Spatial control of neuronal membrane traffic and mitotic abscission by Septin 9

Cell asymmetries drive cell function. Polarized cells such as neurons and epithelia require membrane protein compartmentalization, while fundamental cell processes such as cell division, require precise spatial and temporal control in order to be completed with fidelity. Loss of spatial organization/order is an underlying cause of many diseases ranging from neurodegeneration to cancer. Septins are a family of GTP-binding proteins that associate with membranes and the cytoskeleton. Septins spatially organize the cell membrane, actin and microtubule cytoskeleton, and modulate their dynamics. In this thesis, I examine the role of microtubule-bound septins in the spatial regulation of intracellular transport and membrane-bound septins in the spatiotemporal control of cytokinetic abscission. In the first part of my thesis, I investigated whether microtubule-associated septins influence intracellular transport and how this regulation affects neuronal membrane polarity. Neuronal function depends on maintaining the polarization of axonal pre-synaptic and dendritic post-synaptic proteins. I found that in cultured hippocampal neurons, a microtubule associated septin, SEPT9, localizes predominantly to dendritic microtubules. I discovered that SEPT9 directs sorting into dendrites by halting and reversing axonal motor-cargo, while concurrently promoting dendritic motor-cargo further into dendrites. SEPT9 depletion results in loss of axonal polarization of motor-cargo (e.g. kinesin-1/KIF5 and APP). Conversely, SEPT9 depletion results in a trafficking bottleneck of dendritic motor-cargo (e.g. kinesin 3/KIFA and LDLR) at sites of dendritic entry and at dendritic segments proximal to the cell body. Importantly, cell free in vitro assays revealed that SEPT9 effects on enhancing and impeding the motilities of kinesin-3/KIF1A and kinesin-1/KIF5, respectively, are direct. Furthermore, I found that SEPT9-mediated enhancements of KIF1A and its promotion into dendrites is mediated through a positively charged loop of the motor domain, located at the MT interacting interphase. These results are the first evidence that microtubule-associated septins can directly and differentially affect MT motor motility. Importantly this regulation is critical in maintaining neuronal polarity. In the second part of my dissertation I investigated the role of membrane-bound septins in cytokinesis, the last stage of mitosis. In late cytokinesis, the two daughters physically separate through fission of the membranes of the intracellular bridge (ICB), which is mediated by the ESCRT-III scission machinery. I found that septins form a double membrane bound ring that flanks the midbody and they demarcate the sites of ESCRT assembly. Septin rings partially overlap with ESCRT components at the ICB and dissipate as ESCRT-III rings expand laterally into helical filaments resembling cones to mark the sites of abscission. SEPT9 depletion, which has been previously shown to specifically affect abscission, disrupted the recruitment of VPS25 (ESCRT-II) and CHMP6 (early ESCRT-III) and resulted in improper ESCRT-III ring organization, which appeared dispersed. Notably these ESCRT-III rings failed to constrict the membrane and expand into cones. I discovered that SEPT9 is functionally linked to ESCRT assembly at the ICB by directly interacting with TSG101 (ESCRT-I) and scaffolding the recruitment of downstream ESCRT components. These data further our understanding in the conserved role of septins in cytokinesis and reveal a novel septin interaction with ESCRTs which may have implications for other ESCRT-implicated cell processes such as the multivesicular body-mediated lysosomal degradation. Collectively, these data uncover two novel roles for septins as spatial organizers of the cell. Microtubule-bound septins control the directionality of MT-mediated trafficking, while membrane-bound septin rings control the spatial organization of the ESCRT machinery of cytokinesis. Septins emerge as key spatial regulators from dividing cells to post-mitotic neurons, bearing key insights into the molecular basis and mechanisms that underlie the spatial organization and coordination of eukaryotic cells.