Project Summary The extracellular matrix (ECM) is a complex protein meshwork that constitutes the architectural scaffold of all tissues. In addition to its structural role, the ECM conveys biochemical signals to cells interpreted by receptors, like integrins, and controlling diverse cellular functions including adhesion and migration. The ECM is thus a key regulator of developmental processes and tissue homeostasis. Consequently, alterations in the composition and assembly of the ECM meshwork have been linked to a plethora of diseases including fibrosis, and cancer. Important progress toward understanding how the ECM meshwork is built and how the ECM govern cellular phenotypes have been made by studying major ECM proteins such as fibronectin and collagens. However, using sequence analysis, we have predicted that nearly 300 proteins can contribute to the ECM meshwork. Knowledge regarding the roles of these other ECM proteins remains preliminary. This represents a significant gap in our understanding of the ECM and in our ability to correct disease-causing ECM defects. We have recently become interested in one of these understudied ECM proteins, SNED1, after having found that it was associated with more highly aggressive breast cancers. Of clinical relevance, we found that higher SNED1 expression correlated with a worse prognosis for breast cancer patients. To gain insights into SNED1’s functions, we generated the first knockout (KO) mouse model of Sned1 and showed that Sned1 is an essential gene, since its KO resulted in early neonatal lethality due, in part, to craniofacial malformations. Importantly, we recently identified the first patients with SNED1 variants and they present with craniofacial malformations. Despite these observations, the mechanisms by which SNED1 contributes to embryonic development and cancer metastasis are unknown. Using novel tools we developed (antibodies, mouse models, cell lines, purified proteins), we have shown that SNED1 forms fibers within the ECM scaffold and contributes to its overall organization. In this proposal, we will test the hypothesis that SNED1 assembly in the ECM and its role in regulating ECM architecture depend on SNED1’s interactions with other ECM proteins and integrins. Leveraging our unique toolkit and combining our unique expertise in ECM protein biochemistry and ECM proteomics with state-of-the-art microscopy, we will conduct a time-resolved structure/function analysis to map which domains of SNED1 mediate its incorporation in the ECM (Aim 1); determine the role of ECM proteins/SNED1 interactions in SNED1 ECM assembly (Aim 2); and identify the SNED1 receptors at the cell surface governing SNED1-dependent ECM organization and responsible for the adhesive property of SNED1 (Aim 3)? Our goal is to fill critical gaps in our understanding of the fundamental mechanisms leading to ECM assembly, with respect to SNED1. This is a necessary step toward deciphering how perturbations of these mechanisms can...