Dr Aideen Long

Figure 1 High Content Analysis (HCA). The GE Healthcare INCell Analyser, one of the HCA platforms in use at the IMM may be used to analyse T cell migration at a population level. This equipment has several algorithms which may be used to quantitatively analyse T cell migration.

Dr Aideen Long’s research examines the molecular mechanisms of the inflammatory response. It focuses on the signaling aspects of leukocyteendothelial cell interaction with particular emphasis on the role of the Protein Kinase C (PKC) family of isoenzymes in T lymphocyte signaling and mechanisms of signaling through adhesion molecules on both cell types. We have a model of T cell migration which involves the ligation of either the CD44 or Leukocyte Functionassociated- 1 (LFA-1) molecule on the surface of the T cell using stimulating antibodies or their natural ligands, hyaluronic acid or ICAM-1 respectively. These stimuli result in the polarization of the cell, which occurs as a consequence of reorganization of cytoskeletal elements including actin and tubulin.

Figure 2 Protein Kinase C is constitutively phosphorylated in human T cells. This figure demonstrates that PKC beta, delta and theta are constitutively phosphorylated on their activation loop residue (top panel). Total isoform level is shown on bottom panel.

This polarization and subsequent migration of T cells represents a mechanism whereby leukocytes may extravasate or transmigrate through high endothelial venules and into a site of inflammation. Much of our research focuses on the elucidation of the molecular mechanisms of this process of T cell migration at a single cell level, investigating the signaling molecules/pathways involved, together with their physical and functional association with the cellular cytoskeleton.

Figure 3 Analysis of Rab5 expression (red) and actin localization (green) in HUVEC.

This work involves the use of multiple biochemical, proteomic and microscopy techniques. We use similar techniques to analyse signaling and migration of human umbilical vein endothelial cells (HUVEC) in response to ligation of the CD44 molecule. Using the recently established, High Content Analysis platform, which allows the multi-parametric analysis of cells in multi-well format in a controlled environment, we are using siRNA libraries in combination with the model of T cell migration to screen for potential targets to inhibit the migratory process. This will allow the future identification and design of novel therapeutic anti-inflammatory strategies.

Key Publications
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Loss of PTEN expression does not contribute to PDK-1 activity and PKC activation-loop phosphorylation in Jurkat T cells.
Cellular Signaling (Dec; 19(12):2444- 57. Epub 2007 Aug 3), 2007. Freeley, M., Park, J., Yang, K.-J., Wange, R.L., Volkov, Y., Kelleher, D. & Long, A.

Comment on “PDK1 nucleates T cell receptor-induced signaling complex for NF-kappaB activation”.
Science 312:55-56, 2006. Gruber, T., Freeley, M., Thuille, N., Heit, I,. Shaw, S., Long, A., & Baier, G. CD44 cross-linking induces PKCregulated migration of human T lymphocytes.
International Immunology 17:449-58, 2005.
Fanning A., Volkov, Y. , Freeley, M., Kelleher, D. & Long, A.

Engagement of CD44 modulates cyclooxygenase induction, VEGF generation, and cell proliferation in human vascular endothelial cells. FASEB Journal 19:446-8, 2005.
Murphy, J.F., Lennon, F., Steele, C.,
Kelleher, D., Fitzgerald, D. & Long, A.

The scaffolding protein CGNAP/ AKAP450 is a critical integrating component of the LFA-1-induced signaling complex in migratory T cells. Journal of Immunology 175:7811-8, 2005.
El Din El Homasany, B.S., Volkov, Y., Takahashi, M., Ono, Y., Keryer, G., Delouvee, A., Looby, E., Long, A.,* & Kelleher, D*.

Last updated: May 8, 2008.

Trinity College Dublin

Dr Aideen Long