Filaments, beaded

Movements of single myosin molecules along an actin filament can be measured by means of an optical trap consisting of laser beams focused on polystyrene beads attached to die ends of actin molecules. (Adapted from Finer et at., 1994. Nature 368 113- 119. See also Block, 1995. Nature 378 132 133.)... 

Actin is a 42 kDa bent dumbbell-shaped globular monomer which is found in most eukaryotic cells. It is the primary protein of the thin (or actin) filaments. Also, by mass or molarity, actin is the largest constituent of the contractile apparatus, actually reaching millimolar concentrations. Actins from different sources seem to be more similar than myosins from the same sources. Actin binds ATP which is hydrolyzed to ADP, if the monomeric actin polymerizes. The backbone structure of the actin filament is a helix formed by two linear strands of polymerized actins like two strings of actin beads entwined. 

Actin filaments are thought to exist in a double-stranded, right-hand helix with 14 subunits (per strand) per complete turn (Fig. 4.4), and a crossover distance of 38 nm. This strings of beads appearance is 70 A in diameter and thought to represent the structure of thin filaments. As the new fila-... 

Figure 4.4. Structure of actin filaments (a) shows the beads on a string appearance of an actin filament. This filament comprises actin monomers, which themselves have a polarity, so that they can only assemble head to tail , as shown in (b) thus, the actin filament is polarised, having a barbed and a pointed end.

Beaded filament and anchoring fibril collagens (types VI and VII) 508... 

Figure 6 Model of type VI collagen assembly. Two type VI collagen molecules assemble with 30 nm overlap " with two pairs of disulfide bonds between cysteines, one in collagenous domain and another in the C-terminal globular domain.Two dimers form a tetramer with disulfide bonds presumably in the a3(VI) chains. The tetramers assemble into the long beaded filamentous structure with 105nm periodicity.

An impressive demonstration that myosin heads do move along the actin filaments was provided by Sheetz and Spudich, who found that myosin-coated fluorescent beads 0.7 pm in diameter will move along actin filaments from cells of the alga Nitella in an ATP-dependent fashion at velocities similar to those required in muscle.149 The myosin heads literally glide along the thick cables of parallel actin filaments present in these algae. 

Figure 19-19 Schematic drawing (not to scale) illustrating the use of two optical traps that are focused on beads attached to a single actin filament.

The filament is lowered onto a stationary silica bead sparsely coated with HMM fragments of myosin. In the presence of ATP the myosin heads bind transiently for a few milliseconds to the actin, moving it in one direction and displacing the beads from their positions in the optical traps. An image of one of the beads is projected onto photodiode detectors capable of measuring small displacements. The displacing force can also be recorded. For details of the experiments and of the optical traps and measuring devices see Finer et al.200 Courtesy of J. A. Spudich. 

The radiation pressure exerted by light is very weak. A bright laser beam of several milliwatts of power can exert only a few piconewtons (pN) of force. However, a force of 10 pN is enough to pull a cell of E. coli through water ten times faster than it can swim.213 In about 1986, it was found that a laser beam focused down to a spot of - one K ( 1 pm for an infrared laser) can trap and hold in its focus a retractile bead of 1 pm diameter. This "optical tweezers" has become an important experimental tool with many uses.213 214 For example, see Fig. 19-19. Not only are optical tweezers of utility in studying biological motors but also mechanical properties of all sorts of macromolecules can be examined. For example, DNA can be stretched and its extensibility measured.215 Actin filaments have even been tied into knots 216... 

Actin is a protein that polymerizes at physiologic ionic strength levels into thin filaments. The monomers in actin are oriented to one another by a rotation of 166° and a translation of 275 nm. This gives an assembly that resembles a double-stranded string of beads. 

Goulielmos, G., Gounari, F., Remington, S., Muller, S., Haner, M., Aebi, U., and Georgatos, S. D. (1996). Filensin and phakinin form a novel type of beaded intermediate filaments and coassemble de novo in cultured cells. J. Cell Biol. 132, 643-655. 

In addition, the tetramers are seen to interact to form long chains (beaded filaments). Dimers, tetramers, and polymeric chains all have 30-75-30 nm spacing between the globular ends (Furthmayr et al., 1983 Von Der Mark et al., 1984 Wu et al., 1987). Collagen VI is found to bind to hyaluronan (Kielty et al., 1992), biglycan and decorin (Wiberg et al., 2001), fibrillin (Ueda and Yue, 2003), and other matrix constituents. 

Henderson, M., Polewski, R., Fanning, J. C., and Gibson, M. A. (1996). Microfibril-associated glycoprotein-1 (MAGP-1) is specifically located on the beads of the beaded-filament structure for fibrillin-containing microfibrils as visualized by the rotary shadowing technique./. Histochem. Cytochem. 44, 1389-1397. 

A needle source consists of a hairpin filament (M80 pm diameter), usuafly of a refractory metal such as tungsten, with a short length of smaller diameter (M25 pm) wire spot-welded to it, Figure lb. The tip of the latter wire, the emitter, is electrochemically etched to a point with a radius of curvature at the apex of 2-5 pm the etching technique for tungsten has been described in detail by others (7,29). As quickly as possible after the assembly is thermally cleaned under vacuum (n<10 " Pa), the emitter is dipped into a molten pool of liquid metal and then withdrawn. If done correctly, the junction formed by the bend in the filament and the emitter wire will hold a small bead of metal, and the emitter will appear shiny from the thin film of metal on its surface. 

N-McLhylmorpholine-N-oxidc monohydrate, a tertiary, aliphatic amine N-oxide, is able to dissolve cellulose directly, i.e. without chemical derivatization, which is used on an industrial scale as the basis of the Lyocell process [ 1, 2], This technology only requires a comparatively low number of process steps compared for instance to traditional viscose production. Cellulose material - mainly fibers - are directly obtained from the cellulose solution in NMMO no chemical derivatization, such as alkalization and xanthation for rayon fibers, is required [3]. The main advantage of the Lyocell process lies in its environmental compatibility very few process chemicals are applied, and in the idealized case NMMO and water are completely recycled, which is also an important economic factor. Even in industrial production systems NMMO recovery is greater than 99%. Thus, compared with cotton and viscose the Lyocell process pertains a significantly lower specific environmental challenge [4]. Today, Lyocell fibers are produced on an industrial scale, and other cellulosic products, such as films, beads, membranes and filaments, are also currently being developed or are already produced commercially. 

Bias corrections determined from analysis of standards are applied to the samples under test. Use of such an average bias correction can be viewed only as an approximation to the truth so many factors contribute to bias that it is impossible to control them all. For example, as previously stated, the work function of a rhenium filament is determined by which crystal face is involved One way of loading samples on filaments is through use of single resin beads [56,57]. The beads are 100-200 xm in diameter, which is about the size of rhenium crystallites in a poly crystalline filament [17]. Clearly the work function applicable to the analysis in question may or may not be that operative when instrument calibration was carried out. Another parameter difficult to control in real-world conditions is sample purity, which also affects bias. It is impossible to purify all samples to the same degree, and contaminants adversely affect ionization efficiency low efficiency means higher filament temperatures, which in turn mean a different bias correction. These are only two of sundry variables that can affect ionization efficiency. 

---