Depending on Its Nano-Spacing, ALCAM Promotes Cell Attachment and Axon Growth

<div><p>ALCAM is a member of the <u>c</u>ell <u>a</u>dhesion <u>m</u>olecule (CAM) family which plays an important role during nervous system formation. We here show that the two neuron populations of developing <u>d</u>orsal <u>r</u>oot <u>g</u>anglia (DRG) display ALCAM transiently on centrally and peripherally projecting axons during the two phases of axon outgrowth. To analyze the impact of ALCAM on cell adhesion and axon growth, DRG single cells were cultured on ALCAM-coated coverslips or on nanopatterns where ALCAM is presented in physiological amino-carboxyl terminal orientation at highly defined distances (29, 54, 70, 86, and 137 nm) and where the interspaces are passivated to prevent unspecific protein deposition. Some axonal features (branching, lateral deviation) showed density dependence whereas others (number of axons per neuron, various axon growth parameters) turned out to be an all-or-nothing reaction. Time-lapse analyses revealed that ALCAM density has an impact on axon velocity and advance efficiency. The behavior of the sensory axon tip, the growth cone, partially depended on ALCAM density in a dose-response fashion (shape, dynamics, detachment) while other features did not (size, complexity). Whereas axon growth was equally promoted whether ALCAM was presented at high (29 nm) or low densities (86 nm), the attachment of non-neuronal cells depended on high ALCAM densities. The attachment of non-neuronal cells to the rather unspecific standard proteins presented by conventional implants designed to enhance axonal regeneration is a severe problem. Our findings point to ALCAM, presented as 86 nm pattern, for a promising candidate for the improvement of such implants since this pattern drives axon growth to its full extent while at the same time non-neuronal cell attachment is clearly reduced.</p> </div

Axon branching on various substrates.

<p>(<b>A</b>) Immunofluorescence labeling shows a neuron (identified by β3 tubulin staining) with one branched axon. (<b>B</b>) Quantification of branches per neuron on glass coated with various substrate molecules. (<b>C</b>) Quantification of branches per neuron on various ALCAM nanopatterns. Error bars represent SEM; for statistical analyses, two-way ANOVA followed by post-hoc comparisons using two-tailed Student's t test with Bonferroni-Holm corrections were performed (***P<0.001, **P<0.01, *P<0.05).</p

Growth cone behavior on various substrates.

<p>(<b>A</b>) Growth cone tracks (dark red) on ALCAM nanopatterns or PLL-/ALCAM-coated glass as observed by time-lapse phase contrast microscopy. Each dot represents the position of the growth cone neck (localized every minute); the blue line depicts the distance covered within the 60 min observation time and the dark green line the line of best fit (lobf, overall growth direction). For additional tracks, see Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040493#pone.0040493.s002" target="_blank">Figure S2</a>. (<b>B</b>) Quantification of growth cone velocity (determined every minute as advance on line of best fit) on PLL-/ALCAM-coated glass or on ALCAM nanopatterns for ten growth cones each. (<b>C</b>) Quantification of track lengths and distances (see A) on various substrates for ten growth cones each. (<b>D</b>) Quantification of the lateral track deviations from the distance line (see A) per minute on various substrates for ten growth cones each. (<b>E</b>) Growth cone behavior, i.e. advance, pause, and retraction (green: >1 µm/min, yellow: −1 to +1 µm/min, and red: <−1 µm/min, respectively) on various ALCAM nanopatterns of ten different axons each, plotted for 60 min. Velocity, duration, and frequency of the three types of behavior were determined with respect to the line of best fit. For growth cone behavior on PLL/-ALCAM-coated glass, see Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040493#pone.0040493.s003" target="_blank">Figure S3</a>. Error bars represent SEM; for statistical analyses, two-way ANOVA followed by post-hoc comparisons using two-tailed Student's t test with Bonferroni-Holm corrections were performed (***P<0.001, **P<0.01, *P<0.05).</p

Axon length on various substrates.

