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3627 Journal of Cell Science 112, 3627-3639 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JCS0612 Substitution of flight muscle-specific actin by human β-cytoplasmic actin in the indirect flight muscle of Drosophila Véronique Brault1, Mary C. Reedy2, Ursula Sauder3, Richard A. Kammerer4, Ueli Aebi1 and Cora-Ann Schoenenberger1,* 1M.E. Müller Institute, Biozentrum, University of Basel, CH-4056 Basel, Switzerland 2Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA 3Interdepartmental Electronmicroscopy, Biozentrum, University of Basel, CH-4056, Basel, Switzerland 4Department of Biophysical Chemistry, Biozentrum, University of Basel, CH-4056, Basel, Switzerland *Author for correspondence (e-mail: [email protected]) Accepted 16 August; published on WWW 18 October 1999 SUMMARY The human β-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 βcytoplasmic 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 β-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 β- cytoplasmic actin. Despite the assembly of β-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 β-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. INTRODUCTION 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 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 Key words: Actin, β-Cytoplasmic actin, Isoform, Myofibril, Thin filament 3628 V. Brault and others 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 β-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 βcytoplasmic 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 β-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 selectable white (w) marker gene (Klemenz et al., 1987). The resulting parent pW8Act88F plasmid is shown in Fig. 1A. pW8(5′Act88F)β-cyto construct A PstI-HincII fragment containing ~1.5 kb promoter region of the Act88F gene including the transcription initiation site was excised from the P[ry+;CSB] plasmid and ligated into pW8 at the PstI and StuI sites yielding an intermediate pW8(5′Act88F) construct. The fulllength cDNA encoding human β-cytoplasmic actin (Gunning et al., 1983) was inserted as a blunt end-BamHI fragment into the pW8(5′Act88F) transformation vector downstream of the Act88F transcription initiation site (Okamoto et al., 1986) using the HpaI site adjacent to the former HincII site at the 5′ and the BamHI site at the 3′ end. The resulting pW8(5′Act88F)β-cyto transformation construction is shown in Fig. 1B. pW8Act88F(−3′UTR) construct The KpnI-EcoRI fragment which contains the Act88F 3′UTR was replaced in the parent pW8Act88F (Fig. 1A) construct by a corresponding fragment lacking the 3′UTR. This fragment was obtained by PCR using a sense primer 5′-CGGCGGTACCACCATGTACCCTGG-3′ spanning the upstream KpnI site in Act88F and an antisense primer 5′-CCGGAATTCTTAAAAGCATTTGCGGTG3′, which inserts an EcoRI site 3′ of the TAA stop codon. The final pW8Act88F(−3′UTR) transformation construct is shown in Fig. 1C. pW8(−5′UTR)Act88F construct The parent pW8Act88F was digested with BamHI/EcoRI and the purified Act88F gene fragment subcloned in pUC19 cloning vector (Amersham Pharmacia Biotech). The same BamHI-EcoRI Act88F fragment was used as template in order to introduce a new BamHI site 25 nucleotides upstream of the Act88F translation start site by PCR. The sense primer 5′-CGGGATCCCAGATAAACAACTGCCAAGATG-3′ and the antisense primer 5′CGCACGGTGTGGGAGACACCAT-3′, which spans the DraIII site in the Act88F coding sequence, were used. The amplified BamHIDraIII fragment was cloned into the pUC19Act88F construct in place of the endogenous BamHI-DraIII fragment. Subsequently, a BamHIEcoRI fragment of this construct was ligated into pW8(5′Act88F) at the respective sites yielding the pW8(−5′UTR)Act88F transformation construct which lacks the 5′UTR (Fig. 1D). pW8(StuI)Act88F(EcoRI) construct To replace the Act88F coding sequence with the human β-cytoplasmic actin cDNA, two unique restriction sites were generated at opposite ends of the Act88F coding sequence in pW8Act88F by the following procedure. First, the EcoRI site at the 3′ end of pW8Act88F was eliminated by DNA fill-in synthesis. A new EcoRI site was obtained 3′ of the TAA stop codon by inserting a corresponding linker (5′GAATTCGCCCGCCGCGAAAGCTCTTCAAG-3′) between the stop codon and the SapI site in the 3′UTR of Act88F. At the 5′ end, changing the original nucleotide sequence at positions −11 to −9 upstream of the ATG translation start site from CAA to GGC was achieved by site-directed mutagenesis by PCR using 5′CCTTCCAGATAAAGGCCTGCCAAGATGTGTG-3′ as sense, and 5′-CACACATCTTGGCAGGCCTTTATCTGGAAGG-3′ as antisense primer. Mutating the sequence yielded a unique StuI site without changing the splice acceptor sequence. The resulting pW8(StuI)Act88F(EcoRI) construct (Fig. 1E) served as a control to verify wild-type levels of Act88F actin expression. pW8(StuI)β-cyto(EcoRI) construct The human β-cytoplasmic actin cDNA was modified by PCR in order to add a StuI site at its N terminus and an EcoRI site at its C terminus. The sense primer (5′-GAAGGCCTGCCAAGATGGATGATGATATCGCCGC-3′) was designed to obtain a StuI site followed by 5′GCCAAG-3′ which corresponds to the sequence immediately 5′ of the start ATG in Act88F. The 3′ antisense primer (5′-CGGAATTCC TAGAAGCATTTGCGGTGGA 3′) was designed to introduce an EcoRI site 3′ of the TAG stop codon. Finally, the endogenous StuIEcoRI Act88F fragment of pW8(StuI)Act88F(EcoRI) (Fig. 1C) was replaced with the StuI-EcoRI β-cytoplasmic actin PCR product, yielding the pW8(StuI)β-cyto(EcoRI) transformation construct (Fig. 1F). All constructs were confirmed by DNA sequencing. Germ line transformation Germ line transformation was carried out essentially as described by Rubin and Spradling (1982) using the helper P element plasmid pπ25.7∆2-3 wc. The recipient strain for all constructs was the KM88 null mutant (w; Act88F KM88) (Hiromi and Hotta, 1985; Okamoto et al., 1986). The posterior ends of homozygous KM88 embryos were injected with 100 ng/µl of helper plasmid and 100-300 ng/µl of pW8 Ectopic expression of β-cytoplasmic actin 3629 transformation constructs. Individual adult Go flies were back-crossed to KM88 flies and the progeny was scored for red eyes. For each construct independent homozygous lines were established using balancer chromosomes (Lindsley and Zimm, 1992). Electron microscopy IFMs were prepared for transmission electron microscopy according to the method of Reedy and Beall (1993) with minor modifications. The part containing the dorsal longitudinal IFMs was dissected from thoraces of 24- to 48-hour-old females and directly immersed in a freshly prepared fixative consisting of 3% glutaraldehyde and 0.2% tannic acid in MOPS buffered Drosophila Ringer’s solution (Fyrberg et al., 1990) without phosphate (pH 6.8) for 2 hours at room temperature. After primary fixation, the dorsal thoraces were rinsed in MOPS-buffered