To begin to characterize the regulation of L1CAM–cytoskeleton interactions, we examined the lateral mobility of cell surface L1CAM in cultured cell lines. Full-length rat L1CAM including the neuron-specific RSLE exon was expressed in ND-7 cells (rat DRG/neuroblastoma hybrid; Dunn et al., 1991
) to provide a controlled background on which to characterize L1CAM function. These adherent cells express both endogenous L1CAM and ankyrin B (). Cells were transfected transiently with a wild-type rat L1CAM cDNA construct encoding an amino-terminal myc epitope to permit the detection of the transgene product in the context of endogenous L1CAM. The distribution of the epitope-tagged protein was similar to that of endogenous L1CAM, suggesting that mycL1CAM was appropriately transported and distributed on the cell surface (). 1-μm latex beads coated with an anti-myc antibody (9E10; Evan et al., 1985
) were placed and held with an optical gradient laser trap on the cell surface between 0.5 and 1 μm from the leading edge for 2 s. To identify cells expressing the L1CAM transgene, cells were transfected with a bicistronic vector encoding both mycL1CAM and EGFP (CLONTECH Laboratories, Inc.). mycL1CAM expression, detected by indirect immunofluorescence, was well correlated with EGFP expression (unpublished data). Bead binding to the cell surface varied with antibody concentration and fell off dramatically between 0.037 and 0.0073 mg/ml beads (, white bars). Additionally, binding of beads coated with a high concentration of 9E10 (0.58 mg/ml beads) to cells transfected with L1CAM lacking the myc epitope was 0–20% (for each individual experiment), suggesting that bead binding is selective for myc-tagged L1CAM on the cell surface. Bound beads were subject to a second pulse from the laser trap (see Materials and methods) to test the resistance of L1CAM–bead complex to lateral displacement, a strong indicator of cytoskeletal attachment (Choquet et al., 1997
; Felsenfeld et al., 1999
). At the highest concentration of antibody (0.37 mg Ab/ml beads), the majority of beads were resistant to lateral displacement (, black bars “rigid”). The percentage of beads that were bound but not resistant to displacement (, gray bars, “loose”) was inversely proportional to antibody concentration. These results suggest that extracellular oligomerization of L1CAM by antibody regulates the association between L1CAM and the actin cytoskeleton, consistent with results from analyses of other L1 family members (Dubreuil et al., 1996
Figure 1. Wild-type or mutant rat L1CAM is expressed on the surface of ND7 cell lines. ND7 cells expressing cDNA constructs encoding either wild-type (B) or mutant L1CAM (C and D), tagged with a myc epitope. Wild-type myc-tagged L1CAM (B) detected by indirect immunofluorescence (more ...)
Figure 2. L1CAM–cytoskeleton interactions depend on L1CAM cross-linking. 1-μm latex microspheres coated with anti-myc antibodies recognize cell surface L1CAM tagged with a myc epitope. Bead binding to the cell surface after placement with a laser (more ...)
L1CAM engages in three distinct classes of movement on the cell surface
To analyze directly the behavior of L1CAM on the upper surface of the cell, we recorded the movement of beads bound by antibody to cell surface L1CAM. Beads coated with 9E10 (0.58 mg/ml beads) bound to the cell surface and underwent rapid diffusion (), retrograde movement (), or remained stationary (). For diffusing beads, the trajectory lacked any detectable directed movement with respect to the leading edge ( D). Similarly, mean square displacement for diffusing beads ( G) was linear with respect to time, consistent with diffusion in the absence of directed movement. The average rate of diffusion (0.082 μm2
= 13) suggests that the random movement of L1CAM in the bilayer occurs largely in the absence of cytoskeletal or other interactions that would immobilize the protein(s) bound to the bead. In contrast, beads bound to retrograde-moving L1CAM ( B) showed little or no diffusion, moving away from the leading edge of the cell with a uniform velocity and direction. This directed movement occurred largely in the absence of movement parallel to the leading edge ( E). For all experiments, cells were selected at a wide variety of expression levels (based on coexpression of EGFP) to reduce the possibility of biases that might arise from overexpression. Nonetheless, we cannot preclude the possibility that L1CAM transgene overexpression may affect bead binding and lateral mobility. As with integrins (Felsenfeld et al., 1996
), retrograde movement of L1CAM was often proceeded by a brief (<10 s), diffusive latency period (unpublished data). The velocity of retrograde-moving beads on cells expressing wild-type L1CAM ranged from 0.6 to 2.2 μm min−1
, similar to the velocity of other cell surface adhesion proteins (Felsenfeld et al., 1996
; Lambert et al., 2002
). Therefore, the velocity and direction of bead movement is consistent with an interaction between L1CAM and treadmilling actin in the cytosol of the lamella.
