Research Article 2649 Basigin (EMMPRIN/CD147) interacts with to affect cellular architecture

Kathryn D. Curtin1,3,*, Ian A. Meinertzhagen2 and Robert J. Wyman1 1Department of Molecular, Cellular and Developmental Biology, Yale University, 266 Whitney Avenue, New Haven, CT 06511, USA 2Life Sciences Centre, Dalhousie University, 1355 Oxford Street, Nova Scotia, B3H 4J1, Canada 3Department of Biological Sciences, Fulbright College of Arts & Sciences, University of Arkansas, Fayetteville, AR 72701, USA *Author for correspondence (e-mail: [email protected])

Accepted 29 March 2005 Journal of Cell Science 118, 2649-2660 Published by The Company of Biologists 2005 doi:10.1242/jcs.02408

Summary Basigin, an IgG family glycoprotein found on the surface changes in intracellular structure suggest cytoskeletal of human metastatic tumors, stimulates fibroblasts to disruptions. These defects can be rescued by either fly or secrete matrix metalloproteases that remodel the mouse basigin. Basigin and integrin colocalize to cultured extracellular matrix. Using Drosophila melanogaster we cells and to the visual system. Basigin-mediated changes in identify intracellular, matrix metalloprotease-independent, the architecture of cultured cells require integrin binding roles for basigin. Specifically, we found that basigin, activity. Basigin and integrin interact genetically to affect interacting with integrin, is required for normal cell cell structure in the animal, possibly by forming complexes architecture in some cell types. Basigin promotes at cell contacts that help organize internal cell structure. cytoskeletal rearrangements and the formation of lamellipodia in cultured insect cells. Loss of basigin from photoreceptors leads to misplaced nuclei, rough ER and Key words: Basigin, EMMPRIN, CD147, Integrin, Cell structure, mitochondria, as well as to swollen axon terminals. These Drosophila, Gelded

Introduction induction and migration of tumor cells through a reconstituted Basigin or EMMPRIN (extracellular matrix metalloproteinase basement membrane; anti-basigin antibodies block both of inducer, CD147, OX47, 5A11) is a cell-surface glycoprotein of these activities (Kanekura et al., 2002). Fourth, expression of

Journal of Cell Science the IgG superfamily, found on a variety of metastatic tumors basigin in slow-growing breast cancer cells lines that are then (Biswas et al., 1995; Muraoka et. al., 1993; Polette et al., 1997; injected into mouse mammary tissue, leads to larger and more Bordador et al., 2000; Kanekura et al., 2002). Basigin can invasive tumors than controls (Zucker et al., 2001). Lastly, promote matrix metalloprotease (MMP) secretion from basigin promotes adhesion-independent cell growth and this fibroblasts (Kataoka et al., 1993; Li et al., 2001) and also from may contribute to secondary tumor formation (Marieb et al, tumor cells (Sun and Hemler, 2001). The extracellular domain 2004). alone can stimulate fibroblasts to secrete MMPs by interacting Basigin is one of a three-member family in mammals that with an unidentified receptor (Guo et al., 1997). Basigin includes embigin and neuroplastin (SDR1, gp55/gp65). In oligomerizes with itself (Fadool and Linser, 1996; Yoshida et vertebrates, basigin is expressed in a variety of tissues including al., 2000) and it also interacts physically with α3β1 integrin at the developing retina, -brain barrier, CNS, thymus, points of cell-cell contact, but not at focal adhesions epithelial tissues and a variety of immune cells (Fadool and (Berditchevski et al., 1997). are cell-surface dimers Linser, 1994; Fan et al., 1998b). Embigin is expressed in mouse composed of one α and one β subunit that bind to a variety of embryos and many tissues in the adult (Huang et al., 1990; Fan extracellular matrix molecules, as well as to some cell-surface et al., 1998a). Neuroplastin is expressed in the nervous system receptors (Arnaout et al., 2002). They play roles in cell in the cortex, cerebellum and hippocampus (Langnaese et al., attachment, cell migration and cell-cell interactions. The 1997), and in some non-neural tissues. for Drosophila basigin (previously known as gel) has been In mammals, the basigin gene encodes two nearly identical shown to interact genetically with integrins in the Drosophila isoforms both with two IgG-C2 domains (Kanekura et embryo (Reed et al., 2004). al., 1991), as well as an isoform with three IgG-C2 domains Several lines of evidence suggest a role for basigin in (Ochrietor et al., 2003). The neuroplastin gene also encodes metastasis. First, basigin expression and metastasis correlate in two isoforms, a two-IgG protein (gp55) and a three-IgG protein human melanoma (Kanekura et al., 2002). Second, basigin (gp65). Both forms are expressed in the brain, but the gp65 stimulates MMPs from fibroblasts adjacent to tumors (Zucker protein is brain specific (Langnaese et al., 1997). The gp55 et al., 2001) and MMPs remodel the ECM, allowing tumor form is expressed along the axon whereas the gp65 protein is invasion (Nabeshima et al., 2002). Third, co-culture of basigin- concentrated at postsynaptic densities and may play a role in expressing melanoma cells with fibroblasts results in MMP long-term potentiation (Smalla et al., 2000). 2650 Journal of Cell Science 118 (12)

