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Genetics. 2005 Dec; 171(4): 1757–1765.
PMCID: PMC1456101

Mutations in the Drosophila Orthologs of the F-Actin Capping Protein α- and β-Subunits Cause Actin Accumulation and Subsequent Retinal Degeneration


The progression of several human neurodegenerative diseases is characterized by the appearance of intracellular inclusions or cytoskeletal abnormalities. An important question is whether these abnormalities actually contribute to the degenerative process or whether they are merely manifestations of cells that are already destined for degeneration. We have conducted a large screen in Drosophila for mutations that alter the growth or differentiation of cells during eye development. We have used mitotic recombination to generate patches of homozygous mutant cells. In our entire screen, mutations in only two different loci, burned (bnd) and scorched (scrd), resulted in eyes in which the mutant patches appeared black and the mutant tissue appeared to have undergone degeneration. In larval imaginal discs, growth and cell fate specification occur normally in mutant cells, but there is an accumulation of F-actin. Mutant cells degenerate much later during the pupal phase of development. burned mutations are allelic to mutations in the previously described cpb locus that encodes the β-subunit of the F-actin capping protein, while scorched mutations disrupt the gene encoding its α-subunit (cpa). The α/β-heterodimer caps the barbed ends of an actin filament and restricts its growth. In its absence, cells progressively accumulate actin filaments and eventually die. A possible role for their human orthologs in neurodegenerative disease merits further investigation.

THE cytoskeleton extends throughout the cytoplasm of a cell and comprises a network of filaments including microtubules (containing tubulin), microfilaments (containing actin), and intermediate filaments (containing a variety of proteins such as keratin, vimentin, and neurofilaments, depending on cell type). It contributes to the structural integrity of the cell, and its proper function is necessary for cell motility and for vesicular trafficking within the cell. Cytoskeletal abnormalities are hallmarks of many neurodegenerative diseases, such as Alzheimer's disease (AD) (Goldman and Yen 1986; Jellinger 2001; McMurray 2000; Vickers et al. 2000), Huntington's disease (HD) (Goldman and Yen 1986; Huntington's Disease Collaborative Research Group 1993; McMurray 2000; Jellinger 2001), Parkinson's disease (PD) (Goldman and Yen 1986; Polymeropoulos et al. 1997; McMurray 2000; Jellinger 2001), and amyotrophic lateral sclerosis (ALS) (Goldman and Yen 1986; McMurray 2000; Jellinger 2001; Bruijn et al. 2004; Lariviere and Julien 2004). The neurofibrillary tangles observed in AD and PD consist of an accumulation or aggregation of the microtubule-associated protein, tau, as either a mutant or a hyperphosphorylated form (Goldman and Yen 1986; McMurray 2000; Garcia and Cleveland 2001; Geschwind 2003). Neurofilaments accumulate in PD and ALS (Goldman and Yen 1986; Bruijn et al. 2004; Lariviere and Julien 2004). Hirano bodies, which are observed in AD and ALS, contain actin and actin-binding proteins (Goldman and Yen 1986; Fechheimer et al. 2002). Furthermore, many of the proteins encoded by genes implicated in neurodegenerative diseases interact with cytoskeletal proteins. For instance, presenilin1 (PS1), associated with familial AD, may link to the cytoskeleton via its interaction with δ-catenin (Tanahashi and Tabira 1999; McMurray 2000). Huntingtin, the product of the gene mutated in HD, associates with microtubules and has been shown to interact with the cytoskeletal regulators/interactors HIP1 (Wanker et al. 1997), Duo (Colomer et al. 1997), and Sla1 (Bailleul et al. 1999; McMurray 2000). For most neurodegenerative diseases, the precise mechanism of cell death is not well understood. Although it is well accepted that cytoskeletal abnormalities frequently accompany neurodegeneration, it is not known whether these abnormalities actually promote tissue degeneration and cell death or whether they are simply characteristics of cells dying for other unrelated reasons.

