Graduation Date

Summer 8-19-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Steve Caplan

Abstract

Endocytic trafficking is a critical process for cellular homeostasis, and multiple ailments that include cardiovascular disease and cancer are related to the dysregulation of endocytic transport. As vesicles and target membranes are key to endocytic transport, lipids are essential for the regulation of endocytic trafficking pathways. We have shown that the tubular recycling endosomes (TRE) are essential for the regulation of endocytic recycling pathways. However, the mechanisms by which TRE are biosynthesized and carry out their functions remain unsolved. Studies from our lab have shown that phosphatidic acid (PA) recruits Molecule Interacting with Casl-Like protein 1 (MICAL-L1) as well as Syndapin2, and subsequently Eps15 homology domain containing protein EHD1 to the membrane of TREs as a complex, and this complex is essential for the biogenesis of TRE and efficient recycling of internalized receptors back to the plasma membrane. However, the involvement of PA in endocytic trafficking has not been well characterized. Diacylglycerol kinase (DGK) α is one of ten DGK isoforms that converts diacylglycerol (DAG) to PA. We showed that depletion of DGKα, a kinase devoid of a clathrin-dependent adaptor protein complex 2 binding site, results in an impaired biogenesis of TRE. As a consequence, we observed a delay in MHC I recycling to the plasma membrane. On the other hand, the rate of MHC I internalization remained unaffected. By Co-immunoprecipitation assay, we showed that DGKα forms a complex with the TRE hub protein, MICAL-L1. Given that MICAL-L1 and the F-BAR-containing membrane-tubulating protein Syndapin2 associate selectively with PA, whose generation is majorly mediated by DGKα, we propose a positive feedback loop in which DGKα generates PA to drive its own recruitment to TRE via its interaction with MICAL-L1. Our data support a novel role for the involvement of DGKα in TRE biogenesis and MHC I recycling.

While the molecular mechanism of MICAL-L1 decorated TRE biogenesis is further revealed by our studies of the involvement of DGKα, major questions with regards to their function in endocytic recycling still remained unsolved, such as what cargos travel through these tubular membrane structures, where is the destination of these cargos, etc. We showed that TRE preferentially traffic cargos internalized via clathrin-independent endocytosis (CIE), and may originate from the sorting endosomes (SE). Since MICAL-L1 TRE is a major component of the endocytic recycling compartment (ERC), the understanding of the composition and cargo distribution within the ERC would further add to our knowledge of TRE functions. We used 3D Structured Illumination Microscopy, dual-channel and 3D direct Stochastic Optical Reconstruction Microscopy (dSTORM) to obtain new information about ERC morphology and cargo segregation. For the first time, we discovered that cargo internalized either via clathrin-mediated endocytosis (CME) or CIE remains segregated in the ERC, likely on distinct carriers. This suggests that no further sorting occurs upon cargo exit from SE. Moreover, 3D dSTORM data support a model in which some, but not all ERC vesicles are tethered by contiguous ‘membrane bridges’. These findings support a significantly altered model for endocytic recycling in mammalian cells, in which cargos are sorted in the peripheral endosomes and carried by MICAL-L1 decorated TRE to the ERC, while segregation is maintained at the ERC.

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