T cell activation is initiated by direct contact between T cells and antigen-presenting cells (APCs), leading to polarization of the T cell towards the APC and the formation of the immunological synapse (IS). It is now generally accepted that Ca2+ signals are required for T cell activation. Further, reorganization of Ca2+ signaling proteins during IS formation has been widely reported, although the underlying mechanisms driving this event are poorly described as is the physiological significance of this reorganization. Unpublished observations from our group, reveal that translocation to the IS is highly dependent upon a polybasic region at its C-terminus, with neutralization of any positive charges sufficient to block STIM1 polarization. Further, whereas cytosolic Ca2+ entry was normal in cells expressing these STIM1 mutants, mitochondrial Ca2+ loading and mROS production were defective. Further, Septins, a family of GTP-binding and complex forming proteins known to affect STIM1 localization through reorganization of phosphoinositides around ER-PM junctions, localize to the IS in a STIM1-dependent manner. Based on these preliminary findings, we propose the following hypothesis: Septin-mediated reorganization of Phosphatidylinositides control polarization of STIM1 and associated proteins towards the IS, driving mitochondrial Ca2+ loading, critical for metabolic reprogramming and T cell differentiation. This proposal is organized into 3 aims:
Aim 1 : Define the mechanisms and role of STIM1 translocation during IS formation. Here, we will i. define the role of STIM1 polarization on IS formation, including examining the translocation of STIM1 target proteins towards the IS and ii. determine the impact of loss of polarization on mitochondrial function.
Aim 2 : Assess interdependence between STIM1 and septins during IS formation. Here, we will use Septin4/5-KO cells to determine their contribution to Ca2+ entry and clearance and downstream signaling during T cell activation. In addition, we will use site-directed mutagenesis to eliminate GTPase function and then reexpress in KO cells. Implications to the localization of STIM1, STIM1 mutants and STIM1-associated proteins Orai1, PMCA4 and POST will be determined. Finally, we will determine the relationship between Septins and STIM1 on phosphoinositide localization during T cell activation.
Aim 3 : Role of STIM1 polarization on primary T cell activation. Here, we will use adoptive transfer to generate primary T cells expressing either STIM1WT or STIM1K672M. This will facilitate extending our work to include assessing how the failure of STIM1 to translocate to the IS affects T cell differentiation, cytokine production, expansion and elimination. These investigations will provide new insights into cellular mechanisms regulating STIM1 localization and function, particularly within the context of T cell activation. Completion of this work could have numerous applications, particularly given recent progress in immunology. Hence, the efficiency of IS formation determines the quality and nature of the immune response, with potential implications to the treatment of numerous autoimmune diseases and cancer immunology.
Defining STIM1 function at the Immunological Synapse ? NARRATIVE STIM1-dependent Ca2+ signals represent the primary means of Ca2+ entry in T cells and are required for adaptive immunity. An immunological synapse is a temporary interaction between T cells and antigen-presenting cells and leads to a profound re-organization of multiple signaling proteins within the T cell. The current proposal focuses on defining the underlying mechanisms controlling localization of proteins at the immunological synapse and determining their impact on T cell activation.
