Paul C. Letourneau

Neurite extension by peripheral and central nervous system neurons in response to substratum-bound fibronectin and laminin☆

Dissociated neurons from embryonic chick dorsal root and sympathetic ganglia (peripheral neurons) and from spinal cord and retina (central nervous system neurons) were cultured on plastic substrata treated with purified fibronectin and laminin. Both central and peripheral neurons attached to and extended neurites on laminin. In contrast, only peripheral neurons initiated neurites on fibronectin; central neurons cultured under identical conditions aggregated into clusters and did not extend neurites. Neurite length, number of neurites initiated, and extent of neurite branching on fibronectin- and laminin-treated substrata were evaluated and compared with similar measurements of neuronal response to poly-L-lysine-treated plastic. Poly-L-lysine provides an adhesive surface for neurite elongation, but fibronectin and laminin appear to promote more rapid neurite elongation. Our observations suggest that neuronal interaction with these glycoproteins may involve neuron-specific cell surface components. These responses to laminin and fibronectin in vitro may be related to the presence or absence of these glycoproteins in specific extracellular environments during specific developmental stages.

GUIDED NEURITE ELONGATION AND SCHWANN CELL INVASION INTO MAGNETICALLY ALIGNED COLLAGEN IN SIMULATED PERIPHERAL NERVE REGENERATION

High-strength magnetic fields were used to align collagen gel formed into 4-mm-diameter rods during the self-assembly of type I collagen monomers into fibrils. We developed an in vitro assay to study neurite elongation into the magnetically aligned collagen gel rods from dorsal root ganglia (DRG) explants placed onto one end of the rods. The depth of neurite elongation from chick embryo DRG neurons into these rods was found to be substantially greater than that observed in controls and increased with an increase in magnetic field strength, as did the collagen gel rod birefringence, indicative of collagen fibril alignment along the rod axis. Moreover, the axial bias of neurite elongation became more pronounced with an increase in magnetic field strength, presumably due to a contact guidance response of growth cones at the neurite tips. Coinvasion of Schwann cells from neonatal rat DRG was also studied in these assays using double immunolabeling. In the absence of serum, Schwann cells were highly associated with, and often trailed, elongating neurites. In the presence of serum, Schwann cells showed significantly higher rates of invasion and formed axially aligned chords reminiscent of bands of Büngner. These results may translate into an improved method of entubulation repair of transected peripheral nerves by directing and stimulating axonal growth through a tube filled with magnetically aligned collagen gel.

Ligand-induced changes in integrin expression regulate neuronal adhesion and neurite outgrowth

Receptors of the integrin family are expressed by every cell type and are the primary means by which cells interact with the extracellular matrix. The control of integrin expression affects a wide range of developmental and cellular processes, including the regulation of gene expression, cell adhesion, cell morphogenesis and cell migration. Here we show that the concentration of substratum-bound ligand (laminin) post-translationally regulates the amount of receptor (α6β1 integrin) expressed on the surface of sensory neurons. When ligand availability is low, surface amounts of receptor increase, whereas integrin RNA and total integrin protein decrease. Ligand concentration determines surface levels of integrin by altering the rate at which receptor is removed from the cell surface. Furthermore, increased expression of integrin at the cell surface is associated with increased neuronal cell adhesion and neurite outgrowth. These results indicate that integrin regulation maintains neuronal growth-cone motility over a broad range of ligand concentrations, allowing axons to invade different tissues during development and regeneration.

Immunoreactivity for laminin in the developing ventral longitudinal pathway of the brain.

