The villin-like protein encoded by the Drosophila quail gene is required for actin bundle assembly during oogenesis

Cell, Vol. 78, 291-301,

July 29, 1994, Copyright

0 1994 by Cell Press

The Villin-like Protein Encoded by the Drosophila quail Gene Is Required for Actin Bundle Assembly during Oogenesis Shalina Mahajan-Miklos and Lynn Dooley Department of Genetics Yale University School of Medicine New Haven, Connecticut 06510

Summary Mutations in the Drosophila quail gene result in female sterility due to the disruption of cytoplasmic transport from the nurse cells into the oocyte late In oogenesis. Nurse cells from quad/ mutant egg chambers fail to assemble cytoplasmic actin filament bundles correctly. We have cloned the qua/i gene and found that it encodes a protein with homology to the vertebrate actln-regulating protein villin. Unlike vertebrate villin, which is restricted to specialized absorptive eplthelial cells, the vlllin-like protein encoded by quail is germline specific in adult flies. Antibodies directed against the quail protein show a striking colocalization with filamentous actln in the nurse cells and the oocyte. Our results demonstrate that the villin-like product of quail is required for the formation of cytoplasmic actin filament bundles In nurse cells, possibly by regulating both the polymerization and organization of actin filaments as demonstrated for vertebrate villin in vitro. Introduction The fundamental processes of shape change, movement, and the generation of force within a cell are mediated by the reorganization of the underlying actin cytoskeleton. Changes in the distribution and assembly of actin are in turn driven by the concerted effects of multiple actinbinding proteins. Although thefunctionsof many such proteins have been extensively characterized in vitro, the complex nature of their cellular functions remains to be unraveled. One system in which the in vivo analysis of actin-binding proteins has been extremely informative is the Drosophila ovary (for review see Knowles and Cooley, 1994). Genetic analysis of the actindriven process of cytoplasm transport between the nurse cells and the oocyte during oogenesis has facilitated the study of proteins like profilin and fascin that are required for actin filament bundle assembly in the ovary, without affecting the viability of the fly (Cooley et al., 1992; Cant et al., 1994). Analysis of the mutant phenotypes of the genes encoding these proteins has allowed the convergence of biochemical and genetic approaches, providing insight into their cellular functions (Cant et al., 1994; Verheyen and Cooley, 1994). Oogenesis in Drosophila has been divided into 14 stages and begins with four mitotic divisions of a germline stem cell daughter to form a 16cell cluster (for oogenesis review see King, 1970; Spradling, 1993). This 16-cell cluster is enveloped by somatic follicle cells to form an egg chamber. Incomplete cytokinesis during the four mitotic divisions results in the 16 germline cells being intercon-

netted by a series of cytoplasmic bridges or ring canals, through which cytoplasm can flow. Early in oogenesis, 1 of the 16 germline cells becomes an oocyte while the remaining 15 differentiate into nurse cells. The highly polyploid nurse cells synthesize many of the components required for oocyte maturation and subsequent embryonic development. Cytoplasm transport between the nurse cells and the oocyte takes place in two distinct phases. During the first phase(stages l-lOa), nursecell cytoplasm gradually streams into the oocyte, a process that continues for approximately 2 days. Late in oogenesis, during stages lob and 11, nurse cell contraction rapidly forces the cytoplasmic contents of the nurse cells, excluding the nuclear remnants, into the oocyte. This rapid, terminal phase of transport results in the doubling of oocyte volume and the regression of the nurse cell cluster in about 30 min. Drug inhibitor studies have demonstrated that the actin cytoskeleton is crucial for the generation of the force responsible for rapid transport into the oocyte (Gutzeit, 1966). The actin cytoskeleton undergoes dramatic rearrangements to accommodate the changes in cellular morphology of the nurse cell cluster that occur during rapid transport. Throughout oogenesis, two populations of actin filament networks can be observed in egg chambers. First, filamentous actin is found associated with the inner rim of ring canals (Warn et al., 1965). In addition, dense membrane-associated actin filament networks are found subcortically in the nurse cells, the oocyte, and the follicle cells. Just prior to the onset of rapid transport, a third extensive network of actin filament bundles assembles within the cytoplasm of the nurse cells. These thick actin filament bundles radiate from the nurse cell membranes into the cytoplasm and terminate at or close to nuclear membranes, often reaching lengths of approximately 30 pm warn et al., 1965; Gutzeit, 1966). Previously, mutations in the chickadee and s&red genes have been shown to result in defects in the assembly of cytoplasmic actin filament bundles in nurse cells (Cooley et al., 1992; Cant et al., 1994). Molecular characterization of chickadee and singed revealed that they encode proteins with homology to the actin-binding proteins profilin (Cooley et al., 1992) and fascin (Bryan et al., 1993; Paterson and O’Hare, 1991), respectively. It has been suggested that profilin is required to promote the rapid polymerization of actin in nurse cells (Verheyen and Cooley, 1994), while fascin is required to bundle newly formed filaments into a stable network (Cant et al., 1994). In this paper we present the cloning and characterization of a third gene in this class, quail, whose mutant phenotype in the ovary closely resembles that of chickadee and singed. The product of the quail gene has homology to the vertebrate actin-binding protein villin. Extensive in vitro studies have demonstrated that vertebrate villin can cap, nucleate, sever, and bundle actin filaments in a calciumdependent manner (for review see Mooseker, 1965). In vivo, villin is capable of reorganizing the actin cytoskeleton

