Strength in the Periphery: Growth Cone Biomechanics and Substrate Rigidity Response in Peripheral and Central Nervous System Neurons

Abstract

There is now considerable evidence of the importance of mechanical cues in neuronal development and regeneration. Motivated by the difference in the mechanical properties of the tissue environment between the peripheral (PNS) and central (CNS) nervous systems, we compare substrate-stiffness-dependent outgrowth and traction forces from PNS (dorsal root ganglion (DRG)) and CNS (hippocampal) neurons. We show that neurites from DRG neurons display maximal outgrowth on substrates with a Young's modulus of ∼1000 Pa, whereas hippocampal neurite outgrowth is independent of substrate stiffness. Using traction force microscopy, we also find a substantial difference in growth cone traction force generation, with DRG growth cones exerting severalfold larger forces compared with hippocampal growth cones. The traction forces generated by DRG and hippocampal growth cones both increase with increasing stiffness, and DRG growth cones growing on substrates with a Young's modulus of 1000 Pa strengthen considerably after 18-30 h. Finally, we find that retrograde actin flow is almost three times faster in hippocampal growth cones than in DRG. Moreover, the density of paxillin puncta is significantly lower in hippocampal growth cones, suggesting that stronger substrate coupling of the DRG cytoskeleton is responsible for the remarkable difference in traction force generation. These findings reveal a differential adaptation of cytoskeletal dynamics to substrate stiffness in growth cones of different neuronal types, and highlight the potential importance of the mechanical properties of the cellular environment for neuronal navigation during embryonic development and nerve regeneration.

The mechanical control of nervous system development

“…These substrates are chosen to be linearly elastic (see Glossary, Box 1), which means that g ϰ s. By contrast, many biological materials tend to be non-linearly elastic (see Glossary, Box 1). In traction-force microscopy (Munevar et al, 2001; Betz et al, 2011;Koch et al, 2012), a compliant substrate is deformed by cells and deformation fields are tracked using fluorescent nanoparticles embedded within the substrate. In an alternative approach, stiffer elastomeric substrates are structured as arrays of needle-like posts (Tan et al, 2003).…”

“…Forces (tension) might not only be involved in the generation of axons (Bray, 1984). Many neuronal cell types adapt their morphology, and particularly the number, length and branching patterns of their neurites, to the stiffness of their substrate in vitro, including mammalian dorsal root ganglion cells, spinal cord and hippocampal neurons, but not always cortical neurons (Georges et al, 2006;Jiang et al, 2008;Norman and Aranda-Espinoza, 2010;Koch et al, 2012). Neurite outgrowth is a mechanical process, and as such it might well be influenced by the interaction between neurites and the mechanical environment in vivo.…”

“…The actin cytoskeleton is also coupled to the substrate via point contacts, which are made up of protein complexes containing integrins, vinculin, talin and many others (Renaudin et al, 1999). These point contacts form molecular 'clutches' (Suter and Forscher, 1998), which allow growth cones to transmit forces to their substrate, which may lead to its deformation (Franze et al, 2009; Betz et al, 2011;Koch et al, 2012). Accordingly, inhibition of actin polymerization leads to a reduction in the maximum force and velocity of growth cone protrusion, and a reduction in membrane stiffness results in larger forces and increased velocity (Amin et al, 2012).…”

Swelling and Mechanical Properties of Alginate Hydrogels with Respect to Promotion of Neural Growth

“…30,31 Neurons plated on softer substrates showed decreased branching relative to neurons grown on stiffer substrates. 32,33 Mesenchymal stem cells and primary neural stem cells differentiate into neurons on soft hydrogels with stiffnesses < 1 kPa.…”

Real-Time Observation of Polymerase- Promoter Contact Remodeling during Transcription Initiation

“…To study initiation, we developed a hairpin unzipping assay that is conceptually similar to assays previously used to interrogate proteinnucleic-acid contacts by single-molecule force spectroscopy [23][24][25] . The assay consists of two polystyrene beads, each held in a separate optical trap 26 , and attached to dsDNA handles flanking a single DNA hairpin that carries a promoter with a transcription initiation site (Fig.…”