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.
Highlights
IntroductionPIP2 is a central substrate for second messenger generation and a well-established regulator of cytoskeletal dynamics in many cell types [8,9]
We address roles of Phosphatidylinositol (4,5) bisphosphate (PIP2) in T cell activation
It allowed us to manipulate PIP2 with spatial definition by overexpressing distinct phosphatidylinositol 4-phosphate 5-kinase (PIP5K) isoforms. For such studies we focused on the two distal PIP5K isoforms, c90 and b, as it of interest to understand how regulation of PIP2 levels at the distal pole, away from the location of T cell signaling at the interface, would impact T cell activation
Summary
PIP2 is a central substrate for second messenger generation and a well-established regulator of cytoskeletal dynamics in many cell types [8,9]. 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. We had to investigate whether changes in PIP2 levels primarily affected T cell activation through altered second messenger generation or through altered cytoskeletal dynamics
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