Medial prefrontal cortex serotonin 1A and 2A receptor binding interacts to predict threat-related amygdala reactivity

Background\ud The amygdala and medial prefrontal cortex (mPFC) comprise a key corticolimbic circuit that helps shape individual differences in sensitivity to threat and the related risk for psychopathology. Although serotonin (5-HT) is known to be a key modulator of this circuit, the specific receptors mediating this modulation are unclear. The colocalization of 5-HT1A and 5-HT2A receptors on mPFC glutamatergic neurons suggests that their functional interactions may mediate 5-HT effects on this circuit through top-down regulation of amygdala reactivity. Using a multimodal neuroimaging strategy in 39 healthy volunteers, we determined whether threat-related amygdala reactivity, assessed with blood oxygen level-dependent functional magnetic resonance imaging, was significantly predicted by the interaction between mPFC 5-HT1A and 5-HT2A receptor levels, assessed by positron emission tomography.\ud \ud Results\ud 5-HT1A binding in the mPFC significantly moderated an inverse correlation between mPFC 5-HT2A binding and threat-related amygdala reactivity. Specifically, mPFC 5-HT2A binding was significantly inversely correlated with amygdala reactivity only when mPFC 5-HT1A binding was relatively low.\ud \ud Conclusions\ud Our findings provide evidence that 5-HT1A and 5-HT2A receptors interact to shape serotonergic modulation of a functional circuit between the amygdala and mPFC. The effect of the interaction between mPFC 5-HT1A and 5-HT2A binding and amygdala reactivity is consistent with the colocalization of these receptors on glutamatergic neurons in the mPFC

A Model for the Interplay of Receptor Recycling and Receptor-Mediated Contact in T Cells

Orientation of organelles inside T cells (TC) toward antigen-presenting cells (APC) ensures that the immune response is properly directed, but the orientation mechanisms remain largely unknown. Structural dynamics of TC are coupled to dynamics of T-cell receptor (TCR), which recognizes antigen on the APC surface. Engagement of the TCR triggers its internalization followed by delayed polarized recycling to the plasma membrane through the submembrane recycling compartment (RC), which organelle shares intracellular location with the TC effector apparatus. TCR engagement also triggers TC-APC interface expansion enabling further receptor engagement. To analyze the interplay of the cell-cell contact and receptor dynamics, we constructed a new numerical model. The new model displays the experimentally observed selective stabilization of the contact initiated next to the RC, and only transient formation of contact diametrically opposed to the RC. In the general case wherein the TC-APC contact is initiated in an arbitrary orientation to the RC, the modeling predicts that the contact dynamics and receptor recycling can interact, resulting effectively in migration of the contact to the TC surface domain adjacent to the submembrane RC. Using three-dimensional live-cell confocal microscopy, we obtain data consistent with this unexpected behavior. We conclude that a TC can stabilize its contact with an APC by aligning it with the polarized intracellular traffic of TCR. The results also suggest that the orientation of TC organelles, such as the RC and the effector apparatus, toward the APC can be achieved without any intracellular translocation of the organelles

Activation of adherent vascular neutrophils in the lung during acute endotoxemia

BACKGROUND: Neutrophils constitute the first line of defense against invading microorganisms. Whereas these cells readily undergo apoptosis under homeostatic conditions, their survival is prolonged during inflammatory reactions and they become biochemically and functionally activated. In the present study, we analyzed the effects of acute endotoxemia on the response of a unique subpopulation of neutrophils tightly adhered to the lung vasculature. METHODS: Rats were treated with 5 mg/kg lipopolysaccharide (i.v.) to induce acute endotoxemia. Adherent neutrophils were isolated from the lung vasculature by collagenase digestion and sequential filtering. Agarose gel electrophoresis, RT-PCR, western blotting and electrophoretic mobility shift assays were used to evaluate neutrophil activity. RESULTS: Adherent vascular neutrophils isolated from endotoxemic animals exhibited decreased apoptosis when compared to cells from control animals. This was associated with a marked increase in expression of the anti-apoptotic protein, Mcl-1. Cells isolated 0.5–2 hours after endotoxin administration were more chemotactic than cells from control animals and expressed increased tumor necrosis factor-alpha and cyclooxygenase-2 mRNA and protein, demonstrating that they are functionally activated. Endotoxin treatment of the animals also induced p38 and p44/42 mitogen activated protein kinases in the adherent lung neutrophils, as well as nuclear binding activity of the transcription factors, NF-κB and cAMP response element binding protein. CONCLUSION: These data demonstrate that adherent vascular lung neutrophils are highly responsive to endotoxin and that pathways regulating apoptosis and cellular activation are upregulated in these cells

