Two Distinct Filopodia Populations at the Growth Cone Allow to Sense Nanotopographical Extracellular Matrix Cues to Guide Neurite Outgrowth

Abstract

BackgroundThe process of neurite outgrowth is the initial step in producing the neuronal processes that wire the brain. Current models about neurite outgrowth have been derived from classic two-dimensional (2D) cell culture systems, which do not recapitulate the topographical cues that are present in the extracellular matrix (ECM) in vivo. Here, we explore how ECM nanotopography influences neurite outgrowth.Methodology/Principal FindingsWe show that, when the ECM protein laminin is presented on a line pattern with nanometric size features, it leads to orientation of neurite outgrowth along the line pattern. This is also coupled with a robust increase in neurite length. The sensing mechanism that allows neurite orientation occurs through a highly stereotypical growth cone behavior involving two filopodia populations. Non-aligned filopodia on the distal part of the growth cone scan the pattern in a lateral back and forth motion and are highly unstable. Filopodia at the growth cone tip align with the line substrate, are stabilized by an F-actin rich cytoskeleton and enable steady neurite extension. This stabilization event most likely occurs by integration of signals emanating from non-aligned and aligned filopodia which sense different extent of adhesion surface on the line pattern. In contrast, on the 2D substrate only unstable filopodia are observed at the growth cone, leading to frequent neurite collapse events and less efficient outgrowth.Conclusions/SignificanceWe propose that a constant crosstalk between both filopodia populations allows stochastic sensing of nanotopographical ECM cues, leading to oriented and steady neurite outgrowth. Our work provides insight in how neuronal growth cones can sense geometric ECM cues. This has not been accessible previously using routine 2D culture systems.

The Effect of Including the C2 Insert of Nonmuscle Myosin II-C on Neuritogenesis

“…Possibly, other isoforms of NM II-C compensate for the loss of NM II-C1C2, or the cells are exposed to external cues that bypass the requirement for NM II-C1C2. All isoforms of NM II, including possible alternatively spliced isoforms of NM II-B and NM II-C, are found in neuronal cells (31,33,43), giving rise to complexity as to whether these isoforms have different or redundant functions in neuronal cells. Previously, NM II-C was shown to be involved in multitasking in neuronal cells (33).…”

“…Shortening of Filopodial Length-Filopodia play a significant role in neurite protrusion (42,43). We used actin staining to visualize filopodia following inhibition of NM II-C1C2 protein in Neuro-2a cells during differentiation.…”

Nanoscale Surface Topography Reshapes Neuronal Growth in Culture

“…An accelerated directional neurite elongation was reported on sub-micrometric grooves formed on polyurethane acrylate substrates, providing a one-dimensional anisotropic adhesive environment to cells 26 . This phenomenon was associated to the presence of a stable, fully adherent filopodia population aligned with the grooves and a relative destabilization of the perpendicular filopodia that experience the substrate striations transversally.…”

Biomaterials for Enhancing Neuronal Repair

“…Neuronal migration and neurite extension or directionality can be guided by the addition of topographical cues to a substrate, which enhances control by providing a Abbreviations: DALYs, Disability-adjusted life years; ECM, Extracellular matrix; PLLA, Polylactic acid; DRG, Dorsal root ganglia; ESCs, Embryonic stem cells; iPSCs, Induced pluripotent stem cells; hBMSCs, Human bone marrow stromal cells; S-RuMs, Self-rolled-up membranes; NGCs, Nerve-guide-conduits or nerveguidance-channels; PHB-HV, Poly(3-hydroxybutyrate-co-3-hydroxyvalerate); EFs, Electric fields; DC, Direct current; AC, Alternating current; PPy, Polypyrrole; E, Elastic modulus; NPCs, Neural progenitor cells; CSF, Cerebral spinal fluid. recognizable path (Jang et al, 2010;Baranes et al, 2012). In the case of damaged spinal neurons, a 3D scaffold can provide a sturdy framework to support directional neurite regrowth.…”