Genetics: Published Articles Ahead of Print, published on September 2, 2005 as 10.1534/genetics.105.049213

Title: Mutations in the Drosophila orthologs of the F- capping alpha and

beta subunits cause actin accumulation and subsequent retinal degeneration

Authors: Ivana Delalle*,1,2, Cathie M. Pfleger*, 1,3, Eugene M. Buff1, Paula Lueras1, and

Iswar K. Hariharan1,3.

* These authors contributed equally to this work and are listed in alphabetical order.

1. Massachusetts General Hospital Cancer Center Massachusetts General Hospital Charlestown, MA 02129

2. Department of Pathology Boston University School of Medicine Boston, MA! 02118

3. Department of Molecular and Cell Biology University of California Berkeley, CA 94720

1 Running Head: Loss of F-actin capping activity results in degeneration.

Key Words or phrases: actin capping, neural degeneration, actin accumulation

Corresponding Author: Iswar K. Hariharan 361A Life Sciences Addition University of California Berkeley CA 94720 Phone: 510-643-7438 FAX: 510-643-7448 E-mail: [email protected]

2 ABSTRACT

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 a eyes where the mutant patches appeared black and where the mutant tissue appears 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 cpb locus that encodes the beta subunit of the F-actin capping protein while scorched mutations disrupt the encoding its alpha subunit (cpa). The alpha/beta 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.

3 INTRODUCTION

The extends throughout the cytoplasm of a cell and comprises a network of filaments including (containing ),

(containing actin), and intermediate filaments (containing a variety of such as , , and , 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; THE HUNTINGTON'S DISEASE COLLABORATIVE

RESEARCH GROUP 1993; JELLINGER 2001; MCMURRAY 2000), Parkinson’s Disease (PD)

(GOLDMAN and YEN 1986; JELLINGER 2001; MCMURRAY 2000; POLYMEROPOULOS et al.

1997), and Amyotrophic Lateral Sclerosis (ALS) (BRUIJN et al. 2004; GOLDMAN and

YEN 1986; JELLINGER 2001; LARIVIERE and JULIEN 2004; MCMURRAY 2000). The neurofibrillary tangles observed in AD and PD consist of an accumulation or aggregation of the -associated protein, tau, either as a mutant or hyper-phosphorylated form (GARCIA and CLEVELAND 2001; GESCHWIND 2003; GOLDMAN and YEN 1986;

MCMURRAY 2000). Neurofilaments accumulate in PD and ALS (BRUIJN et al. 2004;

GOLDMAN and YEN 1986; LARIVIERE and JULIEN 2004). Hirano bodies which are observed in AD and ALS contain actin and actin-binding proteins (FECHHEIMER et al.

2002; GOLDMAN and YEN 1986). Furthermore, many of the proteins encoded by implicated in neurodegenerative diseases interact with cytoskeletal proteins. For instance, Presenilin1 (PS1), associated with familial AD, may link to the cytoskeleton via

4 its interaction with delta- (MCMURRAY 2000; TANAHASHI and TABIRA 1999).

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 have been taken to study neurodegeneration in Drosophila. 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

(BONINI 2001; JACKSON et al. 1998; KIM et al. 2004; KRETZSCHMAR et al. 2005; MARSH et al. 2000; O'KANE 2003; SHULMAN et al. 2003; WARRICK et al. 1998) and to induce some features of Parkinson’s disease by overexpression of alpha-synuclein (FEANY and

BENDER 2000). The other approach has been to screen for mutations in Drosophila that result in neurodegeneration.

5 One way of identifying mutations that elicit neurodegeneration in Drosophila has been to screen for mutations that result in reduced lifespan 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 Cretzfeldt-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 2001; MIN and BENZER 1997). 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 (HARRIS and STARK 1977; HOTTA and BENZER

1970; MASAI et al. 1993; VIHTELIC et al. 1991). 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 non-essential tissue such as the eye. In such a screen, heterozygous animals are generated following random mutagenesis. Using a FLP-recombinase expressed from an eye-specific promoter, marked patches of homozygous mutant tissue are generated in

6 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 beta and alpha 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.

MATERIALS AND METHODS

Fly Stocks. w; FRT40A or w; FRT42D males were mutagenized with ethylmethanesulfonate 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, 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], ywhsFLP; 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

7 FRT chromosomes by Berger et al.(BERGER et al. 2001). SNP analysis of recombinants between scrd1 and the EP0755 chromosome narrowed the interval to 423Kb between

57B3 and 57D1 comprising approximately eighty 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 186Kb region containing approximately thirteen genes. 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 female. 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

8 producing progeny with mosaic eyes must have retained the FRT. Several excision lines were obtained that all gave mosaic eyes with approximately equal red and white ratios and normal eye morphology (no degeneration). 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 wildtype 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 as follows: 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], y w eyFLP; FRT42D tsrKo56332/FRT42D P[mini-w, armLacZ].

