Actin Self-Assembly

Actin is one of the most abundant cellular proteins and is present in all mammalian cells. F-actin is physically crosslinked to form the cellular cytoskeleton and its contraction allows cell deformation and movement 

G-actin is soluble in water and is transformed into F-actin in neutral salt solutions. Conversion of G-actin to F-actin is associated with hydrolysis of ATP leading to the formation of filaments that are up to several micrometers long and 7 to 10 nm wide. The process has been characterized as containing two steps, that is, nucleation and growth, and as is polymerization of other macromolecules, entropy driven. In muscle actin filaments tropomyosin binds to F-actin and mechanically stabilizes them. 

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Figure 3. Critical concentration behavior of actin self-assembly. For the top diagram depicting the macroscopic critical concentration curve, one determines the total amount of polymerized actin by methods that measure the sum of addition and release processes occurring at both ends. Examples of such methods are sedimentation, light scattering, fluorescence assays with pyrene-labeled actin, and viscosity measurements. Forthe bottom curves, the polymerization behavior is typically determined by fluorescence assays conducted under conditions where one of the ends is blocked by the presence of molecules such as gelsolin (a barbed-end capping protein) or spectrin-band 4.1 -actin (a complex prepared from erythrocyte membranes, such that only barbed-end growth occurs). Note further that the barbed end (or (+)-end) has a lower critical concentration than the pointed end (or (-)-end). This differential stabilization requires the occurrence of ATP hydrolysis to supply the free energy that drives subunit addition to the (+)-end at the expense of the subunit loss from the (-)-end.

Pengelly, K. Loncar, A. Perieteanu, A. A. Dawson, J. F. Cysteine engineering of actin self-assembly interfaces. Biochem. Cell Biol. 2009,87, 663-675. 

Unfortunately, the description of amyloid fibrils given above is simplistic since in vitro self-assembly of amyloid peptides and proteins yields polymorphic structures, as has been commonly observed in the past for other protein assemblies such as actin filaments (Millonig et al, 1988) and intermediate filaments (Herrmann and Aebi, 1999). On the one hand, assembly polymorphism complicates the characterization of fibril structure. On the other hand, it offers some insight into fibril formation. For this reason a more rational understanding of amyloid fibril formation at the molecular level is a key issue in the field of amyloidosis. 

The polymer self-assembly theory of Oosawa and Kasai (1962) provides valuable insights into the nature of the nucleation process. The polymerization nucleus is considered to form by the accretion of protomers, but the process is highly cooperative and unfavorable. Indeed, this is strongly suggested by the observation that thousands of actin or tubulin protomers are found in F-actin and microtubule structures if nucleation of self-assembly were readily accomplished and highly favorable, the consequence would be that many more fibers of shorter polymer length would be observed. The Oosawa kinetic theory for nucleation permits one to obtain information about the size of the polymerization nucleus if two basic assumptions can be satisfied in the experimental system. First, the rate of nuclei formation is assumed to be proportional to the loth power of the protomer concentration with io representing the number of protomers required to create the nucleus. Second, the treat-... 

True self-assembly is observed in the formation of many oligomeric proteins. Indeed, Friedman and Beychok reviewed efforts to define the subunit assembly and reconstitution pathways in multisubunit proteins, and all of the several dozen examples cited in their review represent true self-assembly. Polymeric species are also formed by true self-assembly, and the G-actin to F-actin transition is an excellent example. By contrast, there are strong indications that ribosomal RNA species play a central role in specifying the pathway to and the structure of ribosome particles. And it is interesting to note that the assembly of the tobacco mosaic virus (TMV) appears to be a two-step hybrid mechanism the coat protein subunits first combine to form 34-subunit disks by true self-assembly from monomeric and trimeric com-... 

The threshold concentration of monomer that must be exceeded for any observable polymer formation in a self-assembling system. In the context of Oosawa s condensation-equilibrium model for protein polymerization, the cooperativity of nucleation and the intrinsic thermodynamic instability of nuclei contribute to the sudden onset of polymer formation as the monomer concentration reaches and exceeds the critical concentration. Condensation-equilibrium processes that exhibit critical concentration behavior in vitro include F-actin formation from G-actin, microtubule self-assembly from tubulin, and fibril formation from amyloid P protein. Critical concentration behavior will also occur in indefinite isodesmic polymerization reactions that involve a stable template. One example is the elongation of microtubules from centrosomes, basal bodies, or axonemes. 

Although the self-assembly of polymeric structures can involve nucleation and elongation steps (See Actin Assembly Kinetics Microtubule Assembly Kinetics), one can simplify the assembly process through what is known as seeded assembly. At an initial monomer concentration [M], seeded assembly is induced by the addition of pre-assembled polymeric structures consequently the polymer number concentration must remain constant. The rate of monomer incorporation into indefinite length polymers can be written as follows ... 

