• Elizabeth Granger

    Student thesis: Phd


    Elizabeth GrangerThe University of ManchesterThe interplay between dynein, accessory proteins and the endocytic pathwayDoctor of Philosophy, Organelle Function25-09-13Cytoplasmic dynein 1 (dynein) is a multi-subunit complex that transports cargo along microtubules towards their minus ends. These microtubule minus ends are normally located toward the centre of the cell. Dynein is involved in transport of endocytic and autophagic membranes and is tightly regulated by interactions between dynein subunits and by dynein-accessory proteins. Dynein accessory proteins that are involved in a wide range of dynein-driven transport events include dynactin, Lis1 and the paralogues Nde1 and Ndel1. Lis1 and Nde1/Ndel1 interact with each other and are involved in the recruitment of dynein to cargo and in regulating dynein activity. Although much is known about the specific interactions of dynein and accessory proteins, the interplay between dynein and its network of regulators in living cells is not well defined.This project used RNAi to investigate how the dynein subunits light intermediate chain (LIC) and intermediate chain (IC) as well as Lis1 and Nde1/Ndel1 influence the endocytic pathway, autophagy and cargo recruitment. Biochemical analysis of bulk membrane preparations showed that IC is important for dynein and dynactin association with intracellular membranes. In addition, dynein and dynactin recruitment to Rab interacting lysosomal protein (RILP)-positive membranes was shown to require LIC and there was redundancy between LIC1 and LIC2 in this role. Lis1 was also needed for dynactin-dynein recruitment to these membranes, in a context that was Nde1/Ndel1-independent.Loss of LIC, IC, Lis1 and Nde1 had differing effects on endocytic compartment size and distribution, but they all led to mislocalisation of early endosomes and lysosomes and caused lysosomes to become enlarged. Loss of LIC led to a specific phenotype whereby cells formed lamellipodia-like regions in which early endosomes and lysosomes accumulated. Loss of Lis1 prevented traffic from the early endosome to late endosomes and caused a striking enlargement of late endosomes and lysosomes. These enlarged lysosomes were LC3-positive, indicating that they were autophagic. In addition, loss of IC and LIC also led to an increase in LC3 puncta, but the LC3 did not colocalise specifically with lysosomes.In summary, the results from this project show that i) dynactin recruitment to intracellular membranes, including RILP-postivie membranes, requires dynein, ii) Lis1 and LIC1 or LIC2 are necessary but not sufficient, individually, to recruit dynein and dynactin to RILP-positive membranes iii) LIC, IC, Lis1 and Nde1/Ndel1 influence endocytic progression in specific ways, which may in turn affect autophagic flux.
    Date of Award1 Aug 2014
    Original languageEnglish
    Awarding Institution
    • The University of Manchester
    SupervisorViki Allan (Supervisor) & Martin Lowe (Supervisor)

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