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Keratocytes pull with similar forces on their dorsal and ventral surfaces.

Galbraith CG, Sheetz MP - J. Cell Biol. (1999)

Bottom Line: Borisy. 1997.Cell Biol. 139:397-415).Similar forces were generated on both the ventral (0.2 nN/microm(2)) and the dorsal (0.4 nN/microm(2)) surfaces of the lamella, suggesting that dorsal matrix contacts are as effectively linked to the force-generating cytoskeleton as ventral contacts.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
As cells move forward, they pull rearward against extracellular matrices (ECMs), exerting traction forces. However, no rearward forces have been seen in the fish keratocyte. To address this discrepancy, we have measured the propulsive forces generated by the keratocyte lamella on both the ventral and the dorsal surfaces. On the ventral surface, a micromachined device revealed that traction forces were small and rearward directed under the lamella, changed direction in front of the nucleus, and became larger under the cell body. On the dorsal surface of the lamella, an optical gradient trap measured rearward forces generated against fibronectin-coated beads. The retrograde force exerted by the cell on the bead increased in the thickened region of the lamella where myosin condensation has been observed (Svitkina, T.M., A.B. Verkhovsky, K.M. McQuade, and G. G. Borisy. 1997. J. Cell Biol. 139:397-415). Similar forces were generated on both the ventral (0.2 nN/microm(2)) and the dorsal (0.4 nN/microm(2)) surfaces of the lamella, suggesting that dorsal matrix contacts are as effectively linked to the force-generating cytoskeleton as ventral contacts. The correlation between the level of traction force and the density of myosin suggests a model for keratocyte movement in which myosin condensation in the perinuclear region generates rearward forces in the lamella and forward forces in the cell rear.

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Keratocytes bind to FN-coated surfaces. Individual keratocytes were plated on coverslips coated with either FN 120 kD (5 μg/ml) or BSA (2 mg/ml). The percentage of cells that bound and spread on FN-coated substrata were significantly greater than the fraction that bound to BSA-coated substrata. The binding to FN was significantly inhibited by the addition of 1 mM GRGdSP; moreover, the addition of 1 mM GRGDNP decreased the percentage of cells bound on FN to the same level as control.
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Figure 4: Keratocytes bind to FN-coated surfaces. Individual keratocytes were plated on coverslips coated with either FN 120 kD (5 μg/ml) or BSA (2 mg/ml). The percentage of cells that bound and spread on FN-coated substrata were significantly greater than the fraction that bound to BSA-coated substrata. The binding to FN was significantly inhibited by the addition of 1 mM GRGdSP; moreover, the addition of 1 mM GRGDNP decreased the percentage of cells bound on FN to the same level as control.

Mentions: To examine the ability of keratocytes to bind FN, we performed adhesion assays. Keratocyte explant cultures were trypsinized and plated on surfaces coated with either the 120-kD fragment of FN or BSA. The percentages of cells that bound and spread on the FN-coated substrata were significantly greater than those that bound and spread on BSA-coated substrata (Fig. 4). The binding to FN was significantly inhibited by the addition of 1 mM GRGdSP, a peptide suggested to specifically inhibit cell binding to FN. Moreover, the addition of 1 mM GRGDNP, a peptide that more strongly inhibits cell binding to FN, but has some cross-inhibition with VN (Pierschbacher and Ruoslahti 1987), decreased the percentage of cells bound on FN to the same level as the BSA control (Fig. 4). Although 1 mM GRGdSP has been shown to produce half-maximal inhibition of adhesion by CHO cells to FN (Pierschbacher and Ruoslahti 1987), it was less effective in our assay, possibly due to species differences or differences in the receptor that binds FN in keratocytes. These results indicate that integrin-dependent binding and spreading does occur.


Keratocytes pull with similar forces on their dorsal and ventral surfaces.

Galbraith CG, Sheetz MP - J. Cell Biol. (1999)

Keratocytes bind to FN-coated surfaces. Individual keratocytes were plated on coverslips coated with either FN 120 kD (5 μg/ml) or BSA (2 mg/ml). The percentage of cells that bound and spread on FN-coated substrata were significantly greater than the fraction that bound to BSA-coated substrata. The binding to FN was significantly inhibited by the addition of 1 mM GRGdSP; moreover, the addition of 1 mM GRGDNP decreased the percentage of cells bound on FN to the same level as control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2168090&req=5

Figure 4: Keratocytes bind to FN-coated surfaces. Individual keratocytes were plated on coverslips coated with either FN 120 kD (5 μg/ml) or BSA (2 mg/ml). The percentage of cells that bound and spread on FN-coated substrata were significantly greater than the fraction that bound to BSA-coated substrata. The binding to FN was significantly inhibited by the addition of 1 mM GRGdSP; moreover, the addition of 1 mM GRGDNP decreased the percentage of cells bound on FN to the same level as control.
Mentions: To examine the ability of keratocytes to bind FN, we performed adhesion assays. Keratocyte explant cultures were trypsinized and plated on surfaces coated with either the 120-kD fragment of FN or BSA. The percentages of cells that bound and spread on the FN-coated substrata were significantly greater than those that bound and spread on BSA-coated substrata (Fig. 4). The binding to FN was significantly inhibited by the addition of 1 mM GRGdSP, a peptide suggested to specifically inhibit cell binding to FN. Moreover, the addition of 1 mM GRGDNP, a peptide that more strongly inhibits cell binding to FN, but has some cross-inhibition with VN (Pierschbacher and Ruoslahti 1987), decreased the percentage of cells bound on FN to the same level as the BSA control (Fig. 4). Although 1 mM GRGdSP has been shown to produce half-maximal inhibition of adhesion by CHO cells to FN (Pierschbacher and Ruoslahti 1987), it was less effective in our assay, possibly due to species differences or differences in the receptor that binds FN in keratocytes. These results indicate that integrin-dependent binding and spreading does occur.

Bottom Line: Borisy. 1997.Cell Biol. 139:397-415).Similar forces were generated on both the ventral (0.2 nN/microm(2)) and the dorsal (0.4 nN/microm(2)) surfaces of the lamella, suggesting that dorsal matrix contacts are as effectively linked to the force-generating cytoskeleton as ventral contacts.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
As cells move forward, they pull rearward against extracellular matrices (ECMs), exerting traction forces. However, no rearward forces have been seen in the fish keratocyte. To address this discrepancy, we have measured the propulsive forces generated by the keratocyte lamella on both the ventral and the dorsal surfaces. On the ventral surface, a micromachined device revealed that traction forces were small and rearward directed under the lamella, changed direction in front of the nucleus, and became larger under the cell body. On the dorsal surface of the lamella, an optical gradient trap measured rearward forces generated against fibronectin-coated beads. The retrograde force exerted by the cell on the bead increased in the thickened region of the lamella where myosin condensation has been observed (Svitkina, T.M., A.B. Verkhovsky, K.M. McQuade, and G. G. Borisy. 1997. J. Cell Biol. 139:397-415). Similar forces were generated on both the ventral (0.2 nN/microm(2)) and the dorsal (0.4 nN/microm(2)) surfaces of the lamella, suggesting that dorsal matrix contacts are as effectively linked to the force-generating cytoskeleton as ventral contacts. The correlation between the level of traction force and the density of myosin suggests a model for keratocyte movement in which myosin condensation in the perinuclear region generates rearward forces in the lamella and forward forces in the cell rear.

Show MeSH
Related in: MedlinePlus