Neuronal growth from a volume perspective.

Microfluidic-based fluorescent exclusion method allows to tackle the issue of neuronal growth from a volume perspective. Based on this technology, we studied the two main actin-rich structures accompanying the early stages of neuron development, i.e. growth cones, located at the tip of growing neuronal processes, and propagative actin waves. Our work reveals that growth cones tend to loose volume during their forward motion, as do actin waves during their journey from the cell body to the tip of neuronal processes, before the total transfer of their remaining volume to the growth cone. Actin waves seem thus to supply material to increasingly distant growth cones as neurons develop. In addition, our work may suggest the existence of a membrane recycling phenomena associated to actin waves as a pulsatile anterograde source of material and by a continuous retrograde transport.

Geometrical Determinants of Neuronal Actin Waves.

Hippocampal neurons produce in their early stages of growth propagative, actin-rich dynamical structures called actin waves. The directional motion of actin waves from the soma to the tip of neuronal extensions has been associated with net forward growth, and ultimately with the specification of neurites into axon and dendrites. Here, geometrical cues are used to control actin wave dynamics by constraining neurons on adhesive stripes of various widths. A key observable, the average time between the production of consecutive actin waves, or mean inter-wave interval (IWI), was identified. It scales with the neurite width, and more precisely with the width of the proximal segment close to the soma. In addition, the IWI is independent of the total number of neurites. These two results suggest a mechanistic model of actin wave production, by which the material conveyed by actin waves is assembled in the soma until it reaches the threshold leading to the initiation and propagation of a new actin wave. Based on these observations, we formulate a predictive theoretical description of actin wave-driven neuronal growth and polarization, which consistently accounts for different sets of experiments.

Multiprotein Printing by Light-Induced Molecular Adsorption.

Light-induced molecular adsorption of proteins (LIMAP) allows for quantitative sub-micrometer-resolution printing of multiple biomolecules. Surface-bound gradients are patterned within minutes over an entire glass cover-slip. LIMAP is used to perform selective immuno-assays, to dynamically control the adhesion of individual cells, and to achieve hierarchical co-cultures instrumental for tissue engineering.

Nanoscale surface topography reshapes neuronal growth in culture.

Neurons are sensitive to topographical cues provided either by in vivo or in vitro environments on the micrometric scale. We have explored the role of randomly distributed silicon nanopillars on primary hippocampal neurite elongation and axonal differentiation. We observed that neurons adhere on the upper part of nanopillars with a typical distance between adhesion points of about 500 nm. These neurons produce fewer neurites, elongate faster, and differentiate an axon earlier than those grown on flat silicon surfaces. Moreover, when confronted with a differential surface topography, neurons specify an axon preferentially on nanopillars. As a whole, these results highlight the influence of the physical environment in many aspects of neuronal growth.

How morphological constraints affect axonal polarity in mouse neurons.

Neuronal differentiation is under the tight control of both biochemical and physical information arising from neighboring cells and micro-environment. Here we wished to assay how external geometrical constraints applied to the cell body and/or the neurites of hippocampal neurons may modulate axonal polarization in vitro. Through the use of a panel of non-specific poly-L-lysine micropatterns, we manipulated the neuronal shape. By applying geometrical constraints on the cell body we provided evidence that centrosome location was not predictive of axonal polarization but rather follows axonal fate. When the geometrical constraints were applied to the neurites trajectories we demonstrated that axonal specification was inhibited by curved lines. Altogether these results indicated that intrinsic mechanical tensions occur during neuritic growth and that maximal tension was developed by the axon and expressed on straight trajectories. The strong inhibitory effect of curved lines on axon specification was further demonstrated by their ability to prevent formation of multiple axons normally induced by cytochalasin or taxol treatments. Finally we provided evidence that microtubules were involved in the tension-mediated axonal polarization, acting 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.

Neuronal architectures with axo-dendritic polarity above silicon nanowires.

An approach is developped to gain control over the polarity of neuronal networks at the cellular level by physically constraining cell development by the use of micropatterns. It is demonstrated that the position and path of individual axons, the cell extension that propagates the neuron output signal, can be chosen with a success rate higher than 85%. This allows the design of small living computational blocks above silicon nanowires.