Figure 3. Wild-type L1CAM engages in three distinct classes of kinetic behavior on the cell surface. 1-μm latex microspheres coated with anti-myc antibodies recognize cell surface L1CAM tagged with a myc epitope. Bound beads underwent diffusion (A, D, and (more ...)
In addition to diffusion and retrograde movement, cell surface L1CAM displayed a distinct behavior characterized by a low rate of diffusion in the absence of detectable directed movement (). The stationary behavior was transient and appeared to punctuate periods of diffusion ( C, black circle). Periods of stationary behavior appeared as plateaus with reduced noise in plots of displacement vs. time ( F, black line), consistent with a decreased rate of diffusion. Although it is difficult to analyze the velocity from the mean square displacement for short data segments, analysis of data sets with a high ratio of stationary behavior to diffusion ( C, data in gray circle; F, data over gray bar) revealed a reduced diffusion coefficient compared with freely diffusing particles () with little or no directed component. In cells expressing wild-type L1CAM, 9E10 beads underwent retrograde transport on the cell surface in 67.9% of trials, whereas 28.6% displayed stationary behavior ( A). Together, these results demonstrate that L1CAM is capable of three distinct classes of behavior on the cell surface, including diffusion, retrograde movement, and stationary behavior in the absence of directed movement. These distinct behaviors are consistent with interactions between L1CAM and both dynamic and static components of the cytoskeleton.
Figure 4. L1CAM–cytoskeleton interactions mediate retrograde and stationary behaviors. Bar graphs showing the percentage of trials engaging in stationary behavior, retrograde movement, or diffusion in cells expressing either wild-type or mutant forms of (more ...)
L1CAM retrograde movement depends on the L1CAM cytoplasmic tail and dynamic pools of actin
To begin to examine the mechanism underlying the directed and stationary behavior of L1CAM on the cell surface, we observed the movement of L1CAM in the presence of various cytoskeletal inhibitors. Cytochalasin D, at a concentration that completely suppresses F-actin in the periphery of ND-7 cells (2 μM; unpublished data), abolishes the retrograde movement of L1CAM ( A; n = 12, P < .01). In contrast, stationary behavior was still observed in a small percentage of trials, suggesting that this behavior is either actin independent or mediated by nondynamic pools of actin that are less sensitive to cytochalasin D treatment. In contrast, both retrograde movement and stationary behavior were observed in the presence of 1 μM nocadazole (a concentration that blocks microtubule polymerization in ND-7 cells; unpublished data), although the frequency of retrograde movement was diminished as compared with untreated control cells (n = 16, P < .22). Treatment with DMSO alone at the same concentration used in the dilution of cytochalasin D and nocadazole resulted in a slight increase in retrograde movement (perhaps due to changes in membrane fluidity). These results suggest that retrograde movement of cell surface L1CAM is actin-mediated, although microtubules may also contribute indirectly to this process.
To determine whether the low diffusive states of L1CAM on the cell surface are mediated directly by the L1CAM cytoplasmic tail, we generated a truncation mutant of L1CAM that interrupts the cytoplasmic tail with a stop mutation 4 aa after the predicted transmembrane domain. Beads bound to truncated L1CAM on the cell surface diffused in 100% of trials ( B; n = 17, P < .01), suggesting that both retrograde movement and stationary behavior depend on interactions between the L1CAM cytoplasmic tail and the cytoskeleton.
