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 Unpublished observations from our group,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.