The Ig superfamily cell adhesion molecule, apCAM, mediates growth cone steering by substrate-cytoskeletal coupling - PubMed (original) (raw)

The Ig superfamily cell adhesion molecule, apCAM, mediates growth cone steering by substrate-cytoskeletal coupling

D M Suter et al. J Cell Biol. 1998.

Abstract

Dynamic cytoskeletal rearrangements are involved in neuronal growth cone motility and guidance. To investigate how cell surface receptors translate guidance cue recognition into these cytoskeletal changes, we developed a novel in vitro assay where beads, coated with antibodies to the immunoglobulin superfamily cell adhesion molecule apCAM or with purified native apCAM, replaced cellular substrates. These beads associated with retrograde F-actin flow, but in contrast to previous studies, were then physically restrained with a microneedle to simulate interactions with noncompliant cellular substrates. After a latency period of approximately 10 min, we observed an abrupt increase in bead-restraining tension accompanied by direct extension of the microtubule-rich central domain toward sites of apCAM bead binding. Most importantly, we found that retrograde F-actin flow was attenuated only after restraining tension had increased and only in the bead interaction axis where preferential microtubule extension occurred. These cytoskeletal and structural changes are very similar to those reported for growth cone interactions with physiological targets. Immunolocalization using an antibody against the cytoplasmic domain of apCAM revealed accumulation of the transmembrane isoform of apCAM around bead-binding sites. Our results provide direct evidence for a mechanical continuum from apCAM bead substrates through the peripheral domain to the central cytoplasmic domain. By modulating functional linkage to the underlying actin cytoskeleton, cell surface receptors such as apCAM appear to enable the application of tensioning forces to extracellular substrates, providing a mechanism for transducing retrograde flow into guided growth cone movement.

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Figures

Figure 9

Substrate–cytoskeletal coupling model. Schematic cross-sections through a growth cone demonstrate the cytoskeletal organization of the P and C domain, as well as of the T zone, at different stages of an RBI experiment. Left inset shows potential conventional (a), unconventional (b), and microtubule-associated (c) myosin localizations and details of actin recycling; right inset depicts actin-filament organization in filopodia and lamellipodia as well as actin assembly. Inset in B shows details of a potential molecular clutch. Cross-section and top views of growth cones during RBIs are shown on left and right, respectively. (A) Latency period. The molecular clutch between receptor and actin cytoskeleton exhibits slippage at low levels of apCAM clustering early in the RBI. ApCAM–actin linkage is not strong enough to support significant C–P domain tension, nor attenuate retrograde flow. Retrograde flow is maximal (F-actin flow marker 1 displacement) and growth is slow. (B) Interaction period. When enough functional linkages are engaged by restrained beads, retrograde flow is attenuated (marker bead 2), C–P domain tension increases and the C domain extends toward the restrained bead. Protrusive growth may result directly from continued actin assembly during F-actin flow attenuation.

Figure 6

Inductopodia preferentially start with an anterograde orientation after restraint. (A) DIC image of a growth cone with a 4E8-coated bead which was restrained for 2 min. Star marks the center of the bead at time of initial restraint. (B) After release of restraint bead moves anterogradely (α = 135°) driven by inductopodium formation (arrow). (C) Scheme showing how α was measured. (D) Inductopodia probability of 4E8 beads restrained for various amounts of time. (E) Inductopodia were grouped according translocation angle (α) into four different categories. 69% (n = 35) of all inductopodia analyzed had an anterograde component to their initial movement (90° < α < 180°). (F) Scheme depicting apCAM behavior as a function of clustering density. Free apCAM (left) does not appear to interact with underlying F-actin (white arrowhead), whereas moderate clustering results in coupling to F-actin flow via putative linkage protein(s) (middle). High-density clustering has been shown to trigger nucleation of actin assembly (right) in addition to flow coupling (Thompson et al., 1996). Bar, 5 μm.

Figure 6

Figure 2

Growth cone– steering response evoked by cross-linking of apCAM. (A) DIC image of a growth cone just before bead placement (6 min before start of C domain extension). Axis of growth cone orientation is indicated. (B) 4 min after strong coupling, the C domain has reoriented toward a bead placed roughly 45° off the advance axis. The newly formed lamellipodium distal to the bead moved up the sides of the bead as the adhesive properties of the poly-lysine substrate were reduced, due to the presence of BSA. Bar, 5 μm.

Figure 1

Cross-linking of apCAM triggers C domain extension, protrusive growth, and cytoskeletal remodeling. All images refer to the same growth cone. (A) Beads coated with the anti-apCAM antibody (4E8) were placed on the P domain of an Aplysia bag cell growth cone near the leading edge and restrained from retrograde movement using a microneedle. This video-enhanced DIC image was recorded after the latency period (G) at the start of C-domain extension. The double-headed arrow indicates interaction axis between bead and C domain. (B) After 4 min, the C domain boundary extended to the bead (arrowhead marks initial boundary position). Arrow indicates new leading edge position. (C) Rhodamine phalloidin labeling of F-actin and (D) β-tubulin immunofluorescence after fixation at the 5-min timepoint. Note F-actin accumulation around the bead (C, arrowhead) and typical control staining for actin bundles in adjacent areas (C, arrow). Microtubule extension (D, arrow) was directed toward the bead, visible as a ring (D, arrowhead) as the secondary antibody also binds to protein A and 4E8 on the bead. (E) Pseudocolor overlay of F-actin (red) and tubulin (green) stainings. (F) DIC time course of this interaction. Line indicates needle position at time 0, note rearward displacement over time. (G) C-domain extension rates plotted as a function of time. Bead placement (arrowhead) followed by latency and interaction periods (double-headed arrows) are indicated. Bars, 5 μm.

