Biology subjects

Betts, D. H.

Publications and source records attributed to Betts, D. H..

3 recordsLinked to original sources

bioRxiv2021

Modulation of PKM1/2 levels by steric blocking morpholinos alters the metabolic and pluripotent state of murine pluripotent stem cells

Cellular metabolism plays both an active and passive role in embryonic development, pluripotency, and cell-fate decisions. However, little is known regarding the role of metabolism in regulating the recently described "formative" pluripotent state. The pluripotent developmental continuum features a metabolic switch from a bivalent metabolism (both glycolysis and oxidative phosphorylation) in naive cells, to predominantly glycolysis in primed cells. We investigated the role of pyruvate kinase muscle isoforms (PKM1/2) in naive, formative, and primed mouse embryonic stem cells through modulation of PKM1/2 mRNA transcripts using steric blocking morpholinos that downregulate PKM2 and upregulate PKM1. We have examined these effects in naive, formative, and primed cells by quantifying the effects of PKM1/2 modulation on pluripotent and metabolic transcripts and by measuring shifts in the population frequencies of cells expressing naive and primed cell surface markers by flow cytometry. Our results demonstrate that modulating PKM1 and PKM2 levels alters the transition from the naive state into a primed pluripotent state by enhancing the proportion of the affected cells seen in the "formative" state. Therefore, we conclude that PKM1/2 actively contributes to mechanisms that oversee early stem pluripotency and their progression towards a primed pluripotent state.

cell biology↗

bioRxiv2020

Differential localization patterns of pyruvate kinase isoforms in murine naïve, formative and primed pluripotent states

Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) represent opposite ends of a pluripotency continuum, referred to as naive and primed pluripotent states, respectively. These divergent pluripotent states differ in several ways including growth factor requirements, transcription factor expression, DNA methylation patterns, and metabolic profiles. Naive cells employ both glycolysis and oxidative phosphorylation (OXPHOS), whereas primed cells preferentially utilize aerobic glycolysis, a trait shared with cancer cells referred to as the Warburg effect. Until recently, metabolism has been regarded as a by-product of cell fate; however, evidence now supports metabolism as being a driver of stem cell state and fate decisions. Pyruvate kinase muscle isoforms (PKM1 and PKM2) are important for generating and maintaining pluripotent stem cells (PSCs) and mediating the Warburg effect. Both isoforms catalyze the last step of glycolysis generating adenosine triphosphate and pyruvate, however, the precise role(s) of PKM1/2 in naive and primed pluripotency is not well understood. The primary objective was to characterize the cellular expression and localization patterns of PKM1 and PKM2 in mESCs, chemically transitioned epiblast-like cells (mEpiLCs) representing formative pluripotency, and mEpiSCs using immunoblotting, flow cytometry and confocal microscopy. The results indicate that PKM1 and PKM2 are not only localized to the cytoplasm but also accumulate in differential subnuclear regions of mESC, mEpiLCs and mEpiSCs as determined by a quantitative, confocal microscopy colocalization employing orthogonal projections and airyscan processing. Importantly, we discovered that the subnuclear localization of PKM1/2 shifts during the transition from mESCs, mEpiLCs and mEpiSCs. We have also authenticated a new method of selecting formative pluripotency cells from naive and primed populations using the cell surface markers SSEA1 and CD24. Finally, we have comprehensively validated the appropriateness and power of the Pearsons correlation coefficient and Manders overlap coefficient for assessing nuclear and cytoplasmic protein colocalization in PSCs by immunofluorescence confocal microscopy. We propose that nuclear PKM1/2 assists with distinct pluripotency state maintenance and lineage priming by non-canonical mechanisms. These results advance our understanding of the overall mechanisms controlling naive, formative and primed pluripotency.

cell biology↗

bioRxiv2020

Telomerase inhibition causes premature senescence of fetal guinea pig muscle cells

Premature senescence in low birth weight rodents is associated with later life metabolic disease, including the development of insulin resistance. Telomerase, a reverse transcriptase enzyme with telomeric and non-telomeric functions, is present at high levels during development to maintain and repair long telomere lengths and to protect cells from oxidative stress-induced growth arrest. Adverse In utero environments are often associated with increased reactive oxygen species (ROS), and ROS have been documented to impair/alter telomerase function. We postulate that telomerase protects cells against oxidative stress-induced damage, and its inhibition could lead to premature senescence. A primary cell line of fetal guinea pig muscle cells was differentiated under high (20%) and low (1-2%) oxygen concentrations and telomerase activity was pharmacologically inhibited using a synthetic tea catechin. Following 48 hours, ROS detection was conducted with MitoSOX, Mitotracker and 6-carboxy-2,7-dichlorodihydrofluorescein diacetate staining. Cells cultured at 20% O2 and treated with a telomerase activity inhibitor displayed reduced cell growth rates and increased levels of senescence markers, including p21 and p53. Telomeric DNA damage, measured by phosphorylated-{gamma}H2A.X staining at telomeres, was significantly increased in cells cultured at all oxygen concentrations with telomerase inhibition. Telomerase inhibition altered metabolic signaling (e.g. mTOR, p66Shc) and increased mitochondrial ROS levels. Telomerase may protect cells during development from adverse in utero environments that cause premature senescence.

cell biology↗