Cell 292

Figure

1. Lack of Cytoplasmic

Actin

Filament

Bundles

in quail Nurse

Cells Allows

Nuclei

to Block

Ring Canals

Egg chambers were stained with rhodamine-conjugated phalloidin to visualize filamentous actin or double labeled with propidium iodide and antibodies against the carboxyl terminus of the hu-Ii tai shao protein (Robinson et al., 1994) to visualize nuclei and ring canals, respectively. Images were generated using confocal microscopy. In wild-type egg chambers throughout oogenesis, a dense network of filamentous actin is present subcortically (SC) in the nurse cells, the oocyte (WC), and the follicle cells (fc) (A). During stage lob, an abundant network of actin filament bundles assembles in the cytoplasm of the nurse cells (cy). Nurse cells and the oocyte are connected to each other by a series of ring canals (green rings in [B]). In wild-type egg chambers, the nurse cell nuclei are maintained in a central position away from the ring canals, most likely anchored by the cytoplasmic actin filament bundles (Cooley et al., 1992). In quail mutant egg chambers, the cytoplasmic actin filament network fails to assemble (C), and the nurse cell nuclei can often be seen extending through ring canals (arrows in [D]).

and can cause changes in cellular morphology when introduced into cells that normally lack this protein (Franck et al., 1990; Friederich et al., 1989). We demonstrate here that avillin-like protein is required for actin filament bundle assembly during development. Results Nurse Cell Cytoplasm Transport Is Disrupted in quail Mutant Egg Chambers Females homozygous for mutations in quail are sterile owing to the disruption of nurse cell cytoplasm transport during oogenesis. In all quail alleles analyzed, egg chambers appeared morphologically wild type until early in stage 10, indicating that the initial slow phase of cytoplasm transport was unaffected (data not shown). However, after stage 10, the nurse cells failed to regress and to deliver all of their cytoplasmic contents into the oocyte. Five of the quail alleles analyzed had a completely penetrant, recessive female sterile phenotype and resulted in oocytes that were approximately half that of normal size. The few small eggs laid by these mutant females failed to develop. Females homozygous for the remaining mutation, quaHMt4, were very weakly fertile and produced oocytes that averaged

three-quarters normal size. The few eggs laid by quaHMj4 females developed into morphologically normal adults. Despite the block in cytoplasm transport, follicle cell development proceeded in all quail mutants, resulting in egg shell deposition around mutant oocytes (data not shown). In contrast with the effect seen on female fertility, no apparent reduction in fertility was observed in males homozygous for quail mutations. In addition, adult viability was unaffected in all six quail alleles studied. Transheterozygous flies containing each of the quailethyl methanesulfonate (EMS) alleles over the chromosomal deficiency Df(2LJTw737 (Wright et al., 1976) were generated and found to have the same female sterile phenotype. No evidence was found for either male sterility or a decrease in adult viability. This analysis suggests that the female sterile phenotype represents a complete loss of function of the quail gene. The Absence of Cytoplasmic Actin Bundles in quail Nurse Cells Allows Nuclei to Block Ring Canals during Rapid Transport lmmunofluorescence analysis of filamentous actin in quail mutant egg chambers using rhodamineconjugated phalloidin showed that they had a very specific defect in the

Drosophila 293

quail Ecodes

a Villin-like

Protein

1234567 Figure 3. A 3.3 kb Transcript Alleles Figure 2. Genetic quail Gene

and Molecular

Map of the Interval

Containing

the

(A) The genetic interval 36C, showing the position of the quail locus and the breakpoints of the qua’374 deletion. (B) Restriction map of the genomic DNA between BicfY and the proximal breakpoint of the qua ‘a’4 deletion. The map shows the positions and directions of transcription of BicD (Suter et al., 1989; Wharton and Struhl, 1969) and three other transcription units identified within this region (arrows). The numbers0.9,2.2, and 3.3 indicate the approximate sizes of the transcription units. The 3.3 kb mRNA is transcribed from the quailgene (Figure 3). The hatched line indicates the genomic probe used to isolate quail cDNAs. (C) Detailed restriction map and genomic organization of the quail gene, which contains seven exons spread over 11 kb of genomic DNA. Closed boxes indicate the extent of the quail open reading frame. Restriction enzymes: R, EcoRI; H, Hindlll; L, Sall; P, Pstl; X, Xbal.

actin cytoskeleton, similar to that observed in chickadee and singed mutants (Cooley et al., 1992; Cant et al., 1994). In quailegg chambers, the correct number (15)of morphologically normal ring canals was present, and the subcortical actin underlying the plasma membranes of the nurse cells, the oocyte, and the follicle cells appeared normal early in oogenesis (data not shown). However, the cytoplasmic actin filament bundles that assemble in wild-type nurse cells just prior to the onset of rapid transport (Figure 1A) were missing in sterile quail mutants (Figure 1C). In the weakly fertile allele quaHM”, some cytoplasmic actin bundles were observed. However, these actin bundles were not as abundant or organized as the cytoplasmic actin bundles in wild-type nurse cells. The nurse cell nuclei in quail egg chambers became dramatically rearranged late in stage 10. In wild-type egg chambers, nuclei were positioned centrally in the nurse cells and were clearly separated from the ring canals even during the process of rapid transport (Figure 1 B). In contrast, after stage lob, the nuclei in quailmutant nurse cells assumed a stretched appearance and were often seen clustered around and extending through the ring canals (Figure 1 D). Although this was most apparent for the nurse cell nuclei directly adjoining the oocyte, nuclei in the more anterior nurse cells often stretched through the ring canals. This phenotype further supports the model proposed by Cooley et al. (1992) in which the nurse cell cytoplasmic actin filament bundles function to anchor the nuclei in

