ADF (actin depolymerizing factor) is an M(r) 19,000 actin-binding protein present in many vertebrate tissues and particularly abundant in neuronal cells. We have cloned human ADF and here show it to be identical in sequence to porcine destrin. Human ADF expressed in Escherichia coli behaves like native ADF from porcine brain. It binds to G-actin at pH 8 with a 1:1 stoichiometry and Kd approximately 0.2 microM, thereby sequestering monomers and preventing polymerization. It does not cosediment with F-actin at this pH, but severs actin filaments in a calcium-insensitive manner. The severing activity is only about 0.1% efficient. By contrast, at pH values below 7, ADF binds to actin filaments in a highly cooperative manner and at a 1:1 ratio to filament subunits. When the pH is raised to 8.0, the decorated filaments are rapidly severed and depolymerized.

Abstract. Actophorin is an abundant 15-kD actinbinding protein from Acanthamoeba that is thought to form a nonpolymerizable complex with actin monomers and also to reduce the viscosity of polymerized actin by severing filaments (Cooper et al., 1986. J. Biol. Chem . 261 :477-485) . Homologous proteins have been identified in sea urchin, chicken, and mammalian tissues. Chemical crosslinking produces a 1 :1 covalent complex of actin and actophorin . Actophorin and profilin compete for crosslinking to actin monomers. The influence of actophorin on the steadystate actin polymer concentration gave a Kd of 0.2 fiM T o understand the mechanisms that regulate the assembly and dynamics of the actin cytoskeleton, a catalog of more than 30 types of actin-binding proteins has been compiled in the hope that understanding the parts will give insight into the whole (Stossel et al., 1985;Pollard and Cooper, 1986) . The redundancy of the system is striking at the biochemical level where a variety of proteins can have similar activities. In a given cell type more than one protein can sequester actin monomers or nucleate actin polymerization or cap actin filaments or crosslink actin filaments . Furthermore, an individual protein can have two or more of these activities. The actin filament severing proteins illustrate this redundancy.The best characterized severing proteins are the -90-kD gelsolin group (Yin and Stossel, 1979) and the -42-kD fragmin/severin group (Hasegawa et al., 1980 ;Brown et al ., 1982) . Gelsolin requires Cam to sever actin filaments and is inhibited by phosphoinositides (Janmey and Stossel, 1987) . It also caps the barbed end of actin filaments and forms nuclei for elongation by binding two actin monomers. These proteins consist of multiple functionally specialized domains sharing a common sequence motif. It is generally believed that both groups arose from a precursor of -125 amino acids by a series of gene duplications resulting in fragmin/severin with three of these domains (Ampe and Vandekerckhove, 1987;Andre et al ., 1988) and gelsolin/villin with six domains (Kwaitkowski et al., 1986;Way and Weeds, 1988;Bazari et al ., 1988). The current models for severing by both groups of proteins involve the interaction of multiple domains with actin filaments. for the complex of actophorin with actin monomers. Several new lines of evidence, including assays for actin filament ends by elongation rate and depolymerization rate, show that actophorin severs actin filaments both at steady state and during spontaneous polymerization. This is confirmed by direct observation in the light microscope and by showing that the effects of actophorin on the low shear viscosity of polymerized actin cannot be explained by monomer sequestration . The severing activity of actophorin is strongly inhibited by stoichiometric concentrations of phalloidin or millimolar concentrations of inorganic phosphate .

SummaryHigher eukaryotic chromosomes are organized into topologically constrained functional domains; however, the molecular mechanisms required to sustain these complex interphase chromatin structures are unknown. A stable matrix underpinning nuclear organization was hypothesized, but the idea was abandoned as more dynamic models of chromatin behavior became prevalent. Here, we report that scaffold attachment factor A (SAF-A), originally identified as a structural nuclear protein, interacts with chromatin-associated RNAs (caRNAs) via its RGG domain to regulate human interphase chromatin structures in a transcription-dependent manner. Mechanistically, this is dependent on SAF-A’s AAA+ ATPase domain, which mediates cycles of protein oligomerization with caRNAs, in response to ATP binding and hydrolysis. SAF-A oligomerization decompacts large-scale chromatin structure while SAF-A loss or monomerization promotes aberrant chromosome folding and accumulation of genome damage. Our results show that SAF-A and caRNAs form a dynamic, transcriptionally responsive chromatin mesh that organizes large-scale chromosome structures and protects the genome from instability.

