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First published online September 9, 2005
doi: 10.1242/10.1242/jcs.02547

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Measuring cell adhesion forces of primary gastrulating cells from zebrafish using atomic force microscopy

¹ Center of Biotechnology, TU Dresden, Cellular Machines, Tatzberg 49, 01307 Dresden, Germany
² MPI-CBG, Pfotenhauerstr.108, 01307 Dresden, Germany

View larger version (44K): [in a new window]   Fig. 1. Reaggregation of mesendodermal progenitors cultured on fibronectin. Images of cultures of wild-type (A-C) and slb/wnt11 mutant mesendodermal progenitors (D-F) after plating for 1 hour (A,D) and for 3 hours (B,C,E,F) in control medium (A,B,D,E) or after adding RGD peptides to the culture medium (C,F). (G) Average number of mesendodermal progenitors/aggregate for wild-type and slb/wnt11 cultures in the absence and presence of RGD peptides at different time-points after plating. Bar, 50 µm.

View larger version (12K): [in a new window]   Fig. 2. Schematic diagram illustrating the capture of a single primary gastrulating cell from zebrafish using a ConA-coated cantilever. Using an inverted optical microscope we placed the cantilever on top of a cell (A) and pressed for 1-2 seconds with a force of 350-500 pN (B). (C) The cantilever together with the adhering cell was then withdrawn several micrometers from the surface and the cell was allowed to settle at this position for 5-15 minutes. (D) Optical image of such a captured cell (arrow) taken 50 µm above the fibronectin surface. The full length of the cantilever is 320 µm.

View larger version (16K): [in a new window]   Fig. 3. Schematic diagram of an adhesion experiment and the corresponding typical force curves for approach (red) and retraction (green) from the surface. The cantilever/cell is moved towards the surface and no force is detected (A). When the cell is pressed on the fibronectin surface, the force increases until it reaches a pre-set level (B). After a given contact time, the cantilever/cell is withdrawn from the surface and the bonds that have been formed break sequentially (C) until the cell has completely separated from the surface (D). To quantify the adhesion, one can measure the maximal force needed to separate cell and surface F, the detachment work W, and the number and magnitude of detachment events along the curve (J, T; see also text).

View larger version (32K): [in a new window]   Fig. 4. Mesendodermal progenitor cell shape and elasticity. (A) The most common cell shapes in a wild-type cell culture. (B,C) Percentage distribution in of the different cell shapes for wild-type cultures (B) and slb/wnt11 mutant cultures (C). Shading in the diagram corresponds to the inset labelling of the different cell shapes in A. The cells used for force measurement were of the two most frequent types. (D,E) Distribution of cell diameters for cells resting on glass in wild-type (D) and slb/wnt11 cultures (E). Gaussian fits give mean±width of 17.0±3.0 µm for the wild type and 18.0±3.1 µm for slb/wnt11 mutant cells. (F) Evaluation of the spring constant k (mean±s.d.) of wild-type and slb/wnt11 mutant cells (>20 cells/genotype; 3 curves/cell) resting on glass measured by using the AFM tip as an indentor. There is no gross difference in k between the wild type (0.46±0.21 mN/m) and slb/wnt11 mutant (0.61±0.30 mN/m) cells. SLB, slb/wnt11; WT, wild type. Bar, 10 µm.

View larger version (13K): [in a new window]   Fig. 5. Typical force curves and histograms for the different cell types and experimental conditions. (A) Force curves for wild-type cells, wild-type cells plus RGD peptide and slb/wnt11 cells adhering to a fibronectin substrate for a contact time of 5 seconds. (B) Corresponding relative count histograms of the detachment forces for wild-type and slb/wnt11 cells on fibronectin. As explained in the text and in the legend to Fig. 6, these data were evaluated by extracting the mean and the s.d. SLB, slb/wnt11; RGD, RGD peptide; WT, wild type.

View larger version (27K): [in a new window]   Fig. 6. Statistics of slb/wnt11 mutant and wild-type mesendodermal progenitor cell adhesion to fibronectin. (A) Detachment force. (B) Detachment work. Data are mean±s.d. The difference between the various conditions becomes more apparent for the detachment work, which takes into account the small unbinding (J and T) events (see Table 2). For 1 second contact time and the RGD experiments, the data sets were compared using the Student's t-test. For 2 and 5 second contact times, wild-type and slb/wnt11 data sets were compared using ANOVA followed by a Bonferroni test. Labels indicate the pairs of data sets with no significant differences (P>0.05). FN, fibronectin; SLB, slb/wnt11; RGD, RGD peptide; WT, wild type.

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