Actin chromobody imaging reveals sub-organellar actin dynamics, bioRxiv, 2019-05-16
AbstractThe actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are difficult to detect with fluorescence microscopy. We developed an approach using fluorescent protein-tagged actin nanobodies targeted to organelle membranes to enable live cell imaging of sub-organellar actin dynamics with unprecedented spatiotemporal resolution. These probes reveal that ER-associated actin drives fission of multiple organelles including mitochondria, endosomes, lysosomes, peroxisomes, and the Golgi.<jatsfig id=ufig1 position=float fig-type=figure orientation=portrait><jatsgraphic xmlnsxlink=httpwww.w3.org1999xlink xlinkhref=639278v2_ufig1 position=float orientation=portrait >
biorxiv cell-biology 100-200-users 2019Chromatin accessibility established by Pou5f3, Sox19b and Nanog primes genes for activity during zebrafish genome activation, bioRxiv, 2019-05-16
ABSTRACTIn many organisms, early embryonic development is driven by maternally provided factors until the controlled onset of transcription during zygotic genome activation. The regulation of chromatin accessibility and its relationship to gene activity during this transition remains poorly understood. Here, we generated chromatin accessibility maps from genome activation until the onset of lineage specification. During this period, chromatin accessibility increases at regulatory elements. This increase is independent of RNA polymerase II-mediated transcription, with the exception of the hyper-transcribed miR-430 locus. Instead, accessibility often precedes the transcription of associated genes. Loss of the maternal transcription factors Pou5f3, Sox19b, and Nanog, which are known to be required for zebrafish genome activation, results in decreased accessibility at regulatory elements. Importantly, the accessibility of regulatory regions, especially when established by Pou5f3, Sox19b and Nanog, is predictive for future transcription. Our results show that the maternally provided transcription factors Pou5f3, Sox19b, and Nanog open up chromatin and prime genes for activity during zygotic genome activation in zebrafish.
biorxiv developmental-biology 100-200-users 2019Gene knock-ins in Drosophila using homology-independent insertion of universal donor plasmids, bioRxiv, 2019-05-16
AbstractSite-specific insertion of DNA into endogenous genes (knock-in) is a powerful method to study gene function. However, traditional methods for knock-in require laborious cloning of long homology arms for homology-directed repair. Here, we report a simplified method in Drosophila melanogaster to insert large DNA elements into any gene using homology-independent repair. This method, known as CRISPaint, employs CRISPR-Cas9 and non-homologous end joining (NHEJ) to linearize and insert donor plasmid DNA into a target genomic cut site. The inclusion of commonly used elements such as GFP on donor plasmids makes them universal, abolishing the need to create gene-specific homology arms and greatly reducing user workload. Using this method, we show robust gene-specific integration of donor plasmids in cultured cells and the fly germ line. Furthermore, we use this method to analyze gene function by fluorescently tagging endogenous proteins, disrupting gene function, and generating reporters of gene expression. Finally, we assemble a collection of donor plasmids for germ line knock-in that contain commonly used insert sequences. This method simplifies the generation of site-specific large DNA insertions in Drosophila cell lines and fly strains, and better enables researchers to dissect gene function in vivo.SummaryWe report a new homology-independent genomic knock-in method in Drosophila to insert large DNA elements into any target gene. Using CRISPR-Cas9 and non-homologous end joining (NHEJ), an entire donor plasmid is inserted into the genome without the need for homology arms. This approach eliminates the burden associated with designing and constructing traditional donor plasmids. We demonstrate its usefulness in cultured cells and in vivo to fluorescently tag endogenous proteins, generate reporters of gene expression, and disrupt gene function.
biorxiv genetics 0-100-users 2019Somatosensory-Motor Dysconnectivity Spans Multiple Transdiagnostic Dimensions of Psychopathology, bioRxiv, 2019-05-16
AbstractBackgroundThere is considerable interest in a dimensional transdiagnostic approach to psychiatry. Most transdiagnostic studies have derived factors based only on clinical symptoms, which might miss possible links between psychopathology, cognitive processes and personality traits. Furthermore, many psychiatric studies focus on higher-order association brain networks, thus neglecting the potential influence of huge swaths of the brain.MethodsA multivariate data-driven approach (partial least squares; PLS) was utilized to identify latent components linking a large set of clinical, cognitive and personality measures to whole-brain resting-state functional connectivity (RSFC) patterns across 224 participants. The participants were either healthy (N=110) or diagnosed with bipolar disorder (N=40), attention-deficithyperactivity disorder (N=37), schizophrenia (N=29) or schizoaffective disorder (N=8). In contrast to traditional case-control analyses, the diagnostic categories were not utilized in the PLS analysis, but were helpful for interpreting the components.ResultsOur analyses revealed three latent components corresponding to general psychopathology, cognitive dysfunction and impulsivity. Each component was associated with a unique whole-brain RSFC signature and shared across all participants. The components were robust across multiple control analyses and replicated using independent task functional magnetic resonance imaging data from the same participants. Strikingly, all three components featured connectivity alterations within the somatosensory-motor network, and its connectivity with subcortical structures and cortical executive networks.ConclusionsWe identified three distinct dimensions with dissociable (but overlapping) whole-brain RSFC signatures across healthy individuals and individuals with psychiatric illness, providing potential intermediate phenotypes that span across diagnostic categories. Our results suggest expanding the focus of psychiatric neuroscience beyond higher-order brain networks.
biorxiv neuroscience 0-100-users 2019Cryo-EM structure of the ClpXP protein degradation machinery, bioRxiv, 2019-05-15
AbstractThe ClpXP machinery is a two component protease complex performing targeted protein degradation in bacteria and eukaryotes. The complex consists of the AAA+ chaperone ClpX and the peptidase ClpP. The hexameric ClpX utilizes the energy of ATP binding and hydrolysis to engage, unfold and translocate substrates into the catalytic chamber of tetradecameric ClpP where they are degraded. Formation of the complex involves a symmetry mismatch, since hexameric AAA+ rings bind axially to the opposing stacked heptameric rings of the tetradecameric ClpP. Here we present the first high-resolution cryo-EM structure of ClpXP from Listeria monocytogenes. We unravel the heptamer-hexamer binding interface and provide novel insights into the ClpX-ClpP crosstalk and activation mechanism. The comparison with available crystal structures of ClpP and ClpX in different states allows us to understand important aspects of ClpXP’s complex mode of action and provides a structural framework for future pharmacological applications.
biorxiv molecular-biology 100-200-users 2019metaFlye scalable long-read metagenome assembly using repeat graphs, bioRxiv, 2019-05-15
AbstractLong-read sequencing technologies substantially improved assemblies of many isolate bacterial genomes as compared to fragmented assemblies produced with short-read technologies. However, assembling complex metagenomic datasets remains a challenge even for the state-of-the-art long-read assemblers. To address this gap, we present the metaFlye assembler and demonstrate that it generates highly contiguous and accurate metagenome assemblies. In contrast to short-read metagenomics assemblers that typically fail to reconstruct full-length 16S RNA genes, metaFlye captures many 16S RNA genes within long contigs, thus providing new opportunities for analyzing the microbial “dark matter of life”. We also demonstrate that long-read metagenome assemblers significantly improve full-length plasmid and virus reconstruction as compared to short-read assemblers and reveal many novel plasmids and viruses.
biorxiv bioinformatics 100-200-users 2019