Biology subjects

Nelson, I.

Publications and source records attributed to Nelson, I..

5 recordsLinked to original sources

bioRxiv2026

Preparation and application of YFV-17D-derived Anterograde Trans-neuronal Viral Vectors for Neuroscience Research

Trans-neuronal viruses that spread between synaptically connected neurons have become invaluable tools in neuroscience, enabling circuit mapping and targeted delivery of genetic material to cells within defined pathways. Although most available trans-neuronal viral tracers propagate retrogradely, an anterograde trans-neuronal virus that spread from presynaptic to postsynaptic neurons would substantially expand experimental capabilities. We previously demonstrated that YFV-17D--a live attenuated yellow fever vaccine used clinically for decades--spreads anterogradely along neuronal circuits. We further developed a suite of recombinant YFV-17D-based vectors for diverse applications, including tracing monosynaptic projectome of defined neuronal cell types, multiplex mapping of parallel pathways, and trans-neuronal genetic manipulation with minimal neurotoxicity. We have now tested and optimized procedures for vector production and application. Based on these optimizations, here, we present a two-part guide for preparing and deploying this viral vector system to map brain connectivity. The first part describes the molecular biology and cell culture workflows required to generate high-quality viral vectors, yielding titers of [~]1 x 10^10 to 5 x 10^11 genomic copies per milliliter within approximately two weeks. The second part outlines optimized animal procedures, including intracranial injections, perioperative care, and experimental considerations tailored to specific aims and vector variants. Depending on the application, efficient trans-neuronal tracing can be achieved within 1-4 weeks following delivery.

neuroscience↗

bioRxiv2024

A Structural Mechanism for Noncanonical GPCRSignal Transduction in the Hedgehog Pathway

SUMMARY PARAGRAPHThe Hedgehog (Hh) signaling pathway is fundamental to embryogenesis, tissue homeostasis, and cancer. Hh signals are transduced via an unusual mechanism: upon agonist-induced phosphorylation, the noncanonical G protein-coupled receptor SMOOTHENED (SMO) binds the catalytic subunit of protein kinase A (PKA-C) and physically blocks its enzymatic activity. By combining computational structural approaches with biochemical and functional studies, we show that SMO mimics strategies prevalent in canonical GPCR and PKA signaling complexes, despite little sequence or secondary structural homology. An intrinsically disordered region of SMO binds the PKA-C active site, resembling the PKA regulatory subunit (PKA-R) / PKA-C holoenzyme, while the SMO transmembrane domain binds a conserved PKA-C interaction hub, similar to other GPCR-effector complexes. In contrast with prevailing GPCR signal transduction models, phosphorylation of SMO promotes intramolecular electrostatic interactions that stabilize key structural elements within the SMO cytoplasmic domain, thereby remodeling it into a PKA-inhibiting conformation. Our work provides a structural mechanism for a central step in the Hh cascade and defines a paradigm for disordered GPCR domains to transmit signals intracellularly.

biochemistry↗

bioRxiv2022

DMT1 bridges endosomes and mitochondria to modulate mitochondrial iron translocation

Transient "kiss-and-run" endosome-mitochondria interactions can mediate mitochondrial iron translocation (MIT) but the associated mechanisms are still elusive. We show that Divalent Metal Transporter 1 (DMT1) modulates MIT via endosome-mitochondria interactions in invasive MDA-MB-231, but not in non-invasive T47D breast cancer cells. CRISPR/Cas9-based DMT1 knockout (KO) stable cells were used to demonstrate that DMT1 regulates MIT, endosomal speed, and labile iron pool (LIP) levels only in MDA-MB-231. DMT1 silencing increases PINK1/Parkin mitophagy markers, the autophagy marker LC3B, as well as mitochondrial ferritin in MDA-MB-231, but not in T47D. Strikingly, re-expression of DMT1 in MDA-MB-231 DMT KO cells rescues all protein levels evaluated. DMT1 silencing decreases Tom20 colocalization with PMPCB, a DMT1 interactor that regulates mitophagy hyperactivation. In MDA-MB-231 both mitochondrial metabolism and invasion were impaired by DMT1 silencing and rescued by DMT1 re-expression. DMT1 acts as a bridge between endosomes and mitochondria to support higher MIT/lower LIP levels, which are necessary for sustaining mitochondrial bioenergetics and invasive cancer cell migration. SummaryCellular iron metabolism is tightly regulated, and cancer cells rely on mitochondrial iron for malignancy. Here, we report that the divalent metal transporter DMT1 serves as a bridge between endosomes and mitochondria regulating mitochondrial iron translocation in breast cancer cells.

cell biology↗

bioRxiv2019

MACS: A Novel Concept for High-Magnification Imaging of Stretch-Activated Cells Reveals Divergent Nuclear Behavior to Different Levels of Strain

Mechanical cues from the environment influence cell behavior. Mechanisms of cellular mechanosensation are unclear, partially due to a lack of methods that can reveal dynamic processes. Here, we present a new concept for a low-cost, 3D-printed TENSCell (TENSion in Cells) device, that enables high-magnification imaging of cells during stretch. Using this device, we observed that nuclei of mouse embryonic skin fibroblasts underwent rapid and divergent responses, characterized by nuclear area expansion during 5% strain, but nuclear area shrinkage during 20% strain. Only responses to low strain were dependent on calcium signaling, while actin inhibition abrogated all nuclear responses and increased nuclear strain transfer and DNA damage. Imaging of actin dynamics during stretch revealed similar divergent trends, with F-actin shifting away from (5% strain) or towards (20% strain) the nuclear periphery. Our findings emphasize the importance of simultaneous stimulation and data acquisition to capture rapid mechanosensitive processes and suggest that mechanical confinement of nuclei through actin may be a protective mechanism during high strain loads.\n\nSTATEMENT OF SIGNIFICANCECells can sense and respond to mechanical cues in their environment. These responses can be rapid, on the time scale of seconds, and new methods are required for their acquisition and study. We introduce a new concept for a 3D-printed cell-stretch device that allows for simultaneous high-resolution imaging, while also being low-cost and easy to assemble to enable broad applicability. Using this device, we further demonstrated to importance of simultaneous stimulation and data acquisition to elicit mechanosensitive cell behavior as we observed rapid changes in nuclear size and reorganization of actin filaments around the nuclear border in skin cells. Overall, our results suggest that the rapid reorganization of actin during high loads might protect the genome from strain-induced damage.

cell biology↗

bioRxiv2018

A total synthetic approach to CRISPR/Cas9 genome editing and homology directed repair

CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We compared synthetic, chemically modified sgRNAs to in vitro transcribed sgRNAs and demonstrate the increased activity of synthetic sgRNAs in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to optimize the design of synthetic DNA templates to promote HDR. Utilizing these principles, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic sgRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

molecular biology↗