Impact of neurite alignment on organelle motion

Abstract

Intracellular transport is pivotal for cell growth and survival. Malfunctions in this process have been associated with devastating neurodegenerative diseases, posing a need for deeper understanding of the involved mechanisms. Here, we used an experimental methodology that lead neurites of differentiated PC12 cells in either of two configurations: an one-dimensional, where the neurites align along lines, or a two-dimensional configuration, where the neurites adopt a random orientation and shape on a flat substrate. We subsequently monitored the motion of functional organelles, the lysosomes, inside the neurites. Implementing a time-resolved analysis of the mean-squared displacement, we quantitatively characterized distinct motion modes of the lysosomes. Our results indicate that neurite alignment gives rise to faster diffusive and super-diffusive lysosomal motion in comparison to the situation where the neurites are randomly oriented. After inducing lysosome swelling through an osmotic challenge by sucrose, we confirmed the predicted slowdown in diffusive mobility. Surprisingly we found that the swelling-induced mobility change affected each of the (sub-/super-) diffusive motion modes differently and depended on the alignment configuration of the neurites. Our findings imply that intracellular transport is significantly and robustly dependent on cell morphology, which might be in part controlled by the extracellular matrix.

Effect of G4C2 Repeat Expansions on the Motion of Lysosomes Inside Neurites

“…In the experiments presented here, except for investigating the effect of the ALS-associated HRE mutation on lysosomal trafficking inside neurites, we were in parallel looking into how the neurite geometry interferes with the motion. We have previously detected and quantitatively characterized a perturbation in the intracellular environment that affected lysosomal motion, while in parallel confirming a positive effect of neurite alignment on organelle motion, even in the presence of the perturbation [44]. Here, we verified a disturbance of lysosomal transport but we didn't detect a consistently enhanced transport inside aligned neurites in the presence of the (G 4 C 2 ) 20 or (G 4 C 2 ) 39 RNA and DPR proteins.…”

“…Using this analysis we have previously identified and quantitatively characterized a sucroseinduced perturbation in the cellular environment that affects lysosomal motion, and we have verified the fact that neurite geometry has a distinct impact on organelle transport [44].…”

“…The local Mean Squared Displacement (lMSD) was calculated for each trajectory as described previously [44,46]. Briefly, the MSD was calculated for each data point of the entire trajectory using a rolling window of 2.22 seconds (N=120, in eq.…”

“…In the first configuration, the neurites were guided to grow along chemicallypatterned, with attractant protein, lines, whereas in the second configuration, they were allowed to adopt any orientation and geometry on the two-dimensional surface. The generation of both neurites geometries has been described in detail previously [44]. Representative volume-view images of two differentiated PC12 cells and their neurites adopting the respective geometries are shown in Fig.…”

“…Along the same lines, and aiming to shed more light on the association between the HRE products and defective axonal transport, we used our previously established experimental setup of neuron-like, differentiated PC12 cells, on a chemically-patterned substrate [44,45].…”