Cytoskeletal dynamics in response to tensile loading of mammalian axons

Abstract

In response to an applied tensile load, axons of cultured neurons exhibit a number of morphological responses. We designed and implemented a cell stretching device to study the cellular mechanisms governing these responses. Rat sensory neurons were seeded onto a flexible silicone substrate and imaged during substrate stretch. The positions of stationary mitochondria, docked to the axonal cytoskeleton, were determined before and after 10% stretch, and used to calculate the resulting "instantaneous" strain in regions of the axon. There was dramatic heterogeneity in strain along the length of the stretched axons, particularly in regions shorter than 20 μm. The substrate was then held at 10% strain and the axons imaged for 20 min during "relaxation." Both strain magnitude and variability were larger at small lengths in stretched axons during the initial phase of relaxation, but after 14 min, decreased to levels smaller than those seen in unstretched axons. Mitochondrial pairs in stretched axons showed uncoordinated movement with each other at all lengths, suggesting that cytoskeletal cohesion is reduced after stretch. Collectively, these data present the axonal cytoskeleton as a dynamic structure, which responds to stretch rapidly and locally. Globally, the axon behaves as a viscoelastic continuum. Below a characteristic length, though, it appears to behave as a series of independent linked elements, each with unique mechanical properties which suggests a length scale within which cytoskeletal structural elements may be altered to modulate the biomechanical response of the axon. Finally, testable hypotheses of strain accomodation in the axon are suggested.

Bidirectional actin transport is influenced by microtubule and actin stability

“…Mitochondria were labeled as previously reported using MitoTracker Red CMX-Ros [38], and imaged at a similar frame rate as actin, thus precluding the observation of very fast moving mitochondria [41,42]. Additional movies for specific experiments were captured at a higher frame rate (1/600 ms) using a 1009 objective.…”

“…Additionally, actin ''waves'' move anterogradely in vitro and in vivo [34][35][36], and patches of F-actin give rise to filopodia [20,37]. Finally, indirect evidence suggests that mechanical deformation enhances actin mobility within axons [38]. In short, both the mechanism of actin transport, and the state of actin during that transport remain unclear.…”

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

“…Two predominant approaches have been utilized to apply experimental forces to neurons. In the first approach, forces are applied to the entire neuron (Lu, Franze et al 2006;Chetta, Kye et al 2010;Lindqvist, Liu et al 2010). In the second approach, forces are applied directly to the axon by pulling on the growth cone.…”

Variability in Membrane Continuity Between Schwann Cells and Neurons

“…DRGs were dissected and placed on ice in 400 lL of DRG media (F-12 media, 10% FBS, 1% L-glutamate, and 1% Penicillin/Streptomycin). The samples were supplemented with 20 lg/mL of collagenase type IA (Sigma-Aldrich) and incubated at 37°C for 20 min (adapted from Oh et al 10,33 ). The samples were then centrifuged at 769g for 5 min and the media was removed.…”

“…This study investigated differences in the mobility of the plasma membrane in neurons and SCs, and compared the degree to which and length scale over which the cellular membranes of neurons and SCs behave as a mechanical continuum. In combination with high-resolution fluorescence imaging, we applied two different methods for correlation analysis previously used in other contexts 5,10,29 to quantify the continuity of the cellular membrane at various length scales during neuronal and SC motility. The establishment of neuronal contact was investigated as an additional factor influencing continuity; for neurons, such contact indicates a major step in maturity prior to axonal fasciculation, and for SCs, neuronal contact is required both for elongation along an axon and myelination.…”