Biology subjects

Roth, E. W.

Publications and source records attributed to Roth, E. W..

6 recordsLinked to original sources

bioRxiv2024

Mitochondrial oxidation of the carbohydrate fuel driven by pyruvate dehydrogenase robustly enhances stemness of older and geriatric Intestinal Stem Cells

Background and AimsAging impairs Intestinal Stem Cell (ISC) function and attenuates their regenerative capacity. Although the transcriptional landscape governing ISC fate during aging has been described, almost nothing is known about how metabolite handling regulates ISC renewal and maintains stemness. We investigated how mitochondrial metabolism of glucose and fatty acid-derived carbons, regulated by the gatekeeper, pyruvate dehydrogenase (PDH) rescues ISC stemness in older and geriatric mice and humans. MethodsProximal small intestinal organoids (enteroids) generated from pinch biopsy specimens obtained from young (21-25y) and older individuals (64-75y), and GFP-sorted single ISCs from Lgr5-EGFP mice (2-24 months) were used to examine hallmarks of ISC stemness. Mitochondrial morphology was evaluated using transmission electron microscopy. Mitochondrial oxygen consumption rate (OCR), ATP (mitoATP), and glycolytic ATP production were measured in the presence of full and single metabolic substrates (pyruvate, glutamate, and fatty acids) in whole cell and isolated mitochondria using the high throughput Seahorse XF technology. Carbon flux through TCA cycle was determined by 13C6-glucose tracing and measuring 13C enrichment in TCA cycle intermediates using liquid chromatography mass spectrometry. ResultsAge induced decline in ISC stemness is driven by a dramatic decrease in PDH activity that shuttles pyruvate away from the TCA cycle. Restoring PDH activity by inhibition of pyruvate dehydrogenase kinase 4 (PDK4) drives glucose-derived carbon entry into TCA cycle and subsequently increases mitochondrial OCR and mitoATP, collectively rescuing the decline in stemness in aging ISCs. The observed shift in fuel preference from fatty acids to glucose is unaltered by PDK4 inhibition. ConclusionPDH upregulation rescues age-induced decline in ISC stemness in humans and mice via directing glucose derived carbons to TCA cycle and increasing mitoATP production.

cell biology↗

bioRxiv2021

Vertex protein PduN tunes encapsulated pathway performance by dictating bacterial metabolosome morphology

Engineering subcellular organization in microbes shows great promise in addressing bottlenecks in metabolic engineering efforts; however, rules guiding selection of an organization strategy or platform are lacking. Here, we study compartment morphology as a factor in mediating encapsulated pathway performance. Using the 1,2-propanediol utilization microcompartment (Pdu MCP) system from Salmonella enterica serovar Typhimurium LT2, we find that we can shift the morphology of this protein nanoreactor from polyhedral to tubular by removing vertex protein PduN. Analysis of the metabolic function between these Pdu microtubes (MTs) shows that they provide a diffusional barrier capable of shielding the cytosol from a toxic pathway intermediate, similar to native MCPs. However, kinetic modeling suggests that the different surface area to volume ratios of MCP and MT structures alters encapsulated pathway performance. Finally, we report a microscopy-based assay that permits rapid assessment of Pdu MT formation to enable future engineering efforts on these structures.

bioengineering↗

bioRxiv2021

Chromatin forms nanoscale three-dimensional packing domains with fractal-like scaling behavior in vitro

Chromatin organization over a wide range of length scales plays a critical role in the regulation of transcription and deciphering the interplay of these processes requires high-resolution, three-dimensional, quantitative imaging of chromatin structure in vitro. Herein, we introduce ChromSTEM, a method that utilizes high angle annular dark-field imaging and tomography in scanning transmission electron microscopy combined with DNA-specific staining for electron microscopy. We utilized ChromSTEM to quantify chromatin structure in cultured cells and the statistical packing behavior of the chromatin polymer. Using chromatin mass and density analysis, we observed that chromatin forms spatially well-defined higher-order domains which are around 100 nm in radius, with a radially decreasing mass-density from the center to the periphery. Although the morphological properties of the domains vary within the same cell line, they seem to exhibit greater heterogeneity across cell lines, underlying a potential role of statistical chromatin packing in regulating cell-type-specific gene expression.

genomics↗

bioRxiv2020

Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for accelerating the design of cellular function, on-demand biomanufacturing, portable diagnostics, and educational kits. Many essential biological processes that could endow CFE systems with desired functions, such as protein glycosylation, rely on the activity of membrane-bound components. However, without the use of synthetic membrane mimics, activating membrane-dependent functionality in bacterial CFE systems remains largely unstudied. Here, we address this gap by characterizing native, cell-derived membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membranebound machinery. We first use nanocharacterization techniques to show that lipid vesicles in CFE extracts are tens to hundreds of nanometers across, and on the order of ~3x1012 particles/mL. We then determine how extract processing methods, such as post-lysis centrifugation, can be used to modulate concentrations of membrane vesicles in CFE systems. By tuning these methods, we show that increasing the number of vesicle particles to ~7x1012 particles/mL can be used to increase concentrations of heterologous membrane protein cargo expressed prior to lysis. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving N-linked and O-linked glycoprotein synthesis. We anticipate that our findings will facilitate in vitro gene expression systems that require membrane-dependent activities and open new opportunities in glycoengineering.

synthetic biology↗

bioRxiv2019

Apparent size and morphology of bacterial microcompartments varies with technique

Bacterial microcompartments (MCPs) are protein-based organelles which encapsulate metabolic pathways. Metabolic engineers have recently sought to repurpose MCPs to encapsulate heterologous pathways to increase flux through pathways of interest. As MCP engineering becomes more common, standardized methods for analyzing changes to MCPs and interpreting results across studies will become increasingly important. In this study, we demonstrate that different imaging techniques yield variations in the apparent size of purified MCPs from Salmonella enterica serovar Typhimurium LT2, likely due to variations in sample preparation methods. We provide guidelines for preparing samples for MCP imaging and outline expected variations in apparent size and morphology between methods. With this report we aim to establish an aid for comparing results across studies.

synthetic biology↗

bioRxiv2019

Quantifying Three-dimensional Chromatin Organization Utilizing Scanning Transmission Electron Microscopy: ChromSTEM

Chromatin organization over a wide range of length scales plays a critical role in the regulation of gene expression and deciphering these processes requires high-resolution, three-dimensional, quantitative imaging of chromatin structure in vitro. Herein we introduce ChromSTEM, a method which utilizes high angle annular dark field imaging and tomography in scanning transmission electron microscopy in combination with DNA-specific staining for electron microscopy. We utilized ChromSTEM to quantify chromatin structure in cultured cells and tissue biopsies through local DNA distribution and the scaling behavior of chromatin polymer. We observed that chromatin is densely packed with an average volume concentration of over 30% with heterochromatin having a two-fold higher density compared to euchromatin. Chromatin was arranged into spatially well-defined nanoscale packing domains with fractal internal structure and genomic size between 100 and 400 kb, comparable to that of topologically associated domains. The packing domains varied in DNA concentration and fractal dimension and had one of the distinct states of chromatin packing with differential ratio of DNA content to the chromatin volume concentration. Finally, we observed a significant intercellular heterogeneity of chromatin organization even within a genetically uniform cell population, which demonstrates the imperative for high-throughput characterization of chromatin structure at the single cell level.

genomics↗