Biology subjects

Westbrook, E. R.

Publications and source records attributed to Westbrook, E. R..

3 recordsLinked to original sources

bioRxiv2024

Redox-dependent binding and conformational equilibria govern the fluorescence decay of NAD(P)H in living cells

When probed using fluorescence lifetime imaging microscopy (FLIM), the emission from reduced nicotinamide adenine dinucleotide (NADH) and its phosphorylated form NADPH have shown promise as sensitive intrinsic reporters of metabolism in living systems. However, an incomplete understanding of the biochemical processes controlling their fluorescence decay makes it difficult to draw unambiguous conclusions from NAD(P)H FLIM data. Here we utilised time-resolved fluorescence anisotropy imaging to identify multiple enzyme binding configurations in live cells associated with lifetimes both longer and shorter than unbound NAD(P)H. FLIM, combined with mathematical and computational modelling, revealed that the redox states of the NAD and NADP pools control the steady-state equilibrium of binding configurations, which in-turn determines the observed fluorescence decay. This knowledge will be foundational to developing the accurate interpretation of NAD(P)H FLIM.

biochemistry↗

bioRxiv2024

Pattern formation along signaling gradients driven by active droplet behaviour of cell groups

Gradients of extracellular signals organise cells in tissues. Although there are several models for how gradients can pattern cell behaviour, it is not clear how cells react to gradients when the population is undergoing 3D morphogenesis, in which cell-cell and cell-signal interactions are continually changing. Dictyostelium cells follow gradients of their nutritional source to feed and maintain their undifferentiated state. Using light sheet imaging to simultaneously monitor signaling, single cell and population dynamics, we show that the cells migrate towards nutritional gradients in swarms. As swarms advance, they deposit clumps of cells at the rear, triggering differentiation. Clump deposition is explained by a physical model in which cell swarms behave as active droplets: cells proliferate within the swarm, with clump shedding occurring at a critical population size, at which cells at the rear no longer perceive the gradient and are not retained by the emergent surface tension of the swarm. The droplet model predicts vortex motion of the cells within the swarm emerging from the local transfer of propulsion forces, a prediction validated by 3D tracking of single cells. This active fluid behaviour reveals a developmental mechanism we term "musical chairs" decision-making, in which the decision to proliferate or differentiate is determined by the position of a cell within the group as it bifurcates.

biophysics↗

bioRxiv2023

Collective signalling drives rapid jumping between cell states

Development can proceed in "fits and starts", with rapid transitions between cell states involving concerted transcriptome-wide changes in gene expression. However, it is not clear how these transitions are regulated in complex cell populations, in which cells receive multiple inputs. It is also not clear to what extent these rapid transitions represent developmental commitment. Here we address these issues using Dictyostelium cells undergoing development in their physiological niche. A continuous single cell transcriptomics time series reveals a sharp "jump" in global gene expression marking functionally different cell states. By simultaneously live imaging the physiological dynamics of transcription and signalling over millimetre length scales, we show that the jump coincides with the onset of collective oscillations of cAMP, the positive feedback signal for multicellular development. Different jump genes respond to distinct dynamic features of signalling. The late gene expression changes of the jump are almost completely dependent on cAMP. In contrast, transcript changes at the onset of the jump require additional input. The spatial boundary marking the jump divides cells separated by only a few minutes of developmental time, with cells missing a jump then waiting several hours for the onset of the next wave of cAMP oscillations. This timing variability contrasts the prevailing developmental paradigm of a timed synchronous process and is associated with substantial pre-jump transcriptome variability. The coupling of collective signalling with gene expression is a potentially powerful strategy to drive robust cell state transitions in heterogeneous signalling environments. Based on the context of the jump, we also conclude that sharp gene expression transitions may not be sufficient for commitment.

developmental biology↗