Here we identify previously unknown functions for basigin instructions); biotinylated secondary antibodies were from Vector family by examining the Drosophila homologue, D- Labs. Alexa-568 anti-chicken, Alexa-488 anti-mouse and Alexa-568 basigin. In particular, we find that D-basigin has dramatic phalloidin were all from Molecular Probes. effects on internal cell architecture, both in culture and in vivo, and that it mediates these effects through interactions with Flies and mosaics integrins. This function appears to be independent of MMPs. Mosaics were prepared by the method of Stowers and Schwarz (Stowers and Schwarz, 1999). P-element insertion P1096 and P1478 were obtained from the Bloomington Drosophila Stock Center, as Materials and Methods δ were EGUF/hid lines for FRT40A. The bsg 265 excision allele was Immunohistochemistry created by crossing P1478 to a fly line containing transposase and Adult fly heads were fixed in 3% paraformaldehyde in 5 phosphate selecting for a loss of the eye color marker encoded within the buffer for 5 hours, washed three times for 10 minutes in PBS, engineered P-element. Integrin alleles and integrin monoclonal incubated overnight in PBS plus 20% sucrose and subsequently antibodies were obtained from Daniel L. Brower (University of mounted in TissueTek (Fischer Scientific) and quick frozen in liquid Arizona). nitrogen. Sections 10-15 µm thick were collected onto slides pre- treated with poly-L-lysine (Sigma). Samples were blocked for 1 hour in PBS plus 1% Triton X-100 and either 2% BSA or normal goat Electron microscopy serum (Vector Labs), then incubated overnight in primary antibody en The lamina, innervated by either control or bsgδ265 mutant bloc. Slides were washed three times for 30 minutes in PBS plus 0.5% photoreceptors, was prepared for electron microscopy (EM) using Tween 20. Secondary antibody was applied for 1 hour. Biotinylated previously reported methods (Meinertzhagen, 1996; Meinertzhagen goat anti-rat, anti-mouse or anti-chicken secondary antibodies (Vector and O’Neil, 1991). Single sections containing cartridge profiles cut in Labs) were resuspended in 1 ml PBS and diluted 1:200 in blocking cross-section were examined and digital montages collected from solution. Slides were washed as before. The ABC HRP (Vector images obtained with a Philips Tecnai 12 operated at 80 kV, using a Labs, PK-6100) was used according to the manufacturer’s Kodak Megaview II camera with software (AnalySIS, Soft Imaging instructions. Visualization was via the Vector VIP stain (SK4600). System, Münster). Slides were mounted with Permount and photographed with a digital camera. Immunofluorescence images were prepared from material fixed and treated the same way, but using fluorescent secondary Northern blots antibodies (described below). These samples were visualized by Total mRNA was isolated from animals at different developmental fluorescence microscopy and the images converted to black and white stages using a kit (Qiagen) and RNA was quantified by running in Adobe Photoshop. samples on a gel and estimating the relative intensity of rRNA bands. Blotting was done by standard techniques (Sambrook and Russell, 2000). A radioactive probe was made to the cloning region of basigin Cell culture and labeling by isolating the basigin gene from an agarose gel and using the gene S2 cells were grown in Schneider’s medium with 10% FBS as a template in a random primer reaction made using a random primer (Invitrogen). High Five cells were grown in HyQ serum-free media kit (New England Biolabs). The probe was labeled with [32P]dCTP 5 supplemented with 10% calf serum (Invitrogen). Between 2 10 and and used to probe the mRNA, as described (Sambrook and Russell, Journal of Cell Science 5 5 10 cells were plated on 25-mm-square coverslips pre-treated with 2000). 0.1 mg/ml poly-L-lysine (Sigma). They were allowed to attach for 18 to 36 hours before fixation for 20 minutes in 4% paraformaldehyde. Fixative was washed out with four rinses in PBS. Cells were BLAST analysis permeabilized for 10-20 minutes with PBS plus 0.3% Triton X-100. BLAST analyses of mammalian using the Drosophila genome Cells were blocked for 15 minutes in blocking buffer (PBS containing as a database for comparison were carried out at the Berkeley 0.3% Triton X-100 and 2% BSA, Sigma). Primary and secondary Drosophila Genome Project (http://www.fruitfly.org/blast/ antibodies were diluted in blocking buffer and applied for 1 hour each. blast_form.html.) using full protein sequences for neuroplastin, Each antibody incubation was followed by three washes with 0.3% basigin and embigin. Default settings for the site were used. An amino Triton X-100 in PBS. Coverslips were dried and mounted in glycerol acid-based search was chosen and the database chosen included all gelatin (Sigma) with 1 mg/ml p-phenylene diamine (Sigma). predicted proteins for the genome. Predicted proteins that had basigin S2 cells with and without integrin genes were obtained from Daniel homology were examined by following available links to see if these L. Brower (University of Arizona). High Five cells and the pIZT proteins were of similar length to basigin and had characteristic expression vector with the V5 tag were obtained from Invitrogen and features of the basigin protein family, including transmembrane the bsg265 transgene from Research Genetics. Cells were transfected domains, IgG domains and high in or near the with Cellfectin (Invitrogen). transmembrane domain.

