Article |
Address correspondence to Tapio I. Heino, Developmental Biology Program, Institute of Biotechnology, Viikki Biocenter, P.O. Box 56 (Viikinkaari 9), FIN-00014, University of Helsinki, Finland. Tel.: 358-9-19159590. Fax: 358-9-19159366. E-mail: tapio.heino{at}helsinki.fi
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Abstract |
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Key Words: actin; Drosophila; twinfilin; ADF/cofilin; bristle
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Introduction |
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Actin monomerbinding proteins also have a central role in regulating cytoskeletal dynamics. For example, profilin is a small actin monomerbinding protein found in all eukaryotes that promotes the assembly of actin filaments and sequesters actin monomers in the absence of free filament ends (Vinson et al., 1998). Profilin also catalyzes the exchange of the nucleotide bound to an actin monomer, and at least in yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, this activity is important in the in vivo actin filament turnover (Wolven et al., 2000; Lu and Pollard, 2001). In Drosophila, profilin is ubiquitously expressed throughout development, and mutations in the profilin-encoding chickadee gene lead to defects in actin-dependent processes such as cytoplasmic transport during oogenesis (Cooley et al., 1992), bristle formation (Verheyen and Cooley, 1994), and motor axon outgrowth (Wills et al., 1999).
Small actin monomerbinding proteins of the thymosin family also regulate actin dynamics in various organisms. Thymosin ß4 is an actin monomersequestering protein in vertebrates, and is involved in maintaining the cellular ATPactin monomer pool (Yu et al., 1994). Ciboulot is a Drosophila brain-specific protein with three thymosin ß4like repeats. Unlike vertebrate thymosin ß4, ciboulot promotes the assembly of actin monomers at the barbed ends of filaments in a manner similar to profilin (Boquet et al., 2000).
ADF/cofilins regulate actin dynamics by depolymerizing actin filaments at their pointed ends and bind actin monomers with high affinities (Carlier et al., 1997). Mutations in the Drosophila ADF/cofilin homologue, twinstar, lead to defects in centrosome migration, cytokinesis, and border cell migration (Edwards et al., 1994; Gunsalus et al., 1995; Chen et al., 2001).
Twinfilin is an 40-kD actin monomerbinding protein found in eukaryotes ranging from yeast to mammals, and is composed of two ADF-H domains that are homologous to ADF/cofilins (Lappalainen et al., 1998); however, twinfilin binds actin monomers with a 1:1 molar ratio and prevents their assembly into filaments and does not bind or depolymerize actin filaments like ADF/cofilins. In yeast and cultured mammalian cells, twinfilin is diffusely distributed in the cytoplasm, and concentrated at the cortical actin cytoskeleton. Twinfilin is involved in the in vivo regulation of actin dynamics, because overexpression of twinfilin in yeast and mammalian cells results in the formation of abnormal actin structures (Goode et al., 1998; Vartiainen et al., 2000). However, the lack of a clear phenotype in twinfilin deletion strains of budding yeast has hampered the elucidation of this ubiquitous actin-binding protein's biological role (Goode et al., 1998).
We show that the Drosophila twf gene encodes a homologue of yeast and mammalian twinfilins. A mutation in the twf gene leads to several developmental defects, including aberrant bristle morphology, that results from misorientation of actin filaments in developing bristles. Our findings demonstrate that twinfilin is essential for actin-dependent morphological processes in multicellular organisms.
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Results |
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We also used the antiserum in whole mount immunostainings of wild-type (Fig. 4 A) and twf 3701 embryos (Fig. 4 B). The preimmune serum did not stain the wholemounts (Fig. 4 C) and the twinfilin amount in mutant embryos is strongly reduced. In the wild-type embryos, the twinfilin protein is uniformly distributed throughout embryogenesis (Fig. 4, A, D, and E) and is also maternally provided, because the protein is already present in early embryos before the onset of zygotic transcription (Fig. 4 D).
