Classic pulse-chase studies have shown that actin is conveyed in slow axonal transport, but the mechanistic basis for this movement is unknown. Recently, we reported that axonal actin was surprisingly dynamic, with focal assembly/disassembly events (“actin hotspots”) and elongating polymers along the axon shaft (“actin trails”). Using a combination of live imaging, superresolution microscopy, and modeling, in this study, we explore how these dynamic structures can lead to processive transport of actin. We found relatively more actin trails elongated anterogradely as well as an overall slow, anterogradely biased flow of actin in axon shafts. Starting with first principles of monomer/filament assembly and incorporating imaging data, we generated a quantitative model simulating axonal hotspots and trails. Our simulations predict that the axonal actin dynamics indeed lead to a slow anterogradely biased flow of the population. Collectively, the data point to a surprising scenario where local assembly and biased polymerization generate the slow axonal transport of actin without involvement of microtubules (MTs) or MT-based motors. Mechanistically distinct from polymer sliding, this might be a general strategy to convey highly dynamic cytoskeletal cargoes.
Pulse-chase and radio-labeling studies have shown that actin is transported in bulk along the axon at rates consistent with slow axonal transport. In a recent paper, using a combination of live cell imaging, super resolution microscopy and computational modeling, we proposed that biased polymerization of metastable actin fibers (actin trails) along the axon shaft forms the molecular basis of bulk actin transport. The proposed mechanism is unusual, and can be best described as molecular hitch hiking, where G-actin molecules are intermittently incorporated into actin fibers which grow preferably in anterograde direction giving rise to directed transport, released after the fibers collapse only to be incorporated into another fiber. In this paper, we use our computational model to make additional predictions that can be tested experimentally to further scrutinize our proposed mechanism for bulk actin transport. In the previous paper the caliber of our model axon, the density of the actin nucleation sites to form the metastable actin fibers, the length distribution of the actin trails and their growth rate were adapted to the biologic axons used for measurements. Here we predict how the transport rate will change with axon caliber, density of nucleation sites, nucleation rates and trail lengths. We also discuss why a simple diffusion-based transport mechanism can not explain bulk actin transport.
Classic pulse-chase studies have shown that actin is conveyed in slow axonal transport, but the mechanistic basis for this movement is unknown. Recently, we reported that axonal actin was surprisingly dynamic, with focal assembly/dis-assembly events (“hotspots”) and elongating polymers along the axon-shaft (“trails”). Using a combination of live imaging, super-resolution microscopy, and modeling, here we explore how these axonal actin dynamics can lead to processive transport. We found abundant actin nucleation, along with a slow, anterogradely-biased flow of actin in axon-shafts. Starting with first principles of monomer/filament assembly – and incorporating imaging data – we generated a quantitative model simulating axonal hotspots and trails. Our simulations predict that the axonal actin dynamics indeed lead to an anterogradely-biased flow of the population, at rates consistent with slow transport. Collectively, the data point to a surprising scenario where local assembly and biased polymerization generate the slow axonal transport of actin. This mechanism is distinct from polymer-sliding, and seems well suited to convey highly dynamic cytoskeletal cargoes.AcknowledgementsThis work was supported by an NIH grant to SR (R01NS075233). The authors thank Stephanie Gupton (UNC) for the Mena/Vasp constructs.
In this paper, we present an experimental setup and an associated mathematical model to study the synchronization of two self-sustained, strongly coupled, mechanical oscillators (metronomes). The effects of a small detuning in the internal parameters, namely, damping and frequency, have been studied. Our experimental system is a pair of spring wound mechanical metronomes; coupled by placing them on a common base, free to move along a horizontal direction. We designed a photodiode array based non-contact, non-magnetic position detection system driven by a microcontroller to record the instantaneous angular displacement of each oscillator and the small linear displacement of the base, coupling the two. In our system, the mass of the oscillating pendula forms a significant fraction of the total mass of the system, leading to strong coupling of the oscillators. We modified the internal mechanism of the spring-wound "clockwork" slightly, such that the natural frequency and the internal damping could be independently tuned. Stable synchronized and anti-synchronized states were observed as the difference in the parameters was varied in the experiments. The simulation results showed a rapid increase in the phase difference between the two oscillators beyond a certain threshold of parameter mismatch. Our simple model of the escapement mechanism did not reproduce a complete 180° out of phase state. However, the numerical simulations show that increased mismatch in parameters leads to a synchronized state with a large phase difference.
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