Single particle cryo-EM reconstruction of 52 kDa streptavidin at 3.2 Angstrom resolution, bioRxiv, 2018-11-13
AbstractThe fast development of single particle cryo-EM has made it more feasible to obtain the 3D structure of well-behaved macromolecules with molecular weight higher than 300 kDa at ~3 Å resolution. It remains a challenge to obtain high resolution structure of molecules smaller than 100 kDa using single particle cryo-EM, mainly due to the low contrast of the molecules embedded in vitreous ice. In this work, we applied the Cs-corrector-VPP coupled cryo-EM to study 52 kDa streptavidin (SA) protein supported on a thin layer of graphene film and embedded in vitreous ice. We were able to solve both the apo-SA and biotin-bound SA at near-atomic resolution using single particle cryo-EM. We demonstrated that the method is capable to determine the structure of molecule as small as 39 kDa and potentially even smaller molecules. Furthermore, we found that using the graphene film to avoid the adsorption to the air-water interface is critical to maintain the protein’s high-resolution structural information.
biorxiv biophysics 0-100-users 2018Target-specific co-transmission of acetylcholine and GABA from a subset of cortical VIP+ interneurons, bioRxiv, 2018-11-13
AbstractThe modulation of cortex by acetylcholine (ACh) is typically thought to originate from long-range projections arising in the basal forebrain. However, a subset of VIP interneurons express ChAT, the synthetic enzyme for ACh, and are a potential local source of cortical ACh. Which neurotransmitters these VIPChAT interneurons (VCINs) release is unclear, and which post-synaptic cell types these transmitters target is not known. Using quantitative molecular analysis of VCIN pre-synaptic terminals, we show expression of the molecular machinery to release both ACh and GABA, with ACh release restricted to a subset of boutons. A systematic survey of potential post-synaptic cell types shows that VCINs release GABA primarily onto other inhibitory interneuron subtypes, while ACh neurotransmission is notably sparse, with most ACh release onto layer 1 interneurons and other VCINs. Therefore, VCINs are an alternative source of cortical ACh signaling that supplement GABA-mediated disinhibition with highly targeted excitation through ACh.
biorxiv neuroscience 0-100-users 2018Characterization of proprioceptive system dynamics in behaving Drosophila larvae using high-speed volumetric microscopy, bioRxiv, 2018-11-12
SummaryProprioceptors provide feedback about body position that is essential for coordinated movement. Proprioceptive sensing of the position of rigid joints has been described in detail in several systems, however it is not known how animals with an elastic skeleton encode their body positions. Understanding how diverse larval body positions are dynamically encoded requires knowledge of proprioceptor activity patterns in vivo during natural movement. Here we applied high-speed volumetric SCAPE microscopy to simultaneously track the position, physical deformation, and temporal patterns of intracellular calcium activity of multidendritic proprioceptors in crawling Drosophila larvae. During the periodic segment contraction and relaxation that occurs during crawling, proprioceptors with diverse morphologies showed sequential onset of activity throughout each periodic episode. A majority of these proprioceptors showed activity during segment contraction with one neuron type activated by segment extension. Different timing of activity of contraction-sensing proprioceptors was related to distinct dendrite terminal targeting, providing a continuum of position encoding during all phases of crawling. These dynamics could endow different proprioceptors with specific roles in monitoring the progression of contraction waves, as well as body shape during other behaviors. We provide activity measurements during exploration as one example. Our results provide powerful new insights into the body-wide neuronal dynamics of the proprioceptive system in crawling Drosophila, and demonstrate the utility of our approach for characterization of neural encoding throughout the nervous system of a freely behaving animal.
biorxiv neuroscience 200-500-users 2018Simultaneous mesoscopic and two-photon imaging of neuronal activity in cortical circuits, bioRxiv, 2018-11-12
AbstractSpontaneous and sensory-evoked activity propagates across spatial scales in the mammalian cortex but technical challenges have generally precluded establishing conceptual links between the function of local circuits of neurons and brain-wide network dynamics. To solve this problem, we developed a method for simultaneous cellular-resolution two-photon calcium imaging of a local microcircuit and mesoscopic widefield calcium imaging of the entire cortical mantle in awake, behaving mice. Our method employs an orthogonal axis design whereby the mesoscopic objective is oriented downward directly above the brain and the two-photon objective is oriented horizontally, with imaging performed through a glass right angle microprism implanted in the skull. In support of this method, we introduce a suite of analysis tools for relating the activity of individual cells to distal cortical areas, as well as a viral method for robust and widespread gene delivery in the juvenile mouse brain. We use these methods to characterize the diversity of associations of individual, genetically-defined neurons with cortex-wide network motifs.
biorxiv neuroscience 0-100-users 2018An atlas of polygenic risk score associations to highlight putative causal relationships across the human phenome, bioRxiv, 2018-11-11
AbstractThe age of large-scale genome-wide association studies (GWAS) has provided us with an unprecedented opportunity to evaluate the genetic liability of complex disease using polygenic risk scores (PRS). In this study, we have analysed 162 PRS (P<5×l0 05) derived from GWAS and 551 heritable traits from the UK Biobank study (N=334,398). Findings can be investigated using a web application (<jatsext-link xmlnsxlink=httpwww.w3.org1999xlink ext-link-type=uri xlinkhref=httpmrcieu.mrsoftware.orgPRS_atlas>httpmrcieu.mrsoftware.orgPRS_atlas<jatsext-link>), which we envisage will help uncover both known and novel mechanisms which contribute towards disease susceptibility.To demonstrate this, we have investigated the results from a phenome-wide evaluation of schizophrenia genetic liability. Amongst findings were inverse associations with measures of cognitive function which extensive follow-up analyses using Mendelian randomization (MR) provided evidence of a causal relationship. We have also investigated the effect of multiple risk factors on disease using mediation and multivariable MR frameworks. Our atlas provides a resource for future endeavours seeking to unravel the causal determinants of complex disease.
biorxiv genetics 100-200-users 2018Lineage tracing on transcriptional landscapes links state to fate during differentiation, bioRxiv, 2018-11-11
AbstractA challenge in stem cell biology is to associate molecular differences among progenitor cells with their capacity to generate mature cell types. Though the development of single cell assays allows for the capture of progenitor cell states in great detail, these assays cannot definitively link cell states to their long-term fate. Here, we use expressed DNA barcodes to clonally trace single cell transcriptomes dynamically during differentiation and apply this approach to the study of hematopoiesis. Our analysis identifies functional boundaries of cell potential early in the hematopoietic hierarchy and locates them on a continuous transcriptional landscape. We reconstruct a developmental hierarchy showing separate ontogenies for granulocytic subtypes and two routes to monocyte differentiation that leave a persistent imprint on mature cells. Finally, we use our approach to benchmark methods of dynamic inference from single-cell snapshots, and provide evidence of strong early fate biases dependent on cellular properties hidden from single-cell RNA sequencing.
biorxiv systems-biology 100-200-users 2018