Studies in Drosophila have been used to improve our understanding of human neurodegenerative diseases. In humans and in Drosophila, neurodegeneration is generally characterized by (1) a relatively late developmental onset, (2) progressive deterioration of adult nervous system structures, and (3) high levels of neuronal apoptosis. Two main approaches for studying neurodegeneration in Drosophila have been taken. One has been to overexpress a protein implicated in a human neurodegenerative disease in Drosophila so as to mimic the degenerative changes observed in the human disease. This approach has been used successfully to model diseases caused by the expansion of trinucleotide repeats (Jackson et al. 1998; Warrick et al. 1998; Marsh et al. 2000; Bonini 2001; O'Kane 2003; Shulman et al. 2003; Kim et al. 2004; Kretzschmar et al. 2005) and to induce some features of PD by overexpression of α-synuclein (Feany and Bender 2000). The other approach has been to screen for mutations in Drosophila that result in neurodegeneration.

One way of identifying mutations that elicit neurodegeneration in Drosophila has been to screen for mutations that result in reduced life span and then to examine their brains for degenerative changes. Such an approach led to the identification of spongecake, in which aging brains exhibit degenerative changes similar to those observed in the spongiform degeneration of Creutzfeldt-Jakob disease, and eggroll, whose multilamellated structures in the brain are reminiscent of the histological changes found in lipid storage diseases such as Tay-Sachs disease (Min and Benzer 1997; Min 2001). Another has been to identify mutations that result in retinal degeneration by screening for flies with impaired function of the visual system. Some mutations that selectively cause the degeneration of the retina are those that impair the ability of retinal photoreceptors to obtain trophic signals from their targets in the brain. Others such as rdgA and rdgB perturb phototransduction and elicit a degenerative phenotype that can be suppressed by rearing the flies in the absence of light (Hotta and Benzer 1970; Harris and Stark 1977; Vihtelic et al. 1991; Masai et al. 1993). Unfortunately, the ability to identify “neurodegeneration genes” by this approach is restricted to genes that do not have a function in a tissue that is essential for the development of a fly to the adult stage or for its viability as an adult.

One way to overcome such a limitation is to restrict the deleterious effect of the mutation to a nonessential tissue such as the eye. In such a screen, heterozygous animals are generated following random mutagenesis. Using an FLP recombinase expressed from an eye-specific promoter, marked patches of homozygous mutant tissue are generated in the eye (Newsome et al. 2000). After screening the four major autosomal arms of Drosophila using such an approach, we identified mutations in two genes, burned (bnd) and scorched (scrd), that result in degeneration of mutant tissue. These genes encode the Drosophila orthologs of the F-actin capping protein β- and α-subunits. Mutations in either gene result in an abnormal accumulation of actin that eventually results in cell death and tissue degeneration. Thus, we demonstrate that abnormal regulation of the actin cytoskeleton can itself elicit a degenerative phenotype.


Fly stocks:

w; FRT40A or w; FRT42D males were mutagenized with ethyl methanesulfonate and then crossed to y w eyFLP; FRT40A P[mini-w, armLacZ] or y w eyFLP; FRT42D P[mini-w, armLacZ], respectively, or both first to w; CyO/Sco and then individually to y w eyFLP; FRT40A P[mini-w, armLacZ] or y w eyFLP; FRT42D P[mini-w, armLacZ]. Flies with eyes that contained substantial black tissue were retained and maintained as balanced stocks. Three alleles of bnd and two alleles of scrd were identified. Other stocks used in phenotypic analysis include y w eyFLP; FRT40A P[mini-w, UbiGFP], y w eyFLP; FRT42D P[mini-w, UbiGFP], y w hsFLP; FRT40A P[mini-w, armLacZ], y w hsFLP; FRT42D P[mini-w, armLacZ], y w; P[W+]tsrKo5633/CyO, and GMR-p35.

