Architectural regulation of cytotoxic synapse detachment

点击翻译按钮获取中文摘要

Summary Cytotoxic lymphocytes, comprising cytotoxic T lymphocytes (CTLs) and natural killer cells, kill by forming specialized immune synapses with their targets, into which they channel toxic factors that induce apoptosis. Although much is known about how cytotoxic synapses form, the cellular and molecular mechanisms that control their dissolution are poorly understood. Addressing this gap in knowledge is important because cytotoxic lymphocytes must let go of dying cells to kill multiple targets in a serial manner. Efficient synapse disassembly also prevents spurious inflammation by attenuating sustained cytotoxic lymphocyte activation and also by facilitating the clearance of apoptotic corpses by phagocytes. Prior attempts to study this process have focused on the recognition of biochemical features associated with cell death. Our preliminary studies, however, suggest an alternative and conceptually innovative model in which lymphocyte detachment is induced by biophysical changes in dying target cells. We are particularly interested apoptotic contraction, which we have found occurs just before the dissolution of CTL-target cell conjugates. In addition, genetic and pharmacological decoupling of contraction from apoptosis delays the dissociation response. Building upon these findings, we hypothesize that CTLs use the characteristic biophysical features of contracting targets to trigger release. Our proposed studies, which are divided into two Specific Aims, will explore the biophysical basis for CTL dissociation and the molecular pathways that govern the process. Specific Aim 1 will employ single cell mechanobiological methods to assess the effects of apoptotic contraction on cortical rigidity and surface ligand mobility. We will also use an optogenetics approach to determine whether cytoskeletal contraction is sufficient to induce synapse disassembly. Specific Aim 2 will examine the molecular bases for apoptotic contraction and its detection by CTLs, using targeted loss-of-function and CRISPR/Cas9 screening. Our proposed studies will leverage technically innovative methods, including super-resolution imaging, optogenetics, and atomic force microscopy. They will also introduce a novel concept, that mechanosensing can determine the lifetime of an immune cell-cell interaction. The successful completion of this project will address a long-standing enigma in lymphocyte cell biology and likely reveal new avenues for the modulation of cellular cytotoxicity in the clinic. As such, this proposal is highly relevant to the NIH mission in that it will contribute to the advancement of knowledge that could improve human health.