Septin regulation of multivesicular body/late endosome maturation and movement

Benjamin Patrick Robinson

doi:10.17918/00001962

The microtubule cytoskeleton provides a platform for organelle positioning and trafficking within the cell. The tracks that microtubules provide for motility are particularly critical for vesicular compartments traversing the intracellular trafficking pathways, as the delivery of sorted protein cargoes and machinery to proper destinations maintains protein homeostasis, cellular signaling, and degradative/recycling functions within the cell. However, the microtubules are not passive pathways for endosomal trafficking, with distinct post-translational modifications (PTMs), tubulin isotypes, and microtubule-associated proteins (MAPs) influencing the motility and positioning of a variety of endosomes under differing cellular conditions. As the biogenesis and maturation of these endosomes has been linked to the positioning of the organelle during intracellular motility, the role of the cytoskeleton extends far beyond tracks to accurately deliver cargo. However, the mechanisms underlying the interface of the microtubules with trafficking organelles are still largely unknown. In this thesis, I will examine the role of septins in this interface between the cytoskeleton and the endosomes that traffic along it. Septins are a group of GTPases that can homo- or hetero-oligimerize to form their own paired multimers, but also possess the ability to bind to both membranes and microtubules. Additionally, septins have been show to interact with and modulate a number motor proteins that control vesicular trafficking, and have been shown to impact a number of endosomal functions, including lysosomal fusion of macropinosomes and the biogenesis of cargo sorting intraluminal vesicles in multivesicular bodies/late endosomes (MVB/LEs). Given that the interface of the microtubules with endosomal membranes and machinery is critical to understanding the controlled delivery of organelles and their cargo to proper destinations, septins are uniquely positioned to shed light on both sides of this interplay. In chapter 1, I will discuss in detail the microtubule and membrane association features of septins and their known impact on intraceullular trafficking, while highlighting the outstanding questions about the nexus of the cytoskeleton and endosomal motility/positioning that will benefit from a deeper understanding of septin functions. In chapter 2, I will explore how microtubule-associated septin complexes inhibit the motility of CD63-containing MVB/LEs in both in vitro assays and live MDCK cells and how the loss of these septin complexes inhibits the maturation of these compartments. This demonstrates the impact of septins on intracellular trafficking specifically while associated with the microtubule network, which promotes the idea that endosomal motility and maturation is not only affected by alterations of membrane machinery and structure but can specifically be influenced by microtubule-associated proteins and modifications. In chapter 3, I will examine how a single membrane-associated septin, SEPT9, is able to directly bind and recruit dynein to lysosomal membranes during periods of oxidative stress, promoting a perinuclear clustering of lysosomes independent from the canonical recruitment of dynein by GTPases such as RAB7. This work sheds light on how a cytoskeletal element such as septins may interface with the membranes of endosomes, leading to additional levels of regulation outside of the microtubule cytoskeleton. Finally, chapter 4 will discuss how the microtubule-associated septins in chapter 2 and the membrane-associated septins in chapter 3 could potentially interact with each other to exert control over intracellular trafficking. This will include insight into how the inhibition of motility by microtubule-associated septins can be reconciled with membrane-associated septins promoting motor recruitment, how recruitment of motor proteins may be linked to cargo sorting membrane machinery, and the potential of multiple septins within the hetero-octamer leading to differential impacts on motor regulation and membrane traffic.

Septin regulation of multivesicular body/late endosome maturation and movement

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Septin regulation of multivesicular body/late endosome maturation and movement

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