Characterization of the bnd and scrd loci:

Alleles of scrd complemented all existing deficiencies in the 2R deficiency kit. Mapping proceeded utilizing stocks already analyzed for PLP and SNP differences from FRT chromosomes by Berger et al. (2001). SNP analysis of recombinants between scrd1 and the EP0755 chromosome narrowed the interval to 423 kb between 57B3 and 57D1 comprising ∼80 genes. Complementation analysis using lethal P insertions in this interval revealed a failure to complement a lethal P insertion in CG10540, CG10540KG0226. Stocks used were y w eyFLP; FRT42D and y w eyFLP; EP0755. Alleles of bnd failed to complement Df(2L)S2 and Df(2L)ast2 but complemented Df(2L)S3 and Df(2L)al, placing bnd between 22A1 to 22B1, an 866-kb interval. SNP analysis of bnd recombinants further mapped the interval to a 186-kb region containing ∼13 genes, including capping protein beta (cpb). All three bnd alleles failed to complement an existing cpb allele, cpbM143 (Hopmann et al. 1996). P [w+] stocks used to generate recombinants were EP(2)0431 and dbek05428.

Excision of the P element in CG10540KG02261:

w; CG10540KG02261/CyO virgin females were crossed to y w; H{w[+mC]=PDelta2-3}HoP2.1; Dr/TM3 males, and w; CG10540KG02261/CyO males were crossed to y w; H{w[+mC]=PDelta2-3}HoP2.1; Dr/TM3 virgin females. From these crosses, males flies with the genotype CG10540KG02261/H{w[+mC]=PDelta2-3}HoP2.1 (regardless of first and third chromosome markers) were crossed to y w eyFLP; P[mini-w, armLacZ]/CyO virgin females; similarly, w; CG10540KG02261/H{w[+mC]=PDelta2-3}HoP2.1 (regardless of third chromosome markers) virgin females were crossed to y w eyFLP; P[mini-w, armLacZ]/CyO males. From this set of crosses, male flies with mosaic white and red eyes were balanced and retested for the mosaic eye phenotype. In addition, all white-eyed, CyO male flies were crossed to y w eyFLP; FRT42D P[mini-w, armLacZ]/CyO to both balance and test for mosaic eye phenotype. Those lines producing progeny with mosaic eyes must have retained the FRT. Several excision lines that all gave mosaic eyes with approximately equal red and white ratios and normal eye morphology (no degeneration) were obtained. Two of these lines were sequenced using primers 1 kb away from the P-insertion site in CG10540 on either side. In each case, the sequence did not differ from wild-type sequence in the same region, indicating precise excision of the P element, although we cannot rule out an imprecise excision that deleted a region large enough to have deleted one of our primers. However, these lines are fully viable, and such a large deletion would likely yield a lethal line.

Microscopy and immunohistochemistry:

For adult eye pictures and sections, genotypes were y w eyFLP; FRT40Abnd1/FRT40A P[mini-w, armLacZ], y w eyFLP; FRT42Dscrd2/FRT42D P[mini-w, armLacZ], y w eyFLP; FRT40A/FRT40A P[mini-w, armLacZ], y w eyFLP; FRT42D/FRT42D P[mini-w, armLacZ], y w hsFLP; FRT40Abnd1/FRT40A P[mini-w, armLacZ], and y w eyFLP; FRT42D tsrKo56332/FRT42D P[mini-w, armLacZ].

For immunofluorescence, larval discs were dissected from the genotypes y w eyFLP; FRT40Abnd1/FRT40A P[mini-w, armLacZ], y w eyFLP; FRT42Dscrd2/FRT42D P[mini-w, armLacZ], y w eyFLP; FRT40Abnd1/FRT40A P[mini-w, UbiGFP], y w eyFLP; FRT42Dscrd2/FRT42D P[mini-w, UbiGFP], and y w eyFLP; FRT42D tsrKo56332/FRT42D P[mini-w, UbiGFP]. Upon fixation, larval eye discs were incubated overnight at 4° in primary antibodies as follows: elav (1:10), TRITC-phalloidin (1:500; no secondary antibody needed), chaoptin (1:40), C3 (1:100), α-spectrin, α-tubulin, Rho1, β-catenin and profilin (all 1:50), cofilin and tau (1:500). Upon washes in PBS, eye discs were incubated for 2 hr at room temperature in secondary antibody (1:50), washed in PBS, and mounted in glycerol-galactosamine solution. Tau antibody was provided by Nick Lowe (The Wellcome Trust/Cancer Research UK Institute of Cancer and Developmental Biology). C3 antibody was from Calbiochem, and cofilin antibody was from Cytoskeleton. All other antibodies were from the Developmental Studies Hybridoma Bank. Imaginal disc clones were analyzed using the lasso tool with the histogram function in Adobe Photoshop to derive the area of the clone in pixels as well as the mean pixel intensity.

