Biology subjects

Spiller, D. G.

Publications and source records attributed to Spiller, D. G..

8 recordsLinked to original sources

bioRxiv2023

Overexpression of IkappaBalpha modulates NF-kappaB activation of inflammatory target gene expression

Cells respond to inflammatory stimuli such as cytokines by activation of the nuclear factor-{kappa}B (NF-{kappa}B) signalling pathway, resulting in oscillatory translocation of the transcription factor p65 between nucleus and cytoplasm to mediate immune response. We investigate the relationship between p65 and inhibitor-{kappa}B (I{kappa}B) protein levels and dynamic properties of the system, and how this interaction impacts on the expression of key inflammatory genes. Using bacterial artificial chromosomes, we developed new cell models of I{kappa}B-eGFP protein overexpression in a native genomic context. We find that cells with high levels of the negative regulator I{kappa}B remain responsive to inflammatory stimuli and maintain dynamics for both p65 and I{kappa}B. In contrast, canonical target gene expression is dramatically reduced by overexpression of I{kappa}B, but can be partially rescued by overexpression of p65. Treatment with leptomycin B to promote nuclear accumulation of I{kappa}B also suppresses canonical target gene expression, suggesting a mechanism in which nuclear I{kappa}B accumulation prevents productive p65 interaction with promoter binding sites. This causes reduced target promoter binding and gene transcription, which we validate by chromatin immune precipitation and in primary cells. Overall, we show how inflammatory gene transcription is modulated by the expression levels of both I{kappa}B and p65, and that transcription can be partially decoupled from p65 protein dynamics. This results in an anti-inflammatory effect on transcription, demonstrating a broad mechanism to modulate the strength of inflammatory response.

cell biology↗

bioRxiv2022

Single-cell imaging reveals non-cooperative and cooperative infection strategies of Listeria monocytogenes in macrophages

Pathogens have developed intricate strategies to overcome the hosts innate immune responses. In this paper we use live-cell microscopy with a single bacterium resolution to follow in real time interactions between the food-borne pathogen L. monocytogenes and host macrophages, a key event controlling the infection in vivo. We demonstrate that infection results in heterogeneous outcomes, with only a subset of bacteria able to establish a replicative invasion of macrophages. The fate of individual bacteria in the same host cell was independent from each other and non-cooperative, but a higher multiplicity of infection resulted in a reduced probability of replication. Using internalisation assays and conditional probabilities to mathematically describe the multi-stage invasion process, we demonstrate that the secreted Listeriolysin toxin (LLO) of the PrfA regulon regulates replication probability by compromising the ability to phagocytose bacteria. Using strains expressing fluorescent reporters to follow transcription of either the LLO-encoding hly or actA genes, we show that replicative bacteria exhibited higher PrfA regulon expression in comparison to those bacteria that did not replicate, however elevated PrfA expression per se was not sufficient to increase the probability of replication. Overall, this demonstrates a new role for the population-level, but not single cell PrfA-mediated cooperativity to regulate outcomes of host pathogen interactions. Key pointsO_LIL. monocytogenes invasion of innate immune macrophages results in heterogeneous infection outcomes at the single cell level C_LIO_LIFate of individual bacteria in the same host cell is independent from each other and non-cooperative C_LIO_LIBacterial populations coordinate host cell uptake via the rate of phagocytosis to reduce internalization at high MOI C_LIO_LIThe PrfA regulon system is necessary but not sufficient for L. monocytogenes replication, but population-level PrfA virulence regulates single cell outcome probability C_LI

microbiology↗

bioRxiv2022

Post-transcriptional regulatory feedback encodes JAK-STAT signal memory of interferon stimulation