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Morphological and developmental defects in twinfilin mutant flies
The twf 3701 mutant flies can be maintained as a homozygous stock. However, the mutant flies are slightly smaller and appear to be less active than wild-type flies. In flight tests, older twf 3701 mutant flies displayed reduced or completely lost flight ability. The hatching frequency was also slightly reduced in twf 3701 mutants. The twf 3701 mutants also had significantly prolonged larval periods compared with wild-type flies, but the duration of the pupal periods were the same (unpublished data).
The twf 3701 mutant has a rough eye phenotype, and closer examination by scanning electron microscopy (SEM) (Fig. 5, JM) showed that the interommatidial bristles were often tufted (Fig. 5 M), whereas in wild-type eyes the bristles are arranged in regular arrays between the ommatidia (Fig. 5 K). The ommatidia in the twf 3701 mutant were sometimes pitted and occasionally fused. Interestingly, similar phenotypes have been previously reported in Drosophila bifocal mutant eyes, which also result from alterations in the actin cytoskeleton (Bahri et al., 1997).
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Twinfilin shows genetic interaction with twinstar
In yeast, lack of twinfilin does not result in a detectable phenotype, except for slightly enlarged cortical actin patches. In combination with a temperature-sensitive cofilin allele, twinfilin causes lethality at the permissive temperature (Goode et al., 1998). In Drosophila, cofilin is encoded by the twinstar (tsr) gene (Edwards et al., 1994; Gunsalus et al., 1995). To investigate the possible genetic interaction between twinfilin and twinstar, we crossed the P element line tsrk05633, which has a lethal insertion in twinstar, with the twf 3701 homozygotes, and examined the resulting double heterozygotes for a bristle phenotype. Nearly all flies had at least one macrochaete with defects at bristle tip (Fig. 5, NQ). The bilateral posterior scutellar and anterior dorsocentral bristles on the thorax were by far the most frequently affected. We scored these four bristles for abnormalities under higher magnification. In tsrk05633/+; twf 3701/+ flies, 66% of the bristles were split, branched, or had a rough surface, whereas only 2% of the bristles on tsrk05633/+; +/+ flies and none of the bristles on +/+; twf 3701/+ flies had this phenotype. The presence of a single tsrk05633 allele in the twf 3701 homozygous background results in a more dramatic eye defect, whereas the severity of the bristle phenotype appears to be equal to the one in the twf 3701 mutant alone (unpublished data). These results show that twinfilin interacts genetically with the ADF/cofilin encoding gene twinstar during bristle and eye morphogenesis.
Actin bundles are severely misoriented in developing twinfilin mutant bristles
To visualize the actin bundles in developing mutant macrochaetae, we stained thoracic epithelia from twf 3701/twf 3701 and twf 3701/Df(3R)SuHw7 pupae with Texas redconjugated phalloidin and examined them using confocal microscopy. In wild-type bristles, the actin bundles are located at the periphery of the developing bristle (Fig. 6 A) and during the elongation phase, new actin filaments are formed in the bristle tip. These newly formed filaments are visible as phalloidin-positive spots in the tip, as well as between the actin bundles. The filaments are gathered into tiny actin bundles that are added to the preexisting thicker bundles. In fully elongated 48-h-old bristles, actin-containing spots are no longer visible and only the actin bundles can be seen (Tilney et al., 1996).
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Randomly oriented bundles can eventually become reorganized into main bundles that extend the bristle in a new direction. Fig. 6 I shows a macrochaete that was bent twice. The original correctly oriented main bundles that extend from the base seem to end at the bending point, instead another set of bundles grow in the new direction.
