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 τ = 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 μm(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.

Fig. 1.

Rheological measurements of the frequency dependent Young’s modulus of growth cones. Filled circles represent the storage modulus, whereas open circles give the loss modulus. The lines represent the fit according to the extended Voigt model as described in SI Text. It should be noted that for the low frequency measurements (below 1 Hz) the data is less reliable as the active retrograde flow can not be neglected in the long time regime, whereas the fit model does not consider such activity. At low frequencies the loss modulus becomes more important than the storage modulus, thus marking a viscous regime, whereas at higher frequencies, the growth cone behaves more like an elastic object. According to the model, it is possible to identify a plateau for the storage modulus, which gives a steady state Young’s modulus of E = 106 ± 21 Pa. The sketch and the inset illustrate the measurement method. A bead, glued to the cantilever of a SFM, penetrates the lamellipodium with an oscillating force (Inset, red), whereas the resulting deformation (Inset, black) is measured. The amplitude and the phase difference between force and deformation determine the viscoelastic properties at the oscillation frequency.

Fig. 2.

Internal stress within a neuronal growth cone. (A) Distribution of the momentary internal stress field in a neuronal growth cone. The color coding gives the stress magnitude in units of force per area (Pa = pN/μm²), and the black arrows indicate the stress direction. Scale bar is 10 μm. (B) Time averaged mean stress distribution of a full time series of internal stress fields (t = 10 min). The stress is highest in the transition zone, and decays toward the edge. (C) A close-up on the central part of the stress field in (A) shows that the internal stresses are contractile and arranged like dipoles.

Fig. 3.

Traction stress, internal stress, and the combined total stress of a neuronal growth cone. (A) Traction stress distribution of a neuronal growth cone. (B) Internal stress field of growth cone shown in A. Contractile stresses are localized at the central domain of the growth cone. (C) The total stress field of the growth cone reveals that the forces are generated at the central domain, and the cross-linked actin network transmits the stress to the substrate adhesions in the peripheral region. The black arrow represents the pulling force with which the neurite is pulling on the growth cone. (A–C) Color code gives stress magnitude in Pa. Scale bar is 10 μm.

Fig. 4.

Summarizing scheme of the forces involved in the actin cytoskeleton in growth cone motility. External forces such as neurite tension (black arrow) and the substrate stress (white arrows) are transmitted to the contracting myosin motors (blue arrows) by the viscoelastic actin networks of the lamellipodium and the actin cortex of the neurite. In this model, the effective link between these two types of forces are the force generating myosin motors in the transition zone.