Cytoskeleton. The main components of the cytoskeleton include microfilaments, intermediate filaments, and microtubules (Figure 1). Microfilaments are linear polymers of actin subunits that can assemble into helical filaments (F-actin). They resist tension in the cell and can polymerize or depolymerize within minutes, which facilitates efficient cell motility. Microfilaments also main- tain the positioning of organelles in the cell and provide overall resistance to deformation from external stimuli. Intermediate filaments serve largely to provide structure in cells and have been classified into six types according to similarities in amino acid sequences and protein structures. They form an elaborate network in the cytoplasm, extending from the nucleus, whose lamina largely comprises intermediate filaments (lamins), to the plasma membrane. Intermediate fila- ments connect with other cytoskeletal elements, organelles, and the cell membrane to form a fully connected network. They also resist tension and are thought to contribute significantly at larger deformations, otherwise remaining mechanically passive (4). That said, recent work has indicated that keratins (5) and vimentin (6), two types of intermediate filaments, can contribute signifi- cantly to small-deformation responses as well. Microtubules are polymers of tubulin that serve to transport intracellular components throughout the cell during normal biological processes while also contributing to overall cellular structure and behavior. Microtubules resist compression, as opposed to microfilaments and intermediate filaments. The entire cytoskeleton acts in concert to balance tensile and compressive loads, and it is this combined network that contributes to a cell’s mechanical phenotype (7–9).
In addition to the three main cytoskeletal elements, there are many other proteins critical to the overall functionality of the mechanical network. Of particular interest are nucleocytoskele- tal proteins that link the nucleus to the cytoskeleton. Intensive study is looking at the role key molecules play in mechanosensing—such as lamins A, B1, B2, and C; spectrin; kinesin; plectin; emerin; SUN proteins, and KASH proteins (i.e., nesprins 1, 2, 3, and 4)—and, to a lesser extent, how these molecules affect the mechanical phenotype of a cell (reviewed in 2, 3).
Besides the relative content of each cytoskeletal protein, their organization has a significant role in how a cell generates or resists forces (10–12). Likewise, cellular interactions with the microenvironment can dramatically influence measured mechanical properties. When spread on a surface, the cytoskeletal network forms a web of filamentous structures that changes over time as the cell moves. In general, the mechanical properties of adhered cells in this morphology are less compliant than in rounded cells (13–15). However, this relationship may not extend fully to the suspended state unless the structural elements contributing to the exhibited phenotype remain consistent in both states (16). The cytoskeleton makes both passive and active contributions to cellular mechanical proper- ties. In an equilibrium state, when the cell is not dividing or moving, a portion of the cytoskeletal structure will remain largely static, and, hence, will impart a general mechanical phenotype to the cell. This passive contribution is often representative of the cell and can be measured using a number of techniques (described in the section entitled Techniques for Measuring the Mechanical Properties of Single Cells). However, the cytoskeleton is never completely static. Portions will naturally depolymerize and polymerize in response to both intracellular and extracellular stimuli. These biological or mechanical signals can induce structural changes, which, if extensive enough, may significantly alter the overall mechanical characteristics of the cell. This is especially notice- able during the cell cycle, when extensive cytoskeletal remodeling occurs to allow for cell division (17). During cell division, the generation of an internal osmotic pressure also creates a prestress on the cortical actomyosin network, leading to increases in measured stiffness (18). Major rearrange- ments in cytoskeletal structure that correspond to cell–extracellular matrix and cell–cell contacts will also result in significant changes in mechanical properties, which occur in concert with the expression of differentiation-specific markers (19). Since many common biochemical techniques to assay cytoskeletal architecture and activity are destructive and cannot be used with living cells, mechanical phenotyping provides an attractive alternative for determining a cell’s state based on these properties.