Barbed ends

Cytochalasins B and D are used as tools to study F-actin. Cytochalasins bind to the barbed end of F-actin and block the addition as well as dissociation of G-actin at that end. When applied to cultured cells micromolar concentrations of cytochalasins remove stress fibres and other F-actin structures. 

The interaction with myosin motors enables F-actin to transport molecules as well as to change or maintain the shape of the cell by exerting tension. Thus, myosin-I motors move to the barbed end and can transport cargoes such as vesicles. When immobilized at the cargo site... 

The cy tochalasins A, B, C, D, E, and H are found in various species of mould. Mainly cytochalasin B and D are used as experimental tools. Cytochalasin D is 10 times more potent, acting at concentrations between 2 and 35 nM in cell-free systems. Cy tochalasins bind to the barbed end of F-actin and block the addition as well as dissociation of G-actin at that end. At micromolar concentrations, cytochalasin D can bind to G-actin and actin dimers and thus block additional polymerization. When applied to cultured cells, micromolar concentrations of cytochalasins remove stress fibres and other F-actin structures. 

Barbed-end-capping proteins (gelsolin and villin, 95 kD) attach to this specific end of the actin filament and inhibit the further addition of actin molecules. 

Hartwig, J. H., Bokoch, G. M. etal. Thrombin receptor ligation and activated Rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets. Cell 82 643-653,1995. 

Gelsolin blocks barbed ends of actin filaments 84 Ca2+,PIP2... 

Protrusion may be due to growth of new actin filaments, which requires net polymerisation of new filaments, and also by the organisation of actin-binding proteins into higher-order structures. Random movements of flexible membranes away from the filaments may result in gross distortion of actin polymerisation at the barbed ends. Thus, once a critical size is reached, ion pumping (i.e. of Ca2+) may occur at the tip of a pseudopod, which further aids directional changes in the network. 

Cytochalasins bind to the barbed ends of filaments with a A j of 10 7-10 8 M, but with much lower affinity to the sides of filaments or to mono-... 

Thus, assembly of actin filaments may occur at the pointed ends during phagocytosis, but at the barbed ends during locomotion. 

Dufort PA and Lumsden CJ [1996] How profilin/barbed-end synergy controls actin polymerization a kinetic model of the ATP hydrolysis circuit. Cell Motil... 

Figure 2 The actin-ADP-ribosylating toxins, (a) Molecular mode of action. The actin-ADP-ribosylating toxins covalently transfer an ADP-ribose moiety from NAD+ onto Arg177 of G-actin in the cytosol of targeted cells. Mono-ADP-ribosylated G-actin acts as a capping protein and inhibits the assembly of nonmodified actin into filaments. Thus, actin polymerization is blocked at the fast-growing ends of actin filaments (plus or barbed ends) but not at the slow growing ends (minus or pointed ends). This effect ultimately increases the critical concentration necessary for actin polymerization and tends to depolymerize F-actin. Finally, all actin within an intoxicated cell becomes trapped as ADP-ribosylated G-actin.

Figure 2. Scheme for the addition and loss of actin-ATP or actin-ADP from the barbed and pointed ends of an actin filament. The barbed end is the faster growing and more stable end of an actin filament. While the exchange of actin-ADP with ATP to yield actin-ATP and ADP is shown here as a spontaneous process, the actin regulatory protein profilin greatly accelerates the exchange process. Note also that hydrolysis is thought to occur after (and not coincident with) addition of actin-ATP at either end. 

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.

Barbed-End Capping Protein Blocks Net Assembly at Barbed Ends of Filaments... 

A cytoskeletal regulatory protein that binds to one end (usually the (+)-end or so-called barbed end) of... 

Actin filaments grow rapidly within cells, and the clearest evidence of this rapid growth is the ability of the cell s leading edge to move at rates of 0.5 to 1 micrometer per second. Likewise, actin-based motility of Listeria and Shigella can attain rates of nearly 0.5 micrometers per second. Because microfilaments contain about 360 actin monomers per micrometer of length, a motility rate of 0.5 to 1 micrometer per second corresponds to an apparent first-order rate constant (/.e., / apparent = on [Actin-ATP]) of about 180-360 s . The bimolecular rate constant for actin-ATP addition to the barbed end has a nominal value of 2-3 X 10 s . Therefore, one can estimate... 

Figure 2. A diagrammatic representation of those segments of an overall reaction mechanism for barbed-end actin polymerization in the absence and presence of profilin and/or thymosin- 4. For clarity the overall scheme is indicated. Experimental data were obtained for individual segments (indicated in black type), and those parts of the scheme that were not evaluated in a particular experiment are shown in gray.

Kang et al. recently used the KINSIM/FITSIM software to model barbed-end actin polymerization (See Actin Assembly Kinetics) in the absence and presence of profilin and/or thymosin-j84. This data analysis protocol permitted them to derive rate constants from a series of... 

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