Actin polymerisation

Assembly and disassembly of the actin network is controlled at two major steps (i) the length of individual actin filaments and (ii) the extent to which these filaments are cross-linked to each other (Fig. 4.2). Filament length itself is controlled by the rates of  

Several experimental approaches can be employed to determine the pool sizes of polymerised and non-polymerised actin. Firstly, the enzyme DNAse I is inhibited by monomeric (G) actin, but not by polymerised (F) actin. Secondly, polymerised actin can be directly visualised by use of fluorescent derivatives of phalloidin, a cyclic peptide isolated from the toadstool Amanita phalloides that selectively binds to polymerised actin with high affinity. 

Actin comprises about 5-8% of the total protein of the neutrophil, and in resting cells about 50-70% of the actin pool exists as a monomer. This proportion of monomeric G-actin is far in excess of what would be predicted from the critical concentration for actin assembly in vitro. Thus, in vivo actin polymerisation and depolymerisation is regulated by the activities of a number of binding proteins, cations and other regulatory molecules, which are in turn regulated by the activation status of the cell. 

The initial step in actin polymerisation is the activation of actin monomers by binding to divalent cations, a process that causes a conformational change in the monomer. Such activated actin monomers may then either 

20 monomers added per sec per /tM actin monomer and 2 monomers removed per sec per /tM actin monomer. 

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Figure 4.1. Actin polymerisation. Actin monomers (G-actin) may reversibly assemble into actin filaments (F-actin). Profilin binds to G actin (to form profilactin) and thus prevents its polymerisation.

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). 

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. 

Bochsler, P. N., Neilsen, N. R., Dean, D. F., Slauson, D. O. (1992). Stimulus-dependent actin polymerisation in bovine neutrophils. Inflammation 16,383-92. 

Like the film Notorious by Alfred Hitchcock, natural products have found diverse and yet complex roles for targeting actin polymerisation. Motivated by their ability to regulate structure and motility at the cellular level, organisms that produce these metabolites gain access to tools that can be used not only to spy on and inhibit the motility of their potential predators and prey but also terminate them by means of regulating their cellular structure. While few molecular pathways have yet to reach the level of understanding as that of actin dynamics, it is clear that the role of the natural product within this story was not only the key to the plot but also delivered an award winning performance. 

Xanthine oxidase increased the rate of actin polymerisation and accelerated the conversion of F(ATP)actin into F(ADP,Pj)actin (Lanzara et al. 1988). 

HOCl induced a rapidly increasing yield of carbonyl groups in rabbit skeletal muscle actin (Dalle-Donne et al. 2001). However, when carbo-nylation became evident, some cysteine and methionine residues had been already oxidised. HOCl-mediated oxidation induced the progressive disruption of actin filaments and the inhibition of F-actin formation. The molar ratios of HOCl to actin that lead to inhibition of actin polymerisation seemed to have effect only on cysteines and methionines. 

P-Actin 70 - favours G-actin polymerisation, determining the growth of actin 41... 

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