Microfilament assembly

DeBell, K. E., Conti, A., Alava, M. A., Hoffman, T., and Bonvini, E. (1992) Microfilament assembly modulates phospholipase C-mediated signal transduction by the TCR/CD3 in murine T helper lymphocytes. J. Immunol. 149,2271-2280. 

Aullo P, Giry M, Olsnes S et al. (1993) A chimeric toxin to study the role of the 21 kDa GTP binding protein rho in the control of actin microfilament assembly. In EMBO J. 12 921 -31... 

Mauss S, Koch G, Kreye VA, et al. (1989) Inhibition of the contraction of the isolated longitudinal muscle of the guinea-pig ileum by botulinum C2 toxin evidence for a role of G/F-actin transition in smooth muscle contraction. In Noun-Schmiedebergs Archiv Pharmacol. 340 345-51 Melamed I, Downey GP, Aktories K, et al. (1991) Microfilament assembly is required for antigen-receptor-mediated activation of human B lymphocytes. In J Immunol. 147 1139-46... 

Melamed I, Franklin RA, Gelfand EW (1995a) Microfilament assembly is required for anti-IgM dependent MAPK and p90rsk activation in human B lymphocytes. In Biochem and Biophys Res Comm. 209 1102-10 Melamed I, Stein L, Roifman CM (1994) Epstein-Barr virus induces actin polymerization in human B cells. In J Immunol. 153 1998-2003 Melamed I, Turner CE, Aktories K, et al. (1995b) Nerve growth factor triggers microfilament assembly and paxillin phosphorylation in human B lymphocytes. In J Exp Med. 181 1071-9... 

Rossol M, Gartner D, Hauschildt S. Diverse r ulation of microfilament assembly, production of TNF-alpha, and reactive o n intermediates by aedn modulating substances and inhibitors of ADP-ribosylation in human monocytes sdmulated with LPS. Cell Motil Cytoskeleton 2001 48(2) 96-108. 

In this chapter we describe the distribution, assembly, and interaction of microfilaments and microtubules and their functional roles in cell movement and in the maintenance of the spatial organization of the cytoplasm. Also, the relative roles... 

Just as myosins are able to move along microfilaments, there are motor proteins that move along microtubules. Microtubules, like microfilaments, are polar polymeric assemblies, but unlike actin-myosin interactions, microtubule-based motors exist that move along microtubules in either direction. A constant traffic of vesicles and organelles is visible in cultured cells especially using time-lapse photography. The larger part of this movement takes place on micrombules and is stimulated by phorbol ester (an activator of protein kinase C), and over-expression of N-J aj oncoprotein (Alexandrova et al., 1993). 

An intracellular fibrous system exists of filaments with an axial periodicity of 21 nm and a diameter of 8-10 nm that is intermediate between that of microfilaments (6 nm) and microtubules (23 nm). Four classes of intermediate filaments are found, as indicated in Table 49-13. They are all elongated, fibrous molecules, with a central rod domain, an amino terminal head, and a carboxyl terminal tail. They form a structure like a rope, and the mature filaments are composed of tetramers packed together in a helical manner. They are important structural components of cells, and most are relatively stable components of the cytoskeleton, not undergoing rapid assembly and disassembly and not... 

In the resting neutrophil, about 50% of the actin is present in filaments within the cytoskeleton (and hence insoluble in detergents such as Triton X-100), whereas the remainder is detergent soluble and hence is not associated with the cytoskeleton. Data from studies of actin polymerisation in vitro predict that almost all of the actin within the cell should be F-actin (i.e. present in microfilaments). Upon stimulation of neutrophils with agonists such as fMet-Leu-Phe or PMA, actin polymerisation is activated extremely rapidly. There are two important questions Firstly, how is actin maintained in the unpolymerised state in resting cells Secondly, how is it rapidly assembled into the cytoskeleton during activation The answers to these questions lie in understanding the functions of the numerous proteins involved in the assembly and disassembly of actin filaments (Table 4.1). 

Microtubules may function as a form of skeletal support for microfilaments. Agents that increase intracellular cGMP favour the assembly of microtubules, whereas those that increase intracellular Ca2+ and cAMP result in the dissolution of tubulin fibres. Furthermore, the oxidation state of the neutrophil may affect the integrity of the tubulin fibres. Oxidised glutathione (which is increased during oxidative metabolism) regulates tubulin disassembly, and oxidation may increase tubulin tyrosylation, which also promotes disassembly. 

Cytochalasins are mold metabolites (from Zygospohum mansonii and related molds), which inhibit microfilament polymerization by capping the growing end of the filaments and preventing further filament assembly and resulting in shortening (89). For disruption of the actin cytoskeleton, cells are pretreated with CD [1 lOpM (added from a stock in DMSO)] in complete culture medium for 30 minutes to 5 hours (44,80) [cytochalasin B (CB)2pg/mL]. 

