Morphological Changes of Neuronal Growth Cones by Intracellular Signaling and Cell Culture Conditions

Yuan Ren, Purdue University


Proper wiring of neurons is key to the functionality of nervous system. This is achieved by a highly motile structure referred to as the neuronal growth cone located at the tip of axons and dendrites during both development and regeneration. The morphology of growth cone is a product of both intracellular signaling pathways and extracellular factors, and is closely linked with neuronal outgrowth. Studies of how growth cone morphology is changed by key regulatory proteins and by cell culture conditions are therefore important in elucidating the mechanisms of neurite regeneration. In the first part of this thesis, we identified a single tyrosine residue (Y499) in Aplysia cortactin that is important for regulating filopodia formation in growth cone. Overexpression of the 499F phospho-deficient cortactin mutant decreased filopodia length and density, whereas overexpression of the 499E phospho-mimetic mutant increased filopodia length, regardless of the phosphorylation state of Y505 or Y509. Using a custom-made antibody against cortactin phosphorylated at Y499, we showed that phosphorylated cortactin is enriched in the peripheral domain, specifically along the leading edge. We found that treatment with the Src inhibitor PP2 decreased cortactin phosphorylation, while overexpression of Src2 increased cortactin phosphorylation. We demonstrated that the leading edge localization of phosphorylated cortactin is F-actin independent, and important in promoting filopodia formation. Finally, by interfering both with cortactin phosphorylation and Arp2/3 activation, we found that Arp2/3 complex acts downstream of cortactin to regulate filopodia density but not length. In conclusion, we have characterized a tyrosine phosphorylation site in Aplysia cortactin that plays a major role in the Src/cortactin/Arp2/3 signaling pathway controlling filopodia formation. In the second part of this thesis, we offer a comprehensive cellular analysis of the motile behavior of Aplysia growth cones on two-dimensional (2D) culture beyond the pausing state. We found that average growth cone size decreased with cell culture time whereas average growth rate increased. This inverse correlation of growth rate and growth cone size was due to the occurrence of large growth cones with a peripheral domain larger than 100 μm2. The large pausing growth cones had central domains that were less consistently aligned with the direction of growth and could be converted into smaller, faster-growing growth cones by addition of a three-dimensional (3D) collagen gel. We conclude that the significant lateral expansion of lamellipodia and filopodia as observed during these culture conditions has a negative effect on neurite growth. Further, using the novel collagen gel and an easy-to-make microwell device, we developed a simple protocol for 3D culture of Aplysia bag cell neuron. We found that the morphology and growth pattern of bag cell growth cones in 3D culture closely resemble the ones of growth cones observed in vivo, and demonstrated the capability of our device for high-resolution imaging of cytoskeletal and signaling proteins as well as organelles. We expect that our microwell device will facilitate a wider adoption of 3D neuronal cultures to study the mechanisms of neurite regeneration.




Suter, Purdue University.

Subject Area

Neurosciences|Cellular biology

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