Drosophila Ringer’s solution, in 100 mM phosphate buffer (pH 6.0) and then immersed for 1 hour in ice-cold secondary fixative consisting of 1% osmium tetroxide in 100 mM phosphate buffer and 10 mM MgCl2 (pH 6.0). After washing, thoraces were block-stained in aqueous 2% uranyl acetate for 1 hour at 4°C. Dehydrated specimens were embedded in Epon. In some cases, thoraces were split down the midline and the resulting hemithoraces embedded in Araldite 506, allowing staining of the sections in KMnO4. For glycerination of IFMs in situ, hemithoraces were used. IFMs were glycerinated according to the method of Reedy et al. (1989) and subsequently fixed and stained as described above. Electron micrographs were recorded on Kodak (Rochester, NY) SO163 film using a LEO 910 (LEO, Oberkochen, Germany) or a Philips EM 420 transmission electron microscope operated at 80 kV. SDS-PAGE and immunoblot analysis of IFM protein extracts Dorsal longitudinal flight muscles were dissected from thorax halves according to the method of Brault et al. (1999), transferred to SDSPAGE sample loading buffer (2.3% SDS, 62.5 mM Tris-HCl, pH 7.0, 15% glycerol, 2.5% β-mercaptoethanol, 0.05% bromophenol blue), and boiled for 5 minutes. Protein extracts corresponding to the IFMs of half a thorax were separated on 11.5% SDS-polyacrylamide gels Pst I A BamH I ATG TIS Pst I HincII Stop TIS Pst I C D pW8Act88F Stop BamH I pW8(5′Act88F)β-cyto β-cytoplasmic actin cDNA BamH I ATG BamH I/ATG Regulation of transgene expression by only the 5′′ promoter region of Act88F yields low levels of human β-cytoplasmic actin in the IFM In the initial pW8(5′Act88F)β-cyto transformation construct (Fig. 1B), expression of the heterologous human β-cytoplasmic actin in the IFM of transformed Drosophila is regulated by approximately 1.5 kb Act88F promoter region including the Act88F transcription initiation site. Transformants established with the pW8(5′Act88F)β-cyto construct carried the insertion on the third chromosome and were unable to fly. To examine the cause of the flightless phenotype at the Sap I DraIII Kpn I Stop/EcoR I pW8Act88F(-3′UTR) 5′UTR Pst I RESULTS EcoR I 3′UTR HincI HpaI I ATG B Kpn I Stop/EcoR I EcoR I pW8(-5′UTR)Act88F 3′UTR HincII Pst I E Stu I ATG Kpn I Stop/EcoR I EcoR I 3′UTR 5′UTR Pst I F Kpn I 5′UTR (Laemmli, 1970). Gels were electroblotted onto an Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA). Blots were rinsed with PBS and transiently stained with Coomassie Brilliant Blue to confirm that protein extractions, gel loadings and electrophoretic transfer were comparable for each lane. After complete destaining with methanol, blots were washed in PBS and 0.1% Tween-20 (PBS-T), blocked in 5% milk powder in PBS-T, and incubated for 2 hours at room temperature with either a mouse mAb recognizing different actin isoforms (Amersham Pharmacia Biotech; diluted 1:7,500 in PBS-T), or with a mouse mAb which specifically recognizes β-cytoplasmic actin (Sigma; diluted 1:2,500 in PBS-T). Blots were washed for 15 minutes each with 5% milk powder in PBST, in PBS-T, and 1% blocking solution (Roche Molecular Biochemicals) in PBS-T, followed by a 2 hour incubation with a 1:5,000 dilution of a goat anti-mouse IgG alkaline phosphatase conjugated secondary antibody (Sigma). Blots were washed 3 times for 15 minutes with PBS-T, once with 100 mM Tris (pH 9.5), 100 mM NaCl2, and 5 mM MgCl2, and then developed with Western Blue stabilized substrate for alkaline phosphatase (Promega). Stu I 5′UTR ATG pW8(StuI)Act88F(EcoRI) Stop/EcoR IEcoR I β-cytoplasmic actin cDNA 3′UTR pW8(StuI)β-cyto(EcoRI) Fig. 1. Act88F and β-cytoplasmic actin constructs for Drosophila transformation. (A) The endogenous Act88F gene inserted into the pW8 vector (pW8Act88F) was used to generate all subsequent constructs. (B) In the pW8(5′Act88F)β-cyto actin construct, expression of β-cytoplasmic actin is regulated by ~1.5 kb of the 5′ promoter region from the Act88F gene including the autologous transcription initiation site (TIS). (C) In pW8Act88F(−3′UTR), the 3′ untranslated region downstream of the TAA stop codon was deleted. (D) In pW8(−5′UTR)Act88F, the Act88F gene including 18 nucleotides upstream of the translation start codon and the Act88F-specific 3′UTR was linked to the Act88F regulatory region at the HincII site 5′ of the transcription initiation site. (E) In pW8(StuI)Act88F(EcoRI), a StuI and an EcoRI restriction site flank the Act88F coding sequence at the 5′ and 3′ end, respectively. (F) In pW8(StuI)βcyto(EcoRI), the Act88F actin coding sequence was replaced with the cDNA encoding human βcytoplasmic actin. Solid black boxes represent translated regions of the Act88F gene, whereas untranslated sequences are shown as white boxes with stippled regions representing intron sequences. The βcytoplasmic actin cDNA is shown as a shaded box. TIS, Act88F transciption initiation site. 3630 V. Brault and others Ectopic expression of β-cytoplasmic actin 3631 Fig. 2. Ultrastructure of IFM from initial pW8(5′Act88F)β-cyto transformants compared to pW8(StuI)Act88F(EcoRI) control IFM. (A) In longitudinal sections of IFM from transformants expressing low amounts of β-cytoplasmic actin, sarcomeric organisation and Zdiscs are absent. M-line-like structures (arrowhead and inset) indicate some degree of thick filament register. (B) Cross-sections of IFM from pW8(5′Act88F)β-cyto transformants suggest that thin filaments are virtually absent. Accordingly, there is no ordered lattice of thin and thick myofilaments. (C) Control transformants display regular sarcomeres marked by Z-discs (Z) and M-lines (M). (D) Myofibrils of control transformants have a wild-type appearance. The higher magnification (inset) reveals the almost crystalline hexagonal array of myofilaments. Bars: (A) 500 nm (inset, 500 nm); (B) 500 nm (inset, 100 nm); (C) 500 nm (inset, 200 nm); (D) 500 nm (inset, 100 nm). ultrastructural level, the thoraces of young adult transformants were fixed in situ and processed for transmission electron microscopy (TEM). Longitudinal IFM sections from the pW8(5′Act88F)β-cyto transformant reveal only some imperfectly aligned skeins of thick filaments (Fig. 2A). In essence, sarcomeric organisation including Z-discs is completely missing and thin filaments appear to be absent. Occasionally, indications of an M-line-like structure (arrowhead) can be discerned. In contrast, control transformants expressing wild-type amounts of Act88F actin Fig. 3. The 5′ promoter region of Act88F yields low levels of βcytoplasmic actin in the IFM of transformants. (A) Immunoblot revealing the accumulation of β-cytoplasmic actin in thoracic extracts from pW8(5′Act88F)β-cyto transformants. (B) Immunoblot with a mAb that recognizes different actin isoforms. The amounts of β-cytoplasmic actin in the IFM of the pW8(5′Act88F)β-cyto transformant are significantly lower than the amounts of Act88F actin in wild-type flies and in