Is Greatly

Reduced

in Four quail EMS

A Northern blot containing 2 rg of poly(A)+ RNAisolated from wild-type and quail mutant females is probed with a genomic probe (hatched line in Figure 28). In wild-type females, three transcription units of sizes 3.3, 2.2, and 0.9 kb are detected. The 3.3 kb mRNA is greatly reduced in qua-(lane 3), quePxu(lane 5), quffn’ (lane 6) and qua(lane 7) female extracts. The 2.2 kb and 0.9 kb transcription units are unaffected in all six quail alleles and serve as loading controls for this experiment.

place during rapid transport. The force for nurse cell contraction is thought to be provided by the subcortical actin filament networks, presumably powered by a myosin motor (Cooley et al., 1992; Theurkauf et al., 1993). Since the cytoplasmic actin filament bundles were not formed in quail mutant nurse cells, the untethered nuclei were pushed through the ring canals in the direction of the oocyte, thus blocking the path of cytoplasm flow. The movement of the nuclei suggested that contraction of nurse cells was initiated at the appropriate time by an intact subcortical contractile apparatus. Molecular Characterization of quail The quail gene had been mapped to the genetic interval 36C (Schiipbach and Wieschaus, 1991) between the comD (BicD) and lethal (2) from plementation groups Bicaudal Blond (1(2)6/d) (Steward and NiLsslein-Volhard, 1986). The interval containing the quail locus was previously cloned during genomic walks through the 36C region (Suter et al., 1989; Wharton and Struhl, 1989). To locate the quail locus within the approximately 250 kb of cloned genomic DNA (gift of R. Wharton), a deletion allele, quaf3”, was utilized (see Experimental Procedures). Genetic analysis revealed that the proximal breakpoint of the quaf3” deletion was between quail and /(2)B/d (Figure 2A). Mapping the proximal breakpoint of the quaj3” deletion within the cloned genomic DNA resolved the location of quail to within 30 kb (Figure 28). DNA from this 30 kb interval was screened for regions that were expressed at high levels in ovaries, first using so-called reverse Northern analysis and then using purified restriction fragments as probes for Northern blots. Although BicD and quail were the only two complementation groups mapped to this heavily mutagenized region, at least 10 additional transcription units were identified (data not shown). The transcripts were mapped with re-

Cdl 294

Figure 4. Developmental of the quail mRNA

f

quail

-

4-ffPA7

*>

Expression

Profile

A full-length quail cDNA was used to probe a Northern blot containing 15 pg of total RNA isolated from wild-type adults, testes, ovaries, embryos, larvae, and pupae. Male and female carcasses refer to adults from which the testis and ovaries were removed. First, second, and third instar refer to the three larval molts. High levels of the 3.3 kb quail transcript are present in males and females (lanes 1 and 2), in which the quail transcript is restricted to the testes (compare lanes 3 and 4) and ovaries (compare lanes 5 and 6) respectively. During embryogenesis, significant levelsof the quailtranscript are detected in O-4 hr embryos (lane 7), representing maternallydeposited mRNA. Low levels of the quail transcript are detected in 6-24 hr embryos (lanes 9 and 10) and during pupation (lane 14). A probe for the ribosomal protein RpA7 mRNA was used as a loading control.

0.241

2

3

4

5

6

7

8

9

10 11 12 13

spect to the position of the BicD gene (Suter et al., 1989; Wharton and Struhl, 1989) and single-stranded RNA probes were used to determine the direction of transcription of three of the transcripts (Figure 28). To identify the quail transcript, Northern blot analysis was performed using RNA isolated from quail mutant females. A 3.3 kb mRNA was greatly reduced in four different quail alleles (Figure 3, lanes 3, 5, 6, and 7). Two transcripts adjacent to the 3.3 kb mRNA (Figure 3) and all the other transcription units identified within the 30 kb region (data not shown) were unaffected in the quail alleles. The expression of the 3.3 kb transcript at various stages of development was examined using Northern blot analysis (Figure 4). High levels of expression were found in both adult males and females (Figure 4, lanes 1 and 2). The expression in adults was entirely restricted to the testes in males (Figure 4, lane 3) and the ovaries in females (lane 5). Although maternally deposited transcript was fairly abundant during the first 4 hr of embryonic development (Figure 4, lane 7), very low levels were detected during the subsequent stages of embryogenesis (lanes 8-10). The 3.3 kb transcript was almost undetectable during larval development (Figure 4, lanes 1 l-l 3) and low levels were once again detected during pupation (lane 14). It is unknown whether the 3.3 kb transcript detected during embryogenesis and pupation represents germline or somatic expression. The lack of expression of the 3.3 kb mRNA outside the ovary in adult females showed a strong correlation with the ovary-specific phenotype of quail mutations. Wholemount in situ hybridization demonstrated that the 3.3 kb mRNA was detected only in the germline-derived nurse cells and the oocyte and not in the somatic follicle cells (data not shown). These data correlated with the quail phenotype since only the germline-derived cells were affected in quailmutant egg chambers. These results, together with the finding that the 3.3 kb mRNA was greatly reduced in

