Biology subjects

Rhine, K.

Publications and source records attributed to Rhine, K..

4 recordsLinked to original sources

bioRxiv2025

Imaging Cellular Metabolic Rewiring with SuMMIT-SRS

Cells dynamically rewire their metabolic pathways in response to physiological and pathological cues. Such plasticity is particularly critical in neurons, stem cells, cancer cells, and immune cells, where biosynthetic demands can shift rapidly. However, current metabolic imaging techniques using isotope labeling typically track only one metabolite at a time, limiting their ability to capture the rapid dynamics of complex metabolic networks including coordinated precursor utilization, crosstalk, and turnover. Here, we present Subcellular Multiplexed Metabolic Isotope Tracing Stimulated Raman Scattering microscopy (SuMMIT-SRS), a platform that enables simultaneous visualization of multiple metabolic dynamics at subcellular resolution. By exploiting the distinct vibrational signatures of carbon-deuterium bonds derived from multiple deuterated amino acids, lipids, and monosaccharide tracers, SuMMIT-SRS maps co-regulated DNA, RNA, protein, and lipid synthesis at the same time and resolves various individual amino acid-mediated metabolic pathways within intact cells and tissues. We demonstrate SuMMITs broad utility across Drosophila fat body tissue and developing brain, tumor organoids, aged human neurons, and mouse liver, capturing cell type-specific metabolic rewiring under genetic and pathological perturbations. This approach extends SRS to multiplexed isotope tracing, offering a powerful tool to uncover dynamic and complex biosynthesis programs in development, health, and disease.

bioengineering↗

bioRxiv2025

RNA triggers chronic stress during neuronal aging

Neurodegenerative diseases are linked with dysregulation of the integrated stress response (ISR), which coordinates cellular homeostasis during and after stress events. Cellular stress can arise from several sources, but there is significant disagreement about which stress might contribute to aging and neurodegeneration. Here, we leverage directed transdifferentiation of human fibroblasts into aged neurons to determine the source of ISR activation. We demonstrate that increased accumulation of cytoplasmic double-stranded RNA (dsRNA) activates the eIF2 kinase PKR, which in turn triggers the ISR in aged neurons and leads to sequestration of dsRNA in stress granules. Aged neurons accumulate endogenous mitochondria-derived dsRNA that directly binds to PKR. This mitochondrial dsRNA leaks through damaged mitochondrial membranes and forms cytoplasmic foci in aged neurons. Finally, we demonstrate that PKR inhibition leads to the cessation of stress, resumption of cellular translation, and restoration of RNA-binding protein expression. Together, our results identify a source of RNA stress that destabilizes aged neurons and may contribute to neurodegeneration.

cell biology↗

bioRxiv2023

Large-scale map of RNA binding protein interactomes across the mRNA life-cycle

Messenger RNAs (mRNAs) interact with RNA-binding proteins (RBPs) in diverse ribonucleoprotein complexes (RNPs) during distinct life-cycle stages for their processing and maturation. While substantial attention has focused on understanding RNA regulation by assigning proteins, particularly RBPs, to specific RNA substrates, there has been considerably less exploration leveraging protein-protein interaction (PPI) methodologies to identify and study the role of proteins in mRNA life-cycle stages. To address this gap, we generated an RNA-aware RBP-centric PPI map across the mRNA life-cycle by immunopurification (IP-MS) of [~]100 endogenous RBPs across the life-cycle in the presence or absence of RNase, augmented by size exclusion chromatography (SEC-MS). Aside from confirming 8,700 known and discovering 20,359 novel interactions between 1125 proteins, we determined that 73% of our IP interactions are regulated by the presence of RNA. Our PPI data enables us to link proteins to life-cycle stage functions, highlighting that nearly half of the proteins participate in at least two distinct stages. We show that one of the most highly interconnected proteins, ERH, engages in multiple RNA processes, including via interactions with nuclear speckles and the mRNA export machinery. We also demonstrate that the spliceosomal protein SNRNP200 participates in distinct stress granule-associated RNPs and occupies different RNA target regions in the cytoplasm during stress. Our comprehensive RBP-focused PPI network is a novel resource for identifying multi-stage RBPs and exploring RBP complexes in RNA maturation. HIGHLIGHTSO_LIAn RBP-centric RNA-aware PPI network focuses on the mRNA life-cycle in human cells C_LIO_LIPrey-prey correlation analysis assigns prey proteins to life-cycle stages, of which 536 proteins (half of the network) interact with multiple steps C_LIO_LIERH is highly connected to multiple RNPs to affect nuclear speckle organization and mRNA export C_LIO_LISplicing factor SNRNP200 interacts with stress granule proteins and has distinct RNA occupancy in the cytoplasm C_LI

systems biology↗

bioRxiv2022

Molecular determinants of Karyopherin-β2 chaperone and disaggregation activity

Karyopherin-{beta}2 (Kap{beta}2) is a nuclear-import receptor that recognizes proline-tyrosine nuclear localization signals (PY-NLSs) of diverse cytoplasmic cargo for transport to the nucleus. Kap{beta}2 cargo include several disease-linked RNA-binding proteins (RBPs) with prion-like domains (PrLDs), such as FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2. These RBPs with PrLDs are linked via pathology and genetics to debilitating degenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and multisystem proteinopathy (MSP). Remarkably, Kap{beta}2 prevents and reverses aberrant phase transitions of these cargo, which is cytoprotective. However, the molecular determinants of Kap{beta}2 that enable these activities remain poorly understood, particularly from the standpoint of nuclear-import receptor architecture. Kap{beta}2 is a superhelical protein comprised of 20 HEAT repeats. Here, we design truncated variants of Kap{beta}2 and assess their ability to antagonize FUS aggregation and toxicity in yeast and FUS condensation at the pure protein level and in human cells. We find that HEAT repeats 8-20 of Kap{beta}2 recapitulate all salient features of Kap{beta}2 activity. By contrast, Kap{beta}2 truncations lacking even a single cargo-binding HEAT repeat display reduced activity. Thus, we define a minimal Kap{beta}2 construct for delivery in adeno-associated viruses as a potential therapeutic for ALS/FTD, MSP, and related disorders.

biochemistry↗