STIM1 regulates spatiotemporal calcium signals and the cytoskeleton in motile growth cones

The precise connectivity that underlies neural circuitry in the central nervous system is regulated by axon guidance. This process is regulated by a highly specialized sensory structure at the tip of extending axons called the growth cone, which responds to extrinsic guidance cues in the embryonic environment. Disorders of growth cone motility and axon guidance are thought to underlie a range of neurological disorders such as autism and schizophrenia due to defects in neuronal targeting and connectivity. The spatial and temporal regulation of calcium signalling at the neuronal growth cone is essential for axon guidance and motility, however the exact mechanisms that regulate these localised calcium signals are not fully elucidated. Growth cone filopodia are the first responders‚ÄövÑvp during axon guidance, transducing guidance cues through receptor-mediated calcium transients. However, what regulates and sustains the spatiotemporal calcium signals at filopodia and precisely how these signals are instructional for growth cone motility remains unclear. As a major store of intracellular calcium, the endoplasmic reticulum (ER) would be predicted to have a vital role in growth cone calcium regulation, although ER function in the growth cone and in particular, the filopodia is largely unexplored. An important calcium-regulatory mechanism that occurs in growth cones is store operated calcium entry (SOCE) which is activated when Stromal Interacting Protein 1 (STIM1), an ERembedded calcium-sensing protein, and Orai1 on the plasma membrane form a highly selective calcium channel allowing calcium to enter the cell. STIM1 expression is necessary for transduction of filopodial calcium transients in Xenopus growth cones. In DRG neurons, STIM1 is required for attractive growth cone turning towards BDNF, where STIM1 functions to sustain calcium by refilling depleted ER stores through SOCE. STIM1 also regulates motility in response to a calcium-independent cue Sema-3a, suggesting that STIM1 functions in multiple pathways. In non-neuronal cells, STIM1 has also been reported to act as a microtubule plus-end tracking protein, where it facilitates microtubule-dependent ER remodelling through direct interaction with end-binding protein-1 and 3 (EB-1/3), in a tip-attachment complex. Given these findings, the central hypothesis of this thesis is that STIM1 mediates instructive ER-microtubule dynamics which are necessary to spatially and temporally localise calcium signals required to sustain motile or turning behaviours in pathfinding growth cones. This study tested whether STIM1 functions in a tip-attachment complex to mediate ERremodelling into filopodia of motile growth cones from rodent DRG sensory neurons. STIM1 localised with the microtubule cytoskeleton through an association with EB1/3, that was required for remodelling ER to peripheral areas of steering growth cones. Filopodial protrusion and stabilisation by microtubules is a well-known correlate of directed growth cone motility, but how microtubules are recruited to facilitate SOCE at filopodia has not been determined until now. The data presented in this study supports the hypothesis that microtubule-ER remodelling in sensory neuron filopodia is regulated by STIM1. Reduced STIM1 expression significantly perturbed microtubule assembly and organization in growth cones turning to BDNF and Sema-3a. STIM1 was necessary for appropriate distribution and dynamics of microtubule-associated proteins EB-1/3, as well as expression levels of filamentous-actin, actin-associated proteins and adhesionregulating elements. Additionally, using an ER-targeted low affinity calcium indicator, calcium dynamics and spatiotemporal localization of ER in filopodia were shown to be perturbed in growth cones with reduced STIM1 expression. Taken together, the data presented here demonstrate that STIM1-EB3 interaction represent a direct physical link between ER-derived calcium signals and the cytoskeleton. These data support a mechanism where ER remodelling, particularly in filopodia, supports and sustains crucial spatiotemporal regulation of calcium which is instructive for pathfinding axons during wiring of developing neural circuitry.