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Integrin-specific mechanoresponses to compression and extension probed by cylindrical flat-ended AFM tips in lung cells.

Acerbi I, Luque T, Giménez A, Puig M, Reguart N, Farré R, Navajas D, Alcaraz J - PLoS ONE (2012)

Bottom Line: We found that cell resistance to compression was globally higher than to extension regardless of the tip coating.These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca(2+) signaling.Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Unitat de Biofísica i Bioenginyeria, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain.

ABSTRACT
Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ~1 µm(2) cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca(2+) signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis.

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Illustration of the 4-step protocol based on FE-AFM tips used to probe cell mechanoresponses to compression and extension.(A) Representative SEM images of a nanofabricated cylindrical FE-AFM tip. A whole FIB-milled cylindrical tip is shown in the left panel, and detailed lateral and top view images of the tip are shown in the middle and right panels, respectively. (B) Driving signal of the piezotranslator in z as a function of time (t) used to probe the sample mechanoresponse to compression and extension. Note that the z axis was scaled relative to zc, which is marked with an horizontal dashed line. Although the relative z signal started at 13.5 µm, the z axis was limited to 4 µm above zc to better visualize the range corresponding to step III and IV. A break in the t axis was introduced for the same purpose. Corresponding F recordings as a function of t on a PAA+ gel and a single A549 cell are shown in (C) and (D), respectively. A common t axis was used in (B–D). F* was obtained from step IV as illustrated in (C, D). (E) Cartoon describing the tip-sample mechanical interactions corresponding to the 4-steps of the experimental protocol. EC and ET were calculated using signals from step III and IV. F signals from (C) and (D) were plotted against z in (F) and (G), respectively. The parts of the z and F signals obtained in compression were highlighted in gray. All F data were scaled relative to the corresponding zero force (k⋅d0).
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pone-0032261-g001: Illustration of the 4-step protocol based on FE-AFM tips used to probe cell mechanoresponses to compression and extension.(A) Representative SEM images of a nanofabricated cylindrical FE-AFM tip. A whole FIB-milled cylindrical tip is shown in the left panel, and detailed lateral and top view images of the tip are shown in the middle and right panels, respectively. (B) Driving signal of the piezotranslator in z as a function of time (t) used to probe the sample mechanoresponse to compression and extension. Note that the z axis was scaled relative to zc, which is marked with an horizontal dashed line. Although the relative z signal started at 13.5 µm, the z axis was limited to 4 µm above zc to better visualize the range corresponding to step III and IV. A break in the t axis was introduced for the same purpose. Corresponding F recordings as a function of t on a PAA+ gel and a single A549 cell are shown in (C) and (D), respectively. A common t axis was used in (B–D). F* was obtained from step IV as illustrated in (C, D). (E) Cartoon describing the tip-sample mechanical interactions corresponding to the 4-steps of the experimental protocol. EC and ET were calculated using signals from step III and IV. F signals from (C) and (D) were plotted against z in (F) and (G), respectively. The parts of the z and F signals obtained in compression were highlighted in gray. All F data were scaled relative to the corresponding zero force (k⋅d0).

Mentions: The pyramidal tip of silicon cantilevers (MikroMasch, Tallinn, Estonia) was shaped into flat-ended cylinders by FIB following a slightly modified two-step procedure reported elsewhere [14]. First, a cylinder was obtained by milling the tip applying a ring-like ion beam (1 µm inner radius, 2.5 µm outer radius) centered on the tip apex with a tilt angle of 11°, dwell time of 10 s, and beam intensity of ∼3000 pA. This step was repeated by reducing progressively the outer diameter. Second, a flat ended tip was obtained by milling a straight line pattern (∼1000 pA) perpendicular to the cylinder axis. Finally, Scanning Electron Microscopy (SEM) images of the tips (Figure 1A) were acquired to assess the actual radius, and to measure the height and circularity of the cross-section with SEM software navigation tools.


