Septin complexes differentially regulate microtubule motors and contribute to organelle positioning in a paralog-specific manner

Yani Suber

doi:10.17918/00001747

Spatial control of microtubule-based membrane trafficking is required for many essential cellular functions from long-range signaling in neurons, to cell motility during wound healing or the positioning of organelles throughout development. Intracellular transport is driven by the microtubule motors kinesin and dynein, which through the guidance of many factors, are able to traverse a crowded cellular environment and efficiently deliver cargo. Although the microtubule-associated mechanisms that guide this transport are not fully understood, altered activity or targeting of motors and membrane cargo, is known to cause debilitating neurological diseases, thus highlighting the clinical significance of studying this process. Septins are a family of filamentous GTP-binding proteins, capable of forming complexes containing multiple family members, and are known regulators of membrane trafficking through their association with a subset of microtubules as well as membranes. Recently, more light has been shed on the role of individual microtubule-associated septins in directing intracellular transport, yet it remains unknown whether septin complexes behave similarly and whether there are functional consequences of altering complex composition. In this thesis I show that microtubule-associated septin complexes represent a novel code for the spatial organization of intracellular transport and provide evidence for the differential regulation of kinesin and dynein by three distinct septin complexes; SEPT2/6/7, SEPT2/6/7/9 and SEPT5/7/11. In vitro motility assays revealed that similar to SEPT9 alone, kinesin-1/KIF5C is inhibited by all three septin complexes, indicating that septins may be universal inhibitors of this particular motor family. I also show that kinesin-3/KIF1A is differentially regulated by septin complexes with SEPT5/7/11 and SEPT2/6/7 displaying a very weak and robust inhibitory effect respectively. Previous work established microtubule-associated SEPT9 as an enhancer of kinesin-3/KIF1A motility and I found that while high concentrations of SEPT2/6/7/9 impede KIF1A, motor velocity increases at low nanomolar amounts, with this hybrid effect most likely being due to the presence of SEPT9. Notably, SEPT5/7/11 transiently tethers both motors to microtubules, although the effect is more prominent on dynein. I also discovered that in hippocampal neurons SEPT5 associates with dendritic microtubules and that loss of SEPT5 disrupts Golgi complex polarity establishment and morphology, which are both dynein dependent. Thus, with the existence of multiple complexes with various properties, septin complexes are unique regulators of microtubule motors and provide an additional microtubule-associated code for the organization of membrane trafficking. In the third chapter of my thesis, I describe, in great detail, the materials and methods utilized for the in vitro motility assays I developed, which are referenced in chapter two. I share methods for the purification of recombinant fluorescently tagged septin complex SEPT2/6/7, the preparation of constitutively active kinesin-3/KIF1A motor, the polymerization of microtubules in vitro and a step-step protocol to carry out the assay in its entirety. Importantly, I also provide extensive notes in this chapter about troubleshooting with the intention that by sharing these techniques, as well as my own personal insight, that his work will be more accessible to others in the future.

Septin complexes differentially regulate microtubule motors and contribute to organelle positioning in a paralog-specific manner

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Septin complexes differentially regulate microtubule motors and contribute to organelle positioning in a paralog-specific manner

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