Biology subjects

McColl, J.

Publications and source records attributed to McColl, J..

7 recordsLinked to original sources

bioRxiv2025

T-cell signaling relies on partial CD45-exclusion at sub-micron sized cellular contacts

How cell contact initiates T-cell activation is uncertain. The local exclusion of the receptor-type protein tyrosine phosphatase CD45 at cell contacts is believed to trigger immune receptor signaling but this is yet to be observed for T cells interacting with authentic cellular targets. Here, quantitative imaging of T cells interacting with tumor cells presenting either native or clinically relevant bi-specific TCR ligands, revealed that they form multiple sub-micron sized close contacts with their targets. The contacts were stabilised by the adhesion protein CD2, but efficient ligand detection required both CD2 and integrin ligation. CD45 was excluded from close contacts at the time of ZAP70 recruitment and signaling, but only partially (30- 40%). A single-cell, mass cytometric analysis showed that this change in kinase/phosphatase activity provoked strong T-cell activation and potent cytotoxicity via very small changes in signaling fluxes. Spatial stochastic simulations suggested that the proximal T-cell signaling network is optimised for efficient antigen discrimination in the setting of partial CD45 exclusion. Our work re-frames early T-cell activation as a process initiated by relatively subtle changes in kinase/phosphatase activity acting on small numbers of signaling effectors at minute cellular contacts.

immunology↗

bioRxiv2025

G-quadruplexes represent promising new targets to overcome multidrug-resistant fungal infections

The growing emergence of antifungal resistance has prompted the identification of novel antifungal targets. G-quadruplexes (G4s), four-stranded secondary structures that form in DNA and RNA, have arisen as a drug target to treat bacterial, viral, and parasitic infections. Here, we provide the first demonstration that G4s form in fungi and represent a promising new target for antifungal development. We found that PhenDC3 and pyridostatin (PDS), ligands that bind to and stabilise G4s, potently inhibited the metabolism of fungal pathogens in different ways. These pathogens included the pan azole-resistant Aspergillus fumigatus isolate TR34/L98H and Candida auris. Notably, PhenDC3 could synergise with amphotericin B, was protective in an in vivo model of fungal infection, and was well-tolerated by human cells. However, continuous exposure to PhenDC3 resulted in some cross-resistance to current antifungals and this needs to be explored further. PhenDC3 could also increase the number of RNA G4s in live A. fumigatus. Further, we demonstrated the structural flexibility of DNA sequences found in cyp51A and cyp51B, with these sequences capable of forming duplexes, hairpins, G4s and i-motifs. Finally, PhenDC3, but not PDS, caused duplex DNA structures in cyp51A to transition into antiparallel G4 structures potentially associated with PhenDC3s increased antifungal potency. Taken together, G4s represent an exciting antifungal target, but a more detailed understanding of their biological roles is essential.

microbiology↗

bioRxiv2022

resPAINT: Accelerating volumetric super-resolution localisation microscopy by active control of probe emission

Points for accumulation in nanoscale topography (PAINT) allows the acquisition of practically unlimited measurements in localisation microscopy. However, PAINT is inherently limited by unwanted background fluorescence at high probe concentrations, especially in large depth-of-field volumetric imaging techniques. Here we present reservoir-PAINT (resPAINT), in which we combine PAINT with active control of probe photophysics. In resPAINT, a reservoir of non-fluorescent activatable probes accumulate on the target, which makes it possible to drastically improve the localisation rate (by up to 50-fold) compared to conventional PAINT, without any compromise in contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate volumetric super-resolution imaging of entire cell surfaces. We then generalise the approach by implementing multiple switching strategies, including photoactivation and spontaneous blinking. We also implement alternative volumetric imaging modalities including the double-helix pointspread function, the tetrapod point-spread function and singlemolecule light field microscopy. Finally, we show that resPAINT can be used with a Fab to image membrane proteins, effectively extending the operating regime of conventional PAINT to encompass a larger range of biological interactions.

biophysics↗

bioRxiv2020

Robust T-cell signaling triggered on soft polydimethylsiloxane-supported lipid-bilayers