Adult fly eyes were fixed and embedded in soft resin essentially as described previously (Tomlinson and Ready 1987). One-micrometer sections were cut using a glass knife, stained with toluidine blue, and visualized by light microscopy. Representative regions were processed for transmission electron microscopy (TEM) by cutting thin sections (0.1 μm) with an LKB ultramicrotome and diamond knife. Thin sections were placed on the grids for electron microscopy and stained with lead citrate. Grids were examined in a Philips 301 transmission electron microscope, and images were captured with an Advanced Microscopy Techniques charge-coupled device camera. For scanning electron microscopy (SEM), ethanol-dehydrated adult eyes were prepared for analysis at the SEM core facility at Northeastern University (Boston).


Identification of bnd and scrd:

We have screened the four main autosomal arms of Drosophila, which together compose approximately 80% of the genome, for mutations that alter the growth properties of clones of mutant tissue during eye development. Using an FLP recombinase expressed from the eyeless promoter, we compared the sizes of mutant clones with the wild-type sister clones generated by the same recombination events. We screened at least 50,000 mutagenized chromosomes for each of the four main autosomal chromosome arms (2L, 2R, 3L, and 3R). At relatively high frequency, we recovered mutations that led to either the complete absence or underrepresentation of mutant tissue. At much lower frequency, we identified mutations that enabled mutant cells to outgrow their wild-type neighbors. Mutations we isolated that result in increased growth map to >25 distinct loci, including mutations in genes that we have previously described elsewhere, such as archipelago (Moberg et al. 2001), Tsc1 (Tapon et al. 2001), salvador (Tapon et al. 2002), and hippo (Harvey et al. 2003). In addition to these, we identified mutations in two different loci that elicit a distinct phenotype where much of the mutant tissue appears black, even in recently eclosed flies. We named these two loci burned (bnd) and scorched (scrd).

We identified three alleles of bnd (referred to as bnd1, bnd2, and bnd3) from our screen of the left arm of chromosome 2 (2L). All three alleles are homozygous lethal and lethal in trans to each other. From our screen of the right arm of chromosome 2 (2R), we identified two alleles of scrd (referred to as scrd1 and scrd2). Both alleles of scrd are homozygous lethal and lethal in trans to each other. Flies that are heterozygous for both bnd and scrd (bnd +/+ scrd) are viable and show no obvious phenotypic abnormalities.

Eyes containing either bnd or scrd mutant clones (Figure 1, B and D, respectively) show a similar ratio of mutant (marked white) to wild-type tissue (marked red) when compared to eyes containing clones of their parent chromosomes (Figure 1, A and C, respectively). However, in bnd or scrd clones, much of the mutant tissue appears black and appears to lack recognizable ommatidial facets. This phenotype is fully penetrant and is observed in 100% of mosaic flies (n > 200 for each genotype). However, some regions of mutant tissue, especially those that are adjacent to the borders of the clone, remain white and, at least superficially, appear to retain recognizable ommatidial facets. Scanning electron micrographs of eyes containing bnd clones (Figure 1E, close-up in F and G) show regions that completely lack any semblance of ommatidial architecture as well as regions that have facets of abnormal appearance. Sections through the adult retina of flies with bnd clones (Figure 1H) show no recognizable rhabdomeres or other evidence of ommatidial organization in mutant clones at the level of light microscopy, indicating that the retinal epithelium has either failed to differentiate or undergone degeneration. To examine the structure of mutant tissue at higher resolution, we used TEM (Figure 1I, close-up in J). In the mutant clone, numerous electron-dense bodies were observed that are likely to be the remnants of degenerating rhabdomeres.