<p>(<b>A</b>) Immunofluorescence labeling shows a neuron (identified by β3 tubulin staining) with two branched axons. (<b>B</b>) Quantification of length of the longest axon of a neuron (minimal length: 30 µm) on uncoated glass (UCG: n = 280) and glass coated with various substrate molecules (PLL: n = 833; ALCAM: n = 272; laminin: n = 104) or (<b>C</b>) various ALCAM nanopatterns (29 nm: n = 168; 54 nm: n = 463; 70 nm: n = 265; 86 nm: n = 402). As no differences in axon length on uncoated glass and glass coated with 50 µg/ml ALCAM were observed, axon lengths on glass coated with various ALCAM concentrations were not quantified.</p

Adhesion of DRG cells to conventional substrates and nanopatterns.

<p>Immunofluorescence labeling shows neurons (identified by β3 tubulin staining) and non-neurons (visualized by F-actin labeling) attached to uncoated glass or glass coated with various molecules and to 29 nm or 137 nm nanopatterned ALCAM substrates.</p

Growth cone dynamics on various substrates.

<p>(<b>A</b>) Phase contrast micrographs of growth cones on various ALCAM nanopatterns. Note that duration (d) and frequency (f) of growth cone size changes (spreading, shrinkage) do not differ significantly (between p<0.07 and p<0.6) on the various nanopatterns (ten growth cones each). (<b>B</b>) Quantification of growth cone area, (<b>C</b>) perimeter, and (<b>D</b>) complexity (perimeter/) on PLL-/ALCAM-coated glass or on various ALCAM nanopatterns (155 growth cones quantified for each substrate). (<b>E</b>) Quantification of change in growth cone size (spreading/shrinkage) expressed as percentage of growth cone area on various ALCAM nanopatterns (ten growth cones each; monitored for 300 sec, every 10 sec). (<b>F</b>) Quantification of growth cone width (expressed as percentage of growth cone length) on various ALCAM nanopatterns. Error bars represent SEM; for statistical analyses, two-way ANOVA followed by post-hoc comparisons using two-tailed Student's t test with Bonferroni-Holm corrections were performed (***P<0.001, **P<0.01, *P<0.05).</p

Axon formation on various substrates.

<p>(<b>A</b>) Immunofluorescence labeling shows a neuron (identified by β3 tubulin staining) with two axons. (<b>B</b>) Quantification of axons formed by a neuron on uncoated glass (UCG) or glass coated with various substrate molecules. Table indicates the proportion of neurons with one, two, or more axons (n.d. = not detected). (<b>C</b>) Quantification of axons formed by a neuron on various ALCAM nanopatterns. Error bars represent SEM; for statistical analyses, two-way ANOVA followed by post-hoc comparisons using two-tailed Student's t test with Bonferroni-Holm corrections were performed (***P<0.001, **P<0.01, *P<0.05).</p

ALCAM distribution during DRG development.

<p>(<b>A</b>) Immunofluorescence labeling of ALCAM (NgCAM double staining for visualization of axons) in sections shows that -at E5- ALCAM is present in DRG, central nerve (CN), peripheral nerve (PN), and dorsal root entry zone (DREZ) and is absent from the spinal cord (SC) proper (except of ventral floor plate). At E7, ALCAM is predominantly present in the DREZ, and only at lower levels in PN and CN. At E9, levels of ALCAM are high again in the PN and CN and almost unchanged in the DREZ; within the DRG, ALCAM is only present in the dorso-medial part. At E11, E13, E15, and E20, all regions show only weak levels of ALCAM. Inserts in E15 and E20 show the PN. Scale bars: 250 µm. (<b>B</b>) Quantification of ALCAM levels by immunofluorescence intensity measurements (arbitrary units) in four DRG regions during embryonic development (E5–E20). Error bars represent SEM.</p