Kuba, M. | Search bioRxiv

bioRxiv2025

Super-resolution compatible DNA labeling technique reveals chromatin mobility and organization changes during differentiation

Chromatin dynamics play a crucial role in cellular differentiation, yet tools for studying global chromatin mobility in living cells remain limited. Here, we developed a novel probe for the metabolic labeling of chromatin and tracking its mobility during neural differentiation. The labeling system utilizes a newly developed silicon rhodamine-conjugated deoxycytidine triphosphate (dCSiRTP). We show that this dCTP is efficiently delivered into living human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) via a synthetic transporter (SNTT1). Using correlative confocal microscopy and stimulated emission depletion (STED) super-resolution microscopy, we quantified the sizes of labeled chromatin domains. Time lapse super-resolution microscopy combined with single particle tracking revealed that chromatin mobility decreases during the transition from iPSCs (pluripotent state) to NSCs and neurons (differentiated state). This reduction in mobility correlates with the differentiation state, suggesting a role for chromatin dynamics in cellular plasticity. Concomitant mechanistic insights obtained from MNase digestion assays, chromatin compaction and histone modification analyses revealed a decrease in chromatin accessibility during neuronal differentiation, indicating that chromatin adopts a more constrained and compacted structure. These findings provide new insights into chromatin regulation during neurogenesis.

cell biology↗

bioRxiv2019

Loss of O-linked protein glycosylation in Burkholderia cenocepacia impairs biofilm formation and siderophore production via alteration of quorum sensing regulation

O-linked protein glycosylation is a conserved feature of the Burkholderia genus. For Burkholderia cenocepacia, the addition of the trisaccharide {beta}-Gal-(1,3)--GalNAc-(1,3)-{beta}-GalNAc to membrane exported proteins is required for virulence and resistance to environmental stress. However, the underlying causes of the defects observed in the absence of glycosylation are unclear. This study demonstrates that the global B. cenocepacia proteome undergoes dramatic changes consistent with alterations in global transcriptional regulation in the absence of glycosylation. Using luciferase reporter assays and DNA cross-linking analysis, we confirm the repression of the master quorum sensing regulon CepR/I in response to the loss of glycosylation, which leads to the abolition of biofilm formation, defects in siderophore production, and reduced virulence. The abundance of most of the known glycosylated proteins did not significantly change in the glycosylation-defective mutants except for BCAL1086 and BCAL2974, which were found in reduced amount, suggesting they could be degraded. However, the loss of these two proteins was not responsible for driving the proteomic alterations, as well as for reduced virulence and siderophore production. Together, our results show that loss of glycosylation in B. cenocepacia results in a global cell reprogramming via alteration of the CepR/I regulon, which cannot be explained by the abundance changes in known B. cenocepacia glycoproteins.\n\nIMPORTANCEProtein glycosylation is increasingly recognised as a common protein modification in bacterial species. Despite this commonality our understanding of the role of most glycosylation systems in bacterial physiology and pathogenesis is incomplete. In this work, we investigated the effect of the disruption of O-linked glycosylation in the opportunistic pathogen Burkholderia cenocepacia using a combination of proteomic, molecular and phenotypic assays. We find that in contrast to recent findings on the N-linked glycosylation systems of Campylobacter jejuni, O-linked glycosylation does not appear to play a role in proteome stabilization of most glycoproteins. Our results reveal that virulence attenuation observed within glycosylation-null B. cenocepacia strains are consistent with alteration of the master virulence regulator CepR. The repression of CepR transcription and its associated phenotypes support a model in which the virulence defects observed in glycosylation-null strains are at least in part due to transcriptional alteration and not the direct result of the loss of glycosylation per-se. This research unravels the pleotropic effects of O-linked glycosylation in B. cenocepacia, demonstrating that its loss does not simply affect the stability of the glycoproteome, but also interferes with transcription and the broader proteome.

microbiology↗