Cellular Force Spectroscopy

A broad spectrum of biological processes requires controlled cell adhesion, including embryonic development, assembly of tissues and the nervous system, cellular communication, inflammation and wound healing, tumor metastasis, cell culturing, and viral and bacterial infection. Although much is known about cell adhesion, many questions remain unanswered owing to its multiple facets and complexity. Cell adhesion is commonly defined as the binding of a cell to a substrate, which can be another cell, a surface or an organic matrix. The process is regulated by specific cell-adhesion molecules (CAMs), which are typically transmembrane receptors that comprise an intracellular domain that interacts with cytoplasmic proteins, including the cytoskeleton, and an extracellular domain that specifically binds to adhesion partners. However, the molecular mechanisms by which CAMs work and how they regulate different types of adhesion are open debates. For example, an extensive array of proteins is known to be involved in adhesive assemblies, i.e. focal adhesions [cell–extracellular-matrix (ECM) junctions], but the contributions of these proteins to the strength of adhesion are not quantitatively understood. To understand cell adhesion, therefore, the vast amount of qualitative data that is available must be augmented with quantitative data of the physics of adhesion. Therefore, to obtain quantiative insight into how cells initiate and strengthen adhesion, we develop AFM-based single-cell force spectroscopy (SCFS) tools and apply these tools to very mammalian systems.

Experimental setup of AFM-based SCFS and of functionalized cantilevers. (A) To detect the deflection (force) of a functionalized cantilever (see B) a laser is reflected from the cantilever onto a position sensitive photodiode. To bring the cantilever probe into contact with a target cell or substrate, which is present on a cell culture dish, a piezoelectric scanner vertically moves the cell culture dish by at least 100 lm. The setup is mounted onto a light microscope (i.e., phase contrast, DIC, fluorescence, confocal microscope) to optically characterize the cell. The entire setup is preferably placed into a temperature controlled (37 C) and noise isolated chamber. (B) Light microscopy image of endothelial cells probed by a functionalized AFM tip. (C) Single dictyostelium cell attached to a tipless cantilever. The dictyostelium ‘probe’ cell on the cantilever is placed above other dictyostelium cells plated on a Petri dish. (D) Electron microscopy image of an AFM cantilever that has been functionalized with a micrometer sized bead. The bead can be functionalized with chemical groups or cells to characterize adhesion to a target cell. (E) Monolayer of bone cells cultivated on a bead. Scale bars, 20 µm. Figure taken from J. Friedrichset al. Methods (2013) 60, 169–178. — Experimental setup of AFM-based SCFS and of functionalized cantilevers. (A) To detect the deflection (force) of a functionalized cantilever (see B) a laser is reflected from the cantilever onto a position sensitive photodiode. To bring the cantilever probe into contact with a target cell or substrate, which is present on a cell culture dish, a piezoelectric scanner vertically moves the cell culture dish by at least 100 lm. The setup is mounted onto a light microscope (i.e., phase contrast, DIC, fluorescence, confocal microscope) to optically characterize the cell. The entire setup is preferably placed into a temperature controlled (37 C) and noise isolated chamber. (B) Light microscopy image of endothelial cells probed by a functionalized AFM tip. (C) Single dictyostelium cell attached to a tipless cantilever. The dictyostelium ‘probe’ cell on the cantilever is placed above other dictyostelium cells plated on a Petri dish. (D) Electron microscopy image of an AFM cantilever that has been functionalized with a micrometer sized bead. The bead can be functionalized with chemical groups or cells to characterize adhesion to a target cell. (E) Monolayer of bone cells cultivated on a bead. Scale bars, 20 µm. Figure taken from J. Friedrichs et al. Methods 60 (2013) 169–178.

Cellular Force Spectroscopy

Further reading

Kindlin-2 cooperates with talin to activate integrins and induces cell spreading by directly binding paxillin

In PC3 prostate cancer cells ephrin receptors crosstalk to β1-integrins to strengthen adhesion to collagen type I

Assay for characterizing the recovery of vertebrate cells for adhesion measurements by single-cell force spectroscopy

Dynamic coupling of ALCAM to the actin cortex strengthens cell adhesion to CD6

Single-cell force spectroscopy, an emerging tool to quantify cell adhesion to biomaterials

α_v-integrins are required for mechanotransduction in MDCK epithelial cells

Deciphering teneurin domains that facilitate cellular recognition, cell-cell adhesion and neurite outgrowth using AFM-based single-cell force spectroscopy

A practical guide to quantify cell adhesion using single-cell force spectroscopy

Quantifying cellular adhesion to covalently immobilized extracellular matrix proteins by single-cell force spectroscopy