Nemaline myopathy (NM) is a congenital myopathy characterized by muscle weakness and nemaline bodies in affected myofibers. Five NM genes, all encoding components of the sarcomeric thin filament, are known. We report identification of a sixth gene, CFL2, encoding the actin-binding protein muscle cofilin-2, which is mutated in two siblings with congenital myopathy. The proband's muscle contained characteristic nemaline bodies, as well as occasional fibers with minicores, concentric laminated bodies, and areas of F-actin accumulation. Her affected sister's muscle was reported to exhibit nonspecific myopathic changes. Cofilin-2 levels were significantly lower in the proband's muscle, and the mutant protein was less soluble when expressed in Escherichia coli, suggesting that deficiency of cofilin-2 may result in reduced depolymerization of actin filaments, causing their accumulation in nemaline bodies, minicores, and, possibly, concentric laminated bodies.

Actin depolymerizing factor (ADF) from vertebrates and actophorin from Acanthamoeba castellanii are members of a protein family that bind monomeric and polymeric actin and have been shown by microscopy to sever filaments. Here, we compare the properties of recombinant human ADF and actophorin using rabbit muscle actin. ADF binds tenfold more strongly than actophorin to monomeric actin (G‐actin)‐ATP, and both bind co‐operatively to F‐actin. ADF decorates filaments below pH 7.3 and induces substantial depolymerization at higher pH values [Hawkins, M., Pope, B., Maciver, S. K. & Weeds, A. G. (1993) Human actin depolymerizing factor mediates a pH‐sensitive destruction of actin filaments, Biochemistry 32, 9985−9993], but, at all pH values tested, actophorin binds to filaments in a similar manner to ADF at pH 6.5. Both proteins increase the depolymerization rate at the pointed ends of gelsolin‐capped filaments, but the effect of ADF is more marked at pH 8.0. Both proteins accelerate the nucleating activity when mixed with filamentous actin (F‐actin), but not with gelsolin‐capped filaments, and they rapidly decrease the lengths of filaments as evidenced by electron microscopy. Both of these effects are best explained by a weak severing activity. Our results are discussed in relation to earlier models and to the structural changes observed when ADF binds F‐actin [McGough, A., Pope, B., Chiu, W. & Weeds, A. (1997) Cofilin changes the twist of F‐actin : implications for actin filament dynamics and cellular function, J. Cell Biol. 138, 771−781]. We also discuss the relevance of these observations to their possible roles in facilitating actin turnover in cells, thereby regulating filament dynamics in cell motility.

In pollen development, a dramatic reorganization of the actin cytoskeleton takes place during the passage of the pollen grain into dormancy and on activation of pollen tube growth. A identical to ZmABPI and ZmABP2, respectively, and its expression is suppressed in pollen and germinated pollen. The fundamental biochemical characteristics of the ZmABP proteins has been elucidated using bacterially expressed ZmABP3 protein. This has the ability to bind monomeric actin (G-actin) and filamentous actin (F-actin). Moreover, it decreases the viscosity of polymerized actin solutions consistent with an ability to depolymerize filaments. These biochemical characteristics, taken together with the sequence comparisons, support the inclusion of the ZmABP proteins in the ADF group.

Naegleria fowleri causes an uncommon but deadly disease called primary amoebic meningoencephalitis (PAM). There has been an increase of reported PAM cases, particularly since 2000. Although water is the dominant route of transmission of PAM, infection through soil/dust is a possible alternative route. We have observed differences in epidemiology between the southern US states and the Indian subcontinent (ISC). The patient age range is greater in ISC than the US, and there are more infections in ISC which are not water-associated. We show that PAM is under reported and argue that climate change will increase the incidence of PAM and the geographic range of N. fowleri will spread poleward.

Actophorin from Acanthamoeba castellanii severs actin filaments and sequesters actin monomers. Here we report that actophorin binds ADP-bound monomers with higher aflinity than ATP-bound monomers. Actophorin is therefore much less efficient at severing actin 6lament.s in the presence of ADP compared to ATP, particularly taking account of the higher critical concentration in ADP. Monomer binding is also reduced in the presence of 25 mM inorganic phosphate (which is assumed to form ADP-Pi-a&r). These findings are discussed in the light of observations on the nucleotide specilicity of other monomer binding proteins and related to the role of actin in lamellar protrusion and cell locomotion.