Figure 1.
Degenerative phenotype of bnd and scrd mutants. In mosaic eyes, homozygous mutant tissue usually appears white, as seen in eyes containing clones of the parent chromosome for FRT40A (A) and FRT42D (C). However, in eyes containing bnd (B) or scrd (D) mutant ...

At the boundaries of the clone, the genotype of individual photoreceptor cells can be deduced by the presence or absence of pigment granules in the stalk of the rhabdomere. In 50 ommatidia lacking the full complement of photoreceptor cells, all morphologically normal photoreceptors were scored as bnd+, indicating a cell-autonomous requirement for bnd to maintain the integrity of photoreceptor cells. However, we cannot exclude the possibility that some bnd+ photoreceptor cells have also degenerated. Taken together, these findings suggest that bnd function is unnecessary for cells to proliferate and generate clones of normal size but that bnd function is required at a later stage of eye development to prevent photoreceptor degeneration.

To test whether the degeneration was light dependent, we reared flies in complete darkness. Mutant clones still displayed the same range of phenotypic abnormalities. We also examined the retinas of bnd/+ heterozygotes up to 14 days after eclosion by TEM and found no evidence of degeneration (Figure 1, K and L).

To determine whether bnd function is required in tissues other than the eye, we generated clones in other parts of the fly by expressing FLP under the control of the heat-shock promoter. Under these conditions, in addition to abnormalities in the eye, we also observed patches of blackened tissue in the wing (Figure 1M), fissures in the abdomen, and abnormalities in the patterning of bristles (Figure 1, N and P) indicating that bnd function is required in many tissues. While eye and bristle abnormalities were observed in all flies examined (n > 100), the blackened spots in adult wings were observed at a much lower frequency (∼5%).

bnd and scrd are required for regulation of the actin cytoskeleton:

To examine bnd and scrd mutant tissue at earlier stages of development, we dissected and examined eye-imaginal discs from third instar larvae. Staining with anti-ELAV, which stains nuclei of cells recruited to a neuronal fate, showed that the recruitment of cells to developing ommatidial clusters occurs normally and without delay in bnd clones (Figure 2, A–C) and scrd clones (not shown). Similarly, chaoptin, an early marker of photoreceptor differentiation, is expressed normally in bnd tissue (not shown) and scrd tissue (Figure 2, D–F). Thus, the early stages of neuronal cell fate specification and photoreceptor differentiation occur normally in bnd and scrd mutant cells.

Figure 2.
Mutation in bnd causes actin accumulation but does not interfere with neuronal recruitment and photoreceptor differentiation. (A–L) Third instar larval eye discs mosaic for bnd1. Anterior is to the left. (A–C) Wild-type tissue is visualized ...

In contrast, visualization of the actin cytoskeleton using phalloidin reveals obvious abnormalities in bnd (Figure 2, G–I, close-up in J–L) and scrd (Figure 2, M–O, close-up in P–R) tissue even in the imaginal disc of the third instar larva. Mutant clones show much higher levels of staining, particularly in the region of the cell cortex and the apical tufts of the photoreceptor cells. We also examined the expression level of a variety of other cytoskeletal components. In contrast to the obvious accumulation of F-actin, the expression patterns of α-spectrin, α-tubulin, tau, Rho1, profilin, cofilin, and β-catenin were indistinguishable from that of wild-type tissue (not shown). We also observed increased staining with phalloidin in mutant clones in the optic lobes.

bnd and scrd encode subunits of an F-actin capping protein:

