Biology subjects

Kafkia, E.

Publications and source records attributed to Kafkia, E..

7 recordsLinked to original sources

bioRxiv2026

Suspension culture drives metabolic rewiring in human pluripotent stem cells

Human pluripotent stem cells (hPSCs) are increasingly expanded in 3D suspension culture to support scalable cell culturing, yet how the transition from 2D to 3D cultures affects hPSCs remains poorly understood. Here, we compare hPSCs in 2D and 3D cultures using transcriptomics, proteomics, spent medium analysis, and stable-isotope tracing. hPSCs maintained pluripotency in both formats, although suspension culture showed altered growth dynamics and increased initial cell loss. Multi-omics analysis identified culture format as the dominant driver of molecular variation and revealed changes in adhesion, cytoskeletal organization, glycolysis, and oxidative phosphorylation. Functional analyses showed increased lactate production from glucose and stable-isotope tracing revealed increased glucose-derived pyruvate formation and altered partitioning of glucose-derived carbon within the TCA cycle in 3D suspension cultures. Glutamine contribution to the TCA cycle was reduced, whereas relative reductive carboxylation was increased in 3D suspension cultures. Together, these findings show that transition from 2D to 3D suspension culture induces metabolic rewiring in hPSCs.

cell biology↗

bioRxiv2025

SCAF1 driven polyadenylation site usage regulates mRNA isoform expression and neuronal differentiation

Accurate co-transcriptional processing is required for correct gene expression of mRNA transcript isoforms under unperturbed conditions, but particularly during development, to ensure tissue-specific mRNA isoform expression. Here we show that the poorly studied SR-related CTD-associated factors SCAF1 protein regulates polyadenylation site usage towards the end of genes. SCAF1 interacts directly with the phosphorylated C-terminal domain (CTD) of RNA polymerase II (RNAPII), in a complex enriched with elongation and 3 end processing factors. While SCAF1 knockout in HEK293 cells is innocuous, it leads to a shift towards expression of shorter mRNA transcripts by co-transcriptional usage of early polyadenylation sites. SCAF1 deficiency induced via auxin-dependent degradation in neuron differentiating mouse embryonic stem cells (mESCs) results in neuronal commitment defects, mediated by altered mRNA isoform usage that impacts expression of key neuronal genes. These findings highlight the importance of mRNA isoform usage and underscores the key role for SCAF proteins in its regulation though polyadenylation site selection.

molecular biology↗

bioRxiv2025

TCA cycle rewiring underpins implantation and histone acetylation programming

Metabolism has emerged as a key regulator of stem cell differentiation and their epigenomes. This coupling is particularly evident during the exit from naive pluripotency in vitro. However, our understanding of the dynamics of the metabolic rewiring especially at implantation remains rudimentary. In this study, we reconstruct the intracellular metabolite routings in pre- and post-implantation mouse embryos and during dynamic pluripotency transitions of cultured stem cells. Our findings reveal that, instead of a simple TCA cycle shutdown, there is a spatio-temporally programmed rewiring of the TCA cycle at implantation. Focusing on the spectrum of pluripotent cells, we identify pyruvate as a key metabolic nexus. Indeed, pyruvate carboxylase and malic enzyme activity establish cyclical carbon flow, which is essential for maintaining a balanced metabolic and transcriptional state and timely exit from naive pluripotency. Additionally, we discover that formative and primed pluripotent cells exhibit increased glutamine contribution to the TCA cycle, reduced oxidative TCA activity, and reciprocal reductive glutamine metabolism. This metabolic rewiring supports increased histone acetylation turnover, primarily using glutamine as a carbon source, supplemented by pyruvate cycling. Thus, we uncover diverse nutrient strategies that are functionally coupled to epigenome programming and dynamic pluripotency cell state transitions at the time of implantation.

