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Title: Substrate stiffness tunes the neuronal response to the chemical guidance cue Semaphorin 3A
Author: Foster, Sarah Katherine
ISNI:       0000 0004 8507 6070
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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During development, neurons extend long axons which must navigate towards specific synaptic targets in the brain. This navigation is accomplished by a sensorimotor apparatus called the neuronal growth cone, located at the tip of the growing axon. Growth cones rely on signals present in their environment to navigate. Previous research has primarily examined how growth cones sense and respond to chemical guidance cues. However, the nervous system presents a rich and varied mechanical environment, and neuronal growth cones also respond strongly to physical stimuli. Previous work from the Franze research group identified that tissue stiffness and the mechanosensitive ion channel Piezo1 are important for proper pathfinding of the axons of retinal ganglion cell neurons (RGCs). In an in vitro culture system comprising explanted Xenopus laevis eye primordia, I found that application of the Piezo1 agonist Yoda1 triggered axonal calcium transients in RGC axons, while Piezo1 knockdown with an antisense morpholino resulted in increased axonal outgrowth. This mechanosensitive ion channel-dependent modulation of intracellular calcium, which is a crucial second messenger in many chemical signalling pathways, indicated that piezo1 signalling may significantly impact the response of growth cones to chemical signals. I therefore used compliant polyacrylamide hydrogels of defined stiffness to control the mechanical environment of RGCs in vitro, and studied if the mechanical environment of neurons impacts their response to Semaphorin 3A (Sema3A), a highly conserved guidance molecule that classically functions as a chemorepellant in this system. Indeed, I found that the response of Xenopus RGC growth cones to Sema3A was significantly reduced on softer substrates. This dramatic change in the response to Sema3A indicated that substrate stiffness may strongly and chronically alter biochemical components that act downstream of chemical guidance cue receptors. In order to test this hypothesis, I used a variety of imaging and perturbation approaches to examine the dependence of critical regulators of the Sema3A response, including cyclic nucleotides, calcium, and resting membrane potentials, on the stiffness of the neuronal environment. CGMP levels were elevated, calcium flickers more dynamic, and resting membrane potential more depolarized in neurons grown on soft, as compared to stiff, substrates. Given what is known about the activity of these second messengers in the Sema3A signalling cascade, each of these changes is consistent with the less repulsive response to Sema3A observed in the soft condition. Piezo1 is thought to be activated by high lateral membrane tension, and membrane tension is presumed to correlate with increased substrate stiffness. In collaboration with Jeffrey Mc Hugh in Ulrich Keyser’s group at the Cavendish Laboratory, I used optical tweezers to measure membrane tension in neurons grown on soft and stiff substrates by pulling membrane tethers from axons. We found no difference in bulk membrane tension as a function of substrate stiffness, while easy sliding of the tethers along axons indicated low membrane-cytoskeletal coupling on all substrates studied. Thus, while Piezo1 is critically involved in neuron growth and pathfinding, variations in substrate stiffness do not seem to invoke changes in membrane tension, suggesting that Piezo1 activity is modulated differently than currently thought. Taken together my results indicate that substrate stiffness is a critical variable in determining how growth cones respond to chemical guidance cues, and that cells integrate chemical and mechanical signals. I have identified several second messengers upon which Sema3A signalling and mechanotransduction cascades converge. Importantly, these second messengers are also master regulators of numerous other axon guidance cue responses (such as netrins and ephrins), and more generally of a wide variety of crucial biochemical signalling cascades. Variations in local tissue mechanics occur naturally during development and disease states, and could therefore be important in determining cellular responses to chemical signals during these pivotal events.
Supervisor: Franze, Kristian Sponsor: Herchel Smith Foundation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Axon Guidance ; Mechanics ; Semaphorin 3A ; Sema 3A ; cGMP