Project Summary/Abstract Eukaryotic cells organize their interior into a set of membrane-enclosed compartments, referred to as organelles or the endomembrane system, that enable cell growth and viability. Each organelle exhibits a unique biochemical composition, complex dynamics, and a distinct morphology. How organelles are shaped, how their shape is linked to their functions, and how individual organelles engage in specific contacts are important open questions that are the focus of the proposed research. Understanding of the spatial organization of human cells is currently experiencing a revolution, with the realization that components of the cytoplasm can undergo a “liquid-liquid” phase separation from the rest of the cytoplasm, forming dynamic and functionally specialized domains that lack any membrane. Our recent research raises the novel possibility that membrane-containing organelles are structured by a two-dimensional variation of this principle, in which ‘rod-like’ proteins (‘golgins’ and golgin-like proteins) self-assemble into lamellar liquid geometries. We aim to test and develop this new organizing principle in context of the organization of the early secretory pathway, where two organelles, the ER and the Golgi stack, form a ‘synapse-like’ interface that is conserved across taxa. It is poorly understood how the spatial organization of the ER-Golgi interface is achieved, and why this specific organization is required. It is also a mystery how this junction can exhibit structural integrity while resisting a high throughput of material, yet exhibit dynamic properties under specific regulatory cues. Our goal is to understand the mechanisms that establish the specific morphology of the ER-Golgi interface in order to enable efficient processing and sorting of cargo within this space. Our motivating hypothesis is that this interface represents a dynamic membrane contact site organized by local phase separation proteins. We will employ a ‘bottom-up’ approach in which we purify individual components to homogeneity and probe them in a model membrane environment, seeking out the minimal components and mechanisms needed to reconstitute morphology and function. We will complement this approach with super-resolution and electron microscopy, genetic perturbations and functional assays in both human cell lines and in the model organism C. elegans. Through this dual strategy, we expect to elucidate the principles by which the ER-Golgi interface is formed, maintained, and how its spatial organization impacts cellullar functions.