developmental biology↗

bioRxiv2021

Glycolytic flux-signaling controls mouse embryo mesoderm development

How cellular metabolic state impacts cellular programs is a fundamental, unresolved question. Here we investigated how glycolytic flux impacts embryonic development, using presomitic mesoderm (PSM) patterning as the experimental model. First, we identified fructose 1,6-bisphosphate (FBP) as an in vivo sentinel metabolite that mirrors glycolytic flux within PSM cells of post-implantation mouse embryos. We found that medium-supplementation with FBP, but not with other glycolytic metabolites, such as fructose 6-phosphate and 3-phosphoglycerate, impaired mesoderm segmentation. To genetically manipulate glycolytic flux and FBP levels, we generated a mouse model enabling the conditional overexpression of dominant active, cytoplasmic Pfkfb3 (cytoPfkfb3). Overexpression of cytoPfkfb3 indeed led to increased glycolytic flux/FBP levels and caused an impairment of mesoderm segmentation, paralleled by the downregulation of Wnt-signaling, reminiscent of the effects seen upon FBP-supplementation. To probe for mechanisms underlying glycolytic flux-signaling, we performed subcellular proteome analysis and revealed that cytoPfkfb3 overexpression altered subcellular localization of certain proteins, including glycolytic enzymes, in PSM cells. Specifically, we revealed that FBP supplementation caused depletion of Pfkl and Aldoa from the nuclear-soluble fraction. Combined, we propose that FBP functions as a flux-signaling metabolite connecting glycolysis and PSM patterning, potentially through modulating subcellular protein localization.

developmental biology↗

bioRxiv2020

Operation of a TCA cycle subnetwork in the mammalian nucleus

Nucleic acid and histone modifications critically depend on central metabolism for substrates and co-factors. Although a few enzymes related to the formation of these required metabolites have been reported in the nucleus, the corresponding metabolic pathways are considered to function elsewhere in the cell. Here we show that a substantial part of the mitochondrial tricarboxylic acid (TCA) cycle, the biosynthetic hub of epigenetic modification factors, is operational also in the nucleus. Using 13C-tracer analysis, we identified activity of glutamine-to-fumarate, citrate-to-succinate, and glutamine-to-aspartate routes in the nuclei of HeLa cells. Proximity labeling mass-spectrometry revealed a spatial vicinity of the involved enzymes with core nuclear proteins, supporting their nuclear location. We further show nuclear localization of aconitase 2 and 2-oxoglutarate dehydrogenase in mouse embryonic stem cells. Together, our results demonstrate operation of an extended metabolic pathway in the nucleus warranting a revision of the canonical view on metabolic compartmentalization and gene expression regulation.

systems biology↗

bioRxiv2020

Oncogenic memory underlying minimal residual disease in breast cancer

Tumor relapse is responsible for most breast cancer related deaths1,2. The disease recurrence stems from treatment refractory cancer cells that persist as minimal residual disease (MRD) for years following initial therapy3. Yet, the molecular characteristics defining the malignancy of MRD remain elusive due to difficulties in observing these rare cells in patients or in model organisms. Here, we use a tractable organoid system and multi-omics analysis to show that the dormant MRD cells retain metabolic peculiarities reminiscent of the tumor state. While the MRD cells were distinct from both normal and tumor cells at a global transcriptomic level, their metabolomic and lipidomic profile markedly resembled that of the tumor state. The MRD cells particularly exhibited a de-regulated urea cycle and elevated glycolysis. We find the latter being crucial for their survival and could be selectively targeted using a small molecule inhibitor of glycolytic activity. We validated these metabolic peculiarities of the MRD cells in corresponding tissues obtained from the mouse model as well as in transcriptomic data from patients following neo-adjuvant therapy. Together, our results show that the treatment surviving MRD cells retain features of the tumor state over an extended period suggestive of an oncogenic memory. In accord, we found striking similarity in DNA methylation profiles between the tumor and the MRD cells. The distinction of MRD from normal breast cells comes as a surprise, considering their phenotypic similarity with regards to proliferation and polarized epithelial organization. The metabolic aberrances of the MRD cells offer a therapeutic opportunity towards tackling emergence of breast tumor recurrence in post-treatment care.

cancer biology↗

bioRxiv2019

Emergence of stable coexistence in a complex microbial community through metabolic cooperation and spatio-temporal niche partitioning

Microbial communities in nature often feature complex compositional dynamics yet also stable coexistence of diverse species. The mechanistic underpinnings of such dynamic stability remain unclear as system-wide studies have been limited to small engineered communities or synthetic assemblies. Here we show how kefir, a natural milk-fermenting community, realizes stable coexistence through spatio-temporal orchestration of species and metabolite dynamics. During milk fermentation, kefir grains (a polysaccharide matrix synthesized by kefir microbes) grow in mass but remain unchanged in composition. In contrast, the milk is colonized in a dynamic fashion with early members opening metabolic niches for the followers. Through large-scale mapping of metabolic preferences and inter-species interactions, we show how microbes poorly suited for milk survive in, and even dominate, the community through metabolic cooperation and uneven partitioning between the grain and the liquid phase. Overall, our findings reveal how spatio-temporal dynamics promote stable coexistence and have implications for deciphering and modulating complex microbial ecosystems.

microbiology↗