The first long tract to form in the brain of a vertebrate embryo is the ventral longitudinal pathway. In order to investigate what chemical cues may guide nerve growth cones along this pathway, affinity-purified antibodies to laminin and collagen type IV were used to stain sections of mouse embryos from Embryonic Days 8 through 17. A monoclonal anti-neurofilament antibody was used to show the development of the ventral longitudinal pathway in relationship to immunoreactivity for laminin and collagen type IV. At Day 8 fluorescent immunoreactivity for laminin is bright in the external limiting membrane of the neural tube, but the neuroepithelium does not show bright laminin or neurofilament immunoreactivity. At E9 the ventral longitudinal pathway is forming and punctate immunoreactivity for laminin is present on the surfaces of neuroepithelial cells in the marginal zone, through which axons of the ventral pathway extend. Punctate immunofluorescence for laminin remains concentrated in the marginal zone on Days E10 through E14, but on E16 punctate immunofluorescence was much reduced, although immunoreactivity for laminin remained bright in the maturing pial and arachnoid membranes and on blood vessels in the brain. Immunoreactivity for collagen type IV was strong in the external limiting membrane and on blood vessels, but never showed concentrated punctate immunofluorescence in the marginal zone. These results indicate that laminin may be available on cell surfaces and in extracellular spaces as an adhesive ligand for growth cones during the formation of the ventral longitudinal pathway.

Characterization of spontaneous calcium transients in nerve growth cones and their effect on growth cone migration

This study examines the mechanisms of spontaneous and induced [Ca2+]i spiking in nerve growth cones and the effect of spikes on growth cone migration. Over a 10-20 min observation period, 29% of DRG growth cones undergo spontaneous and transient elevations in physiological extracellular Ca2+ ((Ca2+)o; 2 mM), whereas 67% of growth cones exposed to 20 mM (Ca2+)o exhibit similar [Ca2+]i spikes. Spontaneous [Ca2+]i spiking was not observed in neuronal cell bodies or nonneuronal cells. Ca2+ influx through non-voltage-gated Ca2+ channels was required for spontaneous [Ca2+]i spikes in growth cones, since removal of (Ca2+)o, or addition of the general Ca2+ channel blockers La3+ or Ni2+, reversibly blocked [Ca2+]i spiking, while blockers of the voltage-gated Ca2+ channels did not. Experiments using agents that influence intracellular Ca2+ stores suggest that Ca2+ stores may buffer and release Ca2+ during growth cone [Ca2+]i spikes. Growth cone migration was immediately and transiently inhibited by [Ca2+]i spikes, but eventually returned to prespike rates.

Neuronal contact guidance in magnetically aligned fibrin gels: effect of variation in gel mechano-structural properties

Neurite outgrowth from chick dorsal root ganglia entrapped in isotropic and magnetically aligned fibrin gels was studied, and the dependence on the diameter of the fibrin fibrils was characterized. The fibrin fibril diameter was varied, as inferred from turbidity measurements, by using different Ca2+ concentrations in the fibrin-forming solution, but this variation was accomplished without affecting the degree of magnetic-induced alignment, as directly visualized in fluorescently spiked gels. Magnetically aligned fibrin gels possessing different fibril diameters but similar alignment resulted in drastic changes in the contact guidance response of neurites, with no response in gels formed in 1.2 mM Ca2+ (having smaller fibril diameter, ca. 150 nm), but a strong response in gels formed in 12 and 30 mM Ca2+ (having larger fibril diameter, ca. 510 nm) with an attendant two-fold increase in neurite length. These changes are attributed to variation of the mechano-structural properties of the network of aligned fibrils as the fibril diameter is varied.

Micropatterning gradients and controlling surface densities of photoactivatable biomolecules on self-assembled monolayers of oligo(ethylene glycol) alkanethiolates

BACKGROUND Bioactive molecules that are covalently immobilized in patterns on surfaces have previously been used to control or study cell behavior such as adhesion, spreading, movement or differentiation. Photoimmobilization techniques can be used, however, to control not only the spatial pattern of molecular immobilization, termed the micropattern, but also the surface density of the molecules--a characteristic that has not been previously exploited. RESULTS Oligopeptides containing the bioactive Arg-Gly-Asp cell-adhesion sequence were immobilized upon self-assembled monolayers of an oligo(ethylene glycol) alkanethiolate in patterns that were visualized and quantified by autoradiography. The amount and pattern of immobilized peptide were controlled by manipulating the exposure of the sample to a UV lamp or a laser beam. Patterns of peptides, including a density gradient, were used to control the location and number of adherent cells and also the cell shape. CONCLUSIONS A photoimmobilization technique for decorating surfaces with micropatterns that consist of variable densities of bioactive molecules is described. The efficacy of the patterns for controlling cell adhesion and shape has been demonstrated. This technique is useful for the study of cell behavior on micropatterns.