Antibodies and labels D-basigin peptide antibody was raised in chickens (Alpha Results Diagnostics) to LIADENKFIIDKTDTNDDGKYSC, a peptide Structure of the Drosophila basigin gene uniquely found in D-basigin. Other antibodies were obtained from the In a screen for genes that act in the eye we discovered the gene β following sources: Anti- -gal, Promega (#Z378A) used 1:1000; anti- for the Drosophila homologue of mammalian basigin. This V5 (Invitrogen) used 1:500, anti-αPS1 (monoclonal DK.1A4), anti- gene maps to cytological band 28E3, spans 25 kb and had been βPS integrin (monoclonal CF.6G11) and anti-αPS2 (CF.2C7) were obtained from Daniel L. Brower (University of Arizona); anti-elav previously designated gelded (gel) (Castrillon et al., 1993), but (mouse and rat monoclonal antibodies), anti-repo and 24B10 from the has been renamed basigin (bsg). We used all three mammalian Developmental Studies Hybridoma Bank (University of Iowa); anti- basigin protein sequences to search the Drosophila genome for tubulin (Sigma T4026, used according to the manufacturer’s predicted proteins related to basigin and found that bsg is the Basigin and integrin affect cell structure 2651 only Drosophila gene that encodes a basigin family member in the dish). The second class of cells showed elaborated flies. According to data from the Drosophila genome project microfilaments (Fig. 3C, Fig. 4F) and microtubules (Fig. 3B) (available at Flybase, http://flybase.bio.indiana.edu/), the bsg throughout the cytoplasm. These cells appeared flattened to the gene encodes nine distinct transcripts that appear to fall into dish in microscopy. three structural classes (Fig. 1). These transcripts encode two When D-basigin protein was expressed in these cells, the predicted protein isoforms of D-basigin, 265 and 298 amino number of each cell type changed noticeably. About 85% of acids, each with two IgG-C2 domains, a transmembrane domain control High Five cells showed cortical actin microfilaments and a short internal tail (Fig. 2A). Class 1 and 2 bsg transcripts (Fig. 3C and similar to Fig. 4F) and a round morphology with contain distinct 5′ non-coding exons that splice onto common a nuclear concentration of tubulin (Fig. 3B), whereas only 15% coding exons to code for D-basigin 265. The class 3 bsg of cells showed elaborate microfilaments and microtubules and transcript encodes D-basigin 298. The two isoforms are a flattened appearance by light microscopy. By contrast 80% identical over 240 amino acids, differing only at the N- and C- of D-basigin-expressing cells showed an elaboration of termini. microfilaments (Fig. 3A, Fig. 4E) and microtubules (Fig. 3B) A northern blot showed that bsg was expressed in all stages whereas only 20% showed a rounded morphology with cortical of Drosophila development tested (Fig. 1B). Only two sizes of actin and a nuclear concentration of tubulin. Thus basigin bsg message were seen. The larger, more diffuse band expression in High Five cells led to a fivefold increase in the corresponded to class 1 and 2 transcripts that have a nearly number of cells showing elaborated microfilaments and identical predicted size of ~2.5 kb (Fig. 1B). Around microtubules and a flattened appearance. This change in embryonic stage 5 a slightly smaller transcript was expressed cytoskeletal rearrangement seemed to result from the cell- that corresponded more closely to the predicted size of ~2 kb autonomous expression of D-basigin. First, these changes were for the class 3 transcript. As no other transcript sizes were seen, independent of cell contact, as physically isolated basigin- we concluded that the Drosophila bsg gene probably encodes expressing cells were just as likely to show the altered only the two predicted isoforms of D-basigin. The expression cytoskeletal arrangement as cells that were touching. Second, of this smaller transcript at very early stages may mean that it these changes in cell architecture were not due solely to is maternally contributed.

