Filament assembly

Pollard et al., 1992] Pollard, T. D., Goldberg, I., and Schwarz, W. H. Nucleotide exchange, structure, and mechanical properties of filaments assembled from ATP-actin and ADP-actin. J. Biol. Chem. 267 (1992) 20339-20345... 

Possible modes of regulation of filament assembly may be anticipated from the basic properties of actin. We have shown that the tightly bound divalent metal ion (Ca or Mg ) interacts with the P- and y-phosphates of ATP bound to actin, and that the Me-ATP bidentate chelate is bound to G-actin in the A configuration. The nature of the bound metal ion affects the conformation of actin, the binding kinetics of ATP and ADP, and the rate of ATP hydrolysis. 

HOW PROFILIN CONTROLS MONOMER-POLYMER STEADY-STATE AND PROMOTES ACTIN FILAMENT ASSEMBLY IN THE PRESENCE OF THYMOSYN p4... 

Pantaloni, D. Carlier, M.-F. (1993). How profilin promotes actin filament assembly in the presence of thymosin beta 4. Cell, 75, 1007-1014. 

In a previous section we mentioned the significance of myosin filament structure. In nematodes two forms of myosin-II, myosin A and B, are required for proper filament stmcture (Epstein, 1988). The two forms of myosin are expressed at the proper time to allow for correct filament assembly. An accessory protein called paramyosin is also required for correct filament assembly. In vertebrate cardiac muscle, there are also two isoforms of myosin-II a-myosin and p-myosin. The proper ratio of these two proteins is of utmost importance for proper muscle activity. The incorrect synthesis of a- and P-myosins results in a severe cardiac disorder known as hypertrophic cardiomyopathy. Genetic transmission of the disease occurs in about 55% of families. The inherited condition is called familial hypertrophic cardiomyopathy (FHC), and this condition is a leading cause of sudden death in young athletes. 

Keratins are made of filaments, approximately 10 nm in diameter and hundreds of nanometers in length, via assembly of rod-shaped, coiled-coil proteins. Filament formation is initiated by the creation of a dimer comprising monomeric units 44-54 nm in length. Such dimers may form three types of lateral interactions leading to filament formation from equimolar amounts of acidic and basic dimers. In vitro assembly involves the correct alignment of two, three, or four dimers into a nucleus for further, rapid filament assembly [6]. 

Filamentous tau is hyperphosphorylated. This is an early event that appears to precede filament assembly. It also renders tau unable to interact with microtubules. Much effort has gone into the mapping of phosphorylation sites and the identification of candidate protein kinases and phosphatases. For sites that are also phos-phorylated in normal brain tau, a higher proportion of tau molecules is phosphorylated in filamentous tau. In addition, filamentous tau is phosphorylated at more serine and threonine residues than tau from normal adult brain. Phosphorylation-dependent anti-tau antibodies were instrumental for the study of many phosphorylation sites. In particular, phosphorylation of S214 and S422 was found to be specific for assembled tau. 

Rodents. Mice expressing wild-type and mutant human tau in nerve cells or glial cells have been found to develop numerous tau-immunoreactive cell bodies and processes. Abundant filaments made of hyperphosphorylated tau protein and neurodegeneration were present in some lines expressing single isoforms of mutant human tau protein [36, 37] (Fig. 45-8 and Fig. 45-9). Hyperphosphorylation of tau appeared to precede filament assembly and an... 

Crowther, R.A. et al. Synthetic filaments assembled from C-terminally truncated a-synuclein. FEES Lett. 436 309-312, 1998. 

All fungal prion proteins readily form filaments in vitro (Dos Reis et al., 2002 Glover et al., 1997 Sondheimer and Lindquist, 2000 Taylor et al., 1999). In near-native buffer conditions, filament formation typically occurs in hours to days. Ure2p filaments assembled in vitro have a diameter of —20 nm and like the filaments observed in situ (Section III.A Fig. 4), they are not hollow. 

Filaments of full-length Ure2p are wider than prion domain filaments and they are not smooth-sided (Fig. 5) rather they have a backbone that closely resembles prion domain filaments in width, surrounded by globular domains—presumably, the G-terminal functional domains. This interpretation is supported by the results of protease digestion experiments that trim filaments of full-length Ure2p down to 4-nm core fibrils that closely resemble prion domain filaments assembled de novo (Fig. 5 Baxa et al, 2003). 

The observations summarized in Section III.A and III.B suggests that the filaments detected in vivo are, in fact, the prion and that the filaments assembled in vitro from purified protein are the same as or closely related to the in vivo material. To substantiate this proposition, it is important to show that filaments formed in vitro are, in fact, infectious. Such infectivity has now been demonstrated for HET-s (Maddelein et al, 2002), Sup35p (King and Diaz-Avalos, 2004 Tanaka et al., 2004), and Ure2p (Fig. 6 Brachmann etal., 2005). In vitro -assembled filaments were brought into cells by biolistic... 

Herrmann, H., and Aebi, U. (1999). Intermediate filament assembly Temperature sensitivity and polymorphism. Cell. Mol. Life Sd. 55, 1416-1431. 

Umeda, M., and Emoto, K., 1999, Membrane phospholipid dynamics during cytokinesis regulation of actin filament assembly by redistribution of membrane surface phosphohpid. Chem. Phys. Lipids, 101 81-91. 

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]. 

This system allows one ActA molecule to concentrate up to 96 profilin molecules, and. Listeria may concentrate as much as 0.5-1 mM profilin on its surface. Profilin markedly potentiates growth of actin filaments, and highly concentrated profilin in the polymerization zone would be expected to result in explosive actin filament assembly, thus generating the forces required for Listeria intracellular movement. 

The mechanical properties of actin filament networks depend on the manner in which actin monomer is prepared and stored, as well as how they are polymerized conditions. Differences in mechanical properties are not the consequence of using two different types of forced oscillatory rheometers. Xu et aid found that filaments assembled in EGTA and Mg from fresh, gel-filtered ATP-actin monomer (1 mg/mL) have an elastic storage... 

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