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 .

No abstract

No abstract

The actin filament-associated protein and Src-binding partner, AFAP-110, is an adaptor protein that links signaling molecules to actin filaments. AFAP-110 binds actin filaments directly and multimerizes through a leucine zipper motif. Cellular signals downstream of Src 527F can regulate multimerization. Here, we determined recombinant AFAP-110 (rAFAP-110)-bound actin filaments cooperatively, through a lateral association. We demonstrate rAFAP-110 has the capability to cross-link actin filaments, and this ability is dependent on the integrity of the carboxy terminal actin binding domain. Deletion of the leucine zipper motif or PKC phosphorylation affected AFAP-110's conformation, which correlated with changes in multimerization and increased the capability of rAFAP-110 to cross-link actin filaments. AFAP-110 is both a substrate and binding partner of PKC. On PKC activation, stress filament organization is lost, motility structures form, and AFAP-110 colocalizes strongly with motility structures. Expression of a deletion mutant of AFAP-110 that is unable to bind PKC blocked the effect of PMA on actin filaments. We hypothesize that upon PKC activation, AFAP-110 can be cooperatively recruited to newly forming actin filaments, like those that exist in cell motility structures, and that PKC phosphorylation effects a conformational change that may enable AFAP-110 to promote actin filament cross-linking at the cell membrane.

We used a novel stopped-flow/rapid-freezing machine to prepare the transient intermediates in the actin-myosin adenosine triphosphatase (ATPase) cycle for direct observation by electron microscopy. We focused on the low affinity complexes of myosin-adenosine triphosphate (ATP) and myosin-adenosine diphosphate (ADP)-Pi with actin filaments since the transition from these states to the high affinity actin-myosin-ADP and actin-myosin states is postulated to generate the molecular motion that drives muscle contraction and other types of cellular movements. After rapid freezing and metal replication of mixtures of myosin subfragment-1, actin filaments, and ATP, the structure of the weakly bound intermediates is indistinguishable from nucleotide-free rigor complexes. In particular, the average angle of attachment of the myosin head to the actin filament is approximately 40 degrees in both cases. At all stages in the ATPase cycle, the configuration of most of the myosin heads bound to actin filaments is similar, and the part of the myosin head preserved in freeze-fracture replicas does not tilt by more than a few degrees during the transition from the low affinity to high affinity states. In contrast, myosin heads chemically cross-linked to actin filaments differ in their attachment angles from ordered at 40 degrees without ATP to nearly random in the presence of ATP when viewed by negative staining (Craig, R., L.E. Greene, and E. Eisenberg. 1985. Proc. Natl. Acad. Sci. USA. 82:3247-3251, and confirmed here), freezing in vitreous ice (Applegate, D., and P. Flicker. 1987. J. Biol. Chem. 262:6856-6863), and in replicas of rapidly frozen samples. This suggests that many of the cross-linked heads in these preparations are dissociated from but tethered to the actin filaments in the presence of ATP. These observations suggest that the molecular motion produced by myosin and actin takes place with the myosin head at a point some distance from the actin binding site or does not involve a large change in the shape of the myosin head.