Spatial control of membrane traffic by septin GTPases

University Of Virginia, Charlottesville VA

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Abstract

Project Summary/Abstract Spatial control of membrane traffic is essential for the morphogenesis and maintenance of polarized cell types such as epithelia and neurons, and their physiological functions in tissue homeostasis and neurotransmission. Loss of cell polarity and defects in membrane traffic underlie the pathogenesis of many diseases including cancer, neurodegeneration and metabolic disorders. The rationale for our studies is that a mechanistic knowledge of the spatial organization and regulation of membrane traffic is critical for the design of new treatments and regenerative therapies. Many advances have been made in understanding membrane traffic at points of origin (protein sorting, vesicle formation) and destination (vesicle docking/fusion). However, key challenges remain in understanding how long-range transport is spatially controlled en route to destination. Our central hypothesis is that septins, a unique family of GTP-binding proteins that associate with distinct subsets of microtubules (MTs) and actin filaments comprise a novel regulatory module for the spatial guidance of membrane traffic. We have found that MT-associated septins regulate kinesin and dynein motor movement, controlling the directionality and position of membrane vesicles and organelles. Driven by recent advances, we will address key questions about septin functions and the spatial control of membrane traffic in epithelia and neurons. We will investigate the molecular mechanism and specificity of MT-septin binding. We will test the hypothesis that different septin complexes localize to different MT subsets by recognizing distinct tubulin isotypes and post-translational modifications, and/or by competing with MT-associated proteins (MAPs). We will map different septins to distinct subsets of MTs in polarized epithelia and neurons, and determine septin- specific functions in routes of polarized membrane traffic. We will examine how septins function in the bidirectional traffic of endosomes on MTs, and in transition from actin filaments to MTs. In the primary cilia of epithelial cells, we will examine how axonemal MT-associated septins function in the bidirectionality of intraflagellar transport. Lastly, we will investigate how septin-MT association is regulated. We will focus on the phosphorylation of septin 9 by glycogen synthase kinase beta (GSK3), and its inhibition as a key signaling event for triggering MT-septin binding and function in neuronal morphogenesis. We will pursue these projects collaboratively using innovative, transdisciplinary and cutting-edge approaches including super-resolution expansion microscopy, cryo electron tomography, and single-molecule in vitro reconstitution assays. We will develop and characterize new tools for rapid and acute loss of septin function such as a chemically inducible degron-based method for septin depletion and a new septin-targeting compound. Outcomes will provide new insights into the spatial regulation and organization of membrane traffic at steady state, and in response to morphogenetic signals. The proposed studies will bear significance on diseases triggered and/or exacerbated by abnormalities in septin expression.