Recombination mapping localized the scrd gene to an interval of 423 kb between 57B3 and 57D1 consisting of ∼80 annotated genes. Stocks containing lethal P-element insertions were available for 7 of these genes. A stock containing a lethal P insertion in CG10540, CG10540KG02261, failed to complement both alleles of scrd. CG10540 is the Drosophila ortholog of the F-actin capping protein α-subunit (cpa). We sequenced the CG10540 ORF from both mutant chromosomes and found a point mutation in each case. In scrd1, there is a change of G to A eight bases 5′ to the annotated initiation ATG codon that potentially creates an alternate translational start site. This may cause inappropriate initiation that is out of frame with the main ORF and also may reduce the frequency of translation at the appropriate ATG. In scrd2, there is a T-to-A substitution that results in a change from valine (183) to an aspartic acid (Figure 3A). When we recombined the allele with the P-element insertion CG10540KG02261 onto the FRT42D chromosome and generated clones, the phenotype was similar to that of scrd; in the adult eye, mutant clones contained patches of black tissue (not shown). The P element is inserted in the 5′-untranslated region at base 37 of the annotated transcript (Spradling et al. 1995, 1999; Levis et al. 2001; Celniker et al. 2002) and likely reduces the level of mRNA and protein. The coding region of CG10540 begins at base 232 of the transcript. We performed complementation crosses to an allele of the only other nearby gene, shotgun (shg). CG10540KG02261 complements the X-ray allele shgG119. In addition, both scrd1and scrd2 also complement shgG119. Moreover, precise excision of the P element in CG10540KG02261 reverts the lethality of the stock as well as the degenerative phenotype in the eyes of mosaic flies.

Figure 3.
Molecular characterization of the cpa and cpb loci. (A) Schematic of the cpa gene product indicating the mutations in the two alleles, cpascrd1 and cpascrd2. The appropriate initiation ATG is indicated in the sequence by a light shaded background. The ...

The bnd locus on 2L was mapped by deficiencies to 22A1–22B1, an 866-kb interval, and by finer recombination mapping to a 186-kb region containing ∼13 genes. One of the genes in this interval is the ortholog of the β-subunit of the F-actin capping protein, cpb (Hopmann et al. 1996). Capping of F-actin utilizes an α/β-heterodimer. In mammals, the F-actin capping protein α-subunit (CAPZA, Cappa) is represented by three separate genes and the β-subunit (CAPZB, cappab) by one gene with three separate isoforms (Schafer et al. 1994; Hart et al. 1997a,b). The Drosophila genome encodes only one gene for α, CG10540, and one gene for β, known as capping protein beta (cpb). All three bnd alleles failed to complement an existing cpb allele, cpbM143 (Hopmann et al. 1996). Sequencing of bnd1 revealed a C-to-T substitution causing a premature stop codon at position five of the coding sequence. In bnd2, a G-to-A substitution causes a glutamic acid to lysine change at position 218, and in bnd3, a G-to-A substitution changes a glutamic acid to a lysine at position 221 (Figure 3B). We will henceforth refer to scrd and bnd as cpa and cpb, respectively, and to our alleles as cpascrd1, cpascrd2, cpbbnd1, cpbbnd2, and cpbbnd3.

Under in vitro conditions, the α/β capping protein heterodimer binds and “caps” the barbed end of actin filaments in a reversible and calcium-independent manner (Cooper and Pollard 1985; Caldwell et al. 1989; Yamashita et al. 2003). Capping of actin filaments prevents further chain elongation by preventing addition of actin monomers to the filament. Therefore, a loss of capping activity would be predicted to allow actin filaments to keep growing, resulting in accumulation of actin filaments. In addition, the stability of each subunit seems to depend upon its association with the other, and capping activity depends on a functional heterodimer (Casella and Torres 1994; Wear et al. 2003), suggesting that loss of one subunit or mutations preventing the association of the two would effectively cause loss of both subunits or, at least, a loss of capping activity. Thus, mutations in either cpa or cpb would be expected to result in the loss of F-actin capping activity at the barbed end of actin filaments. This is consistent with the accumulation of F-actin observed in mutant clones in vivo as well as the similarity of the cpa and cpb mutant phenotypes. Indeed, prior reports characterizing cpb mutations have shown bristle abnormalities and aberrant actin organization in cpb6.15/cpbF19 transheterozygotes (Hopmann et al. 1996) and actin accumulation in cpb4.18 mitotic clones in pupal epidermal cells (Hopmann and Miller 2003).