The localization of twinfilin in developing bristles
To examine twinfilin's localization in developing bristles, we dissected wild-type pupae at 32 or 48 h after pupariation and stained them with twinfilin antiserum and phalloidin. Twinfilin was highly abundant in the cytoplasm of all hair- and bristle-producing cells (unpublished data). In addition, twinfilin was distributed throughout the developing bristle shaft. In surface confocal sections from recently sprouted (32 h) bristles (Fig. 7 A), twinfilin was localized in diffuse spots between the actin bundles, corresponding to the sites of new actin filament assembly (Tilney et al., 1996). Sections from the interior of the bristle gave a uniform signal. In fully elongated bristles (48 h) (Fig. 7 B), the twinfilin spots were still present, although they appeared more condensed and less numerous. Many of the spots colocalized with ends of the modules that make up the actin bundles. In regions with large gaps between successive modules, the spots tended to localize on the tipmost end of the module, which correspond to the barbed ends of filaments (Tilney et al., 1996).
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Discussion |
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Actin filaments can assemble into higher order structures via a bundling process that is involved in formation of complex structures with highly specific functions. Examples include the intestinal microvilli and inner ear hair cell stereocilia of mammals, and Drosophila's mechanosensory bristles and ovarian nurse cell-specific actin filament cables and ring canals (Bartles, 2000; DeRosier and Tilney, 2000). Of these, the developing Drosophila bristle, which is formed as a cytoplasmic extension from a single cell, has been extensively studied as a model for actin bundle formation. In the twf 3701 mutant flies, the adult macrochaetae are split, branched, or bent and have a highly irregular ridge pattern. This phenotype is similar to the one seen in singed and forked mutants, which arise from mutations in actin filamentbundling proteins (Cant et al., 1994; Petersen et al., 1994; Tilney et al., 1995, 1998). The twf 3701 bristle phenotype is explained by our observations that in developing mutant bristles, the actin bundles are twisted and misoriented (Fig. 6). However, unlike fascin (singed gene product) and forked, twinfilin is an actin monomersequestering protein with no detectable affinity for actin filaments (Fig. 2 A). Thus, the presence of a severe bristle phenotype in the twf 3701 mutant demonstrates that the accurate regulation of the size and dynamics of the actin monomer pool is essential for actin filament assembly and subsequent bundle formation. This conclusion is also supported by previous experiments in which developing bristles were cultured in vitro together with drugs that affect filament elongation (Tilney et al., 2000b). Furthermore, mutations in Drosophila cofilin (Chen et al., 2001), profilin (Verheyen and Cooley, 1994) or the ß-subunit of capping protein (Hopmann et al., 1996), all of them proteins that are involved in regulation of actin dynamics, lead to defects in bristle morphology. Interestingly, the severely affected bristles in the capping protein mutant (Hopmann et al., 1996) are almost identical to the bristles in the twf 3701 mutant.
In addition to unstable main bundles, we observed ectopic actin filament-containing spots or perpendicular tiny bundles in fully elongated twf 3701 mutant bristles (Fig. 6). In wild-type bristles, F-actincontaining spots are present only during the elongation phase of bristle development (Tilney et al., 1996). To our knowledge, such a mutant phenotype has not been described earlier. One explanation for the origin of the ectopic spots and/or bundles in the twf 3701 mutant is that in the absence of actin monomersequestering twinfilin, spontaneous actin filament nucleation takes place. These filaments may then become cross-linked into tiny bundles that are not integrated with the main bundles. A second possibility is that in the absence of twinfilin, an uncontrolled polymerization of preexisting actin filaments in the main bundles takes place. The perpendicular ectopic bundles may then originate from main bundles that have split lengthwise and become separated into modules, whereas the spots are the result of further fragmentation. However, we favor the first explanation because the F-actin spots in twf 3701 mutant macrochaetae are reminiscent of actin filament spots normally present in young bristles. In addition, the ectopic bundle pattern is clearly different from the fragmented actin bundles observed in elongating bristles treated with cytochalasin D (Tilney et al., 2000b).
