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Synchronized cell attachment triggered by photo-activatable adhesive ligands allows QCM-based detection of early integrin binding.

Iturri J, García-Fernández L, Reuning U, García AJ, del Campo A, Salierno MJ - Sci Rep (2015)

Bottom Line: Larger frequency change in the QCM-D signal was observed for cells with larger spread area, and for cells overexpressing integrin αvβ3 upon stable transfection.This strategy enables quantification of integrin activity which, in turn, may allow discrimination among different cell types displaying distinct integrin subtypes and expression levels thereof.On the basis of these findings, we believe the strategy can be extended to other photoactivatable ligands to characterize cell membrane receptors activity, a relevant issue for cancer diagnosis (and prognosis) as other several pathologies.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany.

ABSTRACT
The Quartz Crystal Microbalance with dissipation (QCM-D) technique was applied to monitor and quantify integrin-RGD recognition during the early stages of cell adhesion. Using QCM-D crystals modified with a photo-activatable RGD peptide, the time point of presentation of adhesive ligand at the surface of the QCM-D crystal could be accurately controlled. This allowed temporal resolution of early integrin-RGD binding and the subsequent cell spreading process, and their separate detection by QCM-D. The specificity of the integrin-RGD binding event was corroborated by performing the experiments in the presence of soluble cyclicRGD as a competitor, and cytochalasin D as inhibitor of cell spreading. Larger frequency change in the QCM-D signal was observed for cells with larger spread area, and for cells overexpressing integrin αvβ3 upon stable transfection. This strategy enables quantification of integrin activity which, in turn, may allow discrimination among different cell types displaying distinct integrin subtypes and expression levels thereof. On the basis of these findings, we believe the strategy can be extended to other photoactivatable ligands to characterize cell membrane receptors activity, a relevant issue for cancer diagnosis (and prognosis) as other several pathologies.

No MeSH data available.


Related in: MedlinePlus

D/f plots modulus and slope may reflect FAs and cell spreading.D/f values of cell spreading (a) with or without 10 μM antibodies directed to integrin αVβ3 on HUVECs. Time progression is indicated with dashed lines. (b) Average of cell spreading progression at different times in the presence or absence of 10 μM antibodies directed to integrin αVβ3. (c) FA per cell after 40 min incubation onto the QCM substrates in the presence or absence of 10 μM antibodies directed to integrin αVβ3. Insets contain representative pictures of HUVEC cells showing vinculin-GFP stained FA in green, DAPI nuclear staining in blue, and TRITC-phalloidin bound to F-actin fibers in red. (d) Representative pictures of cell spreading at 10 and 40 min in the absence (Control) or presence (anti αVβ3) of 10 μM antibodies.
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f5: D/f plots modulus and slope may reflect FAs and cell spreading.D/f values of cell spreading (a) with or without 10 μM antibodies directed to integrin αVβ3 on HUVECs. Time progression is indicated with dashed lines. (b) Average of cell spreading progression at different times in the presence or absence of 10 μM antibodies directed to integrin αVβ3. (c) FA per cell after 40 min incubation onto the QCM substrates in the presence or absence of 10 μM antibodies directed to integrin αVβ3. Insets contain representative pictures of HUVEC cells showing vinculin-GFP stained FA in green, DAPI nuclear staining in blue, and TRITC-phalloidin bound to F-actin fibers in red. (d) Representative pictures of cell spreading at 10 and 40 min in the absence (Control) or presence (anti αVβ3) of 10 μM antibodies.

Mentions: In an attempt to correlate observable parameters of cell adhesion with the D/f plots during spreading, we analyzed the D/f plots of the attachment of HUVEC in presence or absence of anti αVβ3 blocking antibodies (10 μM) (Fig. 5a). In parallel, we analyzed the spreading area and FA formation by fluorescence microscopy. Significantly lower D/f values were obtained in the presence of αVβ3 blocking antibodies, indicating a relationship between D/f at a particular time after exposure and the ability of the cells to adhere and spread. Interestingly, quantification of the area occupied by the cells after spreading (Fig. 5b) showed only ca. 20% lower spreading of the cells treated with the αVβ3 blocking antibodies, while the differences in D/f values were notably larger. The number of FAs was obtained using vinculin-GFP transfected cells in the presence or absence of anti-αVβ3 antibody after 40 min of spreading. A 3.7-fold lower FA density was found in anti-αVβ3 treated cells than in the control (Fig. 5c, d), confirming the unavailability of integrins to cluster in focal contacts. The reduction in the number of FAs seems to correlate better with the observed D/f differences and suggests that the D/f ratio reflects FAs maturation and the associated cytoskeletal changes, more than cell spreading.