I4

four quailEMS alleles, strongly suggested that this mRNA was transcribed from the quail gene. Five cDNA clones corresponding to the 3.3 kb quail mRNA were isolated by screening a cDNA library from O-4 hr Drosophila embryos (Brown and Kafatos, 1988) using a genomic probe (indicated in Figure 28). Three clones were full length (qua72, qua73, and qua20). Genomic and cDNA clones were sequenced, and the sequences were compared to determine the intron-exon boundaries of the quail gene (see Figure 2C). The quail Gene Encodes a Member of the GelsolinNillin Family of Actin-Binding Proteins Complete cDNAs for the quail mRNA contained an open reading frame of 2664 bp. The predicted translation product of this open reading frame was a protein of 887 amino acids that was 30% identical and 50% similar to the chicken actin-binding protein villin. A striking feature of the predicted quail peptide sequence was the presence of six repeats within the protein. These repeats are the hallmark of a large family of actin-regulating proteins that have been identified in most eukaryotes from slime molds to mammals (Weeds and Maciver, 1993). Proteins from this family contain either three or six repeats (Figure 58). Fragmin from Physarum polycephalum (Ampe and Vandekerckhove, 1987) severin from Dictyostelium discoideum (Andre et al., 1988), and the Myc basic homology protein (Mbhl) from mouse fibroblasts (Prendergast and Ziff, 1991) each contain three such repeats. Proteins with six repeats include vertebrate gelsolin (Kwiatkowski et al., 1986; Way and Weeds, 1988) Drosophila gelsolin (Heintzelman et al., 1993; Stella et al., 1994) Drosophila flightless-l (Campbell et al., 1993), vertebrate villin (Arpin et al., 1988; Bazari et al., 1988) and Dictyostelium protovillin (Hofmann et al., 1993). Phylogenetic analysis of vertebrate and invertebrate

Drosophila 295

quail Ecodes

a Villin-like

Protein

Humvil Mousevil Chickvil Hmgel Drosgel m Dlctyprato orosflight

Figure

5. The Predicted

Translation

Product

of quail Is a Villin-like

Protein

(A) Phylogenetic tree of vertebrate and invertebrate proteins containing six villin-like repeats generated by the program CLUSTAL V (Higgins et al., 1992) using the neighbor-joining method (Saitou and Nei, 1987), which sequentially joins pairs of the most closely related sequences. The core region consisting of the six villin-like repeats in the following peptide sequences were compared: human villin, Humvil (Arpin et al., 1988); mouse villin, Mousevil (GenBank accession number M98454); chicken villin, Chickvil (Bazari et al., 1988); human gelsolin, Humgel (Kwiatkowski et al., 1986); Drosophila gelsolin, Drosgel (Heintzelman et al., 1993; Stella et al., 1994); Drosophila quail, Drosqua (GenBank accession number U10070); Dictyostelium protovillin, Dictyproto (Hofmann et al., 1993); and Drosophila flightless-l, Drosflight (Campbell et al., 1993). The sum of the lengths of the branches connecting two sequences is proportional to the percent amino acid divergence between these sequences (indicated by numbers above the branches). The bootstrapping value (the number of times two sequences or groups of sequences joined together out of 1000 trials) is italicized. Bootstrapping values were not available for the branches connecting protovillin and flightless since these sequences were the outliers in this comparison. (B) Schematic representation of a family of actin-binding proteins that contain either three or six characteristic repeats. Severin is an example of a protein containing three such repeats, while gelsolin and villin both contain six. Villin has a unique carboxy-terminal headpiece domain (HP), Arrows indicate the actin monomer (G)- and filament (F)-binding sites identified in chicken villin (for review see Weeds and Maciver, 1993). (C) Alignment among the predicted peptide sequences of four villin-related proteins reveals sequence homology throughout the length of the proteins. Dictysev refers to the Dictyostelium severin protein (Andre et al., 1988). Like the vertebrate villin sequences, the quail protein has six repeated segments (bold numbers in the right margin) and a carboxy-terminal headpiece region (HP). The segments are centered around motifs, labeled B, A, and C (Way and Weeds, 1988), that are highly conserved in the quail protein (stippled boxes). I indicates the isoleucine in human gelsolin that is at the center of the segment 1 actin-binding site as shown in the gelsolin segment 1-actin crystal structure (McLaughlin et al., 1993). The two presumptive PIPTbinding sites are indicated (Weeds and Maciver, 1993). Residues comprising the actin-binding site at the amino terminus of segment 2 in chicken villin are underlined (de Arruda et al., 1992). The unstippled box indicates residues in the headpiece that are required for the morphogenetic effect of vertebrate villin in vivo (Friederich et al., 1992). In several regions outside the repeats, the quail protein contains insertions or small deletions (indicated by ellipses). Note the insertion of 24 amino acids between residues 625-648 that is extremely serine rich. The PILEUP program (Genetics Computer Group sequence analysis software program) was used to generate this figure.

