Mechanisms and function of endosome-derived tubular carriers

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Endocytic trafficking is central to normal cell function, and dysregulation is the underlying cause for diseases as diverse as atherosclerosis, diabetes and cancer. My laboratory has contributed to understanding the mechanisms by which membranes and receptors are recycled via the endocytic recycling compartment (ERC) to the plasma membrane (PM). Endocytic recycling remains one of the least studied endocytic pathways; in particular, the involvement of membrane tubules in the recycling process is poorly understood, and the roles of the various forms of tubular endosomes, and the mechanisms by which these structures are generated and undergo vesiculation remain a major unanswered question. There are multiple `types' of endosome-derived tubular carriers (EDTC), including sorting nexin-BAR (SNX-BAR) domain and retromer-derived tubules, and networks of endosomal tubules, such as tubular recycling endosomes (TRE) decorated by MICAL-L1, Syndapin2 (Synd2) and Eps15 Homology Domain (EHD) proteins. Little is known about how these apparently different EDTC integrate their functions, or even whether retromer and SXN-BAR EDTC are distinct from TRE. Recent studies demonstrate that EHD proteins and MICAL-L1 interact with components of the retromer complex, suggesting that these tubular networks are related. Our central hypothesis is that overlapping and distinct tubular membranes coordinate transport from endosomes to the PM and the Golgi. In this proposal, we will uncover the functional and physical relationships between TRE, retromer and SNX-BAR-derived EDTC. Moreover, we will focus on a mechanistic understanding of the mode by which EDTC are generated, and how they undergo vesiculation to promote transport within the cell. Aim 1: To examine the relationship and functional roles of EDTC. Tubular endosomes play major roles in endocytic membrane trafficking. EDTC include a number of retromer-containing structures, retromer-independent tubules generated by SNX-BAR domain proteins, and TRE decorated by MICAL-L1, the BAR domain protein Synd2, and EHD proteins. We will examine the cross-talk and cross-regulation of these pathways with regard to the generation and fission of recycling tubules, and the control of endocytic recycling. Aim 2: To define and elucidate the spatio-temporal regulation and mechanism of TRE biogenesis and vesiculation. Our working hypothesis is that phosphatidic acid generation leads to recruitment of the membrane hub, MICAL-L1 and the F-BAR protein Synd2, to generate and remodel TRE. We further hypothesize that EHD3 plays an essential role in this process by stabilizing MICAL-L1-Synd2 interactions. Our studies on the cellular, organellar, molecular and atomic levels will use techniques ranging from structural biology to super-resolution microscopy and novel biophysical vesiculation assays, providing crucial knowledge of the functional role of the poorly understood TRE and retromer-SNX-BAR derived tubules, and their biogenesis and vesiculation.