A mutation in cofilin elicits a more severe degenerative phenotype:

The Drosophila genome encodes a number of genes that regulate the actin cytoskeleton. Despite this, in our fairly extensive screen we identified mutations in only two loci, cpa and cpb, that elicited a degenerative phenotype manifest as large black patches in the adult eye. One possibility is that mutations in some other regulators of actin polymerization may be more deleterious to cell viability or proliferation. Thus, homozygous mutant cells may die soon after they are generated by mitotic recombination, and the mutant tissue may never be visible as large clones. Indeed, in our screen we have commonly seen eyes that appear rough and red, with occasional black spots, indicating that almost all mutant tissue has died early, but the black spots likely represent small mutant clones that died later. cpa and cpb mutations appear to allow cells to proliferate normally yet result in degeneration and cell death at a late stage of cell development.

Another protein that negatively regulates actin polymerization is cofilin. Cofilin enhances removal of ADP-bound actin monomers from the pointed end of an actin filament (Bamburg 1999; Maciver and Hussey 2002). The Drosophila ortholog of cofilin is called twinstar (tsr), and mutant alleles of tsr have been isolated. To determine if tsr mutations cause phenotypic abnormalities similar to those observed in cpa and cpb, we generated clones of tsr cells in the eye. Since we used an allele of tsr that is caused by a P[w+] insertion in the tsr gene (tsrk05633), homozygous mutant clones appear red and their wild-type twin-spots appear white. Such an eye is composed almost entirely of wild-type tissue, with only small clones of tsr remaining (Figure 4A), which sometimes contain black dots, indicating death.

Figure 4.
Eye mosaics for tsr show an underrepresentation of mutant tissue overall and actin accumulation in mutant clones. (A) Adult eye mosaic for tsr, the Drosophila cofilin ortholog. In this case, tsr clones are red and wild-type tissue is white. tsr clones ...

We examined these small mutant clones by electron microscopy and found a loss of ommatidial architecture and misshapen rhabdomeres (Figure 4B). In third instar larval discs, mutant cells stain brightly with phalloidin (Figure 4, D and E, close-up in G and H), more so than that seen in cpa and cpb clones. When the intensity of the staining was quantified using the histogram function of Adobe Photoshop, the mean brightness of tsr tissue compared to wild-type tissue in the same disc was 4.5:1 (n = 3 discs). For cpb tissue, the ratio was 2.6:1 (n = 3 discs). In adult eyes and in imaginal discs, tsr tissue is severely underrepresented. In third instar discs, tsr tissue accounted for only 3.8 ± 1.2% (n = 3 discs) of the overall disc area as compared to cpb discs in which the mutant tissue accounted for 49.6 ± 10.1% of the area (n = 3 discs). Thus, unlike cpa and cpb, tsr mutations either severely impede cell proliferation or cause cell death at an early stage of eye development. To examine these possibilities, we stained third instar discs with an antibody directed against an activated form of mammalian caspase-3 (C3) that also recognizes activated effector caspases in Drosophila. Even though the tsr clones account for a small proportion of the area of the disc, they account for the majority of cells expressing activated C3 (Figure 4, I and K). When the number of cells expressing activated C3 per unit area is calculated, the density of staining cells in mutant tissue is 27.7 times that of the wild-type tissue from the same disc. In contrast, although cpa and cpb clones in the third instar larval disc have started to accumulate F-actin, C3 staining is not detected appreciably above background levels (not shown). Consistent with the lack of increased C3 staining, overexpression of the caspase inhibitor p35 from baculovirus under the control of the GMR promoter does not prevent the appearance of large patches of black tissue in a cpb mosaic eye (not shown). The observation of significant cell death in small tsr clones explains why large black patches of degenerating tissue are not observed in adult eyes.


We have shown that mutations in the Drosophila orthologs of the α- and β-subunits of the F-actin capping protein result in tissue degeneration. Mutation of either gene does not appear to interfere with tissue growth or cell fate determination. Rather, mutant tissue undergoes degenerative changes at a later stage of development. The earliest abnormality that we observed in mutant clones is an accumulation of actin, consistent with the known function of the α/β-heterodimer in capping actin filaments and arresting their growth. Indeed, others have previously described disorganized actin filaments in bristles of cpb mutants (Hopmann et al. 1996). Also, reducing the level of either the α- or β-subunit by RNAi in Drosophila cell lines induced cytoskeletal abnormalities (Kiger et al. 2003). Our experiments show that the cytoskeletal abnormalities observed in mutant cells are not sufficiently deleterious to result in their immediate death or even to interfere with their proliferation or with cell fate determination in vivo. Rather, these cytoskeletal perturbations manifest as tissue degeneration at a later stage of development. Intriguingly, in our entire screen of the four main autosomal arms, mutations in only two genes, cpa and cpb, gave rise to a phenotype characterized by large clones of degenerating tissue.

