Recycling of the cell adhesion molecule L1 in axonal growth cones

Abstract

The cell adhesion molecule (CAM) L1 plays crucial roles in axon growth in vitro and in the formation of major axonal tracts in vivo. It is generally thought that CAMs link extracellular immobile ligands with retrogradely moving actin filaments to transmit force that pulls the growth cone forward. However, relatively little is known about the fate of CAMs that have been translocated into the central (C)-domain of the growth cone. We have shown previously that L1 is preferentially endocytosed at the C-domain. In the present study, we further analyze the subcellular distribution of endocytic organelles containing L1 at different time points and demonstrate that internalized L1 is transported into the peripheral (P)-domain of growth cones advancing via an L1-dependent mechanism. Internalized L1 is found in vesicles positioned along microtubules, and the centrifugal transport of these L1-containing vesicles is dependent on dynamic microtubules in the P-domain. Furthermore, we show that endocytosed L1 is reinserted into the plasma membrane at the leading edge of the P-domain. Monitoring recycled L1 reveals that it moves retrogradely on the cell surface into the C-domain. In contrast, the growth cone advancing independently of L1 internalizes and recycles L1 within the C-domain. For the growth cone to advance, the leading edge needs to establish strong adhesive interactions with the substrate while attachments at the rear are released. Recycling L1 from the C-domain to the leading edge provides an effective way to create asymmetric L1-mediated adhesion and therefore would be critical for L1-based growth cone motility.

Fig. 1.

Subcellular distribution of endocytosed L1 in growth cones. DRG neurons cultured on L1 (A–D) or laminin (E–H) were incubated with anti-L1 Fab for 15 min (A, B, E,F) or 30 min (C, D,G, H) to allow for internalization of the Fab bound to L1. The cells were double-labeled for NCAM to outline the growth cone structure. In superimposed images (A, C, E, G), endocytosed L1 is colored red, and NCAM is colored green. To facilitate visualization of endocytosed L1, the red channel only is shown inblack and white (B, D, F, H). Scale bars: A, B, 10 μm; C, D, 10 μm; E, F, 10 μm; G, H, 10 μm.

Fig. 2.

A, Schematic representation of the growth cone showing the P- and C-domains. For analyses of distribution patterns of endocytosed L1, the P-domain was defined as the area consisting of the filopodia and the lamellar regions within 3 μm of the leading edge based on Schmidt et al. (1995). B, Changes of distribution of endocytosed L1 over time in growth cones migrating on L1. Endocytosed L1 was visualized as shown in Figure 1, and the number of L1-positive endocytic organelles in the P-domain was categorized into four groups. Shown are the percentages of growth cones in each group at the 15 min (n = 48) and 30 min (n = 46) time points. There was a statistically significant difference between the two time points (p < 0.0001). C, Changes of distribution of endocytosed L1 over time in growth cones migrating on laminin. Shown are the percentages of growth cones in each group at the 15 min (n = 46) and 30 min (n = 50) time points.

Fig. 3.

A, Localization of microtubules and endocytosed L1 in a growth cone. DRG neurons cultured on L1 were incubated with anti-L1 antibody for 30 min to allow for internalization of the antibody bound to L1. The cells were fixed and double-labeled for microtubules using an antibody against tyrosinated α-tubulin. Shown is a superimposed image in which endocytosed L1 is colored ingreen and microtubules are colored inred. Arrowheads indicate examples of endocytosed L1 in vesicles positioned along the microtubules. B, C, Effects of taxol on microtubule organization in growth cones. DRG neurons cultured on L1 were pretreated with DMSO (B) or 10 nm taxol (C) for 1 hr and labeled for microtubules using an antibody against tyrosinated α-tubulin (red). The cells were double-labeled for NCAM to outline the growth cone structure (green). D, E, An effect of taxol on the subcellular distribution of endocytosed L1 in growth cones migrating on L1. After pretreatment with DMSO (D) or 10 nm taxol (E) for 1 hr, DRG neurons were incubated with anti-L1 antibody for 30 min to allow for internalization of the antibody bound to L1. The cells were double-labeled for endocytosed L1 (red) and NCAM to outline the growth cone structure (green). Scale bars, 10 μm.

Fig. 4.

Taxol-induced changes of distribution of endocytosed L1 in growth cones migrating on L1. Endocytosed L1 at the 30 min time points was visualized as shown in Figure 3,D and E. A total of 60 taxol-treated growth cones and 45 DMSO control growth cones were analyzed, and the number of L1-positive endocytic organelles in the P-domain was categorized into four groups. Shown are the percentages of growth cones in each group. There was a statistically significant difference between the taxol-treated and control growth cones (p < 0.0001).

Fig. 5.

Cell-surface distribution of recycled L1 on growth cones. DRG neurons cultured on L1 (A–G, J) or laminin (H, I, K, L) were allowed to internalize anti-L1 Fab bound to L1 for 30 min, and the cell-surface Fab was blocked. The cells were reincubated for 0 min (A, D, H, K), 30 min (B, E, I, L), 45 min (C, F), or 60 min (G, J) to allow for exocytosis of the L1–Fab complex. Then, recycled L1 was detected by labeling the unblocked Fab that had reappeared on the cell surface. The cells were double-labeled for NCAM to outline the growth cone structure. In superimposed images (A–C, G–I), recycled L1 is colored in red, and NCAM is colored ingreen. To facilitate visualization of recycled L1, thered channel only is shown in black andwhite (D–F, J–L) below the corresponding superimposed image. Scale bars: A, D, 10 μm; B, E, 10 μm; C, F, 10 μm;G, J, 10 μm; H, K, 10 μm; I, L, 10 μm.

Fig. 6.

Changes of distribution of recycled L1 on growth cones over time. Recycled L1 on growth cones was visualized as shown in Figure 5, and the distribution patterns were categorized into three classes: recycled L1 found only along the leading edge (Class 1), on the growth cone body (Class 3), or on both (Class 2). A, Distribution of recycled L1 on growth cones migrating on L1. Shown are the percentages of growth cones in each class at the 30 min (n = 51), 45 min (n = 46), and 60 min (n = 50) time points. There was a statistically significant difference between the 30 and 45 min time points (p < 0.0001) and between the 45 and 60 min time points (p < 0.0001). B, Distribution of recycled L1 on growth cones migrating on laminin. Shown are the percentages of growth cones in each class at the 30 min (n = 50), 45 min (n = 45), and 60 min (n = 50) time points. These data are a compilation of several different experiments that were pooled.

Fig. 7.

A model of L1 trafficking in the axonal growth cone migrating via an L1-dependent mechanism. L1 is internalized from the plasma membrane at the C-domain via clathrin-mediated pathways. Subsequently, endocytosed L1 is transported into the P-domain via sorting and recycling endosomes, a process that is dependent on the dynamic ends of microtubules (not shown in this figure). Then, trafficking L1 is reinserted into the plasma membrane at the leading edge. Recycled L1 on the cell surface moves toward the C-domain most likely by coupling to the retrogradely moving actin filaments via ankyrin or other linker molecules. The L1CD has at least two different states depending on conformation or phosphorylation. L1's interaction with ankyrin is regulated by phosphorylation (Garver et al., 1997), as is its ability to interact with clathrin adaptors (see last paragraph of Discussion for details).