Figure 1

Figure 7

Characterization of an antibody against the cytoplasmic domain of apCAM. (A) Western blot analysis. Triton-extracted membrane proteins from Aplysia nervous system tissue were separated by 10% SDS-PAGE and probed with crude rabbit antiserum against the MBP fusion protein of the cytoplasmic domain peptide (CapCAM, left lane), the affinity-purified antibody (middle lane), and 4E8 (right lane). The affinity-purified antibody recognizes two protein species at 130 and 150 kD, which correspond to the two higher molecular weight bands recognized by 4E8. Molecular weight markers (kD), as indicated. (B–E) Immunolocalization of membrane-spanning apCAM. Triton- extracted growth cones were double-stained for membrane-spanning apCAM with the CapCAM antibody (B and D), and F-actin (C) or β-tubulin (E), respectively. Note generally homogeneous distribution of membrane-spanning apCAM and accumulation at the contact site between two growth cones (arrow in D). The white dashed line in E indicates the leading edge of the growth cones. Bar, 5 μm.

Figure 5

Analysis of RBIs mediated by apCAM antibody, apCAM and Con A–coated beads. (A) RBI probabilities as a function of ligand. (antibodies) Protein A beads coated with anti-apCAM (mAB 4E8), a nonimmune control antibody (mAB RPC5) or no antibody (Prot A). ApCAM beads were tested either in the absence (apCAM) or presence of 4E8 (apCAM/4E8). Lectins: avidin beads coated with biotinylated lectins such as Concanavalin A (Con A), wheat germ agglutinin (WGA), succinyl-wheat germ agglutinin (S-WGA), Sophora japonica agglutinin (SJA) or no lectins (avidin). n indicates the number of experiments. (B) Interaction latencies represent the delay between bead placement and start of C domain extension (strong coupling). (C) C domain extension rates for 4E8, apCAM, and Con A beads. Data represent average values ± SEM.

Figure 3

Retrograde F-actin flow is attenuated specifically along the interaction axis between C domain and restrained bead. (A) F-actin flow markers (small Con A beads) were positioned with a laser tweezer within the interaction corridor (on-axis box) and on adjacent areas (off-axis box) during an interaction experiment using a 4E8 bead. (B) Cumulative displacement over time of on-axis and off-axis beads and C domain boundary during the latency period. Note that before strong coupling occurs, retrograde F-actin flow rates in the interaction axis are equal to off-axis rates. Inset depicts F-actin mark movements and relative bead-restraining force during the latency period shown. (C, top) DIC image sequence of off-axis bead movement (solid line) in area of interest indicated in A. (Bottom) Concurrent time sequence showing on-axis bead movement (solid line) and C-domain extension (dashed line; growth rate = 3.20 μm/min). Note progressive attenuation of F-actin marker bead movement. (D) Displacement over time of on-axis, off-axis beads, and C domain boundary during interaction period. Inset depicts F-actin mark movements and relative increase in bead- restraining force during interaction period. Bar, 5 μm.

Figure 3

Figure 4

SDS-PAGE analysis of purified apCAM. Affinity-purified apCAM (0.2 μg per lane) was analyzed by 7.5% SDS-PAGE and processed for silver staining (left lane) and Western blotting using the 4E8 antibody (right lane). No contaminants were detected by silver staining. All three bands (major band at 100 kD, two minor bands at 130 and 150 kD) are 4E8 positive as reported (Keller and Schacher, 1990). Molecular weight markers (kD) as indicated.

Figure 8

Membrane-spanning apCAM accumulates at Con A bead interaction sites. (A) Start of C domain extension during an interaction with a Con A–coated bead. (B) 4 min later, the C domain (open arrow) extends toward bead. (C) Total apCAM immunofluorescence visualized with 4E8. (D) Membrane-spanning apCAM isoform immunofluorescence visualized with the affinity-purified antibody against the cytoplasmic domain of apCAM. Note the accumulation of membrane-spanning apCAM around the bead interaction site. Bar, 5 μm.

Comment in

Cell crawling: first the motor, now the transmission.
Heidemann SR, Buxbaum RE. Heidemann SR, et al. J Cell Biol. 1998 Apr 6;141(1):1-4. doi: 10.1083/jcb.141.1.1. J Cell Biol. 1998. PMID: 9531543 Free PMC article. Review. No abstract available.

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The Ig superfamily cell adhesion molecule, apCAM, mediates growth cone steering by substrate-cytoskeletal coupling - PubMed (original) (raw)