Immunostainings showed that twinfilin is localized in the cytoplasm and to actin filament structures in bristles (Fig. 7). Similar localizations have been shown previously for yeast and murine twinfilins (Goode et al., 1998; Vartiainen et al., 2000). Interestingly, in fully elongated Drosophila bristles, twinfilin is localized along the actin filament bundles in spots, which may represent the barbed ends of actin filaments (Fig. 7 B). Therefore, it is possible that in addition to sequestering actin monomers, the role of twinfilin in bristles may be to localize monomers at the sites of actin filament assembly. This is also supported by our recent studies showing that interactions with actin monomers and capping protein are essential for localization of twinfilin in Saccharomyces cerevisiae (Palmgren et al., 2001). Also in Drosophila bristles, the localization of twinfilin to actin bundles may be mediated through interactions with capping protein.
tsr/+; twf/+ double heterozygotes display a weak but significant bristle defect (Fig. 5, NQ). A similar genetic interaction between cofilin and twinfilin has previously been demonstrated in yeast (Goode et al., 1998). In yeast cells, cofilin promotes actin dynamics by depolymerizing actin filaments, and a mutation in the cofilin gene that affects its actin filament depolymerization rate shows synthetic lethality with a twinfilin deletion (Lappalainen and Drubin, 1997; Goode et al., 1998). Analogously, we suggest that during bundle formation, the decreased actin filament depolymerization rate due to the tsr/+ mutation, together with uncontrolled filament assembly resulting from the twf/+ mutation, lead to defects in bristle morphogenesis.
This work demonstrates the essential role of twinfilin, an actin monomerbinding protein, in the development of a multicellular organism. Furthermore, we show that the accurate regulation of the size and dynamics of the actin monomer pool is important for assembly of complex actin filament structures in cells.
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Materials and methods |
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Proteins and antibodies
Drosophila twinfilin was purified as a glutathione S-transferase fusion similar to murine twinfilin (Vartiainen et al., 2000). Rabbit muscle actin was prepared from an acetone powder (Pardee and Spudich, 1982). Pyrene actin and human platelet actin were from Cytoskeleton. We immunized one New Zealand White rabbit with purified recombinant Drosophila twinfilin in Freund's adjuvant, and the serum was collected after four immunizations. Preimmune serum was collected from the same rabbit and used as a control in our assays.
Actin filament cosedimentation assays
40-µl aliquots of actin were diluted to desired concentrations in G buffer (20 mM Tris, pH 7.5, 0.2 mM ATP, 0.2 mM DTT, 0.2 mM CaCl2) and polymerized for 30 min by the addition of 5 µl of 10x initiation mix (20 mM MgCl2, 10 mM ATP, 1 M KCl). 5 µl of twinfilin in G buffer was added to filaments and incubated for 30 min. We sedimented actin filaments by centrifugating the samples for 30 min in a Beckman Optima MAX Ultracentrifuge at 217,000 g using a TLA100 rotor. All steps were performed at room temperature. Equal proportions of supernatants and pellets were loaded on 12% SDS-polyacrylamide gels, the gels were stained with Coomassie blue stain and scanned with a FluorSTM-imager (Bio-Rad Laboratories). The intensities of actin and twinfilin bands were quantified with the QuantityOne program (Bio-Rad Laboratories).
Actin filament assembly assays
Kinetics of actin filament assembly and disassembly were monitored by pyrene fluorescence with excitation at 365 nm and emission at 407 nm using BioLogic MOS-250 fluorescence spectrophotometer. For assembly assays, 64 µl of G buffer (5 mM Tris, pH 7.5, 0.2 mM ATP, 0.2 mM DTT, 0.2 mM CaCl2) containing 3.75 µM actin (1:6 pyrene actin:human platelet actin) was mixed with 8 µl of G-buffer or 15/30 µM twinfilin. Polymerization was induced by addition of 8 µl of 10x initiation mix. For disassembly assays, 3.3 µM actin (1:6 pyrene actin:human platelet actin) in G buffer was polymerized by the addition of 1/10 vol of 10x initiation mix for 1 h. Depolymerization of filaments was induced by mixing 72 µl of filaments with 8 µl of G buffer or 15/30 µM twinfilin.
