Walter Witke

Calcium‐sensitive non‐muscle α‐actinin contains EF‐hand structures and highly conserved regions

The F‐actin crosslinking molecule α‐actinin from the slime mould Dictyostelium discoideum carries two characteristics EF‐hand structures at the C‐terminus. The calcium‐binding loops contain all necessary liganding oxygens and most likely form the structural basis for the calcium sensitivity of strictly calcium‐regulated non‐muscle α‐actinins. Furthermore, the sequence exhibits at the N‐terminal site of the molecule a high degree of homology to chicken fibroblast α‐actinin. This stretch of amino acids appears to have remained essentially constant during evolution and might represent the actin‐binding site. The findings have led us to propose a model for the inhibitory action of Ca2+ on non‐muscle α‐actinins.

Redundancy in the microfilament system: Abnormal development of dictyostelium cells lacking two F-actin cross-linking proteins

We generated by gene disruption Dictyostelium cells that lacked both the F-actin cross-linking proteins, alpha-actinin and gelation factor. Several major cell functions, such as growth, chemotaxis, phagocytosis, and pinocytosis, were apparently unaltered. However, in all double mutants, development was greatly impaired. After formation of aggregates, cells were very rarely able to form fruiting bodies. This ability was rescued when mutant and wild-type strains were mixed in a ratio of 70 to 30. The developmental program in the mutant was not arrested, since the expression pattern of early and late genes remained unchanged. Development of the mutant was rendered normal when a functional alpha-actinin gene was introduced and expressed, showing the morphogenetic defect to be due to the absence of the two F-actin cross-linking proteins. These findings suggest the existence of a functional network allowing mutual complementation of certain actin-binding proteins.

Ca2+-Binding Proteins as Components of the Cytoskeleton

One of the major filamentous systems of non-muscle cells is based on actin, a highly conserved protein of 42kD. The polymerization equilibrium between globular (G-) and filamentous (F-) actin determines the viscosity of the cytoplasm and is regulated by cytoplasmic salt conditions and by actin-binding proteins (for review see Stossel et al., 1985, Pollard and Cooper, 1986). Several of these actin-binding proteins are activated or inhibited by micromolar Ca2+-concentrations either through direct binding to Ca2+ or indirectly through modulators such as calmodulin.

Direct photoaffinity labeling of soluble GTP-binding proteins in Dictyostelium discoideum

Major cytoplasmic GTP‐binding proteins in Dictyostelium discoideum were identified by direct photoaffinity labeling with [α‐32P]GTP. Three actin‐binding proteins and a protein with an apparent molecular mass of 24 kDa (p24) could be labeled with [α‐32P]GTP. p24 binds to DEAE‐cellulose, behaves like a monomer during gel filtration and was purified to homogeneity by GTP‐affinity chromatography. In comparison to other nucleotide triphosphates the binding of GTP to p24 is highly specific.

cDNA clones coding for α‐actinin of Dictyostelium discoideum

cDNA clones coding for Dictyostelium α‐actinin were isolated from a library in the expression vector λgt11 using a genomic probe that contains α‐actinin‐specific sequences. The recombinant phages harbored inserts with sizes up to 3.0 kb. They hybridized to a 3.0 kb message in Northern blot analysis and some produced fusion proteins that reacted with monoclonal antibodies directed against different epitopes of D. discoideum α‐actinin. The sizes of the inserts and the reaction with the monoclonal antibodies indicated that two of the phages carry a nearly complete copy of the α‐actinin message.

