Actin fibers

The F-actin helix has 13 molecules of G-actin in six turns of the helix, repeating every 360 A. Oriented gels of actin fibers yield x-ray fiber diffraction patterns to about 6 A resolution. Knowing the atomic structure of G-actin it was possible for the group of Ken Holmes to determine its orientation in the F-actin fiber, and thus arrive at an atomic model of the actin filament that best accounted for the fiber diffraction pattern. 

Monomeric G-actin (43 kDa G, globular) makes up 25% of muscle protein by weight. At physiologic ionic strength and in the presence of Mg, G-actin polymerizes noncovalently to form an insoluble double helical filament called F-actin (Figure 49-3). The F-actin fiber is 6-7 nm thick and has a pitch or repeating structure every 35.5 nm. 

Microfilaments of F actin traverse the microvilli in ordered bundles. The microfila-ments are attached to each other by actin-as-sociated proteins, particularly fimbrin and vil-lin. Calmodulin and a myosin-like ATPase connect the microfilaments laterally to the plasma membrane. Fodrin, another microfila-ment-associated protein, anchors the actin fibers to each other at the base, as well as attaching them to the cytoplasmic membrane and to a network of intermediate filaments. In this example, the microfilaments have a mainly static function. In other cases, actin is also involved in dynamic processes. These include muscle contraction (see p. 332), cell movement, phagocytosis by immune cells, the formation of microspikes and lamellipo-dia (cellular extensions), and the acrosomal process during the fusion of sperm with the egg cell. 

NO also disrupts cytoskeletal protein complex formation and arrangement of actin fibers in ECs (Kroll and Waltenberger 1997 Lackey et al. 2000), resulting in dilation of the cells tight junctions (Kroll and Waltenberger 1997). 

For these experiments, they used a more well-defined method for attaching the myosin to the beads. The beads were clumps of killed bacterial (Staphylococcus aureus) cells. These cells have a protein on their surface that binds to the Fc region of antibody molecules (Fig. 5-2la). The antibodies, in turn, bind to several (unknown) places along the tail of the myosin molecule. When bead-antibody-myosin complexes were prepared with intact myosin molecules, they would move along Nitella actin fibers in the presence of ATP. 

Spudich and colleagues prepared bead-antibody-myosin complexes with varying amounts of myosin, HMM, and SHMM, and measured their speeds along Nitella actin fibers in the presence of ATP. The graph below sketches their results. 

Fig. 9.1. The first biological labeling experiment with colloidal semiconductor nanocrystal quantum dots, reproduced from the 1998 paper by Ahvisatos and Weiss [1]. A larger size of dot, red emitting, has been used to label the actin fibers of the fibroblast cells, while a smaller, green-emitting set of dots is used to label the histone proteins in the nuclei. Today colloidal quantum dots are widely used in biological imaging, demonstrating the important role of nanoparticles in this field...

MFBs synthesize far more actin fibers than standard fibroblasts, and also have myosin fibers that, when interacting with actin, constitute the contractile motor activity of MFBs. MFBs are found in healing tissue, of which they form 40% of the total number of fibroblasts present. A large number of MFBs are also foimd in the periprosthetic capsule of encapsulated breast implants, while there is no evidence of MFBs if no capsule has formed around the prosthesis. In pathology, abnormal quantities of MFBs are found in diseases such as pulmonary fibrosis and Crohn s disease. Many SMCs respond to a kind of paracrine stimulation, where the mediator is released into the environment of the target cells and diffuses towards the cell, where it interacts with a membrane receptor. 

In some cases, a purified protein chemically linked to a fluorescent dye can be mlcrolnjected into cells and followed by fluorescence microscopy. For example, findings from careful biochemical studies have established that purified actin tagged with a flurochrome is Indistinguishable in function from its normal counterpart. When the tagged protein is mlcrolnjected into a cultured cell, the endogenous cellular and Injected tagged actin monomers copolymerize into normal long actin fibers. This technique can also be used to study individual microtubules within a cell. 

It has been demonstrated that dynein has ATPase activity, with binding of ATP associated with the breaking of dynein cross-bridges. Thus, there are similarities between the mechanisms of the beating of cilia and flagella and the ATP-driven walking of myosin heads along the actin fiber, but there appears to be no relationship between the two systems at the level of protein structure. 

Evidence exists for another kind of transport in the cytoplasm. This one involves not microtubules, but actin fibers. Thus, microtubules may represent the "superhighways" for intracellular transport, whereas actin fibers may serve as the "country roads". 

The actin preparations of these authors do actually contain traces of Mg, which are indispensible for the polymerization by alkali salts the process does not occur if Mg is removed by Calgon. Magnesium is not necessary, however, for that part of the polymerization process in which long actin fibers are formed. When G-actin is treated with 2.5 X 10 M Mg++, the viscosity does not change at first, but when this Mg++, together with that originally present, is removed with calgon. 

Shibuya et al. prepared EGF receptor-overexpressing NIH3T3 (ER12) cells (14). EGF can induce transformed phenotypes in ER12 cells. 2,5-MeC inhibited EGF-induced transformation of morphology, actin fiber organization, and fibronectin expression (15). It also inhibited EGF-induced DNA synthesis in quiescent ER12 cells. Pulse... 

Similar to the situation with 2D membranes, the basic molecular characteristics of chitosan such as DD also show great influence on the ability of chitosan scaffolds to modulate stem cell behavior. The chitosan scaffolds with a high DD can maintain the viability and pluripotency of buffalo embryonic stem-like (ES-Uke) cells [15]. However, the cell behavior on 2D and 3D environments are quite different. Comparison of MSC behavior in both 2D plates and chitosan/gelatin/chondroitin scaffolds demonstrates that the 3D microenviromnent can enhance osteogenesis and maintain the viability of cells [161]. The research by Altman et al. [162] found that the apparent elastic modulus and cytoskeleton F-actin fiber density were higher for ADSCs seeded in 3D sflk fibroin/chitosan scaffolds than on 2D glass plates (Fig. 13). 

Fig. 13 Fluorescent Images and the corresponding line profiles of the F-actin fibers red) of ADSCs seeded on (a) glass surface and (b) silk fibroin/chitosan (SFCS) scaffold. F-actin fiber density of ADSCs was quantified and confirmed by line-profile analysis of the fibers using Image software. The x-axis is the distance in microns, and the peaks correspond to the intensity of the rhodamine-phalloidin stain (red), whose peak maximum occurs at the location of the fibers along the line. Nuclei were stained with DAPI (blue) [162]...

An alternative to the explicit analysis of actin fibers is a continuum approach where the cytoskeletal fibers are treated as one of two phases of the cytosol. Dembo and Harlow [1986] have proposed a general model of contractile biological polymer networks based on the analysis of reactive interpenetrating flow. In that model, the cytoplasm was viewed as a mixture of a contractile network of randomly oriented cytoskeletal filaments and an aqueous solution. Both phases were treated as homogeneous Newtonian fluids. Later, Alt and Dembo [1999] have applied a similar approach to the modehng of the motion of ameboid cells. The authors paid a special attention to the boundary conditions. They introduced three boundary surfaces the area of contact between the cell and the substrate, the surface separating the cell... 

---