Homology between mouse and fly basigin Mouse basigin showed 26% identity and 34% similarity with Drosophila basigin protein. The extracellular domains showed 20% identical residues and 28% similar residues, whereas there was 80% identity in or near the transmembrane domains (Fig. 2). Indeed, the transmembrane domains of basigin, neuroplastin and embigin from many different species show very high identity (Fig. 2) (Ochrietor et al, 2003), including Journal of Cell Science spaced leucines, as well as conserved proline and glutamic acid residues. The presence of a charged residue in the transmembrane domain is consistent with the fact that basigin forms complexes (Fadool and Linser, 1996), possibly within the plane of the membrane. There was no homology in the short internal tail between mouse and D-basigin with the exception of the first five cytoplasmic residues (Fig. 2). D- basigin showed 30% similarity to both rat neuroplastin and rat basigin.

D-basigin promotes cytoskeletal rearrangement in cultured cells Fig. 1. Basigin gene structure and developmental northern blots. 265 (A) Structure of the bsg gene encodes nine transcripts according to The bsg transgene that codes for D-basigin 265 was data from the Drosophila genome project. Transcribed portions of introduced permanently into insect High Five cells. These cells the gene are shown as blue boxes. Transcripts fall into classes 1a-d, are derived from the embryo of the cabbage looper 2a-d and 3. Transcripts 1a-d and 2a-d encode the same protein, D- (Trichoplusia ni) and used as a baculovirus expression system. basigin 265 (sequence shown in Fig. 2). Transcript 3 encodes a High Five cells permanently transfected either with empty slightly longer protein, D-basigin 298. The P elements, P1096 and vector or with bsg265 transgene were labeled with Alexa 568- P1478, inserted at precisely the same location (marked by arrow) phalloidin to visualize actin microfilaments (Fig. 3A), or with 1145 bp from the ATG for D-basigin 265 and 981 bp from the start anti-tubulin antibody to visualize microtubules (Fig. 3B). Two of the first coding exon. The location of the start codon for transcript 3 is also indicated. The excision line 19 (black bar) contains a 4 kb classes of cells were seen showing two clearly distinct deletion that removes the first coding exon. (B) Northern blot cytoskeletal arrangements. One class of cells showed actin showing expression of bsg in embryos at stages 5, 9, 13, and 16, first- filaments in an almost exclusively cortical pattern (Fig. 3A, , second- and third-instar larvae and adult heads (A). The major band Fig. 4F). These cells invariably showed a nuclear concentration of ~2.5 kb is consistent with the length of class 1 and 2 bsg of tubulin (Fig. 3B) and were spherical (not flattened to transcripts. 2652 Journal of Cell Science 118 (12)