Mutations that affect ankyrin binding modulate L1CAM movement in the plane of the membrane
To examine directly the role of L1CAM–cytoskeleton interactions in L1CAM movement on the upper surface, we introduced a series of point mutations into the region of the L1CAM tail that has been implicated in ankyrin binding. Mutant constructs were generated encoding single aa substitutions for tyrosine1229 to either phenylalanine, a mutation that induces constitutive ankyrin binding in other vertebrate L1 family members (L1-YF; Garver et al., 1997
; Tuvia et al., 1997
), or to histidine to inhibit ankyrin binding (L1-YH; a naturally occurring MASA mutation in humans; Garver et al., 1997
; Tuvia et al., 1997
; Needham et al., 2001
). Each of these constructs was expressed in ND-7 cells and displayed cell surface distribution comparable to that seen for wild-type L1CAM (, B–D). In culture, 9E10 beads placed on the upper surface of the cell with a laser trap bound with a frequency similar to that seen in cells expressing wild-type mycL1CAM. Like wild-type L1CAM, L1-YF displayed a combination of diffusive, retrograde, and stationary behaviors. However, the ratio of these behaviors was different from that of the wild-type receptor, showing an increase in stationary behavior (62.5%) with a commensurate decrease in retrograde movement (37.5%; B; n
= 16, P < .01). In contrast, beads bound to the L1-YH mutant showed a large increase in the percentage of trials undergoing retrograde movement (92.9%) and a complete loss of stationary behavior ( B; n
= 14, P < .011). These results suggest that L1CAM stationary behavior is mediated by ankyrin binding, whereas the retrograde movement of L1CAM on the cell surface is ankyrin independent.
Growth factor treatment inhibits ankyrin recruitment and L1CAM stationary behavior at the cell surface
To test further this hypothesis, we examined the behavior of wild-type L1CAM (including the myc-epitope tag) after growth factor treatment. It has been reported previously that tyrosine phosphorylation of L1 family members at the FIGQY motif is modulated by activation of a variety of membrane-linked tyrosine kinase receptors, including receptors for NGF, FGF, EGF (Garver et al., 1997
), and by the Eph kinase Cek5 (Zisch et al., 1997
). L1CAM-transfected 293 cells recruited ankyrin to the membrane in an L1CAM-dependent manner (). Treatment of these cells with EGF (50 ng/ml; 1 h) inhibited ankyrin membrane localization (), similar to the behavior of other L1 family members (Zhang et al., 1998
) and consistent with a phosphorylation-dependent inhibition of L1CAM–ankyrin binding. Measurement of ankyrin immunolocalization along a line drawn across the junction of L1CAM-positive cells ( H; Oancea et al., 1998
) demonstrates a quantifiable and significant change in ankyrin–membrane association ( G; P < .01). Similar results were obtained using ND-7 cells treated with NGF (unpublished data), suggesting that these cells, derived from primary sensory neurons, have maintained their sensitivity to NGF.
Figure 5. Growth factor treatment inhibits ankyrin B binding to L1CAM. 293 cells transfected with a cDNA encoding full-length rat L1CAM in the absence (A, C, and E) or presence (B, D, and F) of EGF. Ankyrin (A and B) and L1CAM (C and D) were detected by indirect (more ...)
Treatment of ND-7 cells expressing myc-tagged wild-type L1CAM with NGF caused a shift in the ratio of stationary to retrograde-moving beads similar to that seen in cells expressing L1-YH (18.2% stationary, 100% retrograde; B; n =11, P < .02; for some trials, beads exhibited both forms of behavior). Together, these results suggest that L1CAM–ankyrin interactions mediate the stationary behavior of cell surface L1CAM. Moreover, the increase in the percentage of beads undergoing retrograde movement in conditions that perturb ankyrin binding raises the possibility that ankyrin may negatively modulate L1CAM-mediated traction-force generation.
Peptides derived from the L1CAM tail inhibit ankyrin binding and stationary behavior by L1CAM on the cell surface
To examine independently the role of ankyrin binding in the directed movement of L1, we designed peptides directed at inhibiting L1–ankyrin interactions in live cells. The inhibitory peptide is a fusion between the ankyrin-binding region of the L1CAM tail and the membrane-permeable penetratin domain of antennapedia (Derossi et al., 1998
). The inhibitory region of this peptide was derived from the 12-aa conserved ankyrin-binding domain of the L1CAM tail (Zhang et al., 1998
) including a Y to F substitution (QFNEDGSFIGQF; AP-YF). Peptide activity was compared with that of a peptide in which the inhibitory sequence was reversed (AP-Scramble).