BIOCHEMICAL SELF-ASSEMBLY ACTIN ASSEMBLY KINETICS HEMOGLOBIN S POLYMERIZATION MICROTUBULE ASSEMBLY KINETICS BIOCONJUGATE BIOCONVERSION BIOLEACHING... 

ACTIN ASSEMBLY KINETICS BIOCHEMICAL SELF-ASSEMBLY FAD-dependent enzymes,... 

MICROSCOPIC DIFFUSION CONTROL MACROSCOPIC DIFFUSION CONTROL MICROSCOPIC REVERSIBILITY CHEMICAL REACTION DETAILED BALANCING, RRINCIRLE OF CHEMICAL KINETICS MICROTUBULE ASSEMBLY KINETICS BIOCHEMICAL SELF-ASSEMBLY ACTIN ASSEMBLY KINETICS HEMOGLOBINS POLYMERIZATION... 

ACTIN ASSEMBLY KINETICS BIOCHEMICAL SELF-ASSEMBLY BIOMINERALIZATION PRION PLAQUE FORMATION Nucleation as a highly cooperative process, MICROTUBULE ASSEMBLY KINETICS... 

Fig. 1 Examples of enzyme-controlled supramolecular polymerisation from the biological world (a) formation of collagen fibrils, (b) dynamic self-assembly of actin filaments, and (c) formation of microtubules...

There is a range of biological self-assembled polymer fibres such as amyloids, actins and fibrin that have important positive and negative roles in biochemistry. These biopolymers act as an inspiration for supramolecular design and share many features in common with their abiotic analogues. We will mention them briefly here in the context of supramolecular polymers. 

Wong, G. C. L., Tang, J. X., Lin, A., Li, Y., Janmey, P. A. Safinya, C. R. Hierarchical self-assembly of F-actin and cationic lipid complexes Stacked three-layer tubule networks. Science (Washington, D. C.) 288, 2035-2039 (2000). 

Aggeli, A., Nyrkova, I.A., Bell, M., Harding, R., Carrick, L., McLeish, T.C.M., Semenov, A.N., and Boden, N. "Hierarchical self-assembly of chiral rod-like molecules as a model for peptide-sheet tapes, ribbons, bris and bers". Proc. Nat. Acad. Sci. U.S.A. 98,11857-11862 (2001). Attri, A.K., Lewis, M.S., and Korn, E.D. "The formation of actin oligomers studied by analytical ultracentrifugation". ]. Biol. Chem. 266, 6815-6824 (1991). 

Percolation is widely observed in chemical systems. It is a process that can describe how small, branched molecules react to form polymers, ultimately leading to an extensive network connected by chemical bonds. Other applications of percolation theory include conductivity, diffusivity, and the critical behavior of sols and gels. In biological systems, the role of the connectivity of different elements is of great importance. Examples include self-assembly of tobacco mosaic virus, actin filaments, and flagella, lymphocyte patch and cap formation, precipitation and agglutination phenomena, and immune system function. 

There are several self-assembling macromolecules that are of interest to us in this text. They include (1) collagen, the primary structural material found in the extracellular matrix (2) actin, a component of the cell cytoskeleton that is involved in cell locomotion and in formation of the thin filaments of muscle (3) microtubules, which are involved in cell mitosis, movement, and organelle movement and finally (4) fibrinogen, which forms fibrin networks that minimize bleeding from cut vessels. Self-assembly is important in these systems because the function of these macromolecules can be modified via processes that increase the molecular axial ratio and hence decrease the solubility. 

Actin and tubulin are two important cellular components that are involved in cell shape and movement. Actin is present in all mammalian cells and is involved in cellular transport and phagocytosis (eating of extracellular materials), provides rigidity to cell membranes, and when bonded to tropomyosin and troponin, forms the thin filaments of muscle. Thbulin is the subunit from which microtubules are self-assembled. Microtubules are most commonly known for their role in cell division. The mechanisms of self-assembly of these macromolecules have been well studied and are important models of biological assembly processes. Below we examine each of these processes. 

Actin exists in two states within the cytoplasm of the cell in the monomeric form (G-actin) and in the self-assembled or fibrous form (F-actin). G-actin has been shown to exist as an oblate ellipsoid by electron microscopy whereas F-actin exists as a double-helical structure. G- and F-actin exist in cells in equilibrium with actin-binding proteins. These proteins are involved in the polymerization and depolymerization of F-actin. 

The interaction of actin and myosin is key to the generation of contractile forces in skeletal muscles. Skeletal muscles are composed of thick and thin filaments the thick filaments are composed primarily of myosin and thin filaments contain actin. The interaction between the two is a reversible self-assembly process that is followed by disassembly that separates actin from myosin. This separation initiates movement of myosin with respect to actin and results in shortening of the muscle, causing force generation. It is the assembly and disassembly process that we are interested in in this section. 

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