Calcium-dependent interaction of Lis1 with IQGAP1 and Cdc42 promotes neuronal motility

Lis1 gene defects impair neuronal migration, causing the severe human brain malformation lissencephaly. Although much is known about its interactions with microtubules, microtubule-binding proteins such as CLIP-170, and with the dynein motor complex, the response of Lis1 to neuronal motility signals has not been elucidated. Lis1 deficiency is associated with deregulation of the Rho-family GTPases Cdc42, Rac1 and RhoA, and ensuing actin cytoskeletal defects, but the link between Lis1 and Rho GTPases remains unclear. We report here that calcium influx enhances neuronal motility through Lis1-dependent regulation of Rho GTPases. Lis1 promotes Cdc42 activation through interaction with the calcium sensitive GTPase scaffolding protein IQGAP1, maintaining the perimembrane localization of IQGAP1 and CLIP170 and thereby tethering microtubule ends to the cortical actin cytoskeleton. Lis1 thus is a key component of neuronal motility signal transduction that regulates the cytoskeleton by complexing with IQGAP1, active Cdc42 and CLIP-170 upon calcium influx.

Growth cone guidance by substrate-bound laminin pathways is correlated with neuron-to-pathway adhesivity

Substrate-bound laminin pathways prepared by the method of Hammarback et al. [J.A. Hammarback, S.L. Palm, L.T. Furcht, and P.C. Letourneau (1985). J. Neurosci. Res. 13, 213-220] guided peripheral nervous system neurites (dissociated dorsal root ganglia and sympathetic ganglia) and central nervous system neurites (dissociated spinal cord and brain). Guidance of individual growth cones by 7- to 10-micron-wide laminin pathways was observed using time-lapse video microscopy. Fibronectin pathways, produced by the method used for laminin pathways, did not guide neurites. The guidance effect of laminin pathways was quantified and found to correlate with the concentration of laminin initially applied to the substratum. The concentration of laminin initially applied to the substratum also correlated with increased adhesivity of dorsal root ganglia (DRG) neurons to laminin constituting the pathways relative to uv-irradiated laminin that borders the pathways. The guidance effect of laminin pathways was blocked by anti-laminin antibodies or by laminin but not by anti-fibronectin antibodies. This study demonstrates that guidance of DRG neurites by laminin occurs at the growth cone in a manner consistent with the hypothesis of guidance by differential neuron-to-substratum adhesivity.

Actin turnover is required to prevent axon retraction driven by endogenous actomyosin contractility

Growth cone motility and guidance depend on the dynamic reorganization of filamentous actin (F-actin). In the growth cone, F-actin undergoes turnover, which is the exchange of actin subunits from existing filaments. However, the function of F-actin turnover is not clear. We used jasplakinolide (jasp), a cell-permeable macrocyclic peptide that inhibits F-actin turnover, to study the role of F-actin turnover in axon extension. Treatment with jasp caused axon retraction, demonstrating that axon extension requires F-actin turnover. The retraction of axons in response to the inhibition of F-actin turnover was dependent on myosin activity and regulated by RhoA and myosin light chain kinase. Significantly, the endogenous myosin-based contractility was sufficient to cause axon retraction, because jasp did not alter myosin activity. Based on these observations, we asked whether guidance cues that cause axon retraction (ephrin-A2) inhibit F-actin turnover. Axon retraction in response to ephrin-A2 correlated with decreased F-actin turnover and required RhoA activity. These observations demonstrate that axon extension depends on an interaction between endogenous myosin-driven contractility and F-actin turnover, and that guidance cues that cause axon retraction inhibit F-actin turnover.