Combined single cell AFM manipulation and TIRFM for probing the molecular stability of multilayer fibrinogen matrices

Abstract

Adsorption of fibrinogen on various surfaces produces a nanoscale multilayer matrix, which strongly reduces the adhesion of platelets and leukocytes with implications for hemostasis and blood compatibility of biomaterials. The nonadhesive properties of fibrinogen matrices are based on their extensibility, ensuing the inability to transduce strong mechanical forces via cellular integrins and resulting in weak intracellular signaling. In addition, reduced cell adhesion may arise from the weaker associations between fibrinogen molecules in the superficial layers of the matrix. Such reduced stability would allow integrins to pull fibrinogen molecules out of the matrix with comparable or smaller forces than required to break integrin–fibrinogen bonds. To examine this possibility, we developed a method based on the combination of total internal reflection fluorescence microscopy, single cell manipulation with an atomic force microscope and microcontact printing to study the transfer of fibrinogen molecules out of a matrix onto cells. We calculated the average fluorescence intensities per pixel for wild-type HEK 293 (HEK WT) and HEK 293 cells expressing leukocyte integrin Mac-1 (HEK Mac-1) before and after contact with multilayered matrices of fluorescently labeled fibrinogen. For contact times of 500 s, HEK Mac-1 cells show a median increase of 57% of the fluorescence intensity compared to 6% for HEKWT cells. The results suggest that the integrin Mac-1-fibrinogen interactions are stronger than the intermolecular fibrinogen interactions in the superficial layer of the matrix. The low mechanical stability of the multilayer fibrinogen surface may contribute to the reduced cell adhesive properties of fibrinogen-coated substrates. We anticipate that the described method can be applied to various cell types to examine their integrin-mediated adhesion to the extracellular matrices with a variable protein composition.

Fibrinogen matrix deposited on the surface of biomaterials acts as a natural anti-adhesive coating

“…Such reduced stability would allow integrins to pull fibrinogen molecules out of the matrix with forces comparable to or smaller than required for breaking integrin-fibrinogen bonds. Indeed, using a method based on a combination of total internal reflection fluorescence microscopy and atomic force microscopy-based single-cell force spectroscopy, we have recently shown that HEK293 cells expressing leukocyte integrin Mac-1 have the capability to pull fibrinogen molecules from the fibrinogen matrix (38). However, further studies are needed to determine the capacity of platelets and leukocytes to pull fibrinogen molecules from the matrix.…”

Development of fusogenic glass surfaces that impart spatiotemporal control over macrophage fusion: Direct visualization of multinucleated giant cell formation

“…Cells were allowed to firmly attach for 1 minute. SCFS measurements were conducted at 37 °C in HBSS with 0.1% BSA as described previously [17]. Force curves were recorded on different spots of the surface with a 2 nN trigger force and a 120 s dwell time.…”

Correlated Multimodal Imaging in Life Sciences: Expanding the Biomedical Horizon

“…This approach allows for a so-called touch-and-watch experiment to study the uptake of foreign or active substances for example. Besides, there are a variety of correlative applications, which assessed combinations of the properties depicted in Figure 4: (i) Elasticity and diffusion using for example Förster Resonance Energy Transfer (FRET) between dye molecule [26][27][28][29][30]; (ii) interaction forces and diffusion using single molecule force spectroscopy and Total Internal Reflection Microscopy (TIRF), termed Single Molecule Cut and Paste, to assemble/split nucleotide-based aptamers individually [25,31,32]; (iii) interaction forces and localization using super-resolution FM to resolve the architecture of focal adhesion under physiological relevant conditions [33,34]; and manipulation and localization to assemble single molecules patterns via the AFM to identify blinking parameters and maximal resolvable fluorophore density [35,36].…”