Immune cells fine tune their responses to infection and inflammatory cues. Here, using live-cell confocal microscopy and mathematical modelling, we investigate interferon induced JAK-STAT signalling in innate immune macrophages. We demonstrate that transient exposure to IFN-{gamma} stimulation induces a long-term desensitisation of STAT1 signalling and gene expression responses, revealing a dose- and time-dependent regulatory feedback that controls JAK-STAT responses upon re-exposure to stimulus. We show that IFN-/{beta}1 elicit different level of desensitisation from IFN-{gamma}, where cells refractory to IFN-/{beta}1 are sensitive to IFN-{gamma}, but not vice versa. We experimentally demonstrate that the underlying feedback mechanism involves regulation of STAT1 phosphorylation but is independent of new mRNA synthesis and cognate receptor expression. A new feedback model of the protein tyrosine phosphatase activity recapitulates experimental data and demonstrates JAK-STAT networks ability to decode relative changes of dose, timing, and type of temporal interferon stimulation. These findings reveal that STAT desensitisation renders cells with signalling memory of type I and II interferon stimulation, which in the future may improve administration of interferon therapy.

immunology↗

bioRxiv2021

Quantification of circadian interactions and protein abundance defines a mechanism for operational stability of the circadian clock

The mammalian circadian clock exerts substantial control of daily gene expression through cycles of DNA binding. Understanding of mechanisms driving the circadian clock is hampered by lack of quantitative data, without which predictive mathematical models cannot be developed. Here we develop a quantitative understanding of how a finite pool of BMAL1 protein can regulate thousands of target sites over daily time scales. We have used fluorescent correlation spectroscopy (FCS) to track dynamic changes in CRISPR-modified fluorophore-tagged proteins in time and space in single cells across SCN and peripheral tissues. We determine the contribution of multiple rhythmic processes in coordinating BMAL1 DNA binding, including the roles of cycling molecular abundance, binding affinities and two repressive modes of action. We find that nuclear BMAL1 protein numbers determine corresponding nuclear CLOCK concentrations through heterodimerization and define a DNA residence time of 2.6 seconds for this complex. Repression of CLOCK:BMAL1 is in part achieved through rhythmic changes to BMAL1:CRY1 affinity as well as a high affinity interaction between PER2:CRY1 which mediates CLOCK:BMAL1 displacement from DNA. Finally, stochastic modelling of these data reveals a dual role for PER:CRY complexes in which increasing concentrations of PER2:CRY1 promotes removal of BMAL1:CLOCK from genes consequently enhancing ability to move to new target sites.

systems biology↗

bioRxiv2019

Heat shock response pathways regulate stimulus-specificity and sensitivity of NF-κB signalling to temperature stress

Ability to adapt to temperature changes trough the Heat Shock Response (HSR) pathways is one of the most fundamental and clinically relevant cellular response systems. Here we report that Heat Shock (HS) induces a temporally-coordinated and stimulus-specific adaptation of the signalling and gene expression responses of the Nuclear Factor {kappa}B (NF-{kappa}B) transcription factor. We show that exposure of MCF7 breast adenocarcinoma cells to 43{degrees}C 1h HS inhibits the immediate signalling response to pro-inflammatory Interleukin 1{beta} (IL1{beta}) and Tumour Necrosis Factor (TNF) cytokines. Within 4h after HS treatment IL1{beta}-induced responses return to normal levels, but the recovery of the TNF-induced responses is delayed. Using siRNA knock-down of Heat Shock Factor 1 and mathematical modelling we show that the stimulus-specificity is conferred via the Inhibitory {kappa}B kinase signalosome, with HSR differentially controlling individual cytokine transduction pathways. Finally, using a novel mathematical model we predict and experimentally validate that the HSR cross-talk confers differential cytokine sensitivity of the NF-{kappa}B system to a range of physiological and clinically-relevant temperatures. This quantitative understanding of NF-{kappa}B and HSR cross-talk mechanisms is fundamentally important for the potential improvement of current hyperthermia protocols.

cell biology↗

bioRxiv2019

ER stress-linked autophagy stabilizes apoptosis effector PERP and triggers its co-localization with SERCA2b at ER-plasma membrane junctions

Specific molecular interactions that underpin the switch between ER stress-triggered autophagy-mediated cellular repair and cellular death by apoptosis are not characterized. This study reports the unexpected interaction elicited by ER stress between the plasma membrane (PM)-localized apoptosis effector PERP and the ER Ca2+ pump SERCA2b. We show that the p53 effector PERP, which specifically induces apoptosis when expressed above a threshold level, has a heterogeneous distribution across the PM of un-stressed cells and is actively turned over by the lysosome. PERP is upregulated following sustained starvation-induced autophagy, which precedes the onset of apoptosis indicating that PERP protein levels are controlled by a lysosomal pathway that is sensitive to cellular physiological state. Furthermore, ER stress stabilizes PERP at the PM and induces its increasing co-localization with SERCA2b at ER-PM junctions. The findings highlight a novel crosstalk between pro-survival autophagy and pro-death apoptosis pathways and identify, for the first time, accumulation of an apoptosis effector to ER-PM junctions in response to ER stress.

molecular biology↗

bioRxiv2019

Macrophage-specific NF-kappa B activation dynamics can segregate inflammatory bowel disease patients

The heterogeneous nature of inflammatory bowel disease (IBD) presents challenges, particularly when choosing therapy. Activation of the NF-{kappa}B transcription factor is a highly-regulated, dynamic event in IBD pathogenesis. We expressed the human NF-{kappa}B/p65 subunit in blood-derived macrophages, using lentivirus. Confocal imaging of p65 activation revealed that a higher proportion of macrophages from Crohns patients responded to lipid-A compared to controls. In contrast, cells from ulcerative colitis (UC) patients exhibited a shorter duration of p65 nuclear localisation compared to healthy controls and Crohns donors. Using a similar lentivirus approach, NF-{kappa}B-regulated luciferase was expressed in patient macrophages, isolated from frozen peripheral blood mononuclear cell samples. Following activation, samples could be segregated into three clusters based on the NF-{kappa}B-regulated luciferase response. The majority of UC samples appeared in hypo-responsive cluster 1, with Crohns patients representing the majority of hyper-responsive cluster 3. A positive correlation was seen between NF-{kappa}B-induced luciferase activity and cytokine levels released to medium from stimulated macrophages, but not in serum or biopsy. Analysis of macrophage cytokine responses and patient metadata revealed a strong correlation between Crohns patients who smoked and hyper-activation of p65. These in vitro dynamic assays of NF-{kappa}B activation in blood-derived macrophages segregate IBD patients into groups with different phenotypes and therefore may help determine response to therapy. Significance statementThis manuscript describes two dynamic assays of NF-{kappa}B activation in blood-derived macrophages that can segregate IBD patients into groups with different phenotypes. For the first time we introduce the use of dynamic measurements of a transcription factor activation as a method to stratify patients and we are confident that our approach will lead in future to early patient stratification and prediction of treatment outcome.

immunology↗

bioRxiv2018

Quantitative, real-time, single cell analysis in tissue reveals expression dynamics of neurogenesis

During embryogenesis cells make fate decisions within complex tissue environments. The levels and dynamics of transcription factor expression regulate these decisions. Here we use single cell live imaging of an endogenous HES5 reporter and absolute protein quantification to gain a dynamic view of neurogenesis in the embryonic mammalian spinal cord. We report that dividing neural progenitors show both aperiodic and periodic HES5 protein fluctuations. Mathematical modelling suggests that in progenitor cells the HES5 oscillator operates close to its bifurcation boundary where stochastic conversions between dynamics are possible. HES5 expression becomes more frequently periodic as cells transition to differentiation which, coupled with an overall decline in HES5 expression, creates a transient period of oscillations with higher fold expression change. This increases the decoding capacity of HES5 oscillations and correlates with interneuron versus motor neuron cell fate. Thus, HES5 undergoes complex changes in gene expression dynamics as cells differentiate.

developmental biology↗