Force nanoscopy of living cells

Planar cell polarity signalling coordinates epithelial organ formation by regulating cell adhesion properties in progenitor cells

Control of directed cell migration in vivo by membrane-to-cortex attachment

Blood vessel lumen formation via electrostatic repulsion

Quantifying cellular adhesion to extracellular matrix components by single-cell force spectroscopy

Stimulated single-cell force spectroscopy to quantify cell adhesion receptor crosstalk

Effect of unlocking RGD-motifs in collagen I on pre-osteoblast adhesion and differentiation

Alignment and cell-matrix interactions of human corneal endothelial cells on nanostructured collagen type I matrices

Movement directionality in collective germ layer progenitor migration

Force probing surfaces of living cells to molecular resolution

New frontiers in atomic force microscopy: Analyzing interactions from single-molecules to cells

TPA primes α2β1 integrins for cell adhesion

A bond for a lifetime: Employing membrane nanotubes from living cells to determine receptor-ligand kinetics

Single-cell force spectroscopy

Galectin-3 regulates the kinetics of integrin α2β1 mediated adhesion to collagen-I and -IV

Single-cell force spectroscopy reveals β1-integrin as a central molecule mediating adhesion of 32D-BCR-ABL cells to bone marrow stroma cells

Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology

Quantifying adhesive and tensile cell properties determining germ layer organization during gastrulation

Studying integrin-mediated cell adhesion on the single-molecule level by using AFM force spectroscopy

Galectin-3 and galectin-9 contribute to adhesion and cystogenesis in epithelial MDCK cells

Revealing early steps of α2β1 integrin-mediated adhesions to collagen type I using single-cell force spectroscopy

A new technical approach to quantify cell adhesion forces by AFM

Wnt11 functions in gastrulation by controlling cell cohesion through Rab5c and E-cadherin

Analysing focal adhesion structure by AFM

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

Cellular Force Spectroscopy

Further reading

Kindlin-2 cooperates with talin to activate integrins and induces cell spreading by directly binding paxillin

In PC3 prostate cancer cells ephrin receptors crosstalk to β1-integrins to strengthen adhesion to collagen type I

Assay for characterizing the recovery of vertebrate cells for adhesion measurements by single-cell force spectroscopy

Dynamic coupling of ALCAM to the actin cortex strengthens cell adhesion to CD6

Single-cell force spectroscopy, an emerging tool to quantify cell adhesion to biomaterials

αv-integrins are required for mechanotransduction in MDCK epithelial cells

Deciphering teneurin domains that facilitate cellular recognition, cell-cell adhesion and neurite outgrowth using AFM-based single-cell force spectroscopy

A practical guide to quantify cell adhesion using single-cell force spectroscopy

Quantifying cellular adhesion to covalently immobilized extracellular matrix proteins by single-cell force spectroscopy

Force nanoscopy of living cells

Planar cell polarity signalling coordinates epithelial organ formation by regulating cell adhesion properties in progenitor cells

Control of directed cell migration in vivo by membrane-to-cortex attachment

Blood vessel lumen formation via electrostatic repulsion

Quantifying cellular adhesion to extracellular matrix components by single-cell force spectroscopy

Stimulated single-cell force spectroscopy to quantify cell adhesion receptor crosstalk

Effect of unlocking RGD-motifs in collagen I on pre-osteoblast adhesion and differentiation

Alignment and cell-matrix interactions of human corneal endothelial cells on nanostructured collagen type I matrices

Movement directionality in collective germ layer progenitor migration

Force probing surfaces of living cells to molecular resolution

New frontiers in atomic force microscopy: Analyzing interactions from single-molecules to cells

TPA primes α2β1 integrins for cell adhesion

A bond for a lifetime: Employing membrane nanotubes from living cells to determine receptor-ligand kinetics

Single-cell force spectroscopy

Galectin-3 regulates the kinetics of integrin α2β1 mediated adhesion to collagen-I and -IV

Single-cell force spectroscopy reveals β1-integrin as a central molecule mediating adhesion of 32D-BCR-ABL cells to bone marrow stroma cells

Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology

Quantifying adhesive and tensile cell properties determining germ layer organization during gastrulation

Studying integrin-mediated cell adhesion on the single-molecule level by using AFM force spectroscopy

Galectin-3 and galectin-9 contribute to adhesion and cystogenesis in epithelial MDCK cells

Revealing early steps of α2β1 integrin-mediated adhesions to collagen type I using single-cell force spectroscopy

A new technical approach to quantify cell adhesion forces by AFM

Wnt11 functions in gastrulation by controlling cell cohesion through Rab5c and E-cadherin

Analysing focal adhesion structure by AFM

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

α_v-integrins are required for mechanotransduction in MDCK epithelial cells