Integrin-specific mechanoresponses to compression and extension probed by cylindrical flat-ended AFM tips in lung cells.

Acerbi I, Luque T, Giménez A, Puig M, Reguart N, Farré R, Navajas D, Alcaraz J - PLoS ONE (2012)

Illustration of the 4-step protocol based on FE-AFM tips used to probe cell mechanoresponses to compression and extension.(A) Representative SEM images of a nanofabricated cylindrical FE-AFM tip. A whole FIB-milled cylindrical tip is shown in the left panel, and detailed lateral and top view images of the tip are shown in the middle and right panels, respectively. (B) Driving signal of the piezotranslator in z as a function of time (t) used to probe the sample mechanoresponse to compression and extension. Note that the z axis was scaled relative to zc, which is marked with an horizontal dashed line. Although the relative z signal started at 13.5 µm, the z axis was limited to 4 µm above zc to better visualize the range corresponding to step III and IV. A break in the t axis was introduced for the same purpose. Corresponding F recordings as a function of t on a PAA+ gel and a single A549 cell are shown in (C) and (D), respectively. A common t axis was used in (B–D). F* was obtained from step IV as illustrated in (C, D). (E) Cartoon describing the tip-sample mechanical interactions corresponding to the 4-steps of the experimental protocol. EC and ET were calculated using signals from step III and IV. F signals from (C) and (D) were plotted against z in (F) and (G), respectively. The parts of the z and F signals obtained in compression were highlighted in gray. All F data were scaled relative to the corresponding zero force (k⋅d0).
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Related In: Results  -  Collection

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pone-0032261-g001: Illustration of the 4-step protocol based on FE-AFM tips used to probe cell mechanoresponses to compression and extension.(A) Representative SEM images of a nanofabricated cylindrical FE-AFM tip. A whole FIB-milled cylindrical tip is shown in the left panel, and detailed lateral and top view images of the tip are shown in the middle and right panels, respectively. (B) Driving signal of the piezotranslator in z as a function of time (t) used to probe the sample mechanoresponse to compression and extension. Note that the z axis was scaled relative to zc, which is marked with an horizontal dashed line. Although the relative z signal started at 13.5 µm, the z axis was limited to 4 µm above zc to better visualize the range corresponding to step III and IV. A break in the t axis was introduced for the same purpose. Corresponding F recordings as a function of t on a PAA+ gel and a single A549 cell are shown in (C) and (D), respectively. A common t axis was used in (B–D). F* was obtained from step IV as illustrated in (C, D). (E) Cartoon describing the tip-sample mechanical interactions corresponding to the 4-steps of the experimental protocol. EC and ET were calculated using signals from step III and IV. F signals from (C) and (D) were plotted against z in (F) and (G), respectively. The parts of the z and F signals obtained in compression were highlighted in gray. All F data were scaled relative to the corresponding zero force (k⋅d0).
Mentions: The pyramidal tip of silicon cantilevers (MikroMasch, Tallinn, Estonia) was shaped into flat-ended cylinders by FIB following a slightly modified two-step procedure reported elsewhere [14]. First, a cylinder was obtained by milling the tip applying a ring-like ion beam (1 µm inner radius, 2.5 µm outer radius) centered on the tip apex with a tilt angle of 11°, dwell time of 10 s, and beam intensity of ∼3000 pA. This step was repeated by reducing progressively the outer diameter. Second, a flat ended tip was obtained by milling a straight line pattern (∼1000 pA) perpendicular to the cylinder axis. Finally, Scanning Electron Microscopy (SEM) images of the tips (Figure 1A) were acquired to assess the actual radius, and to measure the height and circularity of the cross-section with SEM software navigation tools.

Bottom Line: We found that cell resistance to compression was globally higher than to extension regardless of the tip coating.These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca(2+) signaling.Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Unitat de Biofísica i Bioenginyeria, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain.

ABSTRACT
Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ~1 µm(2) cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca(2+) signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis.

Show MeSH
Related in: MedlinePlus