Biology subjects

Aksenov, N. D.

Publications and source records attributed to Aksenov, N. D..

4 recordsLinked to original sources

bioRxiv2026

Complex I is essential for embryonic stem cell proliferation through NADH-mediated redox signaling

Background: Pluripotent embryonic stem cells (ESCs) possess unlimited self-renewal potential and the capacity to generate all cell types of an organism. Mitochondrial metabolism participates in the regulation of ESC self-renewal and differentiation, yet the specific roles of respiratory complex components in these processes remain largely unknown. Methods: Ndufs1-knockout mouse ESCs were generated using the CRISPR/Cas9 method. Proliferation and cell cycle dynamics were assessed via flow cytometry with propidium iodide and Hoechst 33342/Pyronin Y staining. Mitochondrial physiology, including membrane potential, ROS levels, and mass was evaluated using JC-1, MitoSOX Red, DHE, and MitoTracker Red measurements. Cellular ATP and NAD/NADH levels were determined by luminescent and colorimetric assays. Pluripotency was confirmed by immunocytochemistry, Western blotting, teratoma formation, and directed differentiation into EpiSCs and cardiomyocytes. Downstream molecular and transcriptomic shifts were analyzed using RNA-seq and Western blotting. Results: We demonstrate that while Complex I function is dispensable for the maintenance of pluripotent identity, its loss leads to a profound reduction of ESC proliferation rates. Deficiency of the core CI subunit Ndufs1 induces mitochondrial reductive stress, characterized by a decreased cellular NAD/NADH ratio and elevated ROS levels. We find that cytosolic, but not mitochondrial, NADH accumulation acts as the primary signal limiting ESC proliferation, independent of energy depletion or excessive ROS. This process is associated with the activation of the integrated stress response and suppression of the NRF2/PGC-1/SOD2 antioxidant axis. Conclusions: Our findings identify NADH-driven reductive stress as a key checkpoint controlling ESC proliferation and reveal metabolic flexibility that preserves ESC developmental potential despite severe mitochondrial dysfunction. We propose that Complex I functions as a metabolic rheostat, tuning ESC mitotic activity through mitochondrial-to-nuclear redox signaling.

cell biology↗

bioRxiv2025

Pcbp1 orchestrates amino acid metabolism burst during the naive-to-primed pluripotency transition

Embryo implantation is accompanied by the naive-to-primed pluripotency transition in epiblast cells, making them receptive to external differentiation signals. In addition to this developmental program switch, implantation suggests that an anabolic boost is required for this process, as the embryo-uterine connection begins supplying the requisite nutrients. In this study, we show that the DNA-binding protein Pcbp1 plays a key role in intensifying amino acid metabolism during the priming of pluripotent stem cells. Knockout of the Pcbp1 gene leads to embryo growth arrest a few days after implantation. By modeling the naive-to-primed pluripotency transition in vitro, we observe reduced proliferation and induction of apoptosis in cells deficient for Pcbp1. Using multi-omics approaches, we uncover a crucial role for Pcbp1 in driving a transcriptional burst of numerous genes involved in the import and the de novo synthesis of essential and conditionally essential amino acids. Pcbp1 deficiency is consequently associated with a slowdown in protein biosynthesis, explaining the early lethal phenotype of knockout embryos. Our findings thus uncover the molecular mechanisms underlying anabolic changes during the naive-to-primed pluripotency transition and highlight the essential role of Pcbp1 in this process, also pointing to its functions in highly proliferative cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/658314v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1e17b48org.highwire.dtl.DTLVardef@64e940org.highwire.dtl.DTLVardef@10d6b00org.highwire.dtl.DTLVardef@1ec0d23_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2024

Pcbp1 constrains Oct4 expression in the context of pluripotency

Oct4 is a commonly known marker of pluripotent stem cells as well as one of the key factors required for pluripotency induction. Its gene (Pou5f1) is subject to complicated regulation through distal and proximal enhancers. Noteworthy, this protein also plays an important role in primitive endoderm (PrE) specification, though the mechanisms driving its expression during this process are still unknown. Here we show that KH-domain protein Pcbp1 occupies poly(C)- sites of the Pou5f1 enhancers, but Pcbp1 knockout does not affect the Oct4 expression level in ESCs. On the contrary, Pcbp1 is essential for timely Oct4 downregulation upon differentiation signals. Indeed, Pcbp1 loss results in high rate of spontaneous differentiation of ESCs to PrE, and this effect is enhanced upon retinoic acid treatment. This phenotype can be explained by residual Oct4 expression, as Oct4 depletion rescues normal differentiation. Overall, our results point to Pcbp1 is a transcriptional regulator of Pou5f1, purported to synchronize Oct4 expression decline with the pluripotency network shutdown during differentiation. Oct4 being outside of this network loss its functions as factor of pluripotency and acts as PrE specifier.

cell biology↗

bioRxiv2019

Fluorometric intracellular Na+ measurement as compared with flame emission assay: An unexpected problem with gramicidin-based calibration

Fluorescent probes are a popular and indispensable tool for monitoring sodium concentration in living cells in situ. Calibration of fluorescent probes inside cells commonly uses ionophores to equilibrate intracellular and external ion concentrations. Here we test this calibration method using in parallel classical flame emission assay. Suspension human lymphoma cells allow both flow cytometry fluorometric study and flame emission assay. The most sensitive Na+ fluorescent probe ANG-2 and the most common ionophores were tested. Cellular Na+ was altered for calibration in three different ways: by stopping the sodium pump with ouabain, by inducing of apoptosis with staurosporine, and by gramicidin or amphotericin B treatment. We found that ANG-2 fluorescence in cells treated with gramicidin or amphotericin was about two fold lower than in the cells with the same sodium concentration but without ionophores. The equal fluorescence measured in the absence and in the presence of ionophores corresponds to different cell sodium concentrations. No effect of gramicidin on hydrolyzed ANG was observed in vitro. The mechanism, by which gramicidin decreases ANG fluorescence in cells is unlikely to be physical quenching and remains obscure. We conclude that ANG fluorescence does not display realistic cell Na+ if fluorescence in cell is measured in ionophore absence while calibrated in its presence.

physiology↗