proteins

containing

six

villin-like

repeats

using

the

pro-

gram CLUSTAL V (Higgins et al., 1992) demonstrated that the quail protein was clearly a member of this family (Figure 5A). Vertebrate and invertebrate members of this fam-

ily segregated

into distinct

categories

based

on the degree

of sequence conservation within their six repeated segments. Vertebrate villins and gelsolin, being highly conserved, clustered together. This group also included Dro-

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Figure

6. A 97 kDa Protein

Is Missing

4

quail

4

singed

in quaon’

Ovary

Extracts

Western blot analysis of wild-type and quaon ovary extracts. The blot was probed with the quail monoclonal antibody 6C and with the singed monoclonal antibody 7C, which serves as a loading control. Protein molecular mass markers are indicated in kilodaltons. A 97 kDa protein is detected in wild-type, but not qua““‘, ovary extracts.

sophila gelsolin, which was more closely related to the vertebrate gelsolin and villin than to the invertebrate members of this family. Dictyostelium protovillin and Drosophila flightless were highly divergent from the vertebrate proteins and clustered together, forming a separate group. In contrast, the quail protein was unique in that it was equally divergent from both these groups. The alignment between the predicted peptide sequence of quail and four other villin-related proteins showed that in addition to six repeats, the quail protein had a carboxyterminal region similar to the headpiece that is present in villin, but not the other family members (Figure 5C). This headpiece domain allows villin, but not the other family members, to bundle actin filaments (for review see Friederich et al., 1990). Although sequence similarity between the quail protein and other family members extended throughout the length of the protein, several regions of divergence were evident. The most striking differences were two insertions of 24 and 17 amino acids between repeats 5 and 6 in the quail protein. These and other smaller insertions made the quail protein longer (667 residues) when compared with the 826 residue length of vertebrate villins. However, despite the regions of sequence divergence, the motifs defining each of the six repeats were highly conserved in the quail protein (stippled boxes labeled B, A, and C in Figure 5C). The Quail Protein Colocalires with Filamentous Actin in Nurse Cells To investigate further the role of the quail protein during oogenesis, monoclonal antibodies were raised against a bacterially produced quail fusion protein. Western immunoblot analysis of ovary protein extracts demonstrated that the antibodies detected a protein of the predicted size (approximately 97 kDa) that is present in wild-type, but not quaon mutant, extracts (Figure 6). The expression of

the 97 kDa quail protein during development mirrored the expression of the quail mRNA described earlier (data not shown). Wild-type or quail mutant egg chambers were stained with monoclonal antibodies against the quail protein and with rhodamine-conjugated phalloidin tovisualize filamentous actin. The quail protein was first easily detected in stage 8 egg chambers and was present at very low levels during the earlier stages (Figure 78). During stages 8-lOa, the quail protein colocalized with the subcortical actin filament network in the nurse cells and the oocyte and was also present in the cytoplasm of the nurse cells (compare Figure 7A with 78 and Figure 7C with 70). Occasionally, the quail protein was seen localized to ring canals (data not shown). In stage lob, two major changes were observed. First, there was a significant increase in the level of expression of the quail protein (Figure 7F). Second, in addition to colocalizing with subcortical actin, the quail protein localized along the length of the newly assembled actin filament bundles in the nurse cell cytoplasm (compare Figure 7E with 7F and Figure 7G with 7H). In sharp contrast with the staining observed in the nurse cells and the oocyte, the quail protein was conspicuously absent from the follicle cells throughout oogenesis, demonstrating its germline-specific nature. No evidence of quail protein staining was observed in quaon’ mutant egg chambers (data not shown). Discussion The quail Gene Encodes a Germline-Specific Villin-like Protein We have shown that the quailgene encodes a protein with homology to vertebrate villin, a calcium- and phosphoinositide-regulated protein that can cap, sever, nucleate, and bundle actin filaments in vitro (for review see Friederich et al., 1990). Both the quail protein and vertebrate villin contain six homologous repeated segments and a carboxy-terminal headpiece domain. However, the quail protein contains several regions of sequence divergence when compared with the vertebrate villins. Recently, the Dictyostelium protovillin gene was found to encode a villinlike protein with a unique threoninelproline-rich neck region between the six repeats and the headpiece-like domain (Hofmann et al., 1993). In contrast with the vertebrate villins, which share a high degree of sequence homology, the quail protein and protovillin are significantly diverged from each other and vertebrate villins. Such sequence divergence is not the case for all invertebrate members of the gelsolin/villin family. Drosophila secretory gelsolin, which appears to be the homolog of vertebrate plasma gelsolin (Heintzelman et al., 1993; Stella et al., 1994) is more closely related to both vertebrate gelsolin and villin than is the quail protein (Figure 5A). These sequence comparisons suggest that the quail protein may not be the direct homolog of vertebrate villin, but instead a more distant relative. This conclusion is supported by the pattern of expression of quail, which is in sharp contrast with that observed for vertebrate villin. In vertebrates, villin expression is re-