To test the function of the AP-YF in situ, we examined its capacity to inhibit L1CAM-mediated recruitment of ankyrin to the cell membrane. In the presence of peptide AP-YF, ankyrinB was almost entirely absent from sites of cell–cell contact ( A). In contrast, in the absence of peptide or in the presence of the control, scrambled peptide, ankyrinB appeared at the cell membrane where L1CAM was expressed ( A; unpublished data). Quantification of ankyrin colocalization with L1CAM at the membrane revealed a significant reduction in the junctional distribution of ankyrin in AP-YF–treated cells ( A; P < 0.01). These results suggest that the AP-YF peptide is an effective inhibitor of L1CAM–ankyrin interactions in live cells.
Figure 6. FIGQF peptides inhibit L1CAM–ankyrin interactions, stationary behavior of cell surface L1CAM, and increase the velocity of L1CAM retrograde movement. Peptides derived from the sequence of the ankyrin-binding domain of L1CAM conjugated to the membrane-permeant (more ...)
As was the case with the L1-YH mutation, the AP-YF inhibitory peptide reduced the percentage of beads showing stationary behavior on the cell surface with an accompanying increase in the percentage of trials undergoing retrograde movement ( B; n = 18, P < .05). Cells treated with the control, AP-Scramble peptide behaved in a manner similar to untreated cells, although there may be a slight (though not significant) increase in retrograde movement (n = 9; P > .05). These results confirm that experimental treatments that interfere with L1CAM–ankyrin binding inhibit selectively the low diffusion stationary state observed in wild-type L1CAM on the cell surface. This observation strongly suggests that ankyrin mediates L1CAM interactions with stationary components of the cytoskeleton. Moreover, the increase in the percentage of trials undergoing retrograde movement on the cell surface after inhibition of L1CAM–ankyrin interactions raises the possibility that ankyrin binding may also inhibit the directed movement of L1CAM on the cell surface.
To address this question, we quantified the velocity of bead movement in trials undergoing translocation on the cell surface in the presence of AP-YF or control peptides. Cells cultured in the presence of inhibitory peptide showed, on average, a twofold increase in the velocity of L1CAM-directed movement on the cell surface as compared with cells treated with control peptide ( C; P < 0.01) or untreated cells (P < 0.01). The movement of L1CAM in the presence of control peptide was largely unaffected. Similarly, analysis of mutant L1-YH, which is also deficient in ankyrin binding, displays a significant increase in the rate of directed protein movement on the cell surface as compared with untreated cells expressing wild-type L1CAM (P < 0.01). The change in mean velocity does not merely reflect the decrease in the percentage of stationary beads, as beads with a mean velocity of 0 were not included in the calculated average velocity. Together, these results implicate L1CAM–ankyrin interactions in the regulation of L1CAM-directed movement on the cell surface.
Inhibitors of ankyrin binding stimulate L1CAM-mediated neurite outgrowth
The changes in bead kinetics on the upper surface of the cell raise the possibility that the role of ankyrin binding in vivo may be to differentially regulate the adhesion and migration of growing neurons. To address this question directly, we cultured mouse cerebellar granular neurons in the presence of either inhibitory AP-YF or control peptides (). These neurons use cell surface L1CAM as the primary receptor for substrate-bound L1CAM ligands (Dahme et al., 1997
), permitting us to test directly L1CAM function in neurite extension. Neurons grown on NgCAM, a chick homologue of L1CAM, extend 21 μm (± 2) after 24 h in culture in the presence of control peptides. In contrast, neurons cultured in the presence of AP-YF extend 55% above control levels (32 ± 2 μm; P < 0.01). Axon extension on laminin, which promotes outgrowth through interactions with cell surface integrins (Felsenfeld et al., 1994
), was not significantly affected by peptide treatment (P > 0.05). These results suggest that L1CAM-dependent neuronal growth is modulated by changes in L1CAM–ankyrin interactions. Additionally, these results support the idea that traction-force generation in the neuronal growth cone plays a role in neurite extension.
Figure 7. Peptide inhibitors of L1CAM–ankyrin interactions selectively stimulate L1CAM-mediated neuronal growth. Cerebellar cells prepared from P4 mouse (1.25 × 105 cells) were plated on chick NgCAM (A and B) or on mouse laminin (C and D). Cultures (more ...)