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First published online 30 October 2007
doi: 10.1242/jcs.004010

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Distinct kinetic and mechanical properties govern ALCAM-mediated interactions as shown by single-molecule force spectroscopy

Joost te Riet^1,2, Aukje W. Zimmerman¹, Alessandra Cambi¹, Ben Joosten¹, Sylvia Speller², Ruurd Torensma¹, Frank N. van Leeuwen¹, Carl G. Figdor^1,* and Frank de Lange^1,3,

¹ Department of Tumour Immunology (278), Nijmegen Centre for Molecular Life Sciences (NCMLS), Radboud University Nijmegen Medical Centre, PO Box 9101, 6500HB Nijmegen, The Netherlands
² Department of Scanning Probe Microscopy, Institute for Molecules and Materials (IMM), Radboud University Nijmegen, PO Box 9010, 6500GL Nijmegen, The Netherlands
³ Department of Cell Biology (283), Nijmegen Centre for Molecular Life Sciences (NCMLS), Radboud University Nijmegen Medical Centre, PO Box 9101, 6500HB Nijmegen, The Netherlands

View larger version (33K): [in this window] [in a new window]   Fig. 1. ALCAM-mediated adhesion probed by AFM. (A) Homo- and heterotypic ALCAM-mediated interactions. ALCAM contains five Ig domains and the membrane-distal V1 Ig domain mediates homotypic ALCAM-ALCAM interactions (van Kempen et al., 2001). Heterotypic interactions to CD6, a member of the scavenger receptor cysteine rich (SRCR) protein family, are mediated by the ALCAM V1 Ig domain and the third, membrane-proximal, SRCR domain (D3) of CD6 (Bowen et al., 2000). (B) Schematic layout of the AFM experiment. Cells were attached to the AFM cantilever by a ConA-mediated linkage, as detailed in the Materials and Methods. An ALCAM-expressing cell attached to the AFM cantilever interacts with a substrate coated with either ALCAM-Fc or CD6-Fc under the control of the AFM. First (a) the substrate is moved to the cantilever by the piezoelectric scanner until contact is made (b). Then the substrate is pressed onto the cell, causing the cantilever to bend, until a specified force limit is reached. During a preset period of time (interaction time) the cell and substrate are allowed to interact (c). Upon retraction, the cell-substrate adhesion will cause the cantilever to bend in the other direction (d), until the force acting on the molecular bonds are large enough for bond rupture to occur (e). Finally, the cantilever returns to its resting position (f). (C) Example of a single K562-ALCAM cell (arrow), just visible in the shadow of the cantilever, attached to the end of the AFM probe. (D) A typical force-distance curve of an ALCAM-ALCAM interaction, showing single bond ruptures (arrows; K562-ALCAM on ALCAM-coated substrate). a-f correspond to those in B. From the slope just before the final rupture (broken line), the loading rate acting on the bond is calculated. The area enclosed by the approach and retraction curve (shaded) is a measure for the work of de-adhesion under these conditions.

View larger version (30K): [in this window] [in a new window]   Fig. 2. ALCAM is associated with the actin cytoskeleton. (A) Surface expression of ALCAM on K562, K562-ALCAM, KG1a and undifferentiated MUTZ-3 cells was analyzed by flow cytometry. Unfilled histograms represent isotype control staining and shaded histograms represent staining with ALCAM antibody, AZN-L50. The mean fluorescence intensity (MFI) and percentage of positive cells are as indicated. (B) A 42 kDa protein co-precipitates with ALCAM from KG1a cells, as indicated by the arrows. KG1a and undifferentiated Mutz-3 control cells (no ALCAM expression) were incubated overnight with [35S]methionine/cysteine. ALCAM was immunoprecipitated from labeled cell lysates with 1 µg of AZN-L51. Samples that were incubated with protein G beads alone (–) are shown as negative controls. (C) Identification of the co-precipitated protein by western blot analysis. ALCAM was immunoprecipitated from labeled KG1a cell lysates with 1 µg of AZN-L51. ALCAM and β-actin were detected using antibodies AZN-L50 and anti-β-actin (clone AC-15), respectively. As a negative control, lysates were incubated with an irrelevant control antibody (anti-hemagglutinin, clone 12CA5) or with protein G beads alone (–).

