Integrated microscopy for real-time imaging of mechanotransduction studies in live cells

Abstract

Mechanical force is an important stimulus and determinant of many vascular smooth muscle cell functions including contraction, proliferation, migration, and cell attachment. Transmission of force from outside the cell through focal adhesions controls the dynamics of these adhesion sites and initiates intracellular signaling cascades that alter cellular behavior. To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a critical first step is to develop a new technology to investigate cellular behavior at subcellular level that integrates an atomic force microscope (AFM) with total internal reflection fluorescence (TIRF) and fast-spinning disk (FSD) confocal microscopy, providing high spatial and temporal resolution. AFM uses a nanosensor to measure the cell surface topography and can apply and measure mechanical force with high precision. TIRF microscopy is an optical imaging technique that provides high-contrast images with high z-resolution of fluorescently labeled molecules in the immediate vicinity of the cell-coverslip interface. FSD confocal microscopy allows rapid 3-D imaging throughout the cell in real time. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real time.

Force transduction and strain dynamics in actin stress fibres in response to nanonewton forces

“…Importantly, these responses take place well before, and probably govern, the long-term structural reorganization of the SF cytoskeleton in response to force (Trache and Lim, 2009). They consist of a short-term response (20-60 seconds) in which SFs deform as a direct result of an applied force, and a long-term response (60-240 seconds) in which applied forces initiate FA remodelling and movement of SFs far from the point of contact.…”

“…Therefore, the direct response of SFs to force is greatest within 20-120 seconds of contact with the AFM tip. At later times, SF remodelling is no longer a response to a force-induced deformation but due to other processes such as FA movement (Trache and Lim, 2009). Importantly, we found absolutely no evidence that the magnitude of local SF displacement had any dependence on the distance between the SF position and the AFM tip.…”

“…However, the relative influences of the three major filaments systems (actin SFs, MTs and intermediate filaments) on the mechanical properties of the cell are also dependent on physiological state (Kunda et al, 2008;Matzke et al, 2001;Pelling et al, 2009;Maloney et al, 2010). In a previous study (Trache and Lim, 2009), measurements of force-induced SF reorganization were taken over the course of 80 minutes with ,8-to 14-minute time resolution. The application of mechanical force resulted in the reorganization of SFs, although no detailed analysis of the magnitude of the applied force, deformation or strain profiles were performed.…”

“…Atomic force microscopy (AFM) (Binnig et al, 1986) has emerged as a major tool for investigating mechanical signalling pathways within cells Czajkowsky et al, 2000;Haupt et al, 2006;Lehenkari et al, 2000;Radmacher, 2007;Trache and Lim, 2009). Originally intended for use as a topographical tool (Binnig et al, 1986), the ability of AFM to apply known controlled forces to living cells was quickly applied in the field of cell biology (Radmacher et al, 1992).…”

Nck enables directional cell migration through the coordination of polarized membrane protrusion with adhesion dynamics

“…The integrated microscope system used for these studies combines AFM with TIRF and spinning-disk confocal microscopy (Trache and Lim, 2009). AFM experiments were performed with unsharpened silicon nitride cantilevers (MLCT, Bruker Nano-Surfaces Inc., Santa Barbara, CA) coated with fibronectin (FN,Invitrogen,.…”