Motility assay

Figure 13, A schematic diagram of the motility assay. Myosin molecules (HMM or S-1 are also used) stick to glass coverslips coated with nitrocellulose. Actin, in solution, is then added to the glass coverslip and it binds to the myosin molecules. When ATP is added, actin can move over the surface, propelled by the myosin molecules.

Figure 12. Cell motility assay using the computer-controlled LCD images, (a) An image to measure the ratio of Tetrahymena trap. Trapped cells were marked with black arrows, (b) The ratios of aligned Tetrahymena according to the light contrast. At least five rephcates were conducted. (Reproduced with permission from Ref [25] Copyright 2008, American Institute of Physics.)...

Pellegrino, T., et ah. Quantum dot-based cell motility assay. Differentiation 2003, 71, 542-548. 

Klnesln-dependent movement of vesicles can be tracked by In vitro motility assays similar to those used to study myosin-dependent movements. In one type of assay, a vesicle... 

LC17a or skeletal LCl or LC3 isoforms, also suggest that the velocity in the in vitro motility assay is determined by HC and not by the isoform of ELC (Tiybus, 1994). 

Mutations of LC20 coupled with motility assays (see Trybus, Chapter 3 this volume) claim major victories in elucidation of the elementary steps in the contractile interaction between myosin and actin. The scope of this approach is rapidly expanding and new discoveries are expected in the future. Application of these methods to LC17 would be of importance. The first problem to be solved is the reversible removal of LC17 from vertebrate smooth muscle myosin. The question arises whether it is possible to dissect LC17 selectively while LC20 remains bound to the HC. 

RLC phosphorylation also causes myosin to move actin filaments in a motility assay. Dephosphorylated extended myosin adhered to a nitrocellulose substratum does not support movement of actin fila-... 

Functional studies clearly show the presence of high-affinity, TM-dependent sites linked to inhibition. Thus CD inhibition of actin-TM correlates with 1 CD bound per 14 actin (Smith et al., 1987 Marston and Redwood, 1992,1993 Velaz etal., 1989), whereas 1 CD for 1-2 actin is required to inhibit actin filaments in the absence of TM (see Fig. 6) (Marston and Redwood, 1993). In addition, we have observed that CD switches off the actin-TM filament movement in the motility assay with a half-maximal effect at 3 nM CD, corresponding to an affinity of >10 (/ = 0.09 M) (Fra-... 

In the in vitro motility assay, two effects can be identified. Addition of CD promotes the interaction and movement of actin filaments over smooth muscle myosin, presumably because the myosin-CD-actin interaction tethers thick and thin filaments together, permitting interaction (Haeberle etal., 1992b). At higher CD concentrations, CD tethering appears to exert a drag upon movement of actin filaments over myosin that slows down the speed of filament movement (Horiuchi and Chacko, 1995). 

The same effect was observed in motility assays, where restoration of movement of actin over myosin required high concentrations (10 xM) of CaM (Shirinsky et al., 1992). CP can also be phosphoiylated in vitro and this phosphorylation abolishes its inhibitory activity (Winder and Walsh, 1990c). CP phosphorylation is considered in more detail in Section VI. 

In the meantime, the ability to generate large quantities of purified recombinant CP and its subdomains opens the way for the production of crystals for the determination of the molecular structure via X-ray crystallography. This should provide the basis for analyzing in more detail the interaction between CP and its binding partners. We can also anticipate more insight into the in vitro function of CP subdomains from binding studies and in vitro motility assays. 

The Nitella assay served as the inspiration for a second-generation in vitro motility assay. In this assay, which was developed by Kron and Spudich (1986), myosin is bound to a glass coverslip, where it moves actin filaments that are free in solution (see Kg. 

In general, the two motility assays give the same values for velocity and have similar characteristics. In each case the movement is assumed to be "unloaded" since the velocity does not depend on the concentration of myosin bound to the bead or to the glass surface above a certain threshold level (Collins et al., 1990 Sellers et al., 1985 Sheetz et al., 1984). The velocity is also not dependent on the length of actin filaments in the sliding actin assay (Collins et al, 1990 Warshaw et al., 1990). In both systems the direction of movement is determined by the polarity of actin (Sellers and Ka-... 

In this chapter, I will discuss the principle and design of in vitro motility assays and their use for myosin in general and smooth muscle myosin in particular. 

There are several detailed accounts of the equipment and methods required to perform in vitro motility assays (Kron et al., 1991 Sheetz et al., 1986 Warrick et al, 1993 Sellers etal., 1993 Higashi-Fujime, 1991). Because of its ease of use and more general applicability, I will describe only the sliding filament assay in this chapter. A diagram of the equipment needed to measure in vitro motility is shown in Fig. 1. 

FIGURE 1 Schematic of the equipment required for the sliding actin in vitro motility assay. The minimal equipment would be the microscope, an imaging system, and a video monitor. 

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