Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Poster Abstracts

82-POS Board 41 Linking Actin Engagement with Integrin Nanoclustering Using Superresolution in PTPN22 Deficient Migrating T Cells Michael Shannon 1 , Georgina Cornish 2 , Andrew Cope 2 , Dylan Owen 1 . 1 King's College London, London, United Kingdom, 2 King's College London, London, United Kingdom. T cells navigate the body using constant actin flow, which transiently engages with a spatio- temporally controlled ‘molecular clutch’ to translate forward cell movement (Ishibashi et al., 2015). The clutch regulates cell speed through its link to actin and the substrate, using integrin affinity, avidity and colocalization with effectors. We posit that reorganisation of this clutch on the nanoscale results in an observed speed increase in PTPN22 mutant cells, which predispose human patients for autoimmune disease. Nano-adhesions are too small to see with conventional fluorescence microscopy, so we use super resolution localisation microscopy to characterise clustering, and live-TIRF microscopy to image actin flow and engagement. To analyse the point data, we developed new tools to reduce the need for human decision making, allowing us to extract precise metrics on the size, composition and spacing of nanoclusters in cells (Rubin-Delanchy et al., 2015). For actin flow, we adapt Spatio Temporal Image Correlation Spectroscopy (STICS: Hebert, Costantino, & Wiseman, 2005) for fast cells by changing the reference frame. We observe that to migrate, hundreds of ~40 nm membrane LFA-1 clusters gather in the leading edge/front of substrate proximal membrane, which condense and group closer together in the focal zone/middle. This is coupled to a high degree of actin engagement (70 %), retrograde slippage (30 %) and front only anterograde flow. On removal of PTPN22, or treatment of wildtype cells with SDF-1 chemokine, migration speed is increased; this is coupled to reduced actin engagement and a maintained integrin nanoclustering schema. Slow moving cells (Manganese added to increase LFA-1 affinity, or Cytochalasin D to reduce actin polymerisation) show the reverse. Finding out what links an active T cell integrin nanocluster to the cytoskeleton is the next step in this project.

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