pW8(StuI)Act88F(EcoRI) control transformants. Normal amounts of Act88F are present in pW8Act88F(−3′UTR) transformants, whereas truncation of Act88F at the 5′ end abrogates actin accumulation in pW8(−5′UTR)Act88F. The additional ~55 kDa band corresponding to arthrin is present only in wild-type flies and in transformants accumulating wild-type levels of Act88F actin. display the typical, highly ordered sarcomeres (Fig. 2C). A cross-section of the IFM from pW8(5′Act88F)β-cyto transformants (Fig. 2B) reveals the absence of round, highly organised myofibrils which are characteristic of wild-type IFM and the control transformants (Fig. 2D). In contrast to the almost crystalline double-hexagonal pattern of control myofibrils (Fig. 2D, inset), cross-sections of pW8(5′Act88F)βcyto transformant IFM (Fig. 2B, inset) display randomly distributed myosin thick filaments with no apparent hexagonal array of actin containing thin filaments surrounding them. To examine the expression of β-cytoplasmic actin in the IFM of the pW8(5′Act88F)β-cyto transformants, an immunoblot of IFM protein extracts from the transformant, KM88, and wildtype flies was probed with a mAb that specifically recognizes the β-cytoplasmic actin isoform (Gimona et al., 1994). The anti β-actin antibody detected a single 43-kDa band in IFM extracts from pW8(5′Act88F)β-cyto transformants (Fig. 3A). As expected, the β-actin band is absent in IFM extracts from KM88 and wild-type flies. To compare the amount of βcytoplasmic actin that accumulated in the IFM of pW8(5′Act88F)β-cyto transformants to the amount of endogenous Act88F in wild-type IFM, we probed a corresponding blot with a mAb that equally recognizes a number of different actin isoforms (Fig. 3B). This anti-actin antibody revealed that β-cytoplasmic actin in the IFM of pW8(5′Act88F)β-cyto transformants accumulates at a much lower level than the endogenous Act88F actin in wild-type flies. The faint ~55-kDa band which is detected by the antiactin antibody in all lines accumulating wild-type levels of Act88F actin most likely represents arthrin (i.e. ubiquitinated actin; Bullard et al., 1985; Ball et al., 1987). As anticipated, actin was virtually undetectable in IFM extracts from KM88 flies. The 5′′ untranslated region of the Act88F gene is important for proper β-cytoplasmic actin accumulation The coding sequence of Act88F is flanked both at the 5′ and 3′ end by UTRs (Fyrberg et al., 1981; Sanchez et al., 1983; Geyer and Fyrberg, 1986; Okamoto et al., 1986). The 5′UTR contains an intron which is 552 bp in length and spans the region from −568 to −17 upstream of the translation start site (Okamoto et al., 1986). In contrast to the low levels of βcytoplasmic actin in the initial pW8(5′Act88F)β-cyto transformants, transgenic Act88F was expressed at wild-type levels in control transformants that were established with the parent pW8Act88F construct (Fig. 1A), which contains an intact Act88F gene including the homologous 5′UTR and 3′UTRs (data not shown). To examine the role of the 5′UTR and 3′UTR in the expression of IFM actin, we established transformants with the pW8Act88F(−3′UTR) (Fig. 1C) in which the 3′UTR had been removed from the otherwise authentic Act88F gene, and with the pW8(−5′ UTR)Act88F construct missing most of the 5′UTR (from −535 to −20) (Fig. 1D). Transgenic lines homozygous for the 3′UTR-truncated Act88F gene accumulate wild-type amounts of Act88F in their IFMs (Fig. 3B). Moreover, the ultrastructural IFM morphology was indistinguishable from that of wild-type flies and the transformants were able to fly normally (data not shown). In contrast, transformants containing a 5′ truncated Act88F did 3632 V. Brault and others Fig. 4. Regions flanking the Act88F coding sequence confer wild-type levels of βcytoplasmic actin accumulation upon the IFM in transgenic flies. (A) Immuno-blotting IFM protein extracts with an antibody specific for βcytoplasmic actin reveals that the flies transformed with the construct containing the authentic Act88F UTRs accumulate significantly more β-cytoplasmic actin than flies that express the β-cytoplasmic actin under the 5′ promoter region of Act88F gene without the corresponding UTRs. Ubiquitinated β-cytoplasmic actin is detected only in transformants where the Act88F UTRs were included in the construct. (B) If intact Act88F UTRs flank the β-cytoplasmic actin, transformants accumulate wild-type levels of βcytoplasmic actin in the IFM. not accumulate any detectable amount of Act88F actin or arthrin (Fig. 3B) and were unable to fly. This finding indicates that the 5′UTR of the Act88F gene including the intronic sequence is critical for the expression of normal levels of actin and that its absence in the previous pW8(5′Act88F)β-cyto construct probably reduced actin accumulation and thus, affected the IFM structure yielding a flightless phenotype. In the light of these findings, we designed a new control transformation vector, pW8(StuI)Act88F(EcoRI), in which the Act88F UTRs were preserved (Fig. 1E). Generation of a StuI restriction site at position −12 did not affect the splice acceptor site and left the sequence directly upstream of the ATG intact. Control transformants established with the pW8(StuI)Act88F(EcoRI) construct accumulate wild-type level of Act88F actin and their ultrastructural IFM morphology (Fig. 2C and D) corresponds to that of wild-type flies, thus confirming that the sequence modification had no adverse affect. The unique restriction sites allowed us to exclusively exchange the Act88F coding sequence with the coding sequence of β-cytoplasmic actin, while retaining the untranslated sequences of the endogenous Act88F gene. Transformants expressing wild-type amounts of βcytoplasmic actin in the IFM The accumulation of β-cytoplasmic actin protein in six individual transformed lines established with the pW8(StuI)βcyto(EcoRI) construct (Fig. 1F), which for convenience are called β-cyto transformants, is documented in Fig. 4A. In all six transformants, the anti-β antibody recognizes a prominent band with an apparent molecular mass of ~43 kDa which corresponds to the size of actin. The level of expression does not significantly vary between the different lines. In comparison, the amount of β-cytoplasmic actin present in IFM extracts of the initial pW8(5′Act88F)β-cyto transformant which lacks the flanking Act88F UTRs (lane on the right) was strikingly lower. Moreover, the detection of a ~55 kDa arthrin band in β-cyto transformants supports the notion that ubiquitination is not isoformspecific but rather a feature of the IFM (Ball et al., 1987). To directly compare the amount of βcytoplasmic actin present in the β-cyto transformants with endogenous Act88F in wild-type IFM, an equivalent blot was probed with an anti-actin mAb that does not discriminate different isoforms (Fig. 4B). This immunoblot revealed that the amount of β-cytoplasmic actin in β-cyto