An Experimental and Computational Study of Effects of Microtubule Stabilization on T-Cell Polarity

T-killer cells eliminate infected and cancerous cells with precision by positioning their centrosome near the interface (immunological synapse) with the target cell. The mechanism of centrosome positioning has remained controversial, in particular the role of microtubule dynamics in it. We re-examined the issue in the experimental model of Jurkat cells presented with a T cell receptor-binding artificial substrate, which permits controlled stimulation and reproducible measurements. Neither 1-µM taxol nor 100-nM nocodazole inhibited the centrosome positioning at the “synapse” with the biomimetic substrate. At the same time, in micromolar taxol but not in nanomolar nocodazole the centrosome adopted a distinct peripheral rather than the normally central position within the synapse. This effect was reproduced in a computational energy-minimization model that assumed no microtubule dynamics, but only a taxol-induced increase in the length of the microtubules. Together, the experimental and computational results indicate that microtubule dynamics are not essential for the centrosome positioning, but that the fit of the microtubule array in the deformed body of the conjugated T cell is a major factor. The possibility of modulating the T-cell centrosome position with well-studied drugs and of predicting their effects in silico appears attractive for designing anti-cancer and antiviral therapies

Initiation of T cell signaling by CD45 segregation at 'close contacts'.

It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.The authors thank R.A. Cornall, M.L. Dustin and P.A. van der Merwe for comments on the manuscript and S. Ikemizu for useful discussions about the structure. We also thank W. Lu and T. Walter for technical support with protein expression and crystallization, the staff at Diamond Light Source beamlines I02, I03 and I04-1 (proposal mx10627) and European Synchrotron Radiation Facility beamlines ID23EH1 and ID23EH2 for assistance at the synchrotrons, G. Sutton for assistance with MALS experiments, and M. Fritzsche for advice on the calcium analysis. This work was funded by the Wellcome Trust (098274/Z/12/Z to S.J.D.; 090532/Z/09/Z to R.J.C.G.; 090708/Z/09/Z to D.K.), the UK Medical Research Council (G0700232 to A.R.A.), the Royal Society (UF120277 to S.F.L.) and Cancer Research UK (C20724/A14414 to C.S.; C375/A10976 to E.Y.J.). The Oxford Division of Structural Biology is part of the Wellcome Trust Centre for Human Genetics, Wellcome Trust Core Award Grant Number 090532/Z/09/Z. We acknowledge financial support from Instruct, an ESFRI Landmark Project. The OPIC electron microscopy facility was funded by a Wellcome Trust JIF award (060208/Z/00/Z).This is the author accepted manuscript. The final version is available from Nature Publishing Group via https://doi.org/10.1038/ni.339

Tyrosine kinase activity and remodelling of the actin cytoskeleton are co-temporally required for degranulation by cytotoxic T lymphocytes

In this study, we examined the contribution of the actin cytoskeleton to T-cell receptor (TCR)-initiated signalling in cytotoxic T lymphocytes (CTLs). We demonstrate that cytoskeletal remodelling is required for sustaining TCR-stimulated signals that lead to degranulation by CTLs. Disruption of the actin cytoskeleton in CTLs already undergoing signalling responses results in an almost immediate loss of essentially all protein tyrosine phosphorylation. This signal reversal is not restricted to tyrosine phosphorylation, as disruption of the actin cytoskeleton also reverses the phosphorylation of the more downstream serine/threonine kinase extracellular signal regulated kinase (Erk). An intact cytoskeleton and cell spreading are not sufficient for maintaining signals, as stabilization of actin filaments, at a point when peak tyrosine phosphorylation is occurring, also leads to the rapid loss of protein tyrosine phosphorylation. Disruption of tyrosine kinase activity after TCR signals are maximally induced causes the immediate reversal of tyrosine phosphorylation as well as cytoskeletal disruption, as indicated by loss of cell spreading, adhesion and CTL degranulation. Taken together, our results indicate that actin remodelling occurs co-temporally with ongoing tyrosine kinase activity, leading to CTL degranulation. We hypothesize that continuous actin remodelling is important for sustaining productive signals, even after downstream signalling molecules such as Erk have been activated, and that the actin cytoskeleton is not solely required for initiating and maintaining the T cell in contact with its stimulus