TY - JOUR
T1 - Combined single cell AFM manipulation and TIRFM for probing the molecular stability of multilayer fibrinogen matrices
AU - Plochberger, Birgit
AU - Ros, Robert
AU - Ros, Alexandra
AU - Ugarova, Tatiana
AU - Christenson, Wayne
AU - Camacho-Alanis, Fernanda
PY - 2014/1
Y1 - 2014/1
N2 - 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 HEK WT 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.
AB - 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 HEK WT 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.
KW - Single cell force spectroscopy
KW - Atomic force microscopy
KW - Total internal reflection fluorescence microscopy
KW - Cell adhesionIntegrins
KW - Fibrinogen
KW - Single cell force spectroscopy
KW - Atomic force microscopy
KW - Total internal reflection fluorescence microscopy
KW - Cell adhesionIntegrins
KW - Fibrinogen
KW - Cell adhesion
KW - Integrins
KW - Extracellular Matrix/metabolism
KW - Single-Cell Analysis/methods
KW - Macrophage-1 Antigen/metabolism
KW - Humans
KW - Cells, Cultured
KW - Cell Adhesion/physiology
KW - Microscopy, Atomic Force/methods
KW - Fibrinogen/chemistry
KW - HEK293 Cells
KW - Microscopy, Fluorescence/methods
UR - http://www.scopus.com/inward/record.url?scp=84887807885&partnerID=8YFLogxK
U2 - 10.1016/j.ultramic.2013.10.009
DO - 10.1016/j.ultramic.2013.10.009
M3 - Article
C2 - 24239757
SN - 1879-2723
VL - 136
SP - 211
EP - 215
JO - Ultramicroscopy
JF - Ultramicroscopy
ER -