Drosophila 297

9usil Ecodes

a Villin-like

Protein

Rhodamine-Phalloidin

Figure

7. The Quail

Protein

Colocalizes

with Filamentous

Actin in the Nurse

Anti-quailantibody

Cells and the Oocyte

Wild-type egg chambers were double labeled with rhodamine-conjugated phalloidin and quail monoclonal antibody (6C). Images were viewed using confocal microscopy. Scale bar in (A)-(F), 40 pm. Numbers refer to egg chamber stages. Significant levels of the quail protein are first detected in stage 8 (8). Between stages 8-lOa, the quail protein colocalizes with subcortical actin (SC) in the nurse cells and the oocyte and is also present diffusely in the nurse cell cytoplasm (A-D). During stage 1Ob, the quail protein localizes along the length of the newly formed cytoplasmic actin filament bundles (cy) in the nurse cells (compare [E] and [F]). (G) and (H) represent a magnification of the stage 10b egg chamber shown in (15) and (F) (scale bar, 20 pm). As a negative control, each reagent was used individually to test for fluorescence in the complementary channel. No bleedover was observed under these conditions (data not shown).

Cell 29s

stricted to absorptive epithelial cells such as the intestinal and kidney proximal tubule cells (for review see Friederich et al., 1990). Villin is localized at the apical surface of these cells, within a dense population of uniform length microvilli termed the brush border. Lower concentrations of villin have also been detected in epithelial cells lining the pancreatic and bile ducts. Although lacking an organized brush border, these cells are specialized in absorption and share a common embryonic origin with intestinal cells (Robine et al., 1985). Like vertebrate villin, expression of the quailgene is tissue specific. However, the quai/mRNA and protein are restricted to the germline in Drosophila adults. In females, the expression of quail is nurse cell specific. The nurse cells are nonabsorptive and lack brush borders or cell surface projections; thus, it is intriguing to find a villin-like protein specifically expressed here. Abundant brush borders are present in insect larval midguts (Dimitriadis and Kastritsis, 1984) that are extremely similar morphologically and ultrastructurally to those in vertebrates (Bonfanti et al., 1992). Proteins that crossreact with antibodies against vertebrate villin are present in the larval miguts of Manduca sexta (Bonfanti et al., 1992) and Drosophila(Maunouryet al., 1988). However, we have been unable to detect the quail protein in Drosophila larval midgut sections (data not shown; N. S. Morgan and M. B. Heintzelman, personal communication). These results sug gest that there might be two villin genes in Drosophila: an as-yet-unidentified villin-like gene that is expressed in the midgut and quail, which is germline specific in adults. We speculate that vertebrates, like Drosophila, could contain multiple villin-like proteins that are expressed in a tissuespecific manner outside the intestine. Analysis of the Predicted Quail Peptide Sequence Despite regionsof sequence divergence between the quail protein and vertebrate villin and gelsolin, residues that define the repeat motifs in all six segments are strikingly conserved in these proteins (labeled B, A, and C in Figure 5C). The crystal structure of a complex between gelsolin segment 1 and actin has demonstrated the structural significance of residues that comprise the repeat motifs. Gelsolin segment 1 is a globular domain consisting of acentral nonpolar P-sheet core sandwiched between two a helices (McLaughlin et al., 1993). Residues within the segment 1 S, A, and C repeat motifs generate the nonpolar core, thereby dictating the three-dimensional fold of this segment. Since these residues are consewed in all six segments of gelsolin, villin, and the quail protein, they may form similar tertiary structures. However, residues in the gelsolin segment 1 repeat motif do not contact actin and may not directly specify the actin binding properties of this segment. Thus, variations in residues flanking the repeat motifs in the individual segments in gelsolin, villin, and the quail protein could have important effects on the interactions of these proteins with actin. Functional aspects of the homology between the quail protein and gelsolinlvillin can be examined in the context of actin-binding sites that have been defined in the vertebrate proteins. Three actin-binding sites have been identified in gelsolin and the villin core (lacking the headpiece)

through extensive in vitro studies (for review see Weeds and Maciver, 1993). Actin monomer-binding sites are present in segment 1 and within segments 4-6, while an actin filament-binding site is located within segments 2-3 (Figure 5B). In the presence of micromolar calcium, the interactions of these three actin-binding sites are responsible for the actin severing, capping, and nucleating properties of gelsolin and villin. Both severing and capping of actin filaments by these proteins require the segment 1 actinbinding site. Gelsolin segment 1 binds to actin through a nonpolar cluster of residues centered around an isoleutine (McLaughlin et al., 1993). These residues interact with a nonpolar cluster of residues found in the cleft between subdomains 1 and 3of actin. The hydrophobic interaction is strengthened by a ring of intermolecular hydrogen bonds between the two proteins. In the quail protein, the central isoleucine is replaced by a histidine (Figure 5C). Further, many of the residues predicted to be involved in the formation of hydrogen bonds between gelsolin segment 1 and actin are not conserved in the quail protein (data not shown). Thus, if segment 1 of the quail protein does interact with actin, its actin-binding site may be comprised of different residues. This could alter the ability of the quail protein to sever and cap actin filaments. Severing of actin filaments by gelsolin and villin also requires the actin filament-binding site located within segments 2 and 3 (for review see Weeds and Maciver, 1993). In villin, basic residues at the amino-terminal region of segment 2 comprise part of this actin filament-binding site (Figure 5C) (de Arruda et al., 1992). The corresponding region in the quail protein is conserved with the exception of three residues at the amino terminus that are deleted. Nucleation, the third actin binding property of gelsolin and the villin core, requires the segment 2-3 actin filament-binding site and the segment 4-6 actin monomer-binding site (for review see Weeds and Maciver, 1993). Although residues involved in the segment 4-6 actin-binding site have not been defined, insertions in the quail protein sequence, like the stretch of highly polar residues between repeats 5 and 6, might indicate a modification of this function unique to the quail protein. The severing, capping, and nucleating properties of gelsolin and villin are negatively regulated by phosphatidylinositol diphosphate (PIP*) (Weeds and Maciver, 1993). Two presumptive PIPz-binding sites are present in these proteins. One of these sites is conserved in the quail protein, while part of the second site is deleted (Figure 5C). The villin headpiece contains an additional actin filament-binding site (Flgure 58) (for review see Friederich et al., 1990). In vitro, under conditions of physiological calcium (lo-’ M), the actin filament-binding sites present in villin within segments 2-3 and the headpiece cross-link actin filaments into bundles. In contrast, gelsolin, which lacks a headpiece domain and contains only one actin filament-binding site, is unable to bundle actin filaments. The actin-binding site in the villin headpiece has also been implicated in the reorganization of the actin cytoskeleton in vivo (Friederich et al., 1992). Overexpression of villin in fibroblasts results in the transformation of existing rudimentary surface projections into long actin-containing mi-

