Substitution of flight muscle-specific actin by human (beta)-cytoplasmic actin in the indirect flight muscle of Drosophila

Vronique Brault 2 4 Mary C. Reedy 1 4 Ursula Sauder 0 4 Richard A. Kammerer 3 4 Ueli Aebi 2 4 Cora-Ann Schoenenberger 2 4 0 Interdepartmental Electronmicroscopy , Biozentrum , University of Basel , CH-4056, Basel , Switzerland 1 Department of Cell Biology, Duke University Medical Center , Durham, North Carolina 27710 , USA 2 M.E. Muller Institute , Biozentrum , University of Basel , CH-4056 Basel , Switzerland 3 Department of Biophysical Chemistry , Biozentrum , University of Basel , CH-4056, Basel , Switzerland 4 Ectopic expression of b -cytoplasmic actin the indirect flight muscle of Drosophila SUMMARY The human b -cytoplasmic actin differs by only 15 amino acids from Act88F actin which is the only actin expressed in the indirect flight muscle (IFM) of Drosophila melanogaster. To test the structural and functional significance of this difference, we ectopically expressed b cytoplasmic actin in the IFM of Drosophila that lack endogenous Act88F. When expression of the heterologous actin was regulated by ~1.5 kb of the 5 promoter region of the Act88F gene, little b -cytoplasmic actin accumulated in the IFM of the flightless transformants. Including Act88Fspecific 5 and 3 untranslated regions (UTRs) yielded transformants that expressed wild-type amounts of b Multiple isoforms of actin have been described in almost all eukaryotic organisms (Gallwitz and Seidel, 1980; Fidel et al., 1988; Hirono et al., 1987; for review see Rubenstein, 1990; Herman, 1993). Humans have six actin isoforms, four of which are muscle-specific, and two are found in the cytoplasm of nonmuscle cells. Likewise, Drosophila melanogaster expresses two nonmuscle and four muscle-specific actins in a temporally and spatially regulated pattern (Fyrberg et al., 1983). For instance, Act88F, is exclusively expressed in the indirect flight muscle (IFM), and encodes all of the actin contained in the myofibrils of these muscles (Ball et al., 1987). Among mammals, specific isoforms are extremely conserved if not identical, and Drosophila actins share 93 to 97% identical amino acid residues with mammals. Although the high sequence conservation raises the question as to whether there is a functional significance of the multiple actin isoforms, the stageand tissue-specific expression pattern of different actins suggests that isoforms have distinct functions (McKenna et al., 1985; DeNofrio et al., 1989; Sawtell and Lessard, 1989; EppenbergerEberhardt et al., 1990; Peng and Fischman, 1991; Mounier et al., 1997). To date, there is no expression system that produces large quantities of pure, fully functional actin. Therefore, in vitro cytoplasmic actin. Despite the assembly of b -cytoplasmic actin containing thin filaments to which endogenous myosin crossbridges attached, sarcomere organization was deficient, leaving the transformants flightless. Rather than affecting primarily actin-myosin interactions, our findings suggest that the b -cytoplasmic actin isoform is not competent to interact with other actin-binding proteins in the IFM that are involved in the organization of functional myofibrils. studies of actin isoforms have been rather restricted. A further drawback in analyzing the functional significance of the closely related isoforms is the scarcity of specific antibodies that reliably distinguish one isoform from another in mixtures of different actins. Expressing a particular actin isoform or mutant in vivo in order to analyze subtle functional or structural differences is complicated by the toxicity of excess amounts of actin, the presence of more than one type of actin in a given cell, or the frequently disruptive effects of deleting or mutating this essential protein (Hennessey et al., 1992). Recently, substitution of whole actin isoforms has been achieved in yeast and in mouse heart, where the substituted actin isoform rescued lethality and/or improved function (Karlsson et al., 1991; Kumar et al., 1997). In contrast to other organisms in which detailed structural and functional analysis of actin isoform substitution is difficult, the IFMs of Drosophila provide an excellent system for exploring actin isoform diversity. IFMs are dispensable for viability, so disruption of IFM structure and function simply impedes flight performance, providing an easy assay for functional change (Sparrow et al., 1991a; Bernstein et al., 1993). Because the IFMs are not required for viability, a stable strain that is completely null for IFM-specific Act88F actin could be established (e.g. KM88; Hiromi and Hotta, 1985; Mahaffey et al., 1985) which allows expression of another isoform against an IFM background free of wild-type Act88F actin. In addition, IFMs display a high degree of structural order (Reedy and Beall, 1993), thereby providing a sensitive experimental system for examining even subtle structural as well as functional changes resulting from the substitution of one isoform for another. Recently, Fyrberg and co-workers have used the IFM to test the consequences of isoform-specific amino acid replacements in Act88F actin (Reedy et al., 1991; Fyrberg et al., 1998). When a single isoform-specific amino acid of Act88F was substituted with a residue corresponding to another Drosophila actin isoform, the exchange affected myofibrillar function only in one out of ten cases. Flies transformed with chimeric genes containing multiple replacements showed flight impairment, and replacement of all IFM-specific residues with amino acids corresponding to Drosophila nonmuscle Act42A actin, produced flightlessness and disorganized myofibrils. One might expect that cytoplasmic actin isoforms, which function in a very different environment from the highly ordered IFMs, differ most from muscle actin isoforms. However, human b -cytoplasmic actin diverges from the Drosophila IFM-specific isoform by only 15 residues. Insect muscle actins form a distinct family of related proteins characterized by about 10 amino acids which appear to distinguish them from the vertebrate cytoplasmic actins (Mounier et al., 1992; see also Fig. 8). These residues may be critical in tailoring actin to perform its isoformspecific function in a muscle environment. Here we report the ectopic expression of an entire human b cytoplasmic actin isoform in a Drosophila muscle environment free of any other actin. Consistent with the assumption of functional diversity among actin isoforms, we find that human b -cytoplasmic actin does not fully compensate for the endogenous Act88F isoform, even when present in amounts similar to Act88F in wild-type Drosophila. MATERIALS AND METHODS Construction of plasmids A PstI-EcoRI fragment comprising the Act88F gene including the 5 UTR with the first intron (Okamoto et al., 1986), the 3 UTR, and approximately 1.5 kb regulatory sequences upstream of the transcription initiation site was excised from the P[ry+;CSB] plasmid (Hiromi et al., 1986) and cloned into the pW8 Drosophila transformation vector which contains the selec (...truncated)