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Investigation of the role of novel neuronal proteins in T lymphocyte<br/>migration

Final Report Summary - NEUROTCELL (Investigation of the role of novel neuronal proteins in T lymphocyte<br/>migration.)

Summary
The host group identified three novel proteins that demonstrate a polarized distribution in migrating T cells. The scientific aim of the proposed project was to investigate the functional role of these proteins - Synaptopodin, Spectrin beta 1 and Neurofilament Heavy chain (SYNPO, SPTBN1 and NEFH) in T cell polarization and migration. These are cytoskeletal and associated proteins responsible for actin-based cell shape, motility and cell integrity in neurons. The Marie Curie Fellow (MCF) analysed the subcellular localization of the proteins in relation to the actin and microtubule components of the cytoskeleton and a number of candidate proteins known to be involved in T cell migration and/or display a polarized phenotype in migrating cells. Using an siRNA approach she knocked down the expression of each of the proteins (SYNPO, SPTBN1 and NEFH) and investigated their function in the regulation of cell polarity, actin capping, live cell migration, transmigration and cell adhesion. These studies demonstrated that loss of expression of these proteins is associated with a change in migratory properties, in particular speed and persistence. The results suggest that this is due to the role in driving and maintaining cell polarity and their regulation of actin capping. The results demonstrate that SYNPO, SPTBN1 and NEFH are functionally involved in lymphocyte migration. Further elucidation of their function may ultimately aid in the identification of novel therapeutic targets for the treatment of inflammatory disease.

Description of work performed
The T cell migration model was set up using HuT 78 T cell line and was replicated in all experiments with primary T cells. In both models, expression of SYNPO, SPTBN1 and NEFH was individually knocked down using siRNA. The effect of the loss of expression each of the 3 proteins was confirmed on gene (by real-time PCR) and protein (by Western blotting) level. The effect of gene knockdown on cell polarity was investigated by High Content Analysis (including multi-parametric analysis), as was the effect of knockdown of the target genes on LFA-1-stimulated T cell migration (live cell migration) using specific software.
The MCF also investigated the effect of knockdown of target genes on LFA-1-stimulated T cell adhesion and transmigration (adhesion and Boyden-chamber type transwell assays).

Subcellular localization of SYNPO, SPTBN1 and NEFH was analysed using confocal fluorescence microscopy of the proteins at successive time-points following stimulation through LFA-1 (0-90 minutes). Confocal microscopy was also used to analyse of co-localisation with actin and microtubule components of the cytoskeleton (double staining). Identification of interacting proteins was investigated using a candidate approach – co-immunofluorescence analysis (candidates included L-plastin CXCR4, Myosin IIa and RACK-1).

Description of the main results
When cells were stimulated through LFA-1, cells gradually polarize - leading edge followed by a uropod or ‘Tail’. This was followed over a 90 –minute period when SYNPO, SPTBN1 and NEFH re-distributed to the leading edge of migrating cells. Their expression at the leading edge was particularly concentrated at an area rich in cytoskeletal elements, in particular actin. Part of this cytoskeletal reorganization necessary for T cell migration is ‘actin capping’ – the polarization of the actin cytoskeleton that is intrinsically involved in moving the cell forward. To investigate this (in primary T cells), each of the three proteins (SYNPO, SPTBN1 and NEFH) was individually knocked down using siRNA and an actin capping assay was performed. Interestingly, this analysis demonstrated that these proteins (SYNPO, SPTBN1 and NEFH) have a role in actin capping and in subtly different ways as demonstrated using High Content Analysis. Further investigation of the subcellular distribution of these proteins demonstrated that SYNPO, and SPTBN1 co-localised with the scaffolding protein 14-3-3 while NEFH partially co-localises with the adaptor protein RACK1. Both 14-3-3 and RACK1 are functionally important in facilitating protein-protein interactions and in adjusting the subcellular localization of proteins to facilitate their function.
In investigating cell polarity as a whole, knockdown of beta-spectrin had a significant effect on a number of polarity-associated parameters, including nuclear displacement, gyration radius and form factor. Knockdown of either synaptopodin or Neurofilament heavy chain had a slight effect on nuclear displacement but not the other parameters. live-cell migration measurements were made in primary T cells following knockdown of SYNPO, SPTBN1 and NEFH. Knockdown of these three proteins (individually) decreased the speed of the T cells. In addition, the cells demonstrated a difference in their mode of migration in that they did not move in multiple directions but rather moved persistently in the same direction.

Impact
In this project, the work of Dr Skubis-Zegadlo has demonstrated that SYNPO, SPTBN1 and NEFH play a functional role in lymphocyte migration and that the elucidation of their function may ultimately aid in the identification of novel therapeutic targets for the treatment of inflammatory disease. Inflammatory diseases, including autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel diseases pose a huge health problem for the citizens of Europe. In addition, this project has facilitated Dr Skubis-Zegadlo to develop essential practical and theoretical skills in this field of research and has allowed her both to re-establish her scientific career and to return to Poland with important additional research expertise.