Filopodia

Abstract

The processes of neuronal outgrowth and guidance have typically been studied in classic 2D cell culture systems that do not recapitulate topographical cues present in the in vivo extracellular matrix (ECM). Using microfabrication techniques, we mimicked this ECM topography by presenting laminin on a line pattern with nanometric size features. We found that this not only allows neurite orientation but also robust outgrowth. This depends on dynamic stochastic sensing of the line pattern by growth cone filopodia which allows them to probe their surrounding space by measuring the extent of filopodial adhesion surface with the ECM. Filopodium alignment with an ECM line leads to the formation of a robust F-actin network that leads to its stabilization, allowing steady neurite extension along the line pattern. Because this model system allows exquisitely stereotypic filopodial dynamics, this opens up the possibility to easily study the spatio-temporal dynamics of the signaling networks that regulate this prototypic growth cone navigation system.

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Acknowledgments

This work was supported by grants from the Swiss National Science Foundation and Human Frontiers Science Program to Olivier Pertz.

Figures and Tables

Figure 1 Model of neurite guidance in response to nanotopographical cues. (A) Plain substrate. Unrestricted access to ECM leads to a large amount of long filopodia, none of which can be stabilized by a robust F-actin cytoskeleton. This is accompanied with a high frequency of neurite collapse events. (B) Line substrate, unaligned growth cone. Filopodia scan the line substrate through lateral scanning and protrusion/retraction events. Only few filopodia align on the lines and thus almost all filopodia sense only discrete adhesion points to the ECM. (C) Line substrate, aligned growth cone. Through stochastic sensing, multiple filopodia have aligned on the line substrate and have assembled an F-actin rich cytoskeleton that stabilizes them. On the distal part of the growth cone, non-aligned, unstable filopodia continue to operate, suggesting a crosstalk between both filopodia populations. This stabilizes the growth cone leading to steady neurite outgrowth. Figure reproduced with permission from Jang et al.5

Figure 2 Comparison of growth cone dynamics on the line substrate and in a developing retina. (A) Growth cone dynamics of N1E-115 neuron-like cells on the line substrate. N1E-115 were transfected with GFP-lifeact, an F-actin marker. Red and blue lines indicates the lateral movement of non-aligned filopodia. Note the high F-actin content in aligned filopodia. Timescale is in minutes:seconds. Part reproduced with permission from Jang et al.5 (B) Growth cone dynamics of Xenopus retinal axons imaged live in the developping retina. Note streamlined appearance of growth cone and left-right lateral movement of lateral filopodia (indicated by arrows). Part reproduced with permission from.12 Scale bars = 10 mm.

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