Biology subjects

Furth, D.

Publications and source records attributed to Furth, D..

5 recordsLinked to original sources

bioRxiv2024

Detection of protein-protein interactions by bio-orthogonal fluorogenic proximity probes

Detecting protein-protein interactions within cells is challenging. Transgenic approaches risk altering protein function via fluorescent tagging, while in situ methods lack in vivo compatibility. Here, we introduce fluorogenic probes with dual-tetrazine pegylated branched arms linked to xanthene dye. Activation requires both tetrazine arms to interact simultaneously with target proteins, enabling dual-substrate recognition. We applied our method to detect protein-protein interactions in both fixed and living cells, utilizing antibody conjugation for fixed cells and genetic code expansion for real-time detection in living cells. Our strategy ensures versatile applicability and seamless transition between fixed and living systems.

molecular biology↗

bioRxiv2020

Replicability of spatial gene expression atlas data from the adult mouse brain

BackgroundSpatial gene expression is particularly interesting in the mammalian brain, with the potential to serve as a link between many data types. However, as with any type of expression data, cross-dataset benchmarking of spatial data is a crucial first step. Here, we assess the replicability, with reference to canonical brain sub-divisions, between the Allen Institutes in situ hybridization data from the adult mouse brain (ABA) and a similar dataset collected using Spatial Transcriptomics (ST). With the advent of tractable spatial techniques, for the first time we are able to benchmark the Allen Institutes whole-brain, whole-transcriptome spatial expression dataset with a second independent dataset that similarly spans the whole brain and transcriptome. ResultsWe use LASSO, linear regression, and correlation-based feature selection in a supervised learning framework to classify expression samples relative to their assayed location. We show that Allen reference atlas labels are classifiable using transcription, but that performance is higher in the ABA than ST. Further, models trained in one dataset and tested in the opposite dataset do not reproduce classification performance bi-directionally. Finally, while an identifying expression profile can be found for a given brain area, it does not generalize to the opposite dataset. ConclusionsIn general, we found that canonical brain area labels are classifiable in gene expression space within dataset and that our observed performance is not merely reflecting physical distance in the brain. However, we also show that cross-platform classification is not robust. Emerging spatial datasets from the mouse brain will allow further characterization of cross-dataset replicability.

genomics↗

bioRxiv2020

A comparison of dopaminergic and cholinergic populations reveals unique contributions of VTA dopamine neurons to short-term memory

We systematically compared the contribution of two dopaminergic and two cholinergic ascending populations to a spatial short-term memory task in rats. In ventral tegmental area dopamine (VTA-DA) and nucleus basalis cholinergic (NB-ChAT) populations, trial-by-trial fluctuations in activity during the delay period related to performance with an inverted-U, despite the fact that both populations had low activity during that time. Transient manipulations revealed that only VTA-DA neurons, and not the other three populations we examined, contributed causally and selectively to short-term memory. This contribution was most significant during the delay period, when both increases or decreases in VTA-DA activity impaired short-term memory. Our results reveal a surprising dissociation between when VTA-DA neurons are most active and when they have the biggest causal contribution to short-term memory, while also providing new types of support for classic ideas about an inverted-U relationship between neuromodulation and cognition.

neuroscience↗

bioRxiv2019

In Situ Transcriptome Accessibility Sequencing (INSTA-seq)

Subcellular RNA localization regulates spatially polarized cellular processes, but unbiased investigation of its control in vivo remains challenging. Current hybridization-based methods cannot differentiate small regulatory variants, while in situ sequencing is limited by short reads. We solved these problems using a bidirectional sequencing chemistry to efficiently image transcript-specific barcode in situ, which are then extracted and assembled into longer reads using NGS. In the Drosophila retina, genes regulating eye development and cytoskeletal organization were enriched compared to methods using extracted RNA. We therefore named our method In Situ Transcriptome Accessibility sequencing (INSTA-seq). Sequencing reads terminated near 3 UTR cis-motifs (e.g. Zip48C, stau), revealing RNA-protein interactions. Additionally, Act5C polyadenylation isoforms retaining zipcode motifs were selectively localized to the optical stalk, consistent with their biology. Our platform provides a powerful way to visualize any RNA variants or protein interactions in situ to study their regulation in animal development.

genomics↗

bioRxiv2018

High-throughput mapping of mesoscale connectomes in individual mice

Comprehensive analysis of neuronal networks requires brain-wide measurement of connectivity, activity, and gene expression. Although high-throughput methods are available for mapping brain-wide activity and transcriptomes, comparable methods for mapping region-to-region connectivity remain slow and expensive because they require averaging across hundreds of brains. Here we describe BRICseq, which leverages DNA barcoding and sequencing to map connectivity from single individuals in a few weeks and at low cost. Applying BRICseq to the mouse neocortex, we find that region-to-region connectivity provides a simple bridge relating transcriptome to activity: The spatial expression patterns of a few genes predict region-to-region connectivity, and connectivity predicts activity correlations. We also exploited BRICseq to map the mutant BTBR mouse brain, which lacks a corpus callosum, and recapitulated its known connectopathies. BRICseq allows individual laboratories to compare how age, sex, environment, genetics and species affect neuronal wiring, and to integrate these with functional activity and gene expression.

neuroscience↗