transformants is similar to that of Act88F actin in wild-type flies. Consistent with the result from the immunoblot using the β-cytoplasmic actin-specifc antibody (Fig. 4A), the actin level in the IFM of the initial pW8(5′Act88F)β-cyto transformant was at least 10 times lower than in the β-cyto transformants or in wild-type flies. In some instances, the anti-actin antibody also detected a faint 43 kDa band in control extracts from the KM88 null mutant. Although the band could correspond to endogenous cytoplasmic actin present in the IFM, we believe it rather represents other Drosophila actin isoforms from surrounding muscle or nonmuscle tissue, especially since in the absence of a discernible myofibrillar structure in KM88, it is extremely difficult to exclusively dissect IFM. Similar to the anti-β mAb (Fig. 4A), the anti-actin mAb also detected the 55 kDa arthrin band in wild-type flies and in the β-cyto transformants. By flanking the heterologous human β-cytoplasmic actin with the 5′ and 3′ UTRs of the Act88F gene in the transformation vector, we achieved ectopic expression of βcytoplasmic actin in the IFM that is regulated in a manner similar to endogenous Act88F in wild-type flies. Myofibrillar structure of β-cytoplasmic transformants As illustrated in Fig. 5, the IFM of β-cyto transformants contain thin as well as thick filaments, which are bundled together into myofibrillar-like structures. However, these are not marked by regular sarcomeric repeats (Z-discs and Mlines) and frequently split or taper. In contrast to both wildtype and KM88 IFM, the β-cyto myofibrils very rarely display M-lines, which may reflect the lack of thick filament register. At higher magnification, ultra-thin sections of β-cyto transformants clearly reveal that the thin filaments interdigitate with thick filaments in an orderly fashion (see small arrows, Ectopic expression of β-cytoplasmic actin 3633 Fig. 5. Thin filaments containing β-cytoplasmic actin assemble into myofibril-like bundles. (A) Longitudinal sections reveal rudiments of sarcomeric organisation. Large arrows point to two Z-disc-like structures that are formed by alignment of Z-bodies (zb). Note that both Z-disclike structures are by-passed by out-of-register filaments, lying close to the arrowheads. Smaller arrows point to isolated Z-bodies. Mitochondria (MI); bar, 500 nm. (B) Higher magnification of aligned Z-bodies (zb) bracketing rudimentary sarcomere. Arrowhead points to discontinuity. Larger arrow points to an isolated Z-body showing that thin filaments are frequently not linked end-to-end and do not emerge from both sides of Z-bodies. Only a few thick filaments are in-register at Z-bodies. Small arrows point along corridors in which thin filaments can be seen. Bar, 500 nm. (C) Discontinuities between Z-bodies (arrowheads) and lack of thick and thin filament register. Bar, 250 nm. (D) Myosin crossbridges forming angled chevrons along the β-cytoplasmic actin containing thin filaments. Bar, 150 nm. (E) The polarity of rigor chevrons (indicated by arrowheads) is uniform along individual thin filaments. Chevrons on adjacent thin filaments may point in opposite directions. Bar, 200 nm. 3634 V. Brault and others Fig. 5B). Examination of longitudinal sections in rigor (Fig. 5D and E) document that crossbridges bind to β-cytoplasmic actin thin filaments, forming ~45° angled chevrons, which are comparable to those found in wild-type IFM (Reedy and Reedy, 1985). Moreover, the polarity of the chevrons is uniform along the entire length of a particular thin filament (Fig. 5D). However, along an adjacent thin filament (arrowheads in Fig. 5E), chevrons may be oriented in the opposite direction. This finding indicates that the laterally aligned thin filaments do not have uniform polarity, possibly because they are not correctly anchored in Z-discs. Consistent with this notion, the myofibrils of β-cyto transformants display amorphous, dense ‘Z-bodies’ (zb, Fig. 5A,B and C) instead of Z-discs. In some areas, Zbodies are not integrated into myofibrils and occur as isolated dense bodies with tangles of short thin filaments emerging from them (medium size arrows in Fig. 5A and B). Some other Zbodies are narrow and extend axially over two or three hundred nanometers (Fig. 5C), while other, less extended Z-bodies, are so well aligned laterally that they appear to be a Z-disc (zb, Fig. 5A). However, unlike with a normal Z-disc, only a few thin filaments appear achored into each Z-body, which is separated from the neighboring one by a gap (Fig. 5C, arrowheads). In a few cases, axially repeated Z-disc-like structures delineate a rudimentary sarcomere (Fig. 5A and B; delineated by two large arrows in Fig. 5A). Measurements of rudimentary sarcomeres reveal their length to be most often ~2 µm, although examples as short as 1.6 µm could also be found. These significantly shorter sarcomere-like structures reflect the shorter lengths of the thin and thick filaments. Cross-sections (Fig. 6) reveal that β-cyto myofibrils have no regular boundary. Central areas exhibiting hexagonal packing of thin and thick filaments are seen (marked ‘A’ in Fig. 6A), surrounded by a disorganized packing of thin and thick filaments. Areas with ordered β-cyto myofibrils exhibit thin and thick filaments that are laterally well aligned, in which case the hollow profiles of the thick filaments in the A-band (marked ‘A’ in Fig. 6B) can be distinguished from the solid profiles that characterize the bare zone of the thick filament (marked ‘M’ in Fig. 6B). This observation indicates that the apparent lack of an M-line is not due to misassembly of the midregion of the thick filaments, and supports the idea that filament misregister is responsible for the lack of a distinct M-line. Dense Z-body formations of various sizes appear in scattered areas of the βcyto IFM (‘Zb’ in Fig. 6A), consistent with the longitudinal views. At the myotendon junction (MTJ), myofibrils are attached to the cuticle via epithelial tendon cells. Substitution of Act88F actin for β-cytoplasmic actin has a strong impact on this junction. In wild-type flies, each myofibril is anchored to the muscle cell membrane by a modified terminal Z-disc (MTZ) (double-headed arrow in Fig. 7A), which is extended into a meshwork of actin filaments and dense material. A dense zigzag junction (‘*’ in Fig. 7A) is formed between the muscle membrane at the MT-Z and the apposed membrane of the epithelial tendon cell. Microtubule bundles (arrowheads in Fig. 7) extend from the tendon cell membrane opposite the MT-Z to the tendon cell membrane apposed to the cuticle, where dense tonofibrils (t) penetrate the cuticle and anchor the Fig. 6. Cross-sections of β-cyto transformant IFMs. (A) Myofibrils of β-cyto transformants are irregular in size and shape. Even the best ordered areas, such as the extensive region of hexagonal overlap seen in one A-band (A), are surrounded