Abstract

AbstractNeuronal differentiation is under the tight control of biochemical and physical information arising from micro-environment. Here, through a panel of poly-L-lysine micropatterns, we wished to assay how external geometrical constraints of neurons may modulate axonal polarization. Constraints applied to either the cell body or to the neurite directions revealed the existence of a differential mechanical tension between the nascent axon and other neurites. Also, we show that centrosome location is not predictive of axonal polarization but responds to the force exerted by the nascent axon. Using curved trajectories for neurite growth inhibited axonal differentiation and prevented formation of multiple axons normally induced by cytochalasin or taxol treatments. Finally we provide evidence that microtubules act as curvature sensors during neuronal differentiation. Thus, biomechanics coupled to physical constraints might be the first level of regulation during neuronal development, primary to biochemical and guidance regulations.

Highlights

  • By varying the orientation of the directions of neurite growth, we show that the neurite that displays the highest tension becomes the axon, suggesting that axonal specification may result from the achievement of the highest mechanical tension

  • Curved lines conflicted multiple-axon-promoting effect of cytoskeleton drugs We further investigated the inhibitory role of curved lines toward axonal polarization by performing experiments using pharmacological compounds known to promote the formation of multiple axons (MA) in hippocampal neurons grown on flat unconstrained substrates[21, 22]

  • Neuronal polarization is sensitive to external physical constraints Neuronal differentiation in vivo and axonal specification are both under the control of a large number of parameters including adhesion[23, 24] to the extra-cellular matrix, complex responses to guidance molecules[8], and physical constraints[11, 12]

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Summary

Methods

Micro-pattern fabrication Poly-L-lysine patterns were transferred on glass substrates silanized with 3GPS40 using UV classical photolithography steps, including Shipley S1805 photoresist spinning (4000 rpm, 0.5μm thickness, 115°C annealing step for 1 min), insulation through a mask, development (Microposit concentrate 1:1, Shipley), PLL deposition (1mg/ml one night), and lift-off using an ultra-sound ethanol bath. Neurons were fixed and permeabilized for 30 min in 3.7% formaldehyde/0.5% glutaraldehyde/0.1% triton X100. For Ankyrin G immunostaining 6-7 days in vitro (DIV), neurons were fixed for 6 min in methanol (-20°C). Primary antibodies (mouse mAbs against Ankyrin G (Santa Cruz); Tau (clone tau-1, Millipore); MAP2 (clone AP-20, Sigma); rat mAb against tubulin (cloneYL1/2), and rabbit γ tubulin Centriole analysis Image sortings were performed using Labview vision software (National Instrument) and a semi-automatic interface that positioned the datum lines associated with each pattern. Density maps of centriole positions were made by a custommade Matlab program using an algorithm for smoothing of two-dimensional histograms[42]. Statistics All percentage comparisons were performed using χ2 tests as implemented in Prism 4.0 (GraphPad Software, La Jolla, USA)

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