Non-invasive Neurite Mechanics in Differentiated PC12 Cells

Fernanda Gárate, María Pertusa, Yahaira Arana and Roberto Bernal

Thermal Fluctuations Spectroscopy (TFS) in combination with novel optical-based instrumentation was used to study mechanical properties of cell-cultured neurites with a spatial resolution limited only by the light diffraction. The analysis of thermal fluctuations together with a physical model of cellular elasticity allow us to determine relevant mechanical properties of neurite as axial tension σ, flexural rigidity B, plasma membrane tension γ, membrane bending rigidity K, and cytoskeleton to membrane-coupling ρbk, whose values are consistent with previously reported values measured using invasive approaches. The value obtained for the membrane-coupling parameter was used to estimate the average number of coupling elements between the plasma membrane and the cytoskeleton that fell in the range of 30 elements per area of the laser spot used to record the fluctuations. Furthermore, to expand the TFS analysis, we investigate the correlation between F-actin linear density and the mechanical features of PC12 neurites. Using a hybrid instrument that combines TFS and a simple fluorescent technique, our results show that the fluctuations are related with the F-actin concentration. These measurements have an advantage of not requiring the application of an external force, allowing as to directly establish a correlation between changes in the mechanical parameters and cytoskeleton-protein concentrations. The sensibility of our method was also tested by the application of TFS technique to PC12 neurite under Paraformaldehyde and Latrunculin-A effect. These results show a dramatic modification in the fluctuations that are consistent with the reported effect of these drugs, confirming the high sensitivity of this technique. Finally, the thermal fluctuation approach was applied to DRG axons to show that its utility is not limited to studies of PC12 neurites, but it is suitable to measure the general characteristic of various neuron-like cells.

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Cell Mechanotransduction With Piconewton Forces Applied by Optical Tweezers

Fabio Falleroni, Vincent Torre and Dan Cojoc

Mechanical stresses are always present in the cellular environment and mechanotransduction occurs in all cells. Although many experimental approaches have been developed to investigate mechanotransduction, the physical properties of the mechanical stimulus have yet to be accurately characterized. Here, we propose a mechanical stimulation method employing an oscillatory optical trap to apply piconewton forces perpendicularly to the cell membrane, for short instants. We show that this stimulation produces membrane indentation and induces cellular calcium transients in mouse neuroblastoma NG108-15 cells dependent of the stimulus strength and the number of force pulses.

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Cdc42 and RhoA reveal different spatio-temporal dynamics upon local stimulation with Semaphorin-3A

Federico Iseppon, Luisa M. R. Napolitano, Vincent Torre and Dan Cojoc

Small RhoGTPases, such as Cdc42 and RhoA, are key players in integrating external cues and intracellular signaling pathways that regulate growth cone (GC) motility. Indeed, Cdc42 is involved in actin polymerization and filopodia formation, whereas RhoA induces GC collapse and neurite retraction through actomyosin contraction. In this study we employed Förster Resonance Energy Transfer (FRET) microscopy to study the spatio-temporal dynamics of Cdc42 and RhoA in GCs in response to local Semaphorin-3A (Sema3A) stimulation obtained with lipid vesicles filled with Sema3A and positioned near the selected GC using optical tweezers. We found that Cdc42 and RhoA were activated at the leading edge of NG108-15 neuroblastoma cells during spontaneous cycles of protrusion and retraction, respectively. The release of Sema3A brought to a progressive activation of RhoA within 30 s from the stimulus in the central region of the GC that collapsed and retracted. In contrast, the same stimulation evoked waves of Cdc42 activation propagating away from the stimulated region. A more localized stimulation obtained with Sema3A coated beads placed on the GC, led to Cdc42 active waves that propagated in a retrograde manner with a mean period of 70 s, and followed by GC retraction. Therefore, Sema3A activates both Cdc42 and RhoA with a complex and different spatial-temporal dynamics.

DOI