Synchronized cell attachment triggered by photo-activatable adhesive ligands allows QCM-based detection of early integrin binding.

Iturri J, García-Fernández L, Reuning U, García AJ, del Campo A, Salierno MJ - Sci Rep (2015)

D/f plots modulus and slope may reflect FAs and cell spreading.D/f values of cell spreading (a) with or without 10 μM antibodies directed to integrin αVβ3 on HUVECs. Time progression is indicated with dashed lines. (b) Average of cell spreading progression at different times in the presence or absence of 10 μM antibodies directed to integrin αVβ3. (c) FA per cell after 40 min incubation onto the QCM substrates in the presence or absence of 10 μM antibodies directed to integrin αVβ3. Insets contain representative pictures of HUVEC cells showing vinculin-GFP stained FA in green, DAPI nuclear staining in blue, and TRITC-phalloidin bound to F-actin fibers in red. (d) Representative pictures of cell spreading at 10 and 40 min in the absence (Control) or presence (anti αVβ3) of 10 μM antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4379501&req=5

f5: D/f plots modulus and slope may reflect FAs and cell spreading.D/f values of cell spreading (a) with or without 10 μM antibodies directed to integrin αVβ3 on HUVECs. Time progression is indicated with dashed lines. (b) Average of cell spreading progression at different times in the presence or absence of 10 μM antibodies directed to integrin αVβ3. (c) FA per cell after 40 min incubation onto the QCM substrates in the presence or absence of 10 μM antibodies directed to integrin αVβ3. Insets contain representative pictures of HUVEC cells showing vinculin-GFP stained FA in green, DAPI nuclear staining in blue, and TRITC-phalloidin bound to F-actin fibers in red. (d) Representative pictures of cell spreading at 10 and 40 min in the absence (Control) or presence (anti αVβ3) of 10 μM antibodies.
Mentions: In an attempt to correlate observable parameters of cell adhesion with the D/f plots during spreading, we analyzed the D/f plots of the attachment of HUVEC in presence or absence of anti αVβ3 blocking antibodies (10 μM) (Fig. 5a). In parallel, we analyzed the spreading area and FA formation by fluorescence microscopy. Significantly lower D/f values were obtained in the presence of αVβ3 blocking antibodies, indicating a relationship between D/f at a particular time after exposure and the ability of the cells to adhere and spread. Interestingly, quantification of the area occupied by the cells after spreading (Fig. 5b) showed only ca. 20% lower spreading of the cells treated with the αVβ3 blocking antibodies, while the differences in D/f values were notably larger. The number of FAs was obtained using vinculin-GFP transfected cells in the presence or absence of anti-αVβ3 antibody after 40 min of spreading. A 3.7-fold lower FA density was found in anti-αVβ3 treated cells than in the control (Fig. 5c, d), confirming the unavailability of integrins to cluster in focal contacts. The reduction in the number of FAs seems to correlate better with the observed D/f differences and suggests that the D/f ratio reflects FAs maturation and the associated cytoskeletal changes, more than cell spreading.

Bottom Line: Larger frequency change in the QCM-D signal was observed for cells with larger spread area, and for cells overexpressing integrin αvβ3 upon stable transfection.This strategy enables quantification of integrin activity which, in turn, may allow discrimination among different cell types displaying distinct integrin subtypes and expression levels thereof.On the basis of these findings, we believe the strategy can be extended to other photoactivatable ligands to characterize cell membrane receptors activity, a relevant issue for cancer diagnosis (and prognosis) as other several pathologies.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany.

ABSTRACT
The Quartz Crystal Microbalance with dissipation (QCM-D) technique was applied to monitor and quantify integrin-RGD recognition during the early stages of cell adhesion. Using QCM-D crystals modified with a photo-activatable RGD peptide, the time point of presentation of adhesive ligand at the surface of the QCM-D crystal could be accurately controlled. This allowed temporal resolution of early integrin-RGD binding and the subsequent cell spreading process, and their separate detection by QCM-D. The specificity of the integrin-RGD binding event was corroborated by performing the experiments in the presence of soluble cyclicRGD as a competitor, and cytochalasin D as inhibitor of cell spreading. Larger frequency change in the QCM-D signal was observed for cells with larger spread area, and for cells overexpressing integrin αvβ3 upon stable transfection. This strategy enables quantification of integrin activity which, in turn, may allow discrimination among different cell types displaying distinct integrin subtypes and expression levels thereof. On the basis of these findings, we believe the strategy can be extended to other photoactivatable ligands to characterize cell membrane receptors activity, a relevant issue for cancer diagnosis (and prognosis) as other several pathologies.

No MeSH data available.


Related in: MedlinePlus