Phosphatidylinositol (4,5) Bisphosphate Controls T Cell Activation by Regulating T Cell Rigidity and Organization Yi Sun 1 , Radhika D. Dandekar1, Yuntao S. Mao2, Helen L. Yin2, Christoph Wu¨ lfing1,3* 1 Department of Immunology, UT Southwestern Medical Center, Dallas, Texas, United States of America, 2 Department of Physiology, UT Southwestern Medical Center, Dallas, Texas, United States of America, 3 Department of Cell Biology, UT Southwestern Medical Center, Dallas, Texas, United States of America Abstract Here we investigate the role of Phosphatidylinositol (4,5) bisphosphate (PIP2) in the physiological activation of primary murine T cells by antigen presenting cells (APC) by addressing two principal challenges in PIP2 biology. First, PIP2 is a regulator of cytoskeletal dynamics and a substrate for second messenger generation. The relative importance of these two processes needs to be determined. Second, PIP2 is turned over by multiple biosynthetic and metabolizing enzymes. The joint effect of these enzymes on PIP2 distributions needs to be determined with resolution in time and space. We found that T cells express four isoforms of the principal PIP2-generating enzyme phosphatidylinositol 4-phosphate 5-kinase (PIP5K) with distinct spatial and temporal characteristics. In the context of a larger systems analysis of T cell signaling, these data identify the T cell/APC interface and the T cell distal pole as sites of differential PIP2 turnover. Overexpression of different PIP5K isoforms, as corroborated by knock down and PIP2 blockade, yielded an increase in PIP2 levels combined with isoform-specific changes in the spatiotemporal distributions of accessible PIP2. It rigidified the T cell, likely by impairing the inactivation of Ezrin Moesin Radixin, delayed and diminished the clustering of the T cell receptor at the cellular interface, reduced the efficiency of T cell proximal signaling and IL-2 secretion. These effects were consistently more severe for distal PIP5K isoforms. Thus spatially constrained cytoskeletal roles of PIP2 in the control of T cell rigidity and spatiotemporal organization dominate the effects of PIP2 on T cell activation. Citation: Sun Y, Dandekar RD, Mao YS, Yin HL, W u¨lfing C (2011) Phosphatidylinositol (4,5) Bisphosphate Controls T Cell Activation by Regulating T Cell Rigidity and Organization. PLoS ONE 6(11): e27227. doi:10.1371/journal.pone.0027227 Editor: Jean Kanellopoulos, University Paris Sud, France Received August 19, 2011; Accepted October 12, 2011; Published November 11, 2011 Copyright: 2011 Sun et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the National Institutes of Health to C.W. (R01 CA96733) and H.L.Y. (P50-GM21681) and a Robert A. Welch Foundation grant to H.L.Y. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Christoph.Wuelfing@UTSouthwestern.edu Introduction Here we address roles of Phosphatidylinositol (4,5) bisphosphate (PIP2) in T cell activation. Physiological T cell activation occurs in the cellular interaction between a T cell and an antigen presenting cell (APC). T cells polarize upon APC contact as driven by the cytoskeleton [1,2,3], yielding a complex organization of T cell signaling in dynamic and diverse spatiotemporal patterns [4,5,6]. Prominent is the sustained accumulation of the T cell receptor (TCR) at the center of the T cell/APC interface [5], an accumulation pattern that can be associated with efficient T cell activation [6,7]. A critical outcome of T cell activation is cytokine secretion, prominently that of the autocrine growth factor IL-2. PIP2 is a central substrate for second messenger generation and a well-established regulator of cytoskeletal dynamics in many cell types [8,9]. Hydrolysis of PIP2 by phospholipase Cc (PLCc) yields diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (IP3) [10], two signaling intermediates critical for the induction of T cell IL-2 secretion. In cytoskeletal regulation, PIP2 controls cytoskeleton- plasma membrane adhesion, the activity of actin severing proteins, and assembly of endocytic vesicles [9,11,12]. Ezrin Radixin Moesin (ERM) proteins are a critical mediator of PIP2 function in the regulation of cytoskeleton-plasma membrane adhesion, as binding of ERM to PIP2 in the plasma membrane activates them to strengthen the association of the plasma membrane with the underlying cortical actin cytoskeleton [13,14]. A first general challenge in understanding the function of PIP2 in any cell type is to determine whether the role of PIP2 as a substrate for second messenger generation or cytoskeletal roles dominate the effects of PIP2 on cellular activation. In other words, we had to investigate whether changes in PIP2 levels primarily affected T cell activation through altered second messenger generation or through altered cytoskeletal dynamics. PIP2 is turned over rapidly. The principal biosynthetic pathway of PIP2 involves phosphorylation of phosphatidylinositol 4- phosphate by the type I phosphatidylinositol 4-phosphate 5- kinases (PIP5K) [15]. There are three PIP5K isoforms, a, b, and c The nomenclature for the a and b isoforms is switched between humans and mice. We use the more widely employed human nomenclature. The c isoform has multiple splice variants. The predominant isoforms are PIP5K c87 (also called c635) and c90 (c661) with the isoforms being denoted by their molecular weight (87 or 90 kDa) or the number of amino acids (635 or 661). PIP2 is metabolized through hydrolysis by PLCc or phosphorylation by phosphatidylinositol 3-kinase (PI3K). Additionally, PIP2 synthe- sized locally will be dissipated by diffusion [16] unless captured by scaffolding molecules [17]. PIP2 is also dephosphorylated by phosphatidylinositol phosphatases [18]. A second key challenge in PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27227