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.

High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices.

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs).

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.

Multiwall carbon nanotubes directly promote fibroblast-myofibroblast and epithelial-mesenchymal transitions through the activation of the TGF-ß/Smad signaling pathway.

A number of studies have demonstrated that MWCNTs induce granuloma formation and fibrotic responses in vivo, and it has been recently reported that MWCNT-induced macrophage activation and subsequent TGF-ß secretion contribute to pulmonary fibrotic responses. However, their direct effects against alveolar type-II epithelial cells and fibroblasts and the corresponding underlying mechanisms remain largely unaddressed. Here, MWCNTs are reported to be able to directly promote fibroblast-to-myofibroblast conversion and the epithelial-mesenchymal transition (EMT) through the activation of the TGF-ß/Smad signaling pathway. Both of the cell transitions may play important roles in MWCNT-induced pulmonary fibrosis. Firstly, in-vivo and in-vitro data show that long MWCNTs can directly interact with fibroblasts and epithelial cells, and some of them may be uptaken into fibroblasts and epithelial cells by endocytosis. Secondly, long MWCNTs can directly activate fibroblasts and increase both the basal and TGF-ß1-induced expression of the fibroblast-specific protein-1, α-smooth muscle actin, and collagen III. Finally, MWCNTs can induce the EMT through the activation of TGF-ß/Smad2 signaling in alveolar type-II epithelial cells, from which some fibroblasts involved in pulmonary fibrosis are thought to originate. These observations suggest that the activation of the TGF-ß/Smad2 signaling plays a critical role in the process of the fibroblast-to-myofibroblast transition and the EMT induced by MWCNTs.

Tuning the adhesive geometry of neurons: length and polarity control.

Neurons acquire their functional and morphological axo-dendritic polarity by extending, from competing minor processes (neurites), one long axon among numerous dendrites. We employed complementary sets of micropatterns built from 2 and 6 µm wide stripes of various lengths to constrain hippocampal neuron shapes. Using these geometries, we have (i) limited the number of neuronal extensions to obtain a minimal in vitro system of bipolar neurons and (ii) controlled the neurite width during growth by the generation of a progressive cell shape asymmetry on either side of the cellular body. From this geometrical approach, we gained a high level of control of each neurite length and of the localization of axonal specification. To analyze these results, we developed a model based on a width and polarization dependent neurite elongation rate and on the existence of a critical neurite length that sets the axonal fate. Our data on the four series of micro-patterns developed for this study are described by a single set of growth parameters, well supported by experiments. The control of neuronal shapes by adhesive micro-patterns thereby offers a novel paradigm to follow the dynamical process of neurite lengthening and competition through the process of axonal polarization.