by out-of-register filaments. (Zb), Zbodies; scale bar, 500 nm. (B) Region of hexagonal overlap; in A, thick filaments are hollow, whereas the narrow, irregularly shaped region ‘M’ displays solid thick filament profiles that characterize the bare zone of the thick filament. Bar, 100 nm. Ectopic expression of β-cytoplasmic actin 3635 Fig. 7. Longitudinal sections through the myotendon junction (MTJ) of wild type, KM88, and the β-cyto transformant IFM. (A) At the wild-type MTJ, each myofibril is linked to the cuticle (C) via an epithelial tendon cell. Myofibrils (mf) maintain sarcomere organization right up to the modified terminal Z-disc, a dense meshwork of actin filaments linking each myofibril to the muscle membrane (large double-headed arrow). The muscle membrane forms the characteristic dense zig-zag junction (asterisks) with the tendon cell membrane. Bundles of microtubules (arrowheads) link to tonofibrils (t) that extend into the cuticle (C). (B) At the MTJ of the β-cyto transformant, myofibrils (mf) terminate near the muscle membrane (arrows) and no modified terminal Z-disc is formed. However, dense junctional plaques between the membranes of the muscle and tendon cells are formed (asterisks), although not necessarily opposite the terminus of a myofibril (longest arrow, bottom left). The microtubule bundles (arrowheads) are fairly dense in this example and link to the tonofibrils (t) which connect to the cuticle (C). (C) In KM88, irregular myofibrillar bundles (mf) simply terminate near the muscle membrane and no modified terminal Zdisc is formed (vacant location indicated by double headed arrow). The membranes of the muscle (upper asterisk) and tendon cell (lower asterisk) are widely separated. In this example, the microtubule bundles in the tendon cell, and a junction between tendon cells, are quite dense, and at low magnification, may give the misimpression that the dense membrane junction is present. The tonofibrils (t) connect to the cuticle (C) in the normal manner. Bars, 1 µm. muscle in it (see Reedy and Beall, 1993, for a more complete description). In the β-cyto transformants (Fig. 7B), MT-Zs are absent, leaving the myofibrils unconnected to the tendon cell and thus, to the cuticle. However, the transformants exhibit dense junctions between the membranes of the muscle and tendon cells (arrows in Fig. 7B), and the tendon cell morphology is nearly normal. This is an important detail, because in the Act88F actin null IFM (Fig. 7C), both the MT-Z and the dense 3636 V. Brault and others junctions between the tendon and muscle cell membranes are missing (double-headed arrow in Fig. 7C). The membrane surrounding the muscle cells in the Act88F actin null strain balloons and blebs, thereby creating vacuoles between the muscle and tendon cells (‘*’ in Fig. 7C), whereas the tendon cell morphology remains almost normal. DISCUSSION Sequences that influence the accumulation of actin in the IFM Our Act88F control transformants establish which sequences of the Act88F gene affect actin protein accumulation in the IFM. These experiments demonstrate that the Act88F 5′UTR including its intronic sequence is essential for the accumulation of wild-type amounts of actin protein in the IFM. In our initial pW8(5′Act88F)β-cyto transformants, expression of βcytoplasmic actin cDNA was regulated by the 5′ Act88F promoter region because this region has been reported to contain the sequences required for wild-type mRNA accumulation in the IFM (Geyer and Fyrberg, 1986). However, compared to the amounts of Act88F actin in the IFM of wildtype flies and pW8Act88F control transformants, the pW8(5′Act88F)β-cyto transformants accumulated very little βcytoplasmic actin, and their IFM ultrastructure resembled that of the KM88 null mutant. Recently, Fyrberg et al. (1998) suggested that both the intronic sequence in the 5′UTR and the intron within codon 307 of the protein-coding region (Fig. 1A) of the Act88F gene were required for orderly temporal and spatial actin expression. They based their conclusion primarily on transformants expressing a chimeric actin gene, which after the first 84 Act88F-specific amino acids encodes human αactin and thus lacks the downstream intron interrupting the coding region in theAct88F gene. Since our corresponding pW8(StuI)β-cyto(EcoRI) transformants also did not include the downstream intron and still showed accumulation of wildtype amounts of β-cytoplasmic actin, we conclude that the deficient expression might be related to the α-actin isoform rather than the absence of the intron residing within the coding region of Act88F. By flanking the β-cytoplasmic actin cDNA with the Act88F UTRs in the pW8(StuI)β-cyto(EcoRI) construct, we achieved accumulation of wild-type levels of heterologous actin in the IFM of β-cyto transformants. There is evidence that the amount of Act88F actin plays a critical role in the proper structure and function of the IFM (Mahaffey et al., 1985). Although immunoblot analysis with an anti-actin antibody that does not distinguish between Act88F and β-cytoplasmic actin suggests that the latter accumulates at wild-type level, we cannot exclude that there are minor differences which imbalance the critical stoichiometry of thin and thick filaments (Beall et al., 1989). However, if any minor reduction in the amount of actin occurs, it has not impeded the ability of the β-cytoplasmic actin to polymerize and form thin filaments in the IFM of the β-cyto transformants. Isoform-specific amino acid differences The amino acid differences between IFM-specific Act88F actin, human β-cytoplasmic actin, and rabbit skeletal muscle actin are listed in Fig. 8A. N-terminally processed Act88F differs by 15 amino acids from mammalian β-cytoplasmic actin which, in turn, differs by 25 amino acids from mammalian skeletal muscle actin. The majority of the replacements are conservative. Residues 232, 278, and 368 (boxed in Fig. 8A) of Act88F, which belong to the small group of muscle-specific residues in Drosophila (bold in Fig. 8A; Mounier et al., 1992), are replaced by polar amino acids in β-cytoplasmic actin. To visualize the approximate position of the differences with respect to the intersubunit contacts and the surface of the F-actin filament, we have mapped the residues that are different in β-cytoplasmic actin onto the ribbon of a trimer representing Act88F (Fig. 8B). Based on this stereo representation, differences in residues 325 and 169 possibly affect the intersubunit contacts along the two longpitch helical strands. The most likely candidates involved in intersubunit contacts between the two long-pitch helical strands appear to be residues 169 and 76. Slight changes in geometry and/or conformation of the filament due to isoform-specific differences could in turn interfere with the proper interaction of F-actin with actin-binding proteins. Amino acids that are exposed on the surface of the