In addition to the actin-capping proteins, a number of other proteins also regulate the actin cytoskeleton in vivo. They do so by a variety of mechanisms including the regulation of bundling, cross-linking, severing, polymerizing-depolymerizing, and by sequestering actin monomers. We therefore also examined the phenotype of mutations in tsr, a Drosophila cofilin/actin depolymerizing factor homolog that also inhibits actin polymerization. Mutations in tsr have previously been shown to result in defects in centrosome migration and cytokinesis accompanied by abnormal accumulations of F-actin (Gunsalus et al. 1995). In addition to the strong accumulation of F-actin in tsr clones, we also observed high levels of apoptosis. In contrast, we did not see increased apoptosis in cpa or cpb clones in the developing eye discs. A likely explanation is that the disruption of the cytoskeleton in tsr clones is severe enough to cause cell death almost immediately, whereas the less severe abnormalities in cpa or cpb clones are not. An alternate possibility is that the mechanism of cell death that occurs in tsr clones differs from that which is activated in cpa and cpb clones. In either case, the delayed cell death observed in cpa and cpb mutant clones is more similar to the type of death that occurs in degenerative diseases.

How do the cytoskeletal abnormalities found in cpa and cpb mutations eventually result in cell death? The accumulation of F-actin could cause the mislocalization or altered regulation of a number of actin-binding activities within the cell. In addition, the uncontrolled and undirected accumulation of actin could interfere with directed cell movement, cell polarity, or cell protrusions, thereby disrupting crucial signaling events. Alternatively, physical stresses due to uncontrolled actin polymerization may simply cause the cells to break apart and undergo lysis. Indeed, cells may be particularly susceptible to such stresses when they undergo significant shape changes during the later stages of pupal development.

Our findings raise the possibility that mutations in genes encoding actin-capping proteins could cause degenerative diseases in humans. Indeed, mutations that result in alterations in the actin cytoskeleton have been implicated in two types of progressive hearing loss. The autosomal dominant deafness DFNA1 syndrome results from mutations in the human ortholog of Drosophila diaphanous, which is a member of the formin gene family and is involved in regulating actin polymerization (Lynch et al. 1997). The DFNA 20/26 syndrome results from mutations in the gamma actin 1 (ACTG1) gene (Zhu et al. 2003). Recently, weakening of the nuclear actin network have been suggested to underlie X-linked Emery-Dreifuss muscular dystrophy characterized by loss of emerin, an LEM-domain protein of the nuclear inner membrane (Holaska et al. 2004). Furthermore, the aggregation of actin and cofilin has been reported in the brains of identical twins with DYT1-negative dystonia (Gearing et al. 2002). There is a single ortholog of cpb and three cpa orthologs in the human genome (Schafer et al. 1994; Hart et al. 1997a,b). Their role in either causing or modifying degenerative disease phenotypes may warrant further investigation.


We thank S. Schelble and L. Madden for technical assistance. We also thank K. Harvey, K. Moberg, J. Walker, B. Pellock, and K. Tseng for advice and helpful conversations. We thank W. Fowle (Northeastern University) for generating the SEM images, Martin Selig (Department of Pathology, Massachusetts General Hospital) for technical assistance and advice in generating TEM images, and Igor Bagayev of the confocal microscope facility (Neuroscience Center, Massachusetts General Hospital). We thank the Bloomington Stock Center and the Developmental Studies Hybridoma bank for fly stocks, antibodies, and reagents. I.D. was funded by the National Institutes of Health (KO8 EY-13639A). C.M.P. was a fellow of the Jane Coffin Childs Memorial Fund for Medical Research during much of this work. I.K.H. was funded in part by the National Institutes of Health (GM-61672 and CA-95281).


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