Characterising the instructive role of nuclear membrane potential in chromatin dynamics

Abstract

Cell generated voltage gradients play a key role in metabolic activity and gene regulation. Electrical gradients, controlled by proteins embedded in sub-cellular membrane bound organelles, such as the nucleus, are likely to contribute to changes in nuclear architecture. The role of nuclear transmembrane potential, in regulating chromatin dynamics and gene expression is yet to be well characterised. This study aims to characterise the instructive role of the inner nuclear membrane potential on chromatin in stem cells. To this end, parameters of three image analysis methods were optimised for assessing chromatin compaction levels – ‘Chromatin Condensation Parameter’, textural analysis using ‘Grey Level Correlation Matrix’, and ‘Hyper-Spectral Phasor’ analysis of Hoechst 33342 emission. All three methods correctly quantified chromatin compaction levels in myoblast cells (L6) treated with chromatin relaxing and condensing conditions (Trichostatin-A/hypo-osmolar media, and hyper-osmolar media, respectively). The use of the anionic dye, DiBAC4(3), was explored for nuclear Vmem characterisation. This dye localised in the nucleus and showed fluorescence intensity changes in response to ouabain or potassium gluconate treatment. Experiments combining the use of chromatin compaction metrics with DiBAC4(3) suggest that nuclear membrane depolarisation leads to chromatin compaction. However, it was not clear whether DiBAC4(3) was reporting the inner, outer or both nuclear membrane potentials. Consequently, efforts were directed towards employing ASAP3-R3, a genetically encoded voltage indicator, to report the inner nuclear membrane potential of Normal Rat Kidney (NRK) cells. To which the nuclear localisation sequence of two natively expressed proteins of the inner nuclear membrane were attached to the amino terminus of ASAP3-R3: Sun2 and the second transmembrane domain of Lamin B Receptor. The results indicate not only localisation of ASAP3-R3, this probe also functionally reports inner nuclear membrane potential. Experiments were then conducted using the chromatin condensation metrics and ASAP3-R3 to explore the relationship between the inner nuclear membrane potential and chromatin dynamics. From which depolarisation of the inner nuclear membrane, induced by ouabain, is associated with higher levels of chromatin relaxation. In contrast, hyperpolarisation induced by pinacidil led to increased chromatin compaction. Moreover, timeseries analysis revealed the existence of significant cross-correlation between nuclear membrane potential and compaction levels. Ouabain appeared to reduce this association, while pinacidil ostensibly increased it. These processes may be involved in chromatin regulation, ultimately interfering with the nuclear membrane potential’s orchestration over chromatin conformation. Taken together, the current research provides evidence for the instructive role of nuclear membrane potential in chromatin regulation. The nuclear membrane potential may serve as a lever of control over genomic architecture and potentially gene expression.

Date of Award	2023
Original language	English
Awarding Institution	Western Sydney University
Supervisor	Mark Jones (Supervisor)