M E A K F L A S A L S F L S I F L A I Y A Q S L D K L V P N Y D N A E H Q M K F Y D I R S *pi45 Droso hg5ila bas in 26 M A A A L F * * * V L L * * G F * * * * * A L L G T H G A S G A A G T V F T T V E D L G S K 3g26 Human Basi in M A A A L L * * * L A L * * A F * * * * * T L L S G Q G A C A A A G T I Q T S V Q E V N S K 3g26 Mouse Basi in M A A A L L * * * L A L * * A F * * * * * T F L S G Q G A C A A A G T I V T S V Q E V D S K 36 Rat Basigni M S G S S L P G A L A L * * S L L L V S G S L L P G P G A A Q N E P R I V T S E E V I I R D 44 Rat gop5pn5/Neur lasti

* * * P L V L S C N V K D G T P G G V * L I W K K N G T A V T D V P S L R G R F K L I A DpiE 87 Droso hg5ila bas in 26 I L L T * * * * C S L N D S A T E V T G H R W L K G G V V L K E D A * * * * * * * L P Gg2Q K 71 Human Basi in T Q L T * * * * C S L N S S G V D I V G H R W M R G G K V L Q E D T * * * * * * * L P D L Hg271 Mouse Basi in T Q L T * * * * C F L N S S G I D I V G H R W M R G G K V L Q E D T * * * * * * * L P D L Qgn71 Rat Basi i S L L P V T L Q C N L T S S S H T L M Y S Y W T K N G V E L T A T R * * * * * * * K N A SpN 83 Rat go5pn5/Neur lasti

N K F I I D K T D T N D D G K Y S C E F D G V S K * * * * * E I E V I A R V V V R V PpiS N T 128 Droso hg5ila bas in 26 T E F K V D S * * D D Q W G E Y S C V * * F L P E P M G T A N I Q L H G P P R V K A V K S S 1g213 Human Basi in T K Y I V D A * * D D R S G E Y S C I * * F L P E P V G R S E I N V E G P P R I K V G K K S 1g213 Mouse Basi in M K Y T V D A * * D D R S G E Y S C I * * F L P E P V G R G N I N V E G P P R I K V G K K S 1gn13 Rat Basi i M E Y R I N K P R A E D S G E Y H C V Y H F V S A P K A N A T I E V K A A P D I T G H K R S 1p29 Rat go5pn5/Neur lasti

A * V V E G E K M S V T C S V V G T K P E L T * W T F A N V T * * * * * * * * * * L T N A Tpi162 Droso hg5ila bas in 26 E H I N E G E T A M L V C K S E S V * P P V T D W A W Y K I T D S * E D K A L M N G Sg2E * * 155 Human Basi in E H S S E G E L A K L V C K S D A S Y P P I T D W F W F K T S D T G E E E A I T N S T E A N 1g259 Mouse Basi in E H A S E G E F V K L I C K S E A S H P P V D E W V W F K T S D T G D Q * T I S N G T E A N 1g58 Rat Basi in E N K N E G Q D A M M Y C K S * V G Y P H * P E W M W R K K * * * * E N G V F E E I S N SpS 170 Rat g 5pn5/Neuro lasti

