The intermediate filament (IF) cytoskeleton plays an important role in integrating biomechanical pathways associated with the actin and microtubule cytoskeleton. Vimentin is a type III IF protein commonly found in fibroblast cells and plays a role in transmitting forces through the cytoskeleton. Employing simultaneous laser scanning confocal and atomic force microscopy (AFM), we developed a methodology to quantify the deformation of the GFP-vimentin-labeled IF cytoskeleton as a function of time in response to force application by the AFM. Over short times (seconds), IFs deformed rapidly and transmitted force throughout the entire cell in a highly complex and anisotropic fashion. After several minutes, mechanically induced displacements of IFs resemble basal movements. In well-adhered cells the deformation of IFs is highly anisotropic as they tend to deform away from the longitudinal axis of the cell. This study demonstrates that simultaneous AFM and LSCM can be employed to track the deformation and dissipation of force through the IF cytoskeleton. 1. Introduction Recent advancements in the field of biophysics, such as the rapid improvement of our understanding of the mechanical roles of the cytoskeleton (CSK) and extracellular matrix (ECM) [1], have led to a picture of the cell from a mechanical point of view, as opposed to the traditional biochemical perspective [1–3]. It is now known that biological processes such as DNA replication are not only affected by the presence of certain biochemical signals in the cell, but also by mechanical forces such as tension on the DNA strand itself [4]. The new perspective involving physical forces at a micro- and nanoscale has allowed for many new discoveries [1]. For one, the process of mechanotransduction occurs by changes in the concentrations of local signaling molecules as a response to the deformation of the CSK induced by externally applied forces [5]. This process depends on the close integration of the three cytoskeletal elements, actin, microtubules (MTs), and intermediate filaments (IFs) [1]. Understanding how these cytoskeletal elements deform will provide insight into how cells both sense and react to their external physical environment, which results in the conversion of mechanical signals from the ECM to the CSK and eventually into cellular signaling [1, 2]. The IF network is one of the three main components of the CSK. However, the intermediate filament network has since been proven to play a major role in the mechanical functions of the cell [6–8]. IFs have been shown to stabilize MTs against
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