Cells must exert traction forces onto the substratum for continuous migration. Molecular dynamics such as actin polymerization at the front of the cell and myosin II accumulation at the rear should play important roles in the exertion of forces required for migration. Therefore, it is important to reveal the relationships between the traction forces and molecular dynamics. Traction forces can be calculated from the deformation of the elastic substratum under a migrating cell. A transparent and colorless elastic substratum with a high refractive index (1.40) and a low Young's modulus (1.0 kPa) were made from a pair of platinum-catalyzed silicones. We used this substratum to develop a new method for simultaneous recording of molecular dynamics and traction forces under a migrating cell in which total internal refractive fluorescence (TIRF) and force microscopies were combined. This new method allows the detection of the spatiotemporal distribution of traction forces produced by individual filopodia in migrating Dictyostelium cells, as well as simultaneous visualization of these traction forces and the dynamics of filamentous myosin II.
Cell migration plays an essential role in the development of most organisms (1,2) and in adult life (3). A migrating cell exerts the traction forces to the substratum to realize its migration. Traction forces are generated by intracellular molecular machinery. At the anterior of the cell, actin polymerization is considered the source of the driving forces for extension of the leading edge (3,4,5,6). On the other hand, the detachment of the rear of the cell from the substratum and its retraction is thought to occur through contraction via a myosin II-dependent process (7,8,9,10,11). At the front of the cell, actin polymerization-mediated counterforces should therefore be exerted onto the substratum in the opposite direction of extension. On the other hand, prior to detachment of the rear of the cell from the substratum, myosin II accumulation and a subsequent increase in traction forces should be exerted onto the substratum via a myosin II-dependent process. To detect the traction forces exerted by migrating cells, Dembo and colleagues (12,13) developed force microscopy, in which the measurement of deformation of the elastic substratum under a migrating cell was converted into stress in the substratum using finite element methods. They showed a detailed map under migrating fibroblasts (14,15,16,17,18,19) and revealed dynamic traction stresses at the leading edge of migrating fibroblasts (15,16). On the other hand, Butler et al. (20) proposed another kind of calculation method, namely Fourier-transform traction cytometry, and showed a detailed traction map under smooth muscle cells.
Fluorescence of green fluorescent protein (GFP)-zyxin in a migrating fibroblast and traction forces were simultaneously observed using fluorescence microscopy (17), and in some cases, traction forces appeared at nascent focal adhesions. Simultaneous observation of more detailed molecular dynamics, such as polymerization of actin at the leading edge or accumulation of filamentous myosin II at the rear end of migrating cells, as well as exertion of traction forces onto the substratum, could provide important information about the relationships between molecular dynamics and their production of traction forces.
Dictyostelium is one of the most appropriate materials to investigate the relationships between cell migration and molecular dynamics because Dictyostelium cells are known as fast moving irregularly shaped cells and molecular genetic approaches can be easily applied to them, such as expressing GFP fusion proteins. Although it had been difficult to measure the traction force of migrating Dictyostelium cells because of their small size, several authors recently showed the distribution of traction forces under a migrating Dictyostelium cell (11,20,21,22,23).
We therefore developed a new method for simultaneous recording of molecular dynamics and traction forces under migrating cells, in which total internal refractive fluorescence (TIRF) and force microscopy were combined. Here we describe the method and its application to simultaneous recording of enhanced GFP-myosin II and traction forces under a migrating Dictyostelium cell, as well as the recording of traction forces produced by individual filopodia.Materials and Methods Cell Culture
Dictyostelium discoideum cells were cultured in HL5 medium (1.3% w/v bacteriological peptone, 0.75% w/v yeast extract, 85.5 mM D-glucose, 3.5 mM Na2HPO412H2O, 3.5 mM KH2PO4, pH 6.4) and developed until the cells became aggregation-competent in a balanced salt solution (BSS; 10 mM NaCl, 10 mM KCl, 3 mM CaCl2). Two cell lines, AX2 (referred to as wild-type in this paper and an axenic derivative of the wild-type strain NC4) expressing GFP-ABD120k (24) and myosin heavy chain null cells (HS1) expressing GFP-myosin II (25), were used. ABD120k is the actinbinding domain of an actin cross-linking protein, ABP120k. ABD120k can bind to filamentous but not globular actin. Therefore, fluorescence imaging of GFP-ABD120k reflects the distribution of filamentous actin. The GFP-ABD120k gene was kindly provided from David A. Knecht (University of Connecticut, Storrs, CT). This fused gene was inserted into the pBIG expression vector by Taro Q. Uyeda (AIST, Tsukuba, Japan).Preparation of Elastic Substrata
To simultaneously observe the dynamics of molecular machinery and traction forces in a migrating cell, we combined TIRF and force microscopy. To create an evanescent field above the surface of elastic substrata, the refractive index of the substrata must be higher than 1.33, the index of the medium surrounding the cells. An evanescent field could not be created on the surface of elastic substrata made with previously described materials such as polyacrylamide (15) and gelatin (26) because of their low refractive indexes and/or low degree of transparency. Therefore, we selected a pair of liquid silicones (CY52-276A and B; Dow Corning Toray, Tokyo, Japan) as materials for the elastic substrata. These are platinum-catalyzed silicones that use a platinum complex to participate in hydrosilylation of a vinyl functional siloxane polymer by a hydride functional siloxane polymer. The gel made from the pair of silicones is clear and colorless, and its refractive index is 1.40. Moreover, the elasticity of the gel can be modulated by changing the mixing ratio of the two liquid silicones.