filament according to the Holmes-Lorenz model of the F-actin filament (Holmes et al., 1990; Lorenz et al., 1993) are indicated in orange in Fig. 8. These residues, i.e. also residues 232 and 368, are more likely to be involved in interactions with actin-binding proteins. For example, it has been proposed that the α-helix extending from residue Asp222 to Ser233 could be part of a contact site between actin and tropomyosin (Kabsch and Vandekerckhove, 1992), and that Asn225 which is specific for muscle actins in mammals directly interacts with tropomyosin (Mounier and Sparrow, 1997). However, both Act88F and β-cytoplasmic actin have Gln, which has a slightly longer side chain, at position 225 and yet they interact with tropomyosin. In their recent publication, Fyrberg et al. (1998) state that exchanging singly residues 232, 278, or 368 of Act88F with the residue corresponding to a Drosophila cytoplasmic actin had no effect on IFM structure and function. This suggests that these muscle-specific residues do not confer a unique function to their respective isoforms. It is noteworthy that the N terminus where several isoformspecific residues are clustered and which protrudes from the filament surface at the largest radius, is directly involved in myosin binding (Rayment et al., 1993). However, the different amino acids at the N terminus of β-cytoplasmic actin did not prevent rigor crossbridge formation of IFM myosin with thin filaments built of β-cytoplasmic actin (Fig. 5D and E). In those three positions (i.e. 76, 153, and 279) where the amino acids in Act88F actin are identical to the mammalian muscle-specific residues, the replacements in β-cyto transformants are rather conservative: IrV; LrM; and YrF. None of these residues have been directly implicated in interactions with actin-binding proteins, albeit their location on the surface of the F-actin filament would favor such interactions. Interestingly, the substitution of Ile at position 76 with Val, which is a residue of similar stereochemistry, produced mild perturbations in the myofibrillar structure of transformants (Fyrberg et al., 1998), in particular, the Z-disc morphology is affected (M. Reedy, unpublished observation). As discussed above, this effect could possibly be mediated Ectopic expression of β-cytoplasmic actin 3637 by modifications of intersubunit contacts in the F-actin filament. Although comparison of the primary sequence does not reveal an obvious basis for the structural and functional differences between the IFM expressing either Act88F or βcytoplasmic actin, one has to keep in mind that the respective actin conformation in vivo may vary from the one predicted by the atomic structure which has been derived from actin complexed with actin monomer-binding proteins (Kabsch et al., 1990; McLaughlin et al., 1993; Schutt et al., 1993). Moreover, not much is known about how isoform differences affect F-actin filament geometry which, in turn, appears to be an important parameter in regulating the interactions of actin with actin-binding proteins (McGough et al., 1997). Structural and functional properties of a nonmuscle actin in a muscle environment The detection of arthrin in the β-cyto transformants documents that ubiquitination is not restricted to IFM-specific Act88F. Consistently, ubiquitinated actin was detected in transformants expressing chimeric Act88F transgenes (Fyrberg et al., 1998). Our findings also agree with other evidence that ubiquitination and filament formation are closely correlated: (i) ubiquitination lags several hours behind Act88F expression, paralleling myofibril formation (Ball et al., 1987), and (ii) in transgenic flies that express tagged variants of Act88F actin, ubiquitination of tagged actin only occurred in transformants that were able to form thin filaments (Brault et al., 1999). However, arthrin was not detected in transformants expressing Fig. 8. Amino acid replacements between different actin isoforms. In A, the position of a given amino acid is indicated by its residue number. White numbers indicate that we predict the residue at this position to be presumably exposed on the filament surface based on the Holmes-Lorenz F-actin filament model (Holmes et al.,1990; Lorenz et al., 1993). A shaded box indicates a partial surface exposure, whereas black numbers are positions of predominantly internal residues. Insect muscle-specific residues are given in bold. Asterisks identify mammalian musclespecific residues. The arrows point to amino acids that differ between Act88F and human βcytoplasmic actin. Orange indicates that we predict surface exposure for βcytoplasmic specific residues, yellow indicates β-cytoplasmic specific substitutions that are likely to be buried in the filament. The isoform-specific replacement that had an effect on IFM ultrastructure (Reedy et al., 1991, Fyrberg et al., 1998) is marked by an arrowhead. (B) Stereo image of a ribbon representation of an F-actin trimer (Lorenz et al., 1993), aligned and transparently overlaid with an averaged and refined 3-D helical reconstruction of a rabbit skeletal muscle F-actin filament (Steinmetz et al., 1997). The 3-D reconstruction is surface-rendered to include 100% of the nominal molecular volume. The Act88F ribbon representation of two neighbouring monomers along one long-pitch helical strand are shown in blue, whereas the ribbon representation of a monomer on the opposite strand is depicted in turquoise. Residues that are different in β-cytoplasmic actin are indicated in orange if they appear to be exposed on the filament surface. Residues that are presumably at least partially buried are indicated in yellow. The molecular modeling work (courtesy of D. Stoffler, M.E. Müller Institute) was performed using Insight II (Biosym/Molecular Dynamics, Inc. San Diego). 3638 V. Brault and others a chimeric Act88F-α-actin (Fyrberg et al., 1998), suggesting that not all isoforms are invariably ubiquitinated. Despite the competence to assemble β-cytoplasmic actin containing thin filaments, with which the endogenous myosin thick filaments can interact via typical rigor crossbridge formation, there are only rudimentary indications of sarcomeric organization in the IFMs of β-cyto transformants. For one, they do not assemble normal Z-discs. Aberrant Zdiscs have been reported for a number of Drosophila myofibrillar protein mutants (Reedy et al., 1989; Fyrberg et al., 1990; Sparrow et al., 1991b, 1992; Miller et al., 1993). For example, transheterozygous tropomyosin mutants contained sarcomeres with multiple, tandemly arrayed Z-discs that often did not span the entire width of the fibril (Kreuz et al., 1996). A transformant with Drosophila Act42A-specific replacements in the Act88F gene was reported by Fyrberg and co-workers (1998) which, although not examined in detail, appears to have disrupted Z-discs. Because of the absence of proper thin filament anchoring in