Investigating Macropinocytosis: The Role of Sorting Nexins in Macropinosome Biogenesis

Tsang-Hsing Wang (2010). Investigating Macropinocytosis: The Role of Sorting Nexins in Macropinosome Biogenesis PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

       
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Author Tsang-Hsing Wang
Thesis Title Investigating Macropinocytosis: The Role of Sorting Nexins in Macropinosome Biogenesis
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2010-03
Thesis type PhD Thesis
Supervisor Rohan Teasdale
Markus Kerr
Total pages 196
Total colour pages 42
Total black and white pages 154
Subjects 06 Biological Sciences
Abstract/Summary Macropinocytosis is an actin-driven endocytic process, whereby membrane ruffles fold back onto the plasma membrane to form large macropinosomes. In comparison to the body of knowledge regarding other endocytic pathways, there is a paucity of information regarding the molecular components and mechanisms required for macropinocytosis. Recently, members of the Sorting Nexin (SNX) family have been implicated in this process, localizing to the cell surface and on the body of the macropinosome very early post formation. This thesis furthers our understanding of macropinocytosis through the systematic examination of members of the SNX family within this endocytic pathway. This study established a sensitive quantitative assay for measuring macropinosome formation, which was systematically evaluated in terms of molecular markers, temporal variables, imaging protocol, and automated image analysis (Chapter 2). This quantitative assay was able to detect an increase in macropinosome number in response to EGF treatment, and decreases in macropinosome formation in response to amiloride and PI(3)K disruption. Disrupting macropinosome and late endosome/lysosome fusion through YM201636 treatment had no effect on macropinosome numbers as measured through this assay, showing that it is specific for the quantitation of early-stage macropinosome formation; moreover, this shows a disconnect between the formation mechanism and the dynamics of macropinosome maturation. This quantitative assay was then applied to the evaluation of SNX5 in macropinosome formation (Chapter 3). In bone-marrow-derived-macrophages, the protein levels of SNX5 decreased along with a concomitant drop in macropinosome formation in response to LPS treatment. Furthermore stable-overexpression of GFP-SNX5 significantly increases macropinosome formation, which is further exaggerated in the presence of EGF. This elevation of macropinosome formation requires the EGF-dependent recruitment of SNX5 to regions of the cell-surface rich in PI(3,4)P2, which is disrupted by treatment with the EGF-signalling inhibitor AG1478. Based upon the observation that SNX5 overexpression elevates macropinosome formation, I conducted a systematic screen of all proteins that share the domain architecture with SNX5 – the SNX-PX-BAR family (Chapter 4). The quantitative assay developed in Chapter 2 was adapted using overexpression screening methodology, and was validated by detecting increases in macropinosome formation following the transient elevation of SNX5, Rab5, and Rabankyrin 5. This screening assay was then applied to the SNX-PX-BAR family and SNX1, SNX5, SNX9, SNX18, and SNX33 overexpression were all found to elevate macropinosome formation; SNX1, SNX5, SNX9, and SNX18 were also found to associate with early-stage macropinosomes. The phosphoinositide-binding capacity of the PX-BAR module within these proteins is likely to be involved in their ability to influence macropinosome formation, as modulating PI(3,4,5)P3 levels by overexpressing PTEN mutants also significantly altered macropinosome numbers. This represents the first systematic analysis of the SNX-PX-BAR family in macropinocytosis, specifically implicating multiple members of the family in macropinosome formation. The last chapter of this thesis focuses on the molecular mechanisms of SNX18, as its overexpression induced the largest quantitative increase in macropinosome formation out of the entire SNX-PX-BAR family. SNX18 overexpression increases macropinosome formation in the presence or absence of EGF, and through fluorescence time-lapse microscopy I was able to determine its association with early-stage macropinosomes and actin-positive linear structures continuous with the plasma membrane. The capacity of SNX18 to elevate macropinosome formation is contingent upon its interaction with dynamin 2, and dynamin 2 inhibition further increased macropinocytosis when SNX18 is overexpressed. This implicates SNX18 in a macropinocytic mechanism to compensate for the loss of cellular intake through dynamin-dependent endocytic pathways. SNX18 also associates with the actin polymerization regulator N-WASP, and can be found together on macropinosomes and actin-positive puncta that elongate into linear structures over time. The overexpression of both of these proteins also resulted in the synergistic elevation of macropinocytosis. SNX18 also colocalizes with Rac1 on linear structures emanating from the cell surface, possibly acting as a scaffold for downstream signalling. Together, this data represents the first report of the role of SNX18 in macropinosome formation. The observations made within this thesis provide novel insight into both the fundamental molecular mechanisms involved in macropinocytosis, as well as the contribution of members of the SNX family towards this process.
Keyword macropinocytosis
sorting nexins
Additional Notes Pages in colour: 19, 26, 28, 30, 33, 35, 37, 39, 50, 53, 55, 56, 67, 70, 79, 81, 83, 86, 89, 102, 103, 105, 107, 109, 111, 113, 117, 145, 146, 148, 149, 151, 152, 153, 154, 179, 180, 182, 184, 186, 187, 190

 
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Created: Fri, 19 Nov 2010, 10:49:12 EST by Mr Tsang-hsing Wang on behalf of Library - Information Access Service