling

STING, an ER resident cyclic dinucleotide (CDN) receptor, plays an important role in innate immune response signaling. Upon binding to CDNs, it activates the TBK1-IRF3 signaling axis, which stimulates gene expression including interferon beta. We hypothesized that the ER localization of STING reflects a role for calcium mobilization in its signaling. To test this hypothesis, we treated mouse cells with two STING agonists, the synthetic drug DMXAA and a natural ligand, cyclic GMP-AMP (cGAMP), and measured intracellular calcium. Both triggered a rapid rise in intracellular calcium that was partially inhibited by STING depletion. Intracellular calcium chelation blocked DMXAA induced signaling downstream of STING activation, but had no effect on cGAMP induced signaling. We propose that intracellular calcium plays an important role in the response of the STING pathway. In response to DMXAA, calcium is mobilized form the ER and required for signaling. In the case of cGAMP calcium is mobilized but not required. This difference could be explained by alternative modes of STING activation for the two ligands, or a combination of STING-dependent and -independent actions of extracellular cGAMP.

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↗

bioRxiv2017

A microbially derived tyrosine sulfated peptide mimics a plant peptide hormone

O_LIThe biotrophic pathogen Xanthomonas oryzae pv. oryzae (Xoo) produces a sulfated peptide named RaxX, which shares similarity to peptides in the PSY (plant peptide containing sulfated tyrosine) family. We hypothesize that RaxX functionally mimics the growth stimulating activity of PSY peptides.\nC_LIO_LIRoot length was measured in Arabidopsis and rice treated with synthetic RaxX peptides. We also used comparative genomic analysis and Reactive Oxygen Species (ROS) burst assay to evaluate the activity of RaxX and PSY peptides.\nC_LIO_LIHere we found that a synthetic sulfated RaxX derivative comprising 13 residues (RaxX13-sY), highly conserved between RaxX and PSY, induces root growth in Arabidopsis and rice in a manner similar to that triggered by PSY. We identified residues that are required for activation of immunity mediated by the rice XA21 receptor but that are not essential for root growth induced by PSY. Finally, we showed that a Xanthomonas strain lacking raxX is impaired in virulence.\nC_LIO_LIThese findings suggest that RaxX serves as a molecular mimic of PSY peptides to facilitate Xoo infection and that XA21 has evolved the ability to recognize and respond specifically to the microbial form of the peptide.\nC_LI

plant biology↗

bioRxiv2018

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

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

cell biology↗