Techniques to stimulate and interrogate cell-cell adhesion mechanics

Abstract

Cell-cell adhesions maintain the mechanical integrity of multicellular tissues and have recently been found to act as mechanotransducers, translating mechanical cues into biochemical signals. Mechanotransduction studies have primarily focused on focal adhesions, sites of cell-substrate attachment. These studies leverage technical advances in devices and systems interfacing with living cells through cell-extracellular matrix adhesions. As reports of aberrant signal transduction originating from mutations in cell-cell adhesion molecules are being increasingly associated with disease states, growing attention is being paid to this intercellular signaling hub. Along with this renewed focus, new requirements arise for the interrogation and stimulation of cell-cell adhesive junctions. This review covers established experimental techniques for stimulation and interrogation of cell-cell adhesion from cell pairs to monolayers.

Keywords: BioMEMS; Cell mechanics; Cell–cell adhesion; FRET; Mechanobiology.

Fig. 1

Cell–cell adhesion in epithelial cells. a. Adherens junctions (AJs) and desmosomes are cadherin-based intercellular junctions, which, along with adhesions at the cell–ECM (HD: hemidesmosome; FA: focal adhesion), are responsible for maintenance of the epithelial phenotype. b. The major components of the desmosome junction are desmocollin (Dsc), desmoglein (Dsg), plakoglobin (PG), plakophilin (PKP), and desmoplakin (DP), which connect to intermediate filaments (IFs). c. The major components of classical AJs are the transmembrane protein E-cadherin, p120, α-, and β-catenin.

Fig. 2

Mechanical stretching of a monolayer of cells. a. Uniaxial stretching; b: Biaxial stretching; c: Substrate flection; d: Stretching with curved template; e: Equiaxial stretching with vacuum suction.

Fig. 3

Fluid shear stimulation of a monolayer of cells. a. Fluid shear in a two-plate based flow chamber; b. Fluid shear with a plate and rotating cone system.

Fig. 4

Cell force interrogation techniques and their use in cell–cell adhesion studies. a. TFM (a1) and micropost arrays (a2); b. Micropipette aspiration for single cells (b1) and cell–cell adhesion (b2); c. Optical trapping and stretcher for single cells (c1) and for cell–cell adhesion (c2); d. MTC for single cells (d1) and for cell–cell adhesion (d2); e. AFM based single mechanical interrogation (e1), single molecule force spectroscopy (SMFS) (e2) and single cell force spectroscopy (SCFS) for cell–cell adhesion (e3).

Fig. 5

Micropost arrays reveal that desmosome/IF linkage regulates cell–cell adhesion forces. a. Micropost arrays 10 μm in height and 2 μm in diameter are fabricated using micromolding of PDMS. b. Force balance established between cell pairs are used to calculate cell–cell tugging force. c, d, e. Distribution of intercellular forces in nN as measured by micropost arrays for DPNTP, S2849G DP and WtDP, respectively. c, d and e are recreated from Fig. 2 in [21], reprinted with permission.).

Fig. 6

AFM imaging reveals cytoskeletal bundles. a. AFM nanomechanical measurements reveal global cell mechanics. b. AFM image is scanned at the cell–cell junction. Scale bar: 2 μm. c. To identify the filaments in the AFM image, an immunofluorescence image is taken at the junction to visualize IF (Red: IF; Blue: plakoglobin; Green: DP). Scale bar: 5 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

MEMS based single cell interrogation and stimulation systems. a. Uniaxial single cell stretching; b: Biaxial single cell stretching. (Reprinted with permission from Fig. 4 of [189].).

Fig. 8

Parallel stimulation and interrogation of cell–cell adhesion with bioMEMS system. a. SEM image of the microfabricated device, composed of folded beams (load sensor), and shuttles to mount the rafts in which cells are cultured (b). c–e. The parylene C rafts are successfully released from the substrate by dissolving a sacrificial layer (PNIPA) in 37 °C water, manipulated using a micropipette and positioned on a device. f. Raft fabrication and release. Top view of the rafts with stamped ECM proteins.