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Protein

crovilli, concomitant with the disappearance of actin stress fibers (Franck et al., 1990; Friederich et al., 1989). This effect on the actin cytoskeleton requires a charged motif, KKEK, that comprises part of the actin filament-binding domain in the headpiece (Friederich et al., 1992). In the headpiece-like domain present in the quail protein, the corresponding residues are KKQF (Figure 5C). Recently, the erythroid bundling protein dematin was found to contain a carboxy-terminal headpiece-like domain, although it does not contain any homology to villin in the remainder of its sequence. The dematin headpiece-like domain, in which the KKEK sequence is replaced by KKKA, can bind actin filaments in vitro (Rana et al., 1993). The conservation of the lysine pair in the quail headpiece implies that this domain could bind to actin filaments in a manner similar to the headpiece domains in villin and dematin. However, the sequence differences between the quail protein and villin in both the headpiece and the core domain might indicate a modulation of protein function unique to requirements in their respective cell types. Analyzing the interactions of the quail protein with actin in vitro should highlight the similarities and differences between the invertebrate and vertebrate forms of villin and should provide insight into important regulatory or functional regions of the protein that are conserved. Function of the Quail Protein In Vivo Despite the obvious differences both in the overall morphology and embryonic origin of Drosophila nurse cells and vertebrate enterocytes, certain parallel features underlie their development. Most significantly, the terminal differentiation of both these cell types is associated with the formation of actin filament bundles. In differentiated enterocytes, actin bundles form the core of the brush border microvilli (for review see Heintzelman and Mooseker, 1992). Just before the regression of Drosophila nurse cells, abundant cytoplasmic actin bundles are assembled. Both the quail protein and villin are expressed before the formation of the specialized actin cytoskeleton in their respective cell types. Early in oogenesis, the quail protein is expressed at low levels and colocalizes with the subcortical actin network in the nurse cells. Similarly, low levels of villin are colocalized with actin in the apical region of immature enterocytes before the formation of the brush border (for review see Louvard, 1989). During terminal differentiation and coincident with the reorganization of actin into projecting bundles, expression levels of both the quail protein and villin increase dramatically, and the proteins localize along the length of the newly assembled actin filament bundles. These similarities suggest that the quail protein and villin could share some cellular functions, albeit in different cell types. Cytoplasmic actin bundles fail to assemble in quail mutant nurse cells, demonstrating that the villin-like protein encoded byquailfunctions to regulate actin bundle assembly in vivo. The homology of the quail protein to villin and its subcellular localization in the nurse cells suggest that the quail protein, like singed-encoded fascin (Cant et al., 1994) could be functioning to bundle cytoplasmic actin filaments into stable arrays. This is supported by the local-

ization of the quail protein along the length of the cytoplasmic actin filament bundles. There is a precedent for actin bundles being organized by two proteins in the brush border, where villin and fimbrin are the two major bundling proteins (for review see Louvard, 1989). Additional functions for the quail protein are suggested by the colocalization of the quail protein with the subcortical actin network prior to the formation of cytoplasmic actin bundles. The quail protein could promote the nucleation of actin filaments at the membrane or provide additional nucleating sites by severing preexisting subcortical filaments in a calcium-dependent manner. Currently, it is not known whether fluxes in intracellular calcium levels occur in the nurse cells prior to regression. Manipulating the intracellular calcium levels in wild-type and quail mutant nurse cells should allow us to test the hypothesis that changes in the actin cytoskeleton can occur in response to the activation of quail protein function in vivo. The actin cytoskeleton in Drosophila egg chambers can be genetically manipulated with relative ease. Extending our analysis of the quail protein by testing the effects of additional quail mutations in vivo should be extremely informative, especially in the context of other actin-binding proteins like profilin and fascin, all of which are required for the assembly of the same population of actin filament bundles in the nursecells. Thus, by studying the seemingly specialized actin cytoskeleton in Drosophila nurse cells, we can begin to unravel the complex interactions between actin and its regulating proteins, which are required to orchestrate changes in the cellular architecture of other diverse cell types. Experimental

Procedures

Drosophila Stocks All fly stocks were maintained under standard culturing conditions. Seven alleles of quail were used in this study. Six alleles (quennzs, qua”“, quapx’*, quaOE2’, quaon’, and qua”“) were induced by EMS mutagenesis (Schiipbach and Wieschaus, 1991). The remaining allele (qua’374) was derived from a P element mutagenesis screen as described below. w”‘~ flies were used as the wild-type control for quail (Lindsley and Zimm. 1992). Generation of quiF’ Since insertions into the quailgene had not been recovered by conventional P element mutagenesis screens, a’local hopping screen”(Tower et al., 1993) was attempted. A P element inserted in the nearby dorsal gene (dfWJ; gift of L. Yue and A. Spradling) was mobilized as described in Verheyen and Cooley (1994) (Figure 2A). Progeny in which new insertion events might have occurred were scored for retention of the ry+ eye color marker carried by the P element. The ry+ progeny were then tested for their failure to complement qua““‘. A total of 1750 lines were analyzed in this screen and one new allele, quaT3”, was identified. Southern blot analysis using DNA isolated from quaT3” flies indicated that the original P element was still present at the dorsal locus and that the mutation in quailwas caused not by a new P element insertion, but by a deletion. Genetic analysis revealed that the deletion in qua’374 extended between dorsal and quail (see Figure 2A). DNA from the proximal breakpoint of qua’374 was isolated using inverse PCR (Sambrook et al., 1989). Nucleic Acid Menlpuletlons Phage DNA was isolated using standard procedures (Sambrook et al., 1989). quail cDNAs were isolated from a O-4 hr embryo library (Brown and Kafatos, 1988). sequenced, and analyzed as described in Xue and Cooley (1993). RNA isolation and formaldehyde-denaturing

Cdl 300

gel electrophoresis were also performed as described in Xue and Cooley (1993). RNA and DNA gels were blotted by capillary transfer to Hybond-N (Amersham) and ultraviolet cross-linked (Stratalinker, Stratagene). %P-labeled probes were prepared using random hexamer priming (Feinberg and Vogelstein, 1983). So-called reverse Northern blot analysis using poly(A)’ selected ovary RNA as a probe was performed according to Scott et al. (1983). Antibody Production The quailopen reading frame was cloned into the T7 RNA polymerasedriven expression vector Petl4b (Novagen, Incorporated). The resultant quail fusion protein contained an additional 20 amino acids at its amino terminus, including a tag of six histidines. BL21(DE3) cells transformed with the quail expression construct were grown at 37% and induced for 2 hr with 1 mM isopropyl-I-thio-8-galactopyranoside (IPTG). The induced fusion protein was isolated from inclusion bodies as described in Cant et al. (1994). Purified fusion protein was used to immunize mice, and antisera were screened for reactivity to the fusion protein using Western immunoblot analysis. In addition, antisera were screened for reactivity to wild-type Drosophila ovaries, but not to quaon’ (a genetic null allele) ovaries, by Western immunoblot analysis and immunofluorescence staining. Hybridoma cell lines were generated using standard techniques (Harlow and Lane, 1988). Western lmmunoblot Analysis Drosophila tissues or whole animals were ground in Laemmli sample buffer, and protein concentration was determined using the Bio-Rad protein assay (Bio-Rad Laboratories). Lysates were separated by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970) and transferred to nitrocellulose membranes. Following transfer. the filters were treated according to Xue and Cooley (1993). Monoclonal antibody supernatants directed against the quail fusion protein or the singed fusion protein (Cant et al., 1994) were used after diluting 1:lO. lmmunotluorescence Staining Ovaries were dissected, fixed, and stained with antibodies or rhodamine-conjugated phalloidin (Molecular Probes) as described in Xue and Cooley (1993). Anti-quail monoclonal antibody was used undiluted or anti-hu-Ii tai shao carboxy-terminal monoclonal antibody was used after diluting 1:l in PBT (Robinson et al., 1994). To visualize nuclei, fixed egg chambers were preincubated in 500 ug/ml ANAase for 2 hr at room temperature and subsequently incubated in 100 @ml propidium iodide for 10 min. Confocal Mlcroscopy Scanning laser confocal images were collected using the Bio-Rad MRCGOO system. All images were collected using a Zeiss 25 x lens with a numerical aperture of 0.8. Optical sections taken at 1 u-2~ intervals were combined using the COMOS program (Bio-Rad). Acknowledgments We thank Trudi Schiipbach for EMS alleles of quail; Robin Wharton for genomic DNA containing the quail locus; Lin Yue and Allan Spradling for the P element insertion allele dP; Marcel0 JacobsLorena for the RpaI probe; Nancy Morgan and Matthew Heintzelman for performing larval midgut staining; Nancy Morgan for help with the phylogenetic analysis of villin-related proteins; Laurent Caron and Spyros Artavanis-Tsakonas for help with confocal microscopy; and Mark Mooseker, Dennis McKearin, Esther Verheyen, Allan Fanning, and members of the Cooley lab for reading this manuscript and for countless valuable discussions. This work was supported by Public Health Service grant GM43301, the Pew Charitable Trusts, and Miles, hCOrDOrted.

Received

April 18, 1994; revised

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