Monitoring in real-time focal adhesion protein dynamics in response to a discrete mechanical stimulus.
The adhesion of cells to the extracellular matrix is a hierarchical, force-dependent, multistage process that evolves at several temporal scales. An understanding of this complex process requires a precise measurement of forces and its correlation with protein responses in living cells. We present a method to quantitatively assess live cell responses to a local and specific mechanical stimulus. Our approach combines atomic force microscopy with fluorescence imaging. Using this approach, we evaluated the recruitment of adhesion proteins such as vinculin, focal adhesion kinase, paxillin, and zyxin triggered by applying forces in the nN regime to live cells. We observed in real time the development of nascent adhesion sites, evident from the accumulation of early adhesion proteins at the position where the force was applied. We show that the method can be used to quantify the recruitment characteristic times for adhesion proteins in the formation of focal complexes. We also found a spatial remodeling of the mature focal adhesion protein zyxin as a function of the applied force. Our approach allows the study of a variety of complex biological processes involved in cellular mechanotransduction.
Centro de Microscopías Avanzadas and Departamento de Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
Catalina
C
von Bilderling
Laboratorio de Electrónica Cuántica, Departamento de Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
Martín
M
Caldarola
Laboratorio de Electrónica Cuántica, Departamento de Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
Martín E
ME
Masip
IFIBA-CONICET-UBA, Buenos Aires, Argentina.
Andrea V
0000000344829059
AV
Bragas
Centro de Microscopías Avanzadas and Departamento de Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
Lía I
0000000227143162
LI
Pietrasanta
D002468:Cell Physiological Phenomena; D022001:Focal Adhesions; D040542:Mechanotransduction, Cellular; D011506:Proteins / Q000737:chemistry*
D011506:Proteins