Western blotting
Wild-type and mutant larvae were washed with PBS containing a dissolved protease inhibitor cocktail tablet (Roche). Larvae were homogenized with a Dounce homogenizer and the resulting cells were lysed with 1% Triton X-100. The proteins in the cell lysates were separated on a 12% SDS-polyacrylamide gel, electroblotted onto a nitrocellulose membrane, and immunoblotted with a 1:2,000 dilution of anti-twinfilin antiserum.
SEM
Whole adult flies were anaesthetized with CO2, and then dehydrated by 24-h incubations in a graded ethanol series. The dehydrated flies were critical point dried, mounted on SEM stubs, sputter coated with platinum, and examined with SEM.
Fly strains and genetics
The strains Df(3R)SuHw7/TM6Tb and tsrk05633/CyO were obtained from the Bloomington Stock Center (Bloomington, IN). The EP(3)3701 strain was identified by the Berkeley Drosophila Genome Project (Berkeley, CA) and obtained from the Szeged Stock Centre (Szeged, Hungary). The putative semilethal element in the original EP(3)3701 strain (Results) was outcrossed against a w strain. The outcrossed homozygous stock has the mini-w eye color marker and bristle phenotype described in the Results section. Genetic interactions between twinfilin and twinstar were examined in progeny from crosses between tsrk05633/CyO males and twf3701 homozygous females. Bristles of the progeny with normal wings were examined by light microscopy and SEM along with tsrk05633/+ and twf3701/+ flies as a control. Canton-S, or w strains were used as wild-type controls in all experiments. The flies were maintained on standard food at 25°C.
Phalloidin staining of bristles
White prepupae were collected and dissected in PBS 3248 h after pupariation. We dissected the head and abdomen from the thorax, and removed the internal organs. The epithelial tissue was flattened after making a ventral incision, then transferred into an Eppendorf tube containing 4% paraformaldehyde in PBS and placed on ice. The specimens were further processed as described in Tilney et al. (1996). Filamentous actin was stained with Texas redconjugated phalloidin at a concentration of 2 U/ml. The samples were mounted in Vectashield (Vector Laboratories), and examined with a Biorad MRC 1024 confocal microscope. Optical sections were combined using the Confocal Assistant 4.02 program.
Immunostainings
Wild-type and twf3701 embryos were collected, fixed, and stained according to standard protocols. The anti-twinfilin antiserum and preimmune serum were diluted to 1:20,000, and all stainings were performed under same conditions. For antibody stainings of bristles, the material was fixed and washed as above, then blocked for 1 h in 1% BSA, 0.1% Triton X-100 in PBS. The anti-twinfilin antiserum was used at a 1:10,000 dilution, and FITC-conjugated secondary antibody at a 1:1,000 dilution. Ovaries were dissected, fixed, and stained with twinfilin antiserum as above, except that the blocking was in 0.5% BSA, 0.1% Triton X-100 in PBS. Ovaries were mounted in Vectashield containing 0.5 µg/ml Hoechst 33258.
Miscellaneous
Protein concentrations were determined with a Hewlett-Packard 8452A diode array spectrophotometer using calculated extinction coefficients for Drosophila twinfilin at 280 nm ( = 34 990 M-1cm-1) and for actin at 290 nm (
= 26 600 M-1cm-1). Total RNA was isolated using the TRIZOL reagent (GIBCO BRL; Life Technologies) according to the manufacturers' instructions and blotted and hybridized according to standard protocols.
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Footnotes |
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Acknowledgments |
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This study was supported by grants from Victoria Foundation (to G. Wahlström), Academy of Finland (to P. Lappalainen), and Biocentrum Helsinki (to P. Lappalainen and T.I. Heino).
Submitted: 6 August 2001
Revised: 9 October 2001
Accepted: 12 October 2001
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References |
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