For immunofluorescence, larval discs were dissected from the following 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], y w eyFLP; FRT42D tsrKo56332/FRT42D P[mini-w, UbiGFP]. Upon fixation larval eye discs were incubated overnight at 4o C in primary antibodies as follows: elav (1:10), TRITC-phalloidin (1:500; no secondary antibody needed), chaoptin (1:40), C3 (1:100), alpha-, alpha- tubulin, Rho1, beta-catenin and (all 1:50), cofilin and tau (1:500). Upon washes

9 in PBS, eye discs were incubated for 2 hrs at RT 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 Inc. All other antibodies were from the Developmental Studies

Hybridoma Bank. Imaginal disc clones were analyzed suing the lasso tool together 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 micron sections were cut using a glass knife, stained with toluidine blue and visualized by light microscopy. Representative regions were processed for TEM by cutting thin sections (0.1 mm) 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 captured with an Advanced Microscopy

Techniques CCD camera.! For Scanning Electron Microscopy, ethanol dehydrated adult eyes were prepared for analysis at the SEM core facility at Northeastern University,

Boston, MA.

RESULTS

Identification of bnd and scrd.

10 We have screened the four main autosomal arms of Drosophila, which together comprise approximately 80% of the genome, for mutations that alter the growth properties of clones of mutant tissue during eye development. Using a 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 4 main autosomal chromosome arms

(2L, 2R, 3L and 3R). At relatively high frequency, we recovered mutations that led either to the complete absence or under-representation of mutant tissue. At much lower frequency, we identified mutations that enabled mutant cells to outgrow their wild-type neighbors. Mutations that result in increased growth map to more than 25 distinct loci and include mutations in genes 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.

11 Eyes containing either bnd or scrd mutant clones (Figure 1B, 1D, 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 1A, 1C, 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 1F,

1G) 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 transmission electron microscopy (Figure 1I, close-up in 1J). In the mutant clone, numerous electron-dense bodies were observed that are likely to be the remnants of degenerating rhabdomeres.

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.

12 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 transmission electron microscopy and found no evidence of degeneration (Figure 1K-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 1N, 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 (approximatley 5%).

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

In order 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

13 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 2A-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 2D-F). Thus the early stages of neuronal cell fate specification and photoreceptor differentiation occur normally in bnd and scrd mutant cells.

In contrast, visualization of the actin cytoskeleton using phalloidin reveals obvious abnormalities in bnd (Figure 2G-I, close-up in 2J-L) and scrd (Figure 2M-O, close-up in 2P-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 pattern of alpha-spectrin, alpha-tubulin, tau, Rho1, profilin, cofilin, and beta- catenin were indistinguishable from 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 approximately eighty annotated genes. Stocks containing lethal P-element insertions were available for seven 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 alpha subunit

14 (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 an 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 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

(CELNIKER et al. 2002; LEVIS et al. 2001; SPRADLING et al. 1999; SPRADLING et al. 1995) 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.

The bnd locus on 2L was mapped by deficiencies to 22A1-22B1, an 866 Kb interval and by finer recombination mapping to a 186Kb region containing approximately thirteen genes. One of the genes in this interval is the ortholog of the beta subunit of the

F-actin capping protein, cpb (HOPMANN et al. 1996). Capping of F-actin utilizes an alpha-beta hetero-dimer. In mammals, the F-actin capping protein alpha subunit

15 (CAPZA, Cappa) is represented by three separate genes, and the beta subunit (CAPZB, cappab) by one gene with three separate isoforms (HART et al. 1997a; HART et al. 1997b;

SCHAFER et al. 1994). The Drosophila genome encodes only one gene for alpha,

CG10540, and one gene for beta, known as capping protein beta (cpb). All three bnd alleles failed to complement an existing cpb allele, cpbM143 (HOPMANN et al. 1996).

Sequencing of bnd1revealed 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 alpha-beta capping protein heterodimer binds and

“caps” the barbed end of actin filaments in a reversible and calcium independent manner

(CALDWELL et al. 1989; COOPER and POLLARD 1985; 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 hetero-dimer (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

16 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 only identified mutations in 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

17 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.

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 4D-E, close-up in G-H), more so than 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 underepresented. 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 4I-K). When the number of cells expressing activated C3

18 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, over-expression 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.

DISCUSSION

We have shown that mutations in the Drosophila orthologs of the alpha and beta 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 alpha-beta 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 alpha or beta 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

19 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/ADF homologue which 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.

20 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 cytoskelton 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, a 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 (HART et al. 1997a; HART

21 et al. 1997b; SCHAFER et al. 1994). Their role in either causing or modifying degenerative disease phenotypes may warrant further investigation.

