PROJECT SUMMARY The significant cellular demand of the hematopoietic system is maintained by a rare pool of tissue-specific, hematopoietic stem and progenitor cells (HSPCs) that are primarily found in a quiescent state. Upon hematopoietic stresses, such as significant bleeding, overwhelming infection, radiation exposure and myelosuppressive therapy, HSPCs are rapidly recruited into cell cycle, but ultimately must return to quiescence. The ability to transiently modulate HSPC return to quiescence has the potential to extend the activation of HSPCs and significantly improve overall patient outcomes from hematopoietic stresses and for transplantation therapies. However, in order to leverage a transient extension of HSPC activation to improve the regenerative response to stress, we must first understand the mechanisms by which the complex network of cell-intrinsic and -extrinsic signaling within the bone marrow are coupled to regulate HSPC quiescence. The objective of our current proposal is to evaluate the tetraspanin membrane-scaffold protein, CD82, as a tractable target to modulate HSPC quiescence signaling within the bone marrow. Tetraspanins are a family of membrane-scaffold proteins with the unique ability to regulate cell-cell/cell-matrix interactions and modulate intracellular signaling, thus linking cell-microenvironment interactions to downstream signaling consequences. Our new preliminary data indicate that the CD82 scaffold promotes a quiescent HSPC phenotype when cells are niche engaged and implicate a role for Transforming Growth Factor (TGF) signaling. Thus, in this proposal, we will test the hypothesis that the CD82 membrane scaffold promotes HSPC quiescence by organizing and enhancing the signaling activity of a TGF receptor complex within the bone marrow niche. In Specific Aim 1, we will determine the molecular mechanisms by which CD82 modulates the TGFβ signaling response of HSPCs. For Specific Aim 2, we will identify the mechanism by which CD82 promotes the spatial activation of TGF signaling locally within the bone marrow niche during hematopoietic stress. In Specific Aim 3, we will evaluate CD82 as a therapeutic target to improve the hematopoietic regeneration response to hematologic injury. In pursuit of these aims, we will apply an innovative combinatorial approach that includes mutational analysis, biochemistry and sophisticated imaging techniques, which will enable us to obtain a multi- scale understanding of the mechanisms by which CD82 regulates TGF signal transduction in the context of hematopoietic stress. Moreover, the successful completion of the proposed aims will be significant because we expect to integrate mechanistic insights across multiple scales to identify the multifaceted contribution of CD82 to the regulation of TGF signaling in the context of hematopoietic stress and quiescence, which will build a foundation for the development of improved therapeutics that locally target the complex TGF signaling c...