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Growth cones as soft and weak force generators.

ABSTRACT: Many biochemical processes in the growth cone finally target its biomechanical properties, such as stiffness and force generation, and thus permit and control growth cone movement. Despite the immense progress in our understanding of biochemical processes regulating neuronal growth, growth cone biomechanics remains poorly understood. Here, we combine different experimental approaches to measure the structural and mechanical properties of a growth cone and to simultaneously determine its actin dynamics and traction force generation. Using fundamental physical relations, we exploited these measurements to determine the internal forces generated by the actin cytoskeleton in the lamellipodium. We found that, at timescales longer than the viscoelastic relaxation time of ? = 8.5 ± 0.5 sec, growth cones show liquid-like characteristics, whereas at shorter time scales they behaved elastically with a surprisingly low elastic modulus of E = 106 ± 21 Pa. Considering the growth cone's mechanical properties and retrograde actin flow, we determined the internal stress to be on the order of 30 pN per ?m(2). Traction force measurements confirmed these values. Hence, our results indicate that growth cones are particularly soft and weak structures that may be very sensitive to the mechanical properties of their environment.

SUBMITTER: Betz T

PROVIDER: S-EPMC3158236 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Growth cones as soft and weak force generators.

Betz Timo T Koch Daniel D Lu Yun-Bi YB Franze Kristian K Käs Josef A JA

Proceedings of the National Academy of Sciences of the United States of America 20110803 33

Many biochemical processes in the growth cone finally target its biomechanical properties, such as stiffness and force generation, and thus permit and control growth cone movement. Despite the immense progress in our understanding of biochemical processes regulating neuronal growth, growth cone biomechanics remains poorly understood. Here, we combine different experimental approaches to measure the structural and mechanical properties of a growth cone and to simultaneously determine its actin dy ...[more]

PMID: 21813757

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Project description:To form operative neuronal networks neurons connect to their synaptic partners by axonal outgrowth guided by the movement of the axon tip, a specialized structure known as the growth cone (GC), which travels along stereotypical paths by sensing extracellular guidance cues. Interestingly, GCs contain complex transcriptomes and different guidance cues can rapidly regulate the translation of specific mRNAs which help to remodel the GC cytoskeleton thus steering axon navigation. However, the full landscape of proteomic changes elicited by different cues is not known and the precise protein synthesis (PS)-dependent molecular mechanisms underlying GC repulsive and attractive turning remain largely elusive. Here we have investigated the cue-induced changes in the nascent proteome by pulse Stable Isotope Labeling by Amino acids in Cell culture (pSILAC) in somaless developing retinal axons derived from Xenopus laevis primary cultures. The experimental approach presented major challenges due to the limited quantity of axonal material (~2 μg per condition) and to the desired rapid (5-15 min) time-scale (no previous work has accomplished SILAC with a pulse less than 20 min). To overcome these difficulties we used a superior protocol, termed Single-Pot Solid- Phase-enhanced Sample Preparation, based on ultrasensitive paramagnetic bead technology which allows high proteomic sample recovery from sub-microgram amounts of material. By employing this method we were able to identify up to 500 newly synthesized proteins after only 5 min of pSILAC. Subsequently, we successfully validated the purity and viability of the axonal samples and the reliability of the method. Finally, to characterize and quantify the global patterns of the axonal translational changes in response to specific guidance cues and to unravel the switch between repulsive and attractive responses, we carried out pSILAC in the presence of different repulsive cues and pharmacological reagents that convert repulsion into attraction. Our results uncovered a surprisingly high level of translational activity within the axonal compartment, even in basal conditions. Remarkably, comparative analysis revealed many changes in the nascent proteome in response to different cues, some distinct and some common. Interestingly, several proteins showed cue-specific downregulation. Taken together, our findings suggest a comprehensive GC PS-dependent chemotropic model and novel cue-specific molecular mechanisms possibly underlying the integration of chemotropic responses.

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Growth cones as soft and weak force generators.

Publications

Growth cones as soft and weak force generators.

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