The T-cell receptor (TCR) is thought to be triggered either by mechano-transduction or local tyrosine phosphatase exclusion at cell-cell contacts. However, the effects of the mechanical properties of activating surfaces have only been tested for late-stage T-cell activation, and phosphatase segregation has mostly been studied on glass-supported lipid bilayers that favor imaging but are orders-of-magnitude stiffer than typical cells. We developed a method for attaching lipid bilayers to polydimethylsiloxane polymer supports, producing soft bilayers with physiological levels of mechanical resistance (Youngs modulus of 4 kPa). Comparisons of T-cell behavior on soft and glass-supported bilayers revealed that early calcium signaling is unaffected by substrate rigidity, implying that early steps in TCR triggering are not mechanosensitive. Robust phosphatase exclusion was observed on the soft bilayers, however, suggesting it likely occurs at cell-cell contacts. This work sets the stage for an imaging-based exploration of receptor signaling under conditions closely mimicking physiological cell-cell contact.

biophysics↗

bioRxiv2020

vLUME: 3D Virtual Reality for Single-molecule Localization Microscopy

Super-Resolution (SR) Microscopy based on 3D Single-Molecule Localization Microscopy (SMLM) is now well established1,2 and its wide-spread adoption has led to the development of more than 36 software packages, dedicated to quantitative evaluation of the spatial and temporal detection of fluorophore photoswitching3. While the initial emphasis in the 3D SMLM field has clearly been on improving resolution and data quality, there is now a marked absence of 3D visualization approaches that enable the straightforward, high-fidelity exploration of this type of data. Inspired by the horological phosphorescence points that illuminate watch-faces in the dark, we present vLUME (Visualization of the Universe in a Micro Environment, pronounced volume) a free-for-academic-use immersive virtual reality-based (VR) visualization software package purposefully designed to render large 3D-SMLM data sets. vLUME enables robust visualization, segmentation and quantification of millions of fluorescence puncta from any 3D SMLM technique. vLUME has an intuitive user-interface and is compatible with all commercial VR hardware (Oculus Rift/Quest and HTC Vive, Supplementary Video 1). vLUME accelerates the analysis of highly complex 3D point-cloud data and the rapid identification of defects that are otherwise neglected in global quality metrics.

biophysics↗

bioRxiv2019

The costs of close contact: Visualizing the energy landscape of cell contacts at the nanoscale

Cell-cell contact often underpins signalling between cells. Contact is mediated by proteins on both cells creating interfaces with gap sizes typically around 14 nm. Protein binding and accumulation leads to the contact becoming crowded, reducing the rate of protein diffusion, even for unbound proteins. Here we show that, by tracking quantum dots of different dimensions for extended periods of time, it is possible to obtain the probability of a molecule entering the contact, the change in its diffusion upon entry and the impact of spatial heterogeneity of adhesion protein density in the contact. By analysing the contacts formed by a T cell interacting with adhesion proteins anchored to a supported lipid bilayer, we find that probes are excluded from contact entry in a size-dependent manner for gap-to-probe differences of 4.1 nm. We also observe probes being trapped inside the contact and a decrease in diffusion of up to 85% in dense adhesion protein contacts. This approach provides new insights into the nature of cell-cell contacts, revealing that cell contacts are highly heterogeneous, due to topography- and protein density-related processes. These effects are likely to profoundly influence signalling between cells.\n\nStatement of SignificanceThe spatial distribution and diffusion of proteins has been shown to be important for various signalling machineries. As such size-dependent reorganisation of proteins in the immune cell-contact has been shown to affect activation of immune cells. While these studies relied on bulk measurements to investigate protein exclusion, small scale topographical changes and protein dynamics could not be evaluated. However, recent studies show that T cell activation is mediated by nanoscale structures. In our study the use molecular probes of various sizes to investigate the energy landscape of single molecules in a cell contact. This provides additional information and insights which cannot be determined by performing bulk experiments alone indicates.

biophysics↗

bioRxiv2018

4D imaging reveals stage dependent random and directed cell motion during somite morphogenesis

Somites are paired embryonic segments that form in a regular sequence from unsegmented mesoderm during vertebrate development. Of fundamental importance, they are transient structures that generate cell lineages of the musculoskeletal system in the trunk such as cartilage, tendon, bone, endothelial cells and skeletal muscle. Surprisingly, very little is known about the morphological transition and cellular dynamics during somite differentiation. Here, we address this by examining cellular rearrangements and morphogenesis in differentiating somites using live multi photon imaging of GFP-transgenic chick embryos. We specifically focussed on the dynamic changes in two principle regions within the somite (the medial and lateral domains) to investigate extensive morphological changes. Furthermore, by using quantitative analysis and cell tracking, we were able to capture for the first time a progenitor cell bulk movement towards the rostral-medial domain of the myotome, where skeletal muscle formation first initiates.

developmental biology↗