Studies on the Transcription, Translation, and Structure of a-Actinin in Dictyostelium discoideum

A clone coding for the F-actin cross-linking protein ct-actinin was obtained by screening a genomic library of Dictyostelium discoideum DNA in ~,gtll with monoclonal antibodies specific for Dictyostelium ¢t-actinin. The 1.2-kilobase (kb) genomic clone was confirmed as containing part of the ¢t-actinin gene by comparing its nudeotide sequencev cith the amino acid sequence of tryptic peptides from purified a-actinin. The clone recognized a 3.0-kb message in a Northern blot. Hybridization to RNA isolated from different developmental stages of several D. discoideum strains indicated that the mRNA content increased during early development. A similar result was obtained when the ¢t-actinin content of the cells was followed by Western blot analysis. Hybridization of the clone to DNA from different wild-type strains of D. discoideum indicated a polymorphism on the DNA level that coincided with a polymorphism on the protein level. The data suggest (a) continuous transcription of the ~t-actinin gene throughout the development of D. discoideum, (b) upand downregulation of the levels of ¢t-actinin mRNA and protein with maximum levels at the onset of aggregation, and (c) a high diversity of ¢t-actinin at the DNA and protein level among different D. discoideum strains. The structural data make it conceivable that the highly conserved nature of ¢t-actinin resides only at the functional sites, whereas the helical portions of the ~t-actinin molecule allow a higher level of diversity throughout evolution. A C T I N is the major component of the microfilament system. Rearrangements of this system d u n g cell moveat ,Ik ment are controlled by myosin and its regulatory proteins, by tropomyosin, and by regulatory proteins that bind to Gor F-actin (9, 20, 45). From their function in vitro one can distinguish four classes among the actin-binding proteins: G-aetin binding proteins (5, 25, 34), F-actin severing proteins (2, 18), F-aetin capping proteins (19, 3"/), and F-aetin cross-linking proteins (1, 4, 7, 8, 10, 13, 16). ~t-Actinin, a cross-linking protein, can be isolated from both muscle and nonmuscle cells, a-Actinin, as isolated from higher organisms, is composed of two subunits of 100 kD (13) compared with 95 kD per subunit in lower organisms (8, 16). A protein that resembles the nonmuscle ¢t-actinin has been described for Dictyostelium discoideum (8, 16). The primary structures of actin and myosin have been determined (6, 14, 24, 35, 40, 41), the corresponding genes have been isolated and characterized, and their transcription and transcriptional regulation are being investigated (3, 35). In contrast, little is known about the primary structure, genes, and transcriptional regulation of the aetin-binding proteins. So far, only eDNA clones coding for erythrocyte spectrin and ankyrin have been described (26). We have isolated a genomic clone that codes for ,050% of the D. discoideum tt-aetinin molecule. This clone and different monoclonal antibodies against ¢t-aetinin enabled us to investigate the transcription of the a-actinln gene in relation to the protein level during the developmental cycle of several D. discoideum strains. In addition, we have sequenced the clone and present the first data on the primary structure of an ¢t-actinin. Materials and Methods Growth and Development of D. discoideum D. discoideum strain AX2, an axenically growing derivative of NC4, and various wild-type strains (NC4, VI2, WS380B, OHIO, AC4, WS2162, WST, WS51, WSU2, WS269A, WS472, WS526, WS576, WS582, WS583, WS584, WS655, WS656, WS1956, WS2054, ZA3A, KI0, MFD, DIM4, and DD61) (15) were used. They were grown at 21oc on solid medium (38) with Klebsiella aerogenes as food source or in axenic medium (for strain AX2) (43). For development on filters, strains were grown in suspension culture on washed E. coli B/r (1 x 10 j° cells/ml) in 17 mM phosphate buffer (pH 6.0). Amebas were collected before the bacteria were consumed, centrifuged free of bacteria, and deposited on Millipore filters (Milllpore Corp., Bedford, MA) (28). The filters were incubated at 21"C. Cells were harvested from the filters at different stages of development, and aliquols were used for isolation of RNA and sample preparation for immunoblots. DNA and RNA Isolation Chromosomal DNA was isolated from partially purified nuclei. To prepare nuclei, whole cells (•5 x 109 cells) were lysed with 100 ml of Nonidet P-40 buffer (10 mM magnesium acetate, 10 mM NaC1, 30 mM Hepes pH © The Rockefeller University Press, 0021-9525/86/09196917 $1.00 The Journal of Cell Biology, Volume 103, September 1986 969-975 969 on O cber 0, 2017 jcb.rress.org D ow nladed fom 7.5, 10% [wt/vol] sucrose, 2% [wt/vol] Nonidet P-40), and the nuclei pelleted by a low-speed spin. The nuclei were then lysed at 60°C with EDTA-sarcosyl (2 % [wt/vol] sareosyl, 0.2 M EDTA; the pH was adjusted with NaOH to 8.4), and the lysate subjected to a CsCl-ethidium bromide density gradient centrifugation (0.92 g CsCI per gram solution) (31). Total cellular RNA olD. discoideum was isolated after lysis of the cells with SDS (0.5 % final concentration) and purified with several phenol-chloroform extractions (30).