Regulation of microtubule organization and microtubule-dependent transport by septin 9

Microtubules (MTs) are a major component of the mammalian cytoskeleton. MTs are essential for cell morphogenesis and cellular functions including cell motility, cell division and intra-cellular transport. MT functions are tightly regulated by post-translational modifications (PTMs) and MT-associated proteins (MAPs). However, the underlying mechanisms are still elusive. Septins are a family of GTP-binding proteins that can form hetero-oligomeric and polymeric structures, and function as scaffolds or diffusion barriers, controlling the localization of membrane and cytoplasmic proteins. Mammalian septins interact with MTs and actin filaments, Septins are also involved in Golgi-to-plasma membrane vesicle transport and chromosome alignment. Interestingly, septins have been shown to interact with the centromere-associated protein E (CENP-E), a mitotic kinesin-like motor that links kinetochores to the ends of spindle MTs. However, it is unknown whether septins interact directly with MTs and how they affect MT organization and intracellular transport. In the first part of this thesis, I studied how MT organization is regulated by septins. I showed that the N-terminal domain of SEPT9 contains the novel repeat motifs K/R-x-x-E/D and R/K-R-x-E, which bind and bundle MTs by interacting with the acidic C-terminal tails of [beta]-tubulin. Alanine scanning mutagenesis revealed that the K/R-R/x-x-E/D motifs pair electrostatically with one another and the C-terminal tails of [beta]-tubulin, enabling septin-septin interactions that link MTs together. SEPT9 is the only gene linked to hereditary neuralgic amyotrophy (HNA), a rare autosomal-dominant neuropathy. SEPT9 isoforms lacking repeat motifs or containing the HNA-linked mutation R88W, which maps to the R/K-R-x-E motif, diminished intracellular MT bundling and impaired asymmetric neurite growth in PC-12 cells. These findings provide the first insight into the mechanism of septin interaction with MTs and the molecular and cellular basis of HNA. In the second part of this thesis, I discovered a novel interaction between SEPT9 and KIF17, a kinesin 2 family motor that is important for learning and memory, mediating the transport of the NMDA glutamate receptor in hippocampal neurons. I found that SEPT9 associates directly with the cargo-binding C-terminal tail of KIF17 and competes with mLin-10/Mint1, a cargo adaptor/scaffold protein, which links KIF17 to the NMDA receptor subunit 2B (NR2B). Significantly, SEPT9 down-regulates NR2B transport into the dendrites of hippocampal neurons. Because SEPT9 does not affect the microtubule-dependent motility of KIF17, my results suggest that SEPT9 modulates the interaction of KIF17 with NR2B cargo. These results provide the first evidence of an interaction between septins and a non-mitotic kinesin, and suggest that SEPT9 modulates specifically the interactions of KIF17 with membrane cargo. These findings advanced our knowledge of how septins associate with MTs and revealed the mechanism of MT binding and bundling by septins. My studies also provided the first clue about the etiology of HNA, which might be helpful in developing therapies for this disease in the future. In addition, the results of this work provided the first insight into how septins may regulate kinesin-dependent transport and the transport of a neurotransmitter to dendrite membrane. This knowledge could potentially be helpful in developing treatment for diseases associated with misregulation of MT organization and MT based transport.