Microfilament cytoskeleton

Several studies suggest an involvement of azaspiracid-1 in the regulation of microfilament cytoskeleton. In Jmkat T cells azaspiracid treatment induced a reduction in the number of pseudopodia (plasmatic membrane protrusions) per cell (Twiner et al. 2005). Additionally, high doses of azaspiracid-1 seemed to reduce the amount of polymerized actin in neuroblastoma cells (Roman et al. 2002). Very recent results obtained in our lab have shown that lower doses of azaspiracid-1 (10-50 nM) induced a reduction in the number of neurites (cell projections) in neuroblastoma cells (Fig. 17.3) and a disorganization of the basal actin cytoskeleton in caco-2 cells (an enterocyle cell line), without affecting the amount of polymerized actin (Vilarino et al. 2006). 

Figure 17.3. Neuroblastoma cells (BE(2)-M17 cell line) treated with azasplracld-1 (50 nM) for 48 hours show a rearrangement of their microfilament cytoskeleton and adopt a round morphology compared to the more flattened controls. (A) Carrier control (DMSO). (B) Azasplracld-1. MIcrofllaments (F-actIn) were labelled with Oregon Green Phalloldin (Molecular Probes) and pictures were taken using a Nikon Eclipse TE2000-E confocal microscope.

The microfilament cytoskeleton must also be intact for differentiation to occur, but it probably does not have a direct role in signal transduction as postulated for the microtubules. 

Microfilaments and Microtubules. There are two important classes of fibers found in the cytoplasm of many plant and animal ceUs that are characterized by nematic-like organization. These are the microfilaments and microtubules which play a central role in the determination of ceU shape, either as the dynamic element in the contractile mechanism or as the basic cytoskeleton. Microfilaments are proteinaceous bundles having diameters of 6—10 nm that are chemically similar to actin and myosin muscle ceUs. Microtubules also are formed from globular elements, but consist of hoUow tubes that are about 30 nm in diameter, uniform, and highly rigid. Both of these assemblages are found beneath the ceU membrane in a linear organization that is similar to the nematic Hquid crystal stmcture. 

The cytoskeleton also contains different accessory proteins, which, in accordance with their affinities and functions, are designated as microtubule-associated proteins (MAPs), actin-binding proteins (ABPs), intermediate-filament-associated proteins (IFAPs), and myosin-binding proteins. This chapter is focused on those parts of the cytoskeleton that are composed of microfilaments and microtubules and their associated proteins. The subject of intermediate filaments is dealt with in detail in Volume 2. 

The Cytoskeleton—Microtubules and Microfilaments Drug Effects on Microtubules... 

The Cytoskeleton—Microtubules and Microfilaments Patterns of Arrangement of Actin Filaments in Animal Cells... 

The Locomotion of Amoeba The Locomotion of Fibroblastic Cell Types The Locomotion of Leukocytes The Behavior of Locomoting Cells The Role of the Cytoskeleton in Cell Locomotion The Microtubule-Based Cytoskeleton The Intermediate Filament-Based Cytoskeleton The Microfilament-Based Cytoskeleton The Organization of Microfilaments in Cells Microfilament Dynamics and Cell Locomotion Sites of Lamellar Protrusion May Be Determined by the Nucleation of Actin Polymerization... 

Nonmuscle cells perform mechanical work, including self-propulsion, morphogenesis, cleavage, endocytosis, exocytosis, intracellular transport, and changing cell shape. These cellular functions are carried out by an extensive intracellular network of filamentous structures constimting the cytoskeleton. The cell cytoplasm is not a sac of fluid, as once thought. Essentially all eukaryotic cells contain three types of filamentous struc-mres actin filaments (7-9.5 nm in diameter also known as microfilaments), microtubules (25 nm), and intermediate filaments (10-12 nm). Each type of filament can be distinguished biochemically and by the electron microscope. 

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... 

Two major types of muscle fibers are found in humans white (anaerobic) and red (aerobic). The former are particularly used in sprints and the latter in prolonged aerobic exercise. During a sprint, muscle uses creatine phosphate and glycolysis as energy sources in the marathon, oxidation of fatty acids is of major importance during the later phases. Nonmuscle cells perform various types of mechanical work carried out by the structures constituting the cytoskeleton. These strucmres include actin filaments (microfilaments), micrombules (composed primarily of a- mbulin and p-mbulin), and intermediate filaments. The latter include keratins, vimentin-like proteins, neurofilaments, and lamins. 

The cellular cytoskeleton, primarily composed of microfilaments, microtubules, and intermediate filaments, provides structural support and enables cell motility. The cytoskeleton is composed of biological polymers and is not static. Rather, it is capable of dynamic reassembly in less than a minute [136], The cytoskeleton is built from three key components, the actin filaments, the intermediate filaments, and the microtubules. The filaments are primarily responsible for maintaining cell shape, whereas the microtubules can be seen as the load-bearing elements that prevent a cell from collapsing [136], The cytoskeleton protects cellular structures and connects mechanotransductive pathways. Along with mechanical support, the cytoskeleton plays a critical role in many biological processes. 

Two of the cytoskeletal components, the actin filaments and the microtubules have been studied with molecular rotors. The main component of the actin filaments is the actin protein, a 44 kD molecule found in two forms within the cell the monomeric globulin form (G-actin) and the filament form (F-actin). Actin binds with ATP to form the microfilaments that are responsible for cell shape and motility. The rate of polymerization from the monomeric form plays a vital role in cell movement and signaling. Actin filaments form the cortical mesh that is the basis of the cytoskeleton. The cytoskeleton has an active relationship with the plasma membrane. Functional proteins found in both structures... 

Actin microfilaments and the membrane cytoskeleton play critical roles in neuronal growth and secretion 129... 

The cytoskeleton is the collective name for all structural filaments in the cell. The cytoskeletal filaments are involved in establishing cell shape, and providing mechanical strength, locomotion, intracellular transport of organelles and chromosome separation in mitosis and meiosis. The cytoskeleton is made up of three kinds of protein filaments actin filaments (also called microfilaments), intermediate filaments and microtubules. 

Actin filaments are the thinnest of the cytoskeletal filaments, and therefore also called microfilaments. Polymerized actin monomers form long, thin fibers of about 8 nm in diameter. Along with the above-mentioned function of the cytoskeleton, actin interacts with myosin ( thick ) filaments in skeletal muscle fibers to provide the force of muscular contraction. Actin/Myosin interactions also help produce cytoplasmic streaming in most cells. 

Perhaps the most dynamic components of the cytoskeleton, the microfilaments are directly involved in cell movement and phagocytosis. They are... 

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). 

Although intermediate filaments are not universally associated with the cytoskeleton, neutrophils possess intermediate filaments of the vimentin type. Vimentin is a rod-shaped molecule of relative molecular mass 57 kDa that readily polymerises under physiological conditions to produce stable filaments 10-12 nm in diameter. Intermediate filaments are more robust than microfilaments and microtubules, and in neutrophils they form an open network of single filaments in the perinuclear space. 

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]. 

Spector I, Shochet NR, Blasberger D, Kashman Y. Latrunculins—novel marine macrolides that disrupt microfilament and affect cell growth L Comparison with cytochalasin D. Cell Motil Cytoskeleton 1989 13 127-144. 

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