Nonmuscle α-Actinin is an EF-Hand Protein

In 1965, Ebashi and Ebashi isolated an F-actin crosslinking protein from muscle cells which they called α-actinin. In addition to actin, myosin and tropomyosin it was the fourth component so far found in muscle cells. Immunofluorescence studies on striated muscle showed that α-actinin is localized in the Z-lines which separate the sarcomers and anchor the actin filaments. Therefore, a role for α-actinin in linking actin filaments to the Z-line has been discussed. It is generally assumed that α-actinin strengthens the structure and helps the sarcomeric unit to withstand the force generated by the actin-myosin contraction. Whether the in vitro-enhanced Mg-ATPase activity of actomyosin in the presence of α-actinin (Maruyama and Ebashi 1965) plays a significant role in vivo is not clear. Ten years later Lazarides and Burridge (1975) identified α-actinin also in nonmuscle cells. Like muscle α-actinin, its main activity in vitro is the gelation of an F-actin solution by cross linking actin filaments under certain conditions. The finding that α-actinin can be incorporated into artificial membranes and could form trimeric complexes with actin in the presence of diacylglycerol and palmitic acid (Burn et al. 1985) makes it conceivable that α-actinin might anchor actin filaments to the plasma membrane of nonmuscle cells. Since then several isoforms of α-actinin have been isolated from skeletal and smooth muscle (Feramisco and Burridge 1980; Endo and Masaki 1984) or from vertebrate nonmuscle cells like brain (Duhaiman and Bamburg 1984), macrophages (Bennett et al. 1984), platelets (Landon et al. 1985) and fibroblasts (Burridge and Feramisco 1981).

Actin and Actin-Binding Proteins in the Motility of Dictyostelium

Dictyostelium discoideum cells are highly motile throughout all stages of development and directed cell motility is essential for morphogenesis to occur (Williams and Jermyn 1991). Cells move by pseudopod extension, and formation and retraction of pseudopods in response to a chemotactic stimulus is critical for directed migration. Folate acts as chemotactic agent during growth, cAMP is the active compound during development. The understanding of chemotactic signaling has advanced considerably (Devreotes 1989), but its linkage to intracellular changes that lead to cell motility is less clear (reviewed in Schleicher and Noegel 1992). Early observations indicated that chemoattractant-induced pseudopod formation correlates temporally and spatially with the polymerization of actin, and peaks of F-actin occur by 15–60 s after stimulation (McRobbie and Newell 1983; Hall et al. 1988). Five different steps in actin changes can be distinguished (Fig. 1), and these steps were found to coincide with retraction of the existing pseudopod and generation of new pseudopods (Dhamaward- hane et al. 1989). Parallel processes are an influx of Ca2+ and an efflux of protons leading to an alkalinization of the cytoplasm. The phosphoinositides are also affected with an increase of IP3 as fast as 6 s after stimulation at the expense of PIP and PIP2 (Europe-Finner et al. 1991).

Chapter 12 Expression and Function of Genetically Engineered Actin-Binding Proteins in Dictyostelium

Publisher Summary Dictyostelium discoideum is a haploid organism. This allows direct isolation of mutants. A parasexual genetic system has been established and has been possible to use molecular genetic methods, because the description of a transformation system. These features have made D. discoideum a favorable organism for the investigation of various aspects of cell motility and the cytoskeleton. This chapter discusses the microfilament system of Dictyostelium discoideum and F-actin cross-linking proteins in Dictyostelium discoideum. Genetic manipulation of α-actinin in Escherichia coli and dictyostelium discoideum, investigation of the actin-binding domain, investigation of the EF-hand domains, and investigation of the in vivo function of α-actinin are discussed in the chapter. The isolation of α-actinin and gelation factor negative cells was facilitated, because of the availability of nitrosoguanidine-generated mutants that lacked, either α-actinin or the gelation factor. By disruption of the α-actinin gene in strain HG1264 and the gelation factor gene in strain HG1130, cells were generated that lacked two F-actin cross-linking proteins. The double mutans were still motile and chemotactically active. Development was initiated normally, as tested with antibodies, directed toward stage-specific proteins and complementary DNA (cDNA) probes and spores and stalk cells were formed. Spores and stalk cells did not undergo the final morphogenetic movements and were only occasionally organized into a normal fruiting body, with a stalk and a spore head. Most of the cells remained in aggregates on the agar surface. The inability of these strains to undergo morphogenesis was because of the lack of the two F-actin cross-linking proteins.