Biology subjects

Carr, A. R.

Publications and source records attributed to Carr, A. R..

5 recordsLinked to original sources

bioRxiv2025

T cells use TNF and IFNγ for paracrine killing with target discrimination programmed by pathogen-derived factors

Cytotoxic T lymphocytes (CTLs) are known to eliminate target cells through perforin-mediated, contact-dependent killing - a process limited by the number of effector T cells despite its serial nature. CTLs likely also possess a mass killing mechanism that can eliminate target cells more efficiently. Using CAR-T cells against B7H3 present on B16 tumor targets, we demonstrate that activated T cells can also kill targets in a perforin-independent manner by releasing diffusible TNF and IFN{gamma} capable of killing nearby targets in a paracrine fashion even if they bear no antigen. Against an unperturbed target, paracrine killing is inefficient but is enhanced by the deletion of TNFR1 signaling molecules such as TAK1, HOIP, or TBK1/IKK{varepsilon}. Notably, these molecules are inhibited naturally by pathogen-encoded antagonists. Expression of a microbial-encoded antagonist, such as the Yersinia-encoded YopJ that antagonizes TAK1 or Ebola-encoded VP35 that antagonizes TBK1/IKK{varepsilon}, alters the target cell response to these cytokines from non-lethal to lethal. We propose that target cells are inherently resistant to killing by TNF and IFN{gamma}, but the presence of a microbial factor alters this sensitivity, providing for the selective elimination of the infected cell while minimizing harm to uninfected bystander cells. We propose the term pathogen-restriction to describe this discriminatory mechanism. Potentially, any microbial-derived factor that crosstalks with the TNF and IFN{gamma} signaling pathways can function as a pathogen-restriction element. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/696308v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@ea6418org.highwire.dtl.DTLVardef@517489org.highwire.dtl.DTLVardef@1ba0e22org.highwire.dtl.DTLVardef@1f5fef8_HPS_FORMAT_FIGEXP M_FIG C_FIG Short SummaryT cells use TNF and IFN{gamma} to kill target cells in a paracrine manner where target elimination is restricted by the presence of pathogen-derived factors.

immunology↗

bioRxiv2024

TBK1 and IKKϵ protect target cells from IFNγ-mediated T cell killing via an inflammatory apoptotic mechanism

Cytotoxic T cells produce interferon gamma (IFN{gamma}), which plays a critical role in anti-microbial and anti-tumor responses. However, it is not clear whether T cell-derived IFN{gamma} directly kills infected and tumor target cells, and how this may be regulated. Here, we report that target cell expression of the kinases TBK1 and IKK{varepsilon} regulate IFN{gamma} cytotoxicity by suppressing the ability of T cell-derived IFN{gamma} to kill target cells. In tumor targets lacking TBK1 and IKK{varepsilon}, IFN{gamma} induces expression of TNFR1 and the Z-nucleic acid sensor, ZBP1, to trigger RIPK1-dependent apoptosis, largely in a target cell-autonomous manner. Unexpectedly, IFN{gamma}, which is not known to signal to NF{kappa}B, induces hyperactivation of NF{kappa}B in TBK1 and IKK{varepsilon} double-deficient cells. TBK1 and IKK{varepsilon} suppress IKK/{beta} activity and in their absence, IFN{gamma} induces elevated NF{kappa}B-dependent expression of inflammatory chemokines and cytokines. Apoptosis is thought to be non-inflammatory, but our observations demonstrate that IFN{gamma} can induce an inflammatory form of apoptosis, and this is suppressed by TBK1 and IKK{varepsilon}. The two kinases provide a critical connection between innate and adaptive immunological responses by regulating three key responses: (1) phosphorylation of IRF3/7 to induce type I IFN; (2) inhibition of RIPK1-dependent death; and (3) inhibition of NF{kappa}B-dependent inflammation. We propose that these kinases evolved these functions such that their inhibition by pathogens attempting to block type I IFN expression would enable IFN{gamma} to trigger apoptosis accompanied by an alternative inflammatory response. Our findings show that loss of TBK1 and IKK{varepsilon} in target cells sensitizes them to inflammatory apoptosis induced by T cell-derived IFN{gamma}. Short SummaryIn the absence of TBK1 and IKK{varepsilon}, target cells are killed by T cells in an IFN{gamma}-dependent manner. In TBK1 and IKK{varepsilon}-deficient cells, IFN{gamma} induces RIPK1-dependent death, as well as hyper-induction of NF{kappa}B-dependent inflammatory genes. This suggests that any inhibition of TBK1/IKK{varepsilon} to block type I IFN expression will result in the demise of the cell accompanied by an alternate inflammatory program. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/606693v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1606b75org.highwire.dtl.DTLVardef@12eebc0org.highwire.dtl.DTLVardef@177c033org.highwire.dtl.DTLVardef@eb5819_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

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

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↗

bioRxiv2018

Membrane potential regulates the dynamic localisation of mammalian proteasomes

Proteasomes are abundant molecular machines distributed throughout the eukaryotic cell to facilitate protein degradation. We previously showed that proteasomes can re-organize and assemble into foci bodies in response to proteotoxic stress1, which we termed transient aggregate-associated droplets (TAADs)2. Here, we use single-molecule localization microscopy to quantify re-organization of mammalian proteasomes in different subcellular regions during proteotoxic stress to engage with invading protein aggregates. We determine that [~]60% of 20S proteasomes are capped by at least one 19S particle, and this ratio remains constant during stress. Using single-particle tracking, we show that cells confine global proteasomal movement when experiencing proteotoxic stress via a cytoskeleton-dependent mechanism. Similar limitations on proteasome movement are also observed upon membrane depolarization and repolarization events by patch clamp, which directly induce proteasomes to move away or towards the plasma membrane, respectively. Together, our experiments reveal distinct modes of proteasome motion dependent on cellular requirements, and propose that proteasome transport is restricted upon aggregate invasion to facilitate their subsequent degradation.

cell biology↗