Calpain Localization Changes in Coordination with Actin-related Cytoskeletal Changes during Early Embryonic Development of DrosophiZu*

Calpain, a calcium-dependent intracellular protease, was identified in Drosophila melanogaater. Drosophila calpain has an amino acid sequence highly homologous to those of mammalian calpains and exhibits a distinct domain structure consisting of cysteine protease and calcium-binding domains. Specific antibodies raised against a recombinant calpain fragment were used to identify the localization of calpain in developing Drosophila embryos. Calpain was first detected near the anterior pole and in posterior region of the embryo just after fertilization. The anterior calpain disappeared during the cleavage cycles. On the other hand, the pos- terior calpain moved to the posterior pole when polar buds were formed, and condensed just below the pole cells. At cleavage cycles 8 and 9, when nuclei reached the egg surface, calpain was localized between the nuclei at the surface beneath the precleavage furrows. Co-stain-ing experiments with anti-actin antibody revealed that calpain condenses specifically at the edge of and between actin caps that underlie the plasma membrane immedi- ately above each nucleus. These results indicate that calpain is involved in the dynamic changes in the embryonic cytoskeleton, especially actin-related structures, during early embryogenesis prior to cellularization. of DAB alone. Alternatively, fluorescein isothiocyanate-conjugated second-ary antibody was used in the case of anti-tubulin antibody staining. For DNA staining, 4,6-diamidino-2-phenylindole (DAPI) was used after antibody staining.

Various cellular phenomena involving changes in cell shape such as cell division and differentiation are dependent on cytoskeletal structures. The surface area of the cell is structurally dependent mainly on actin and its associated proteins, which form the so-called actin network (for a review, see Weeds (1982)). On the other hand, internal structures, including the mitotic apparatus, are mainly dependent on microtubules consisting of tubulins and microtubule-associated proteins (for reviews, see Dustin (1984) and Olmstead (1986)). Dynamic changes in actin-related structures are dependent on Ca2+ concentration and actin-associated proteins. In addition, proteolytic modification and down-regulation of specific proteins are also considered to play main roles in the reorganization of these structures, especially in irreversible pathways. However, a direct demonstration of proteolytic events and the natures of the responsible proteases have not been demonstrated, probably because of the difficulty in investigating degradative processes inside the cell.
Education, Science and Culture of Japan (to Y. E.). The costs of publi-* This work was supported in part by a grant from the Ministry of cation of this article were defrayed in part by the payment of page in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
charges. This article must therefore be hereby marked "advertisement" The nucleotide sequence(s,J reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X78555 $ To whom correspondence should be addressed. Fax: 81-3-5684-2394.
Cytosolic proteolysis is believed to be mediated by strictly regulated proteolytic enzymes including calpains (for reviews, see Suzuki et al. (1987) and Murachi (1989)) and other proteolytic species such as proteasome (for a review, see Rivett (1993)). Among these enzymes, calpain is a leading candidate as a key enzyme for the degradation of receptors, cytoskeletal proteins, transcription factors, and other proteins in accordance with intracellular calcium signaling (Beckerle et al., 1987;Harris et al., 1989;Wang et al., 1989;Wiedmer et al., 1990;Hirai et al., 1991;Oda et al., 1993). In particular, many cytoskeleton-associated proteins are known to be degraded by calpain.
Biochemical and molecular biological analyses have shown that the activity of calpain is dependent on calcium ion (for a review, see Murachi (1989)). An affinity of calpain for phospholipids and its translocation to the cell surface have also been demonstrated (for a review, see Suzuki et al. (1987)). In addition, the molecular structures of calpains from vertebrates have also been studied; it has been shown that calpain contains a catalytic domain in its N-terminal part that is similar in sequence to other cysteine proteinases such as papain and cathepsins B, H, and L, and a calcium-binding regulatory domain in its C terminus similar to other E-F hand proteins such as calmodulin and troponin C (Ohno et al., 1984;Emori et al., 1986;Aoki et al., 1986;Imajoh et al., 1988). In mammals, two ubiquitously distributed calpain species showing different calcium requirement, termed p-calpain and m-calpain, have been known (Murachi, 1989;Ohno et al., 1984;Emori et al., 1986;Aoki et al., 1986;Imajoh et al., 1988). Recently, two tissuespecific calpains, termed nCL-1 and -2, have been identified (Sorimachi et al., l989,1993a(Sorimachi et al., l989, , 1993b, although their biochemical characteristics are not definitely elucidated. However, physiological studies that demonstrate the i n vivo functions of calpain directly have been reported less than studied on its biochemical and structural features.
To resolve these problems, we selected Drosophila melanogaster, for which developmental and genetic approaches are applicable. We targeted here the early Drosophila embryogenesis for the study of calpain functions, because extensive studies about cytoskeletal structures have already been presented. In early Drosophila embryos, it is known that synchronous nuclear division occurs without cytokinesis and that a syncytial blastderm is formed (for details, see Campos-Ortega and Hartenstein (1985)). Cytokinesis takes place after nuclear division cycle 13, and a cellular blastderm is formed. Some of the nuclei move toward the posterior pole and become germline cells (pole cells). During the above processes, both actin-related and tubulin-related cytoskeletons are known to play important roles in nuclear movement and cytokinesis (Warn et al., 1984;Miller et al., 1985Miller et al., , 1989Kellogg et al., 1989) (for a review, see Schejter and Wieschaus (19931311, where protein degradation, possibly catalyzed by proteolytic enzymes such as calpains, Actin-related Cytoskeleton may regulate cytoskeletal structures via irreversible proteolysis against, for example, actin-binding proteins. In this study, we first identified calpain in Drosophila at both the DNA and protein levels. Then we showed that calpain is probably involved in early embryogenesis, especially in the organization of the actin-related cytoskeleton.

MATERIALS AND METHODS
Isolation of Drosophila Calpain cDNA-A Drosophila larval cDNA library was constructed in AgtlO according to a standard procedure (Emori et al., 1987), and screened with a cDNA fragment encoding chicken calpain (Ohno et al., 1984) under less stringent conditions, in that the final wash was carried out in 2 x SSC containing 0.1% SDS. A clone termed ADmCl was shown to code for a protein related to the chicken calpain; this clone was then subjected to nucleotide sequencing and other analyses (Emori et al., 1985).
In Situ Hybridization to Polytene Chromosome-Polytene chromosomes were prepared from salivary glands of D. melanogaster Canton-S and processed for in situ hybridization using biotin-labeled DNA probe according to the standard method (Engels et al., 1986).
Antibody Preparation-A Drosophila calpain cDNA fragment encoding from residue 247 to the C terminus ( Fig. 1) was inserted into the polylinker site downstream of the lac2 promoter of pUC18. Production of the recombinant protein was induced with isopropyl-1-thio-P-D-galactopyranoside, and the product was subjected to 8% SDS-polyacrylamide gel electrophoresis (Emori et al., 1988;Laemmli, 1971). The protein band visualized by Coomassie Brilliant Blue staining was excised and crushed to a paste. The paste was sonicated extensively, mixed with an equal volume of complete Freund's adjuvant, and injected into house rabbits. m e r 3 weeks, booster injections were begun and continued at 2-week intervals; serum was prepared 10 days after each booster. The antibody was affinity-purified as described (Higashijima et al., 1992a(Higashijima et al., , 1992b and adsorbed with a control extract of Escherichia coli not harboring the recombinant plasmid. The quality of the antibody preparations was confirmed by Western blotting as follows. Drosophila extracts were prepared from frozen embryos by homogenizing in five volumes of a buffer containing 20 mM Hepes, pH 7.2, 100 mM KCl, 5% glycerol, 10 mM EDTA, 1 mM dithiothreitol, 1% Triton X-100, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 20 pg/ml aprotinin, and 20 pg/ml leupeptin, and by subsequent centrifugation at 10,000 x g for 5 min. The extracts containing 5-10 pg of protein were electrophoresed in 10% SDS-polyacrylamide gel and blotted onto an Immobilon-P membrane (Millipore); the membrane was immunoreacted with anti-Dm-calpain antibody (1:2000 dilution) using an ABC kit (Vector) and an ECL kit (Amersham Corp.).
Antibodies against actin and tubulin were purchased from ICN. Immunohistochemistry-Drosophila embryos were prepared from a wild type strain (Canton-S) as described previously (Higashijima et al., 1992b). For anti-tubulin antibody staining, taxol was added just prior to fixation (Karr and Alberts, 1986). Fixed embryos were incubated with anti-calpain antibody, prepared as above by a dilution of 1:200, and processed by a standard procedure using an ABC kit (Vector). For double-staining, embryos stained with anti-calpain antibody by 3,3'diaminobenzidine (DAB)' in the presence of NiC1, and CoCl, were further stained with anti-tubulin or anti-actin antibody in the presence of DAB alone. Alternatively, fluorescein isothiocyanate-conjugated secondary antibody was used in the case of anti-tubulin antibody staining. For DNA staining, 4,6-diamidino-2-phenylindole (DAPI) was used after antibody staining.

RESULTS
Drosophila Calpain Is Similar in Overall Structure to Vertebrate Calpains and Contains Distinct Protease and Calciumbinding Domains-Screening of a Drosophila cDNA library with a cDNA probe for chicken calpain (Ohno et al., 1984) yielded several positive clones. One clone was shown to code for a protein with the amino acid sequence characteristics of calpain as shown below;, the encoded protein was termed Dmcalpain. As shown in Fig. l A , the cDNA coded for a protein with 805 residues highly homologous (about 45% identical and about 65% conservative) in sequence to various vertebrate calpains, ' The abbreviations used are: DAB, 3,3'-diaminobenzidine; DAPI, 4,6diamidino-2-phenylindole.
although an insertion sequence of 76 amino acids was found in the C-terminal region. In particular, the catalytic protease domain containing the putative active site showed the highest similarity (about 65% identity, Fig. lA). Additionally, in the C-terminal calcium-binding domain, specific residues important for Ca2+ binding were also homologous and matched the E-F hand criteria (Emori et al., 1986). The highest similarity in the calcium-binding domain was observed in the first E-F hand region (Fig. lA); the similarity in the other E-F hand regions was lower. The encoded protein thus appears to be a calciumdependent proteolytic enzyme.
Regions other than the protease and calcium-binding domains, referred to as domains I and 111, also showed significant sequence similarity to vertebrate calpains. Although the functions of these domains are not fully understood, this similarity suggests common functions of domains I and I11 in Drosophila and vertebrate calpains. Together, the total structure and probable biochemical profile of Dm-calpain is probably equivalent to those of vertebrate calpains.
The sequence homology between Dm-calpain and vertebrate calpains were nearly equal, suggesting that Dm-calpain cannot be classified into a subtype of vertebrate calpains such as mcalpain and p-calpain.
Homology search of the amino acid sequence of Dm-calpain using the SwissProt data base (release 25) yielded known vertebrate calpains as closely related proteins. In addition, several cysteine proteinases such as cathepsins B, H, and L, as well as E-F hand calcium-binding proteins such as calmodulin and troponin C, were shown to have significant similarity, although to much lesser extent than in the case of vertebrate calpains.
In situ hybridization to polytene chromosome revealed that Dm-calpain gene maps at position 50D-E of the right arm of the second chromosome (Fig. 1B 1. Dm-calpain Is Present in Both the Anterior Pole and Posterior Regions of Very Early Embryo-To know the functional features of Dm-calpain, we prepared and affinity-purified specific antibody against Dm-calpain expressed in E. coli. As shown in Fig. 2, this antibody detected a single band with M, -90,000 in a Drosophila extract, consistent with the deduced molecular weight (91,500). Using this antibody as a probe, we investigated the localization of the protein in embryos. As shown in Fig. 3, in the very early embryo, the anterior pole and posterior surface regions of very early embryos show positive signals. Since only a single or a few nuclei (mitotic spindles) visualized by double-staining with anti-tubulin antibody were observed (Fig. 3, A, B, and D), these embryos are believed to represent those just after fertilization.
This anterior expression disappeared rapidly during nuclear division. On the other hand, the posterior signal gradually moved to the posterior pole, and when polar buds can be seen, positive staining was observed around the buds (data not shown). After pole cell formation, the calpain-positive region continues to be observed just below the pole cells (Fig. 3, A and  D). However, it should also be noted that calpain does not seem to be located in the pole cells themselves. This posterior signal gradually becomes weak according to the movement of the pole cells in the dorsal direction (data not shown).
Dm-calpain Co-localizes with Actin-related Cytoskeletal Structure beneath the Precleavage Furrows before Cellularization-When pole cells were formed, the egg surface began gradually to be stained with anti-calpain antibody in hexagonal lattice or circular forms (Fig. 4, A, B , D, and E ) . To know the relationship between the positive regions and the nuclei, double-staining with DAPI was carried out. Comparing the two staining patterns shown in Fig. 4, it can be seen that each calpain-positive lattice surrounds a nucleus. During additional  dilution). Immunoreactive materials were visualized by using an ABC nuclear cycles, a regular staining pattern was observed in the same manner, but the size of the lattice became smaller according to the cycle (Fig. 4, I and J).
Previously, it was reported that actin filaments form cap structure underlying the plasma membrane immediately above the nuclei (Karr and Alberts, 19861, and some actin-binding proteins have been shown to form a transiently hexagonal framework beneath the plasma membrane, especially just below the precleavage furrow (Miller et al., 1989). Double-staining experiments using anti-actin antibody were carried out in order to examine whether the calpain-positive regions are related to the actin structures. Fig. 5 shows that during cycles 9-19, calpain, shown by black or dark brown, localizes significantly between two adjacent actin caps shown by orange or light brown, where reorganization of actin-related cytoskeletons involving several actin-binding proteins as described by Miller et al. (1989) is thought to take place. In addition, the shape of the calpain-positive regions seem to change according to the nuclear cycle visualized as the staining pattern of actin (Karr and Alberts, 1986). "he calpain-positive regions thus seem to be reorganized dependent on nuclear cycles, probably in correlation with actin-related cytoskeletons. DISCUSSION We report here the first identification of a calpain gene in Drosophila. This gene codes for a cysteine protease with a calcium-binding domain that is highly related to known vertebrate calpains. We have also shown that calpain is localized in a specific manner in early Drosophila embryos, probably interacting with actin-related cytoskeletal structures.
Relationship between Dm-calpain and Other Calpains-"he amino acid sequence of Dm-calpain is similar overall to those of previously identified calpains (Ohno et al. , 1984, Emori et al., 1986Aoki et al., 1986;Imajoh et aZ., 1988;Sorimachi et al., 1989). Although the detailed biochemical characteristics of Dm- other hand, the calcium-binding domain shows less similarity to other calpains than the protease domain; thus, the calciumdependence might be somewhat Werent. However, special amino acid residues in the E-F hand structures thought to be important for calcium-binding are well conserved, indicating that, like other calpains, Dm-calpain binds calcium ion (Minami et al. , 1987).
In addition to domains 11 and N, domains I and 111, whose functions have not been definitely established, also show sequence similarity between Dm-calpain and mammalian calpains. This suggests that the total structure of Dm-calpain including domains I and 111 exhibits all of the characteristics observed for calpain molecules.
However, a 76-amino acid insertion is present in domain N ( Fig. 1). A data base search of this sequence yielded no significant similarity to any other known sequences; thus, ita function is unclear at present. It is known that two insertion sequences also exist in a mammalian calpain-related protein, p94, also termed nCL-1 (Sorimachi et al., 1989). One of these sequences is located near, but at a point distinct eom, the Dm-calpain insertion, although no sequence similarity is observed (Fig. 1). This may mean that the insertion points near the bordering regions between domains 111 and Iv are located on the surface of the calpain molecule. Taken together, the structure and biochemical characteristics including proteolytic activity of Dm-calpain should be comparable with those of mammalian calpains. Recently, two novel calpains have been identified in mammals that show tissue-specific expression (Sorimachi et al., l989,1993a(Sorimachi et al., l989, , 1993b; these are in addition to conventional calpains (p-and m-calpains) (Murachi, 1989). Thus, mammals contain ubiquitously distributed p-and m-calpains and tissuespecific calpains termed nCL-1 and nCL-2. In this respect, Dmcalpain belongs to the former type from its expression pattern as shown in Figs. 4 and 5 and from its ubiquitous distribution in later developmental stages (data not shown).
In Drosophila, two calcium-dependent proteolytic activities, termed calpain I and I1 activities, have been identified in adult extracts, and some of their properties have been reported (Pinter and Friedrich, 1988;Pinter et al., 1992). At present, the relationship between these activities and Dm-calpain is obscure (Pinter and Friedrich, 1988). Possible Functions of Dm-calpain in the Early Embryo-Immunohistochemical analysis showed Dm-calpain to be localized in the anterior pole region in very early embryos containing few nuclei (Fig. 3). Although direct evidence was not obtained, this expression may be related to invasion of the sperm nucleus or nuclear fusion or early nuclear division. Namely, it is suggested that Dm-calpain is involved in the rearrangement of the cytoskeleton during fertilization.
On the other hand, the posterior region also contained Dmcalpain from just after fertilization. While the anterior signal