Biology subjects

Thomason, P.

Publications and source records attributed to Thomason, P..

7 recordsLinked to original sources

bioRxiv2026

RNF213 M1-ubiquitinates damaged mitochondria to activate pro-inflammatory NF-κB signaling

Through mitochondrial outer membrane permeabilization (MOMP), mitochondria are central to apoptosis. MOMP is pro-inflammatory, impacting diverse processes including anti-tumor immunity and cellular senescence. Despite this, how damaged mitochondria activate inflammation remains unclear. Recent studies have shown that following MOMP, mitochondria are extensively ubiquitinated activating pro-inflammatory NF-{kappa}B signaling. We identify the E3 ligase, RNF213, as essential for pro-inflammatory mitochondrial ubiquitination following MOMP. RNF213 has an established role in pro- inflammatory, cell-autonomous immunity to bacteria and other pathogens. We discover that RNF213 is required for mitochondrial M1-linked (linear) ubiquitination. Interestingly, this was found to be independent of the canonical M1-ubiquitin ligase complex LUBAC. RNF213 can directly catalyze M1-linked ubiquitination. By promoting mitochondrial M1-linked ubiquitination, RNF213 initiates the recruitment of the essential NF{kappa}B adaptor molecule, NEMO, and subsequently activates pro-inflammatory NF-{kappa}B signaling. Collectively, our findings highlight striking similarities between how cells detect damaged mitochondria and intracellular pathogens, leading to inflammation.

cell biology↗

bioRxiv2023

How Should Eukaryotic Chemotaxis be Measured?

Chemotaxis and directed cell migration are fundamentally important to eukaryotic biology. To understand the mechanisms that drive such complex processes, informative and robust measurements are essential, but the field does not always agree what these should be. Here we identify the most dependable measures of chemotactic steering and the underlying cell migration, and provide tools to test them. Some widely-used metrics can end up misleading, in particular "cos 8" for directional accuracy. We suggest that chemotactic efficiency should be used as a primary metric. Mean squared displacement and directional autocorrelation can be used to unpick different models of random and directed cell migration. Transition matrices are another useful tool for understanding migration mechanisms and avoiding artefacts, and provide a graphical illustration of how well cells maintain each direction. Unexpectedly, the choice of start and end points of tracks strongly affects the measurements and can seriously bias the measured results. This is particularly clear when cells are not homogeneously distributed at the start of an assay. To support straightforward adoption of these methods, we provide a suite of tools as a plugin for the open-source ImageJ program, and describe how they can be used to understand complex scenarios like self-generated chemotactic gradients.

cell biology↗

bioRxiv2022

Competition between chemoattractants causes unexpected complexity and can explain negative chemotaxis

Negative chemotaxis, where eukaryotic cells migrate away from repellents, is important throughout biology, for example in nervous system patterning and resolution of inflammation. However, the mechanisms by which molecules repel migrating cells are unknown. Here, we use a combination of modelling and experiments with Dictyostelium cells to show that competition between different ligands that bind to the same receptor leads to effective chemorepulsion. 8-CPT-cAMP, widely described as a simple chemorepellent, is inactive on its own, and only repels cells if it interacts with the attractant cAMP. If cells degrade either competing ligand, the pattern of migration becomes more complex; cells may be repelled in one part of a gradient but attracted elsewhere, leading to populations moving in different directions in the same assay, or converging in an arbitrary place. More counterintuitively still, two chemicals can each attract cells on their own, but repel cells when combined together. We have thus identified a new mechanism that drives reverse chemotaxis, verified by mathematical models and experiments with real cells, and important anywhere several ligands compete for the same receptors.

cell biology↗

bioRxiv2021

Extracellular signalling modulates Scar/WAVE complex activity through Abi phosphorylation

The lamellipodia and pseudopodia of migrating cells are produced and maintained by the Scar/WAVE complex. Thus, actin-based cell migration is largely controlled through regulation of Scar/WAVE. Here we report that the Abi subunit - not Scar/WAVE - is phosphorylated in response to extracellular signalling. Like Scar, Abi is phosphorylated after the complex has been activated, implying that Abi phosphorylation modulates pseudopodia, rather than causing new ones to be made. Consistent with this, Scar/WAVE complex mutants that cannot bind Rac are also not phosphorylated. Several environmental cues also affect Abi phosphorylation - cell-substrate adhesion promotes it and increased extracellular osmolarity diminishes it. Both unphosphorylatable and phosphomimetic Abi efficiently rescue the chemotaxis of Abi KO cells and pseudopodia formation, confirming that Abi phosphorylation is not required for activation or inactivation of the Scar/WAVE complex. However, pseudopodia and Scar/WAVE patches in the cells with unphosphorylatable Abi protrude for longer, altering pseudopod dynamics and cell speed. Cells in which Scar and Abi are both unphosphorylatable can still form pseudopods, but migrate substantially faster. We conclude that extracellular signals and environmental responses modulate cell migration by tuning the behaviour of the Scar/WAVE complex after it has been activated.

cell biology↗

bioRxiv2019

Seeing around corners: Cells create chemotactic gradients to solve mazes and respond to distant cues in complex environments.

Chemotaxis, in which cells steer using chemical gradients, drives fundamental biological processes like embryogenesis, metastasis and immune responses. Self-generated chemotaxis, where cells break down abundant attractants to create gradients, is an important but under-studied aspect of physiological navigation. Here we show that self-generated gradients allow cells to navigate arbitrarily complex paths and, remarkably, make accurate choices about pathways they have not yet encountered. This enables cells to solve microfluidic mazes, even with initially homogeneous environments and distant correct destinations. We combine computational models and experiments to understand how cells anticipate environmental features, and how decision accuracy is determined by path complexity, attractant diffusibility and cell speed. This permits mazes that are easy or hard for cells to resolve, despite similar appearances. Counterintuitively, slowly-diffusing attractants can generate a "mirage", making cells prefer dead ends over correct paths. In vivo environments resemble complex mazes, and only self-generated gradients realistically explain cell behaviour.

cell biology↗

bioRxiv2019

Cell-substrate adhesion drives Scar/WAVE activation and phosphorylation, which controls pseudopod lifetime

The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVEs proline-rich domain is polyphosphorylated after the complex is activated. Treatments that stop activation block phosphorylation in both Dictyostelium and mammalian cells. This implies that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether a protrusion is initiated. Unexpectedly, activation-dependent phosphorylation is not promoted by chemotactic signalling, or by signal-dependent kinases such as ERKs, but is greatly stimulated by cell:substrate adhesion. Scar/WAVE that has been mutated to be either unphosphorylatable or phosphomimetic is activated normally, and rescues the phenotype of scar- cells, demonstrating that phosphorylation is dispensible for activation and actin regulation. However, pseudopods and patches of Scar/WAVE complex recruitment last substantially longer in unphosphorylatable mutants, altering cell polarisation and the efficiency of migration. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVEs activity, and that phosphorylation acts as a timer, restricting pseudopod lifetime by promoting Scar/WAVE turnover.

cell biology↗

bioRxiv2017

LPP3 mediates self-generation of chemotactic LPA gradients by melanoma cells

Melanoma cells steer out of tumours using self-generated lysophosphatidic acid (LPA) gradients. The cells break down LPA, which is present at high levels around the tumours, creating a dynamic gradient that is low in the tumour and high outside. They then also migrate up this gradient, creating a complex and evolving outward chemotactic stimulus. Here we introduce a new assay for self-generated chemotaxis, and show that raising LPA levels causes a delay in migration rather than loss of chemotactic efficiency. Knockdown of the lipid phosphatase LPP3 - but not its homologues LPP1 or LPP2 - diminishes the cells ability to break down LPA. This is specific for chemotactically active LPAs, such as the 18:1 and 20:4 species. Inhibition of autotaxin-mediated LPA production does not diminish outward chemotaxis, but loss of LPP3-mediated LPA breakdown blocks it. Similarly, in both 2D and 3D invasion assays, knockdown of LPP3 diminishes melanoma cells ability to invade. Our results demonstrate that LPP3 is the key enzyme in melanoma cells breakdown of LPA, and confirm the importance of attractant breakdown in LPAmediated cell steering.

cancer biology↗