Acknowledgements

We would like to thank S. Schelble and L. Madden for technical assistance. We also thank K. Harvey, K. Moberg, J. Walker and K.Tseng for advice and helpful conversations. We would also like to thank W. Fowle (Northeastern University) for generating the SEMs, Martin Selig (Department of Pathology, Massachusetts General

Hospital) for technical assistance and advice in generating TEMs and Igor Bagayev of 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 NIH (KO8 EY13639A).

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 NIH (GM61672 and

CA95281).

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32 Figure Legends

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 clones, the mutant tissue appears dark. Scanning electron micrographs of an eye containing bnd mutant clones (E, F, G) generated using hsFLP in the eye showing a lack of recognizable facets and absence of interommatidial bristles.

Scale bars indicate 100 mm (E), 10 mm (F,G). (H-J) Sections of adult eye containing a bnd clone created using hsFLP. Mutant tissue lacks the refractile pigment granules. (H)

Section of a bnd clone viewed under light microscopy shows no recognizable photoreceptors or ommatidial organization in the mutant tissue. (I, J) Transmission electron micrographs of an adult eye section are shown in (I) and at higher magnification in (J). Scale bars indicate 2 mm (I) or 5 mm (J). The genotype of individual photoreceptors can be inferred by the presence of pigment granules in the stalk of wild- type photoreceptor cells. All morphologically normal photoreceptors were scored as bnd+ indicating a cell-autonomous requirement for bnd to maintain the integrity of photoreceptor cells. (K-L) Transmission electron micrograph of flies heterozygous for bnd taken at one day (K) or 14 days after eclosion (L). Scale bars indicate 10 mm (K,L).

Flies heterozygous for bnd show no loss of photoreceptors or patterning over time.

Anterior is to the left in panels (A-L). (M) Clones of bnd generated in the wing using hsFLP appear black, showing that the requirement for bnd function is not specific to the eye. (N) Scanning electron micrograph of bnd mutant clones generated by hsFLP in the dorsal abdomen shows a range of disruption in patterning and morphology. Bristle

33 abnormalities (P) compared to wild-type bristles (O) include disorganized spatial distribution and irregularities in length and thickness of the mutant bristles. Scale bars indicate 100 mm (N), 20 mm (O,P).

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 in all images. (A-C) Wild-type tissue is visualized with an anti-bgal antibody (red, A) and mutant tissue is dark. Anti-ELAV staining (staining the nuclei of cells recruited to a neuronal fate) appears green (B) and shows no obvious difference between wild-type and mutant clones (merge is shown in

C). (D-F) Wild-type tissue is visualized with GFP (green, D). Staining for Chaoptin, a marker for neural development, is shown in red (E) and shows no obvious difference between wild-type and mutant clones (merge shown in F). (G-R) Wild-type tissue is visualized with GFP and appears green (G, I, J, L, M, O, P, R). Visualization of the actin cytoskeleton using phalloidin (red) (H, I, K, L, N, O, Q, R) shows accumulation of actin in patches mutant for bnd (G-L) and scrd (M-R). Merges are shown in I, L, O, R. J-L show a close-up of the disc shown in G-I, P-R show a close up of the disc shown in M-O.

Scale bars in D-I and M-O indicate 20 mm. Scale bars in J-L indicate 10 mm.

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 grey box. The actin- binding domain in the C-terminus is represented by a shaded box. (B) Schematic of the

34 cpb gene product indicating the positions and nature of the three alleles, cpbbnd1, cpbbnd2, and cpbbnd3. The actin-binding domain is represented by a shaded box.

Figure 4: Eye mosaic for tsr show an under-representation 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 are under-represented in the adult eye. (B) Transmission electron micrograph of an adult eye section through a tsr mutant clone. Loss of patterning and rhabdomere remnants are seen. Scale bar indicates 5 mm. (C-K) Third instar larval eye discs mosaic for tsr show only small mutant clones. Wild-type tissue is visualizd with GFP and appears green while mutant patches are dark (C,E,F,H,I,K). Actin, visualized with phalloidin (D, E, close up in G,H) appears red; actin accumulates in the mutant clones (merge shown in

E,H), more than in scrd and bnd clones. Activation of the caspase DRICE can be visualized with an antibody to caspase-3 (C3) in the larval eye discs (appearing blue in J,

K). In third instar larval eye discs, the small clones of tsr stain intensely for C3 (J, merge shown in K). Scale bars in C-E and I-K indicate 20 mm.

35 A B C D

E F G

H I K

J L

M N O

P A B C

D E F

G H I

J K L

M N O

P Q R A scrd1 scrd2 G A Val183Asp

1 286 AA CPA scrd1 A GTGGAACTATGG

B

bnd2 bnd1 bnd3 Glu218Lys Gln5Stop Glu221Lys

1 276 AA CPB A B

C D E

F G H

I J K