The cytoplasm of eukaryotic cells is traversed by three-dimensional scaffolding structures consisting of filaments (long protein fibers), which together form the cytoskeleton. These filaments are divided into three groups, based on their diameters microfilaments (6-8 nm), intermediate filaments (ca. 10 nm), and microtubules (ca. 25 nm). All of these filaments are polymers assembled from protein components. 

Paclitaxel (Taxol) is a highly complex, organic compound isolated from the bark of the Pacific yew tree. It binds to tubulin dimers and microtubulin filaments, promoting the assembly of filaments and preventing their depolymerization. This increase in the stability of microfilaments results in disruption of mitosis and cytotoxicity and disrupts other normal microtubular functions, such as axonal transport in nerve fibers. 

Three principal components of the cytoskeleton are microfilaments of 6 nm diameter, microtubules of 23-25 nm diameter, and intermediate filaments of 10 nm diameter. A large number of associated proteins provide for interconnections, for assembly, and for disassembly of the cytoskeleton. Other proteins act as motors that provide motion. One of these motors is present in myosin of muscle. This protein is not only the motor for muscular work but also forms thick filaments of 12-16 nm diameter, which are a major structural component of muscle (see Fig. 19-6). 

In the cytosol of eukaryotic cells is an internal scaffold, the cytoskeleton (see Topic E2). The cytoskeleton is important in maintaining and altering the shape of the cell, in enabling the cell to move from one place to another, and in transporting intracellular vesicles. Three types of filaments make up the cytoskeleton microfilaments, intermediate filaments and microtubules. The microfilaments, diameter approximately 7 nm, are made of actin and have a mechanically supportive function. Through their interaction with myosin (see Topic Nl), the microfilaments form contractile assemblies that are involved... 

Eukaryotic cells have an internal scaffolding called the cytoskeleton or cytomatrix that maintains their cellular morphology and enables them to migrate, undergo shape changes, and transport vesicles. Microfilaments, made of actin, intermediate filaments, which are composed of laminin and other proteins, and microtubules, formed from the protein tubulin, along with many different accessory proteins, comprise the cytoskeleton. Both the microfilaments and the microtubules can assemble and disassemble rapidly in the cell, whereas disassembly of intermediate filaments may require their destruction. Although much is known about the molecular composition of the cytoskeleton, the molecular events involved in most cell movements are still unknown. 

Cytoskeleton. Many cellular activities, such as motility, endocytosis, exocytosis, and cell division, rely on microfilaments and microtubules. A number of alkaloids have been detected which can interfere with the assembly or disassembly of microtubules (Table IV), namely, vincristine, vinblastine, colchicine, maytansine, maytansinine, and taxol. 

Kidder, G. M., Rains, J. and McKeon J. (1987). Gap junction assembly in the preimplantation mouse conceptus is independent of microtubules, microfilaments, cell flattening and cytokines. Proc. Natl. Acad. Sci. USA 84, 3718-3722. 

After the procollagen polypeptides are assembled into a triple helix, they are secreted by the classical route. They pass through the smooth endoplasmic reticulum and the Golgi complex, where they are packaged into membranous vesicles and secreted into the extracellular space by exo-cytosis. This process requires ATP and may involve microtubules and microfilaments. The conformation of procollagen markedly affects the rate of secretion. Prevention of the formation of a triple helix (e.g., lack of 4-hydroxylation due to vitamin C deficiency) leads to the accumulation of nonhelical propolypeptides within the cistemae of the rough endoplasmic reticulum and a delayed rate in its secretion. 

The principal targets for facilitated folding by CCT in cooperation with prefoldin are the cytoskeletal proteins actin and tubulin. The actin monomer assembles into microfilaments, while the subunit that forms microtubules is the tubulin heterodimer, which consists of a single a- and a single /f-tubulin polypeptide. Though actin can be folded to the native state via one or more cycles of ATP-dependent interaction with CCT, this is not the case for either a- or /Ttubulin. Tubulin subunits released from CCT are assembled into a/fi heterodimers by interaction with several tubulin-specific chaperones known as cofactors in a reaction that depends on GTP hydrolysis by the cofactor-bound tubulin. 

Microfilaments are assembled from monomeric actin subunits microtubules, from a. -tubulin subunits and Intermediate filaments, from lamin subunits and other tissue-specific proteins. 

Intermediate filaments are assembled into networks and bundles by various intermediate filament-binding proteins, which also cross-link intermediate filaments to the plasma and nuclear membranes, microtubules, and microfilaments. 

Actin exists as a globular monomer called G-actin and as a filamentous polymer called F-actin, which is a linear chain of G-actin subunits. (The microfilaments visualized in a cell by electron microscopy are F-actin filaments plus any bound proteins.) Each actin molecule contains a Mg " ion complexed with either ATP or ADR Thus there are four states of actin ATP-G-actin, ADP-G-actin, ATP-F-actin, and ADP-F-actin. Two of these forms, ATP-G-actin and ADP-F-actin, predominate in a cell. The importance of the interconversion between the ATP and the ADP forms of actin in the assembly of the cytoskeleton is discussed later. 

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