View larger version (31K): [in this window] [in a new window]   Fig. 3. The actin cytoskeleton regulates ALCAM binding avidity. (A) Typical force-distance curves of the homotypic ALCAM-mediated interaction between a KG1a cell and an ALCAM–Fc-coated plate, before (medium) and after treatment with the actin cytoskeleton inhibitor CytD, and after a subsequent blocking step (CytD + mAb AZN-L50). For clarity, the traces are shown with an offset. The substrate retraction speed was set to 2.5 µm/second. The work needed to detach the cell from the substrate (shaded areas), typically between 1x10–16 and 3x10–16 J for untreated cells, was taken as a measure for overall cell adhesion. (B) Whole-cell analyses of the relative work of de-adhesion comparing the situation before treatment (medium) with that after incubation with the ALCAM-function-blocking mAb AZN-L50, or after incubation with CytD alone and followed by a subsequent AZN-L50 incubation. The relative work of de-adhesion was determined from at least 25 traces per cell per condition (medium condition set to 100%). It can be seen that CytD treatment upregulates overall cell adhesion and that this adhesion is ALCAM-specific. (C) Single-bond-level rupture-force analyses. In contrast to the overall cell adhesion, the single-bond rupture forces under these loading conditions were found to be insensitive to the various treatments (relative force-scale; n>30). Error bars represent s.e.m.; * indicates significance to P<0.05; n.s., not significant. Trends were reproducibly observed in three independent experiments.

View larger version (33K): [in this window] [in a new window]   Fig. 4. Single-molecule force measurements on living KG1a cells. (A) Examples of force-distance curves obtained in the low-adhesion regime (50x10–18 J, see text). Final ruptures (arrows) were used for further analyses. The retraction speed was set to 2.5 µm/second. (B) Statistical analysis of the rupture forces. The mean rupture forces (± s.e.m.) determined from these data were 58±3 pN and 73±3 pN for the ALCAM-ALCAM and ALCAM-CD6 bond, respectively (P<0.001, n=85; see also Fig. 5). (C) Statistical analysis of the loading rates. The mean loading rates were found to be similar in magnitude (i.e. 2632±112 pN/second and 2707±115 pN/second for the homo- and heterotypic bonds, respectively) (n>100; not significant). A single Gaussian function (solid lines) could be fitted to the force- and loading-rate distributions, which, in all cases, accounted for >85% of the events.

View larger version (57K): [in this window] [in a new window]   Fig. 5. Force spectra of ALCAM-mediated interactions. (A) The mean rupture forces for the homotypic (circle) and heterotypic (square) ALCAM-mediated interactions were found to increase linearly with the natural logarithm of the loading rate. This behavior is consistent with the Bell model (see text, R2>0.95). The results obtained using KG1a (white symbol) were similar to those using K562-ALCAM cells (black). At loading rates >1500 pN/second, the forces associated with ALCAM-CD6 bond rupture were significantly higher than those for the ALCAM-ALCAM interaction (n>20, P<0.05; error bars indicate s.e.m.). (B) Schematic representation of the significance of the Bell model parameters in terms of the energy barrier between the bound and unbound state. The situation when no force is applied (solid lines) or when external forces are applied to the bonds (dotted lines, see Discussion) is represented. (C) Comparison of the histograms of rupture forces (K562-ALCAM cells, n>70; three loading rates) and the theoretical probability density distributions for the failure of single ALCAM-ALCAM and ALCAM-CD6 bonds.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Kinetic profiles of the ALCAM-mediated interactions placed in context. The kinetic profiles of ALCAM-mediated interactions, based on the derived Bell model parameters, are compared to (A) homotypic E-, N- and VE-cadherin-mediated interactions, and (B) LFA-1–ICAM-1 (low and high affinity), P-selectin–PSGL-1 and E-selectin–sLex interactions (see text).