Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Poster Abstracts

139

82-POS

Board 41

Linking Actin Engagement with Integrin Nanoclustering Using Superresolution in PTPN22

Deficient Migrating T Cells

Michael Shannon

, Georgina Cornish

, Andrew Cope

, Dylan Owen

King's College London, London, United Kingdom,

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.