Z-discs, actomyosin interactions may move thin and thick filaments out of register. The lack of registration, in turn, may prevent interactions necessary for forming mature Z-discs and M-lines. Moreover, the mis-register or absence of regular Z-discs may also affect thin and thick filament length regulation, as suggested by the shorter filament lengths inferred from the sarcomere-like structures in β-cyto transformants versus the ~3.2 µm sarcomere length in wildtype flies. Most of the rudimentary sarcomere repeats in the β-cyto transformants indicate a sarcomere length of ~2 µm or less. This means that not only the thin filaments but also the thick filaments are significantly shorter compared to wildtype myofilaments. In flight muscle assembly during pupation, thick and thin filament lengths are closely coregulated: thin filaments grow from ~1 µm to 1.5 µm whereas thick filaments start at 1.6 µm and grow to 3 µm. Thin and thick filaments add the same amount of length simultaneously at each stage of sarcomere assembly (Reedy and Beall, 1993). The EMs do not clearly show the ends of actin filaments at mid-sarcomere in the IFM of β-cyto transformants, so they may be too long (~2 µm) or too short (1 µm) or of variable length. An alternative explanation for the shorter filaments in the β-cyto transformants is that insufficient amounts of actin during pupation cause thin filaments to become shorter, thereby secondarily affecting thick filament length. Slight imbalances in the relative amounts of actin and myosin caused both thin and thick filaments to assemble to shorter lengths (Beall et al., 1989). However, quantitative analysis of northern blots from mRNA from late pupae indicated that the levels of respective mRNA in β-cyto transformants and wildtype were comparable (data not shown), supporting our observation that the amount of actin protein present in the IFM was also adequate. Another explanation for aberrant Z-discs and filament lengths is that β-cytoplasmic actin cannot interact properly with α-actinin and/or actin capping or severing proteins during sarcomere assembly (for review see Littlefield and Fowler, 1998), so length regulation of thin and thick filaments is disturbed. Consistent with this notion, more extreme tropomoyosin mutants also display shorter sarcomere lengths (Kreuz et al., 1996). Conversely, it is possible that βcytoplasmic actin cannot appropriately interact with the resident tropomyosin and/or troponin, thereby giving rise to deficient sarcomeres. Taken together, these results establish that β-cytoplasmic actin expressed at wild-type levels partially substitutes for muscle-specific Act88F in the IFM and that intrinsic parameters of actin function (e.g. polymerization and interaction with myosin) are retained in β-cytoplasmic actin, even when it is expressed in the IFM environment. However, the failure of β-cytoplasmic actin to fully substitute for the endogenous Act88F isoform in the IFM exemplifies the functional diversity of actin isoforms. We suggest that the functional variation primarily depends on modulation of the interaction of each isoform with particular actin-binding proteins. We are indebted to the members of Professor Walter J. Gehring’s laboratory (Biozentrum, Basel) for the use of their equipment and for their advice on all work involving flies. The cDNA for human βcytoplasmic actin was kindly provided by Prof. P. Gunning (The New Children’s Hospital, University of Sydney). We thank Dr D. Stoffler (M.E. Müller Institute, University of Basel) for preparing Fig. 8B. This work was supported by the Swiss National Science Foundation, the Canton Basel-Stadt, the M.E. Müller Foundation of Switzerland, and an NIH grant to M.K.R. REFERENCES Ball, E., Karlik, C. C., Beall, C. J., Saville, D. L., Sparrow, J. C., Bullard, B. and Fyrberg, E. A. (1987). Arthrin, a myofibrillar protein of insect flight muscle, is an actin-ubiquitin conjugate. Cell 51, 221-228. Beall, C. J., Sepanski, M. A. and Fyrberg, E. A. (1989). Genetic dissection of Drosophila myofibril formation: effect of actin and myosin heavy chain allels. Genes Dev. 3, 131-140. Bernstein, S. I., O’Donnell, P. T. and Cripps, R. M. (1993). Molecular genetic analysis of muscle development, structure and function in Drosophila. Int. Rev. Cytol. 143, 63-152. Bullard, B., Bell, J., Craig, R. and Leonard, K. (1985). Arthrin: a new actinlike protein in insect flight muscle. J. Mol. Biol. 182, 443-454. Brault, V., Sauder, U., Reedy, M. C., Aebi, U. and Schoenenberger, C.-A. (1999). Differential epitope tagging of actin in transformed Drosophila produces distinct effects on myofibril assembly and function of the indirect flight muscle. Mol. Biol. Cell 10, 135-149. DeNofrio, D., Hoock, T. C. and Herman, I. M. (1989). Functional sorting of actin isoforms in microvascular pericytes. J. Cell Biol. 109, 191-202. Eppenberger-Eberhardt, M., Flamme, I., Kurer, V. and Eppenberger, H. M. (1990). Reexpression of α-smooth muscle actin isoform in cultured adult rat cardiomyocytes. Dev. Biol. 139, 269-278. Fidel, S., Doonan, J. H. and Morris, N. R. (1988). Aspergillus nidulans contains a single actin gene which has unique introns and encodes alpha actin. Gene 70, 283-296. Fyrberg, E. A., Bond, B. J., Hershey, N. D., Mixter, K. S. and Davidson, N. (1981). The actin genes of Drosophila: protein coding regions are highly conserved but intron positions are not. Cell 24, 107-116. Fyrberg, E. A., Mahaffey, J. W., Bond, J. and Davidson, N. (1983). Transcript of the six Drosophila actin genes accumulate in a stage- and tissue-specific manner. Cell 33, 115-123. Fyrberg, E., Kelly, M., Ball, E., Fyrberg, C. and Reedy, M. C. (1990). Molecular genetics of Drosophila alpha-actinin: mutant alleles disrupt Z disc integrity and muscle insertions. J. Cell Biol. 110, 1999-2011. Fyrberg, E. A., Fyrberg, C. C., Biggs, J. R., Saville, D., Beall, C. J. and Ketchum, A. (1998). Functional nonequivalence of Drosophila actin isoforms. Biochem. Genet. 36, 271-287. Gallwitz, D. and Seidel, R. (1980). Molecular cloning of the actin gene from the yeast Saccharomyces cerevisiae. Nucl. Acids Res. 8, 1043-1059. Geyer, P. K. and Fyrberg, E. A. (1986). 5′-Flanking sequence required for regulated expression of a muscle-specific Drosophila melanogaster actin gene. Mol. Cell Biol. 6, 3388-3396. Gimona, M., Vandekerckhove, J., Goethals, M., Herzog, M., Lando, Z. and Ectopic expression of β-cytoplasmic actin 3639 Small, J. V. (1994). Beta-actin specific monoclonal antibody. Cell Motil. Cytoskel. 27, 108-116. Gunning, P., Ponte, P., Okayama, H., Engel, J., Blau, H. and Kedes, L. (1983). Isolation and characterization of full-length cDNA clones for human α, β, and γ-actin mRNAs: skeletal but not cytoplasmic actin have an aminoterminal cysteine that is subsequently removed. Mol. Cell Biol. 3, 387-395. Hennessey, E. S., Harrison, A., Drummond, D. R. and Sparrow, J. C. (1992). Mutant actin: a dead end? J. Musc. Res. Cell Motil. 13, 127-131. Herman, I. M. (1993). Actin isoforms. Curr. Opin. Cell Biol. 5, 48-55. Hiromi, Y. and Hotta, Y. (1985). Actin gene mutations in Drosophila; heat shock activation in the indirect flight muscles. EMBO J. 4, 1681-1687. Hiromi, Y., Okamoto, H. Gehring, W. J. and Hotta, Y. (1986). Germline transformation with Drosophila mutant actin genes induces constitutive expression of heat shock genes. Cell 44, 293-301. Hirono, M., Endoh, H., Okada, N., Numata, O. and Watanabe, Y. (1987). Tetrahymena actin: cloning and sequencing of the Tetrahymena actin gene and identification of its gene product. J. Mol. Biol. 194, 181-192. Holmes, K. C., Popp, D., Gebhard, W. and Kabsch, W. (1990). Atomic model of the actin filament. Nature 347, 44-49. Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F. and Holmes, K. C. (1990). Atomic structure of actin: DNase I complex. Nature 347, 37-44. Kabsch, W. and Vandekerckhove, J. (1992). Structure and function of actin. Annu. Rev. Biophys. Biomol. Struct. 21, 49-76. Karlsson, R., Aspenstrom, P. and Bystrom, A. S. (1991). A chicken betaactin gene can complement a disruption of the Saccharomyces cerevisiae ACT1 gene. Mol. Cell. Biol. 11, 213-217. Klemenz, R., Weber, U. and Gehring, W. J. (1987). The white gene as a marker in a new P-element vector for gene transfer in Drosophila. Nucl. Acids Res. 15, 3947-3959. Kreuz, A. J., Simcox, A. and Maughan, D. (1996). Alterations in flight muscle ultrastructure and function in Drosophila tropomyosin mutants. J. Cell Biol. 135, 673-687. Kumar, A., Crawford, K., Close, L., Madison, M., Lorenz, J., Doetschman, T., Pawlowski, S., Duffy, J., Neumann, J., Robbins, J., Bovin, G. P., O’Toole, B. A. and Lessard, J. L. (1997). Rescue of cardiac α-actindeficient mice by enteric smooth muscle γ-actin. Proc. Nat. Acad. Sci. USA 94, 4406-4411. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 227, 680-685. Lindsley, D. L. and Zimm, G. G. (1992). The genome of Drosophila. Academic Press INC. Harcourt Brace Jovanovitch Publishers. Lorenz, M., Popp, D. and Holmes, K. C. (1993). Refinement of the F-actin model against X-ray fiber diffraction data by the use of a directed mutation algorithm. J. Mol. Biol. 234, 826-836. Littlefield, R. and Fowler, V. M. (1998). Defining actin filament length in striated muscle: rulers and caps or dynamic stability? Annu. Rev. Cell Dev. Biol. 14, 487-525. Mahaffey, J. W., Coutu, M. C., Fyrberg, E. A. and Inwood, W. (1985). The flightless Drosophila mutant raised has two distinct genetic lesions affecting accumulation of myofibrillaer proteins in flight muscle. Cell 40, 101-110. McGough, A., Pope, B., Chiu, W. and Weeds, A. (1997). Cofilin changes the twist of F-actin: implications for actin filament dynamics and cellular function. J. Cell Biol. 138, 771-781. McKenna, N., Meigs, J. B. and Wang, Y. L. (1985). Identical distribution of fluorescently labeled brain and muscle actins in living cardiac fibroblasts and myocytes. J. Cell Biol. 100, 292-296. McLaughlin, P. J., Gooch, J. T., Mannherz, H. G. and Weeds, A. G. (1993). Structure of gelsolin segment 1-actin complex and the mechanism of filament severing. Nature 364, 685-692. Miller, R. C., Schaaf, R., Maughan, D. W. and Tansey, T. R. (1993). A nonflight muscle isoform of Drosophila tropomyosin rescues an indirect flight muscle tropomyosin mutant. J. Musc. Res. Cell. Motil. 14, 85-98. Mounier, N., Gouy, M., Mouchiroud, D. and Prudhomme, J. C. (1992). Insect muscle actins differ distinctly from invertebrate and vertebrate cytoplasmic actins. J. Mol. Evol. 34, 406-415. Mounier, N. and Sparrow, J. C. (1997). Structural comparisons of muscle and nonmuscle actins give insights into the evolution of their functional differences. J. Mol. Evol. 44, 89-97. Mounier, N., Perriard, J.-C., Gabbiani, G. and Chaponnier, C. (1997). Transfected muscle and non-muscle actins are differentially sorted by cultured smooth muscle and non-muscle cells. J. Cell Sci. 110, 839-846. Okamoto, H., Hiromi, Y., Ishikawa, E., Yamada, T., Isoda, K., Maekawa, H. and Hotta, Y. (1986). Molecular characterization of mutant actin genes which induce heat-shock proteins in Drosophila flight muscles. EMBO J. 5, 589-596. Peng, I. and Fischman, D. A. (1991). Post-translational incorporation of actin into myofibrils in vitro: evidence for isoform specificity. Cell Motil. Cytoskel. 20, 158-168. Rayment, I., Holden, H. M., Whittaker, M., Yohn, M., Lorenz, M., Holmes, K. C. and Milligan, R. A. (1993). Structure of the actin-myosin complex and its implications for muscle contraction. Science 261, 58-65. Reedy, M. K. and Reedy, M. C. (1985). Rigor crossbridge structure in tilted single filament layers and flared-X formations from insect flight muscle. J. Mol. Biol. 185, 145-176. Reedy, M. C., Beall, C. and Fyrberg, E. (1989). Formation of reverse rigor chevrons by myosin heads. Nature 339, 481-483. Reedy, M. C., Beall, C. and Fyrberg. E. (1991). Do variant residues among the six actin isoforms of Drosophila reflect functional specialization? Biophys. J. 59, 187a. Reedy, M. and Beall, C. (1993). Ultrastructure of developing flight muscles in Drosophila. I. Assembly of myofibrils. Dev. Biol. 160, 443-465. Rubenstein, P. A. (1990). The functional importance of multiple actin isoforms. BioEssays 12, 309-315. Rubin, G. M., and Spradling, A. C. (1982). Genetic transformation of Drosophila with transposable element vectors. Science 218, 348-353. Sanchez, F., Tobin, S. L., Rdest, U., Zulauf, E. and McCarthy, B. J. (1983). Two Drosophila actin genes in detail: gene structure, protein structure and transcription during development. J. Mol. Biol. 163, 533-551. Sawtell, N. M. and Lessard, J. L. (1989). Cellular distribution of smooth muscle actins during mammalian embryogenesis: expression of the αvascular but not γ-enteric isoform in differentiating striated myocytes. J. Cell Biol. 109, 2929-2937. Schutt, C. E., Myslik, J. C., Rozycki, M. D., Goonesekere, N. C. W. and Lindberg, U. (1993). The structure of crystalline profilin-β-actin. Nature 365, 810-816. Sparrow, J., Drummond, D., Peckham, M., Hennessey, E. and White, D. (1991a). Protein engineering and the study of muscle contraction in Drosophila flight muscles. J. Cell Sci. 14, 73-78. Sparrow, J., Reedy, M., Ball, E., Kyrtatas, V., Molloy, J., Durston, J., Hennessey, E. and White, D. (1991b). Functional and ultrastructural effects of a missense mutation in the indirect flight muscle-specific actin gene of Drosophila melanogaster. Mol. Biol. 222, 963-982. Sparrow, J. C., Drummond, D. R., Hennessey, E. S., Clayton, J. D. and Lindegaard, F. B. (1992). Drosophila mutants and the study of myofibrillar assembly and function. In Molecular Biology of Muscle (ed. A. Elhaj), pp. 111-129. SEB Symposium 46. Steinmetz, M., Stoffler, D., Hoenger, A., Bremer, A. and Aebi, U. (1997). Actin: From cell biology to atomic detail. J. Struct. Biol. 119, 295-320.