D R F I L K P D D N G V P N A I L T L D N V T L D D R G E Y K C I G R N A ApiN V Y G G N T T 208 Droso hila bas gin 265 S R F F V S S S Q G R S E L H I E N L N * * M E A D P G Q Y R C N G T S S * *i* * * * * * K 191 Human Bg2as in G K Y V V V S T P E K S Q L T I S N L D * * V N V D P G T Y V C N A T N A * * * * * * * * Q 195 Mouse Basigin 2 S K Y V I I S T P E L S E L I I S D L D * * M N V D P G T Y V C N A T N S * * * * * * * * Q 1gn94 Rat Basi i G R F F I I N K E N Y T E L N I V N L Q * * I T E D P G E Y E C N A T N Sp* * * * * * * * I 206 Rat go5pn5/Neur lasti

T P A S D V T T V R V K G K F A A L W P F L G I C A E V L I L C I I I L I Y E K R R N K S E 2pi54 Droso hg5ila bas in 26 G S D Q A I I T L R V R S H L A A L W P F L G I V A E V L V L V T I I F I Y E K R R K P E D 237 Human Basigin 2 G T T R E T I S L R V R S R M A A L W P F L G I V A E V L V L V T I I F I Y E K R R K P D Qg241 Mouse Basi in 2 G S A R E T I S L R V R S R L A A L W P F L G I V A E V L V L V T I I F I Y E K R R K P D Qgn240 Rat Basi i G S A S V S T V L R V R S H L A P L W P F L G I L A E I I I L V V I I V V Y E K R K R P D E 2p52 Rat g 5pn5/Neuro lasti

* L E E S D T D P Q E Q 2p65 Droso hgila basi in 265 V L D D D D A G S A P L K S S G Q H Q N D K G K N V R Q R N S S 269 Human Basig2in T L D E D D P G A A P L K G S G T H M N D K D K N V R Q R N A T 2g273 Mouse Basi in T L D E D D P G A A P L K G S G S H L N D K D K N V R Q R N A T 2gn72 Rat Basi i V P D D D E P * A G P M K T N S T N * N H K D K N L R 277 Rat gop55/Neur pnlasti L E E S D T D P Q E Q K K * * K R R N YpiD Droso hg7ila bas in 29

Fig. 2. Sequence alignment of basigin protein family members. Identical residues are marked in black and similar residues in yellow. Only the extreme C-terminus of D-basigin 298 is shown (last line). The first 50 amino acids (aa) of D-basigin 298 are not homologous to the first 24 aa of D-basigin 265. Then, the two proteins are identical for the 240 aa from residue 25 in D-basigin 265 (corresponding to residue 50 in basigin 298) through to the end of D-basigin 265. There are eight additional aa at the C-terminus of D-basigin 298. D-basigin 298 accession number CG31605-PG can be found at NCBI or fl[email protected].

secretion of a soluble factor by D-basigin-expressing cells, as changes in cell architecture depended on integrin binding. medium conditioned by such cells did not induce non- Because many integrins bind to ECM molecules, such as Journal of Cell Science transfected High Five cells to spread out and elaborate collagen and fibronectin, at an Arg, Gly, Asp (RGD) target microtubules and microfilaments. sequence (Arnaout et al., 2002), the peptide GRGDS is commonly used as a competitive inhibitor for such integrin binding (Huang et al., 1993). When D-basigin-expressing cells D-basigin colocalizes to the actin cytoskeleton were cultured in the presence of a GRGDS peptide (Fig. 4F), The D-basigin protein expressed in High Five cells had a V5 the cells looked indistinguishable from control High Five cells, epitope tag at its C-terminus. Antibody to this tag was used to showing a rounded morphology with cortical actin filaments. assess the subcellular distribution of D-basigin (Fig. 4A,C). D- By contrast, D-basigin-expressing cells grown without basigin-V5 expression was found in three patterns. First, it was peptide (Fig. 4E) had elaborated microfilaments and a found in a fine granular pattern throughout the cell membrane flattened appearance. D-basigin-expressing cells were much (Fig. 4A,C). Second, D-basigin was expressed in a punctate less affected by a control peptide, GRGES at the same fashion, visible as bright spots (Fig. 4A) seen to be vesicles by concentration of 200 µg/ml (not shown). Cells incubated with phase-contrast microscopy. High Five cells normally contain GRGES showed that 65% of the cells spread compared to 80% many vesicles even when D-basigin is not expressed. Lastly, a of control cells. subset of D-basigin immunolabeling colocalized to the actin cytoskeleton, especially at points of cell-cell contact (Fig. 4A,B) and near cell edges (Fig. 4C,D). The degree of D-basigin partially colocalizes with integrin in integrin- colocalization in isolated cells varied. However, in cells that transfected S2 cells were in physical contact, D-basigin-actin colocalization at cell- Previous work indicated that basigin colocalizes with some cell contacts was invariable (Fig. 4A,B). integrins at cell-cell contacts (Berditchevski et al., 1997). To examine if D-basigin and integrin colocalize within the cell, we generated antibody to a peptide in the extracellular domain D-basigin-mediated changes in cellular architecture of D-basigin. This antibody did not label control High Five require integrin binding cells, but did label D-basigin–V5-expressing High Five cells. Integrins can promote cell attachment and cause cells to spread When these latter cells were double-labeled with both the out in culture. We therefore tested whether D-basigin-mediated peptide antibody and the V5 antibody, nearly identical patterns Basigin and integrin affect cell structure 2653 cell contact. Integrin-specific antibodies also showed a clear line of expression at the basement membrane (arrows, Fig. 5C) whereas D-basigin antibody did not strongly label the membrane (Fig. 5B) in most samples. Integrins are expressed in retinal pigment cells, and this line may represent the focal adhesions that the cells make with the basement membrane (Longley and Ready, 1995). D-basigin was not expressed in these pigment cells (see below).

D-basigin is expressed in photoreceptors and in basal glia The above labeling did not allow us to identify the specific retinal cell types that express D-basigin protein. To identify these, we examined expression from an enhancer trap line in the gene for D-basigin, bsg. Two P-element insertions in bsg (P1096 and P1478, insertion point indicated in Fig. 1A) were Fig. 3. D-basigin expressing cells spread and form lamellipodia. obtained from the Bloomington Drosophila Stock Center. Both (A) Actin cytoskeleton of High Five cell labeled with Alexa-568 contain a bacterial lacZ gene encoding a nuclear form of β- phalloidin. (B) The same cell labeled with anti-tubulin antibody and galactosidase. This lacZ gene contains no regulatory sequences visualized with Alexa-488 conjugated secondary antibody. (C) High and thus the bsg regulatory elements should drive expression Five cell expressing basigin labeled with Alexa-568 phalloidin. (i.e. it should act as an ‘enhancer trap’). Anti-β-gal revealed (D) The same cell as in C labeled with antibody to tubulin. High Five cells have a spherical morphology (A and B) whereas High Five cells expression in photoreceptors and basal glia in adult head- expressing D-basigin form lamellipodia (C and D). Bar, 40 µm. sections from both lines (Fig. 5A). Basigin expression was examined in the larval eye disc, using both the enhancer trap line and in-situ hybridization, and no exception was seen in of labeling were seen, suggesting that this antibody indeed either of these cell types at this stage. recognized Drosophila basigin. We also saw clear labeling of Drosophila S2 cells with D-basigin antibody, consistent with data from the Drosophila genome project indicating that S2 Basigin gene mutations cells express D-basigin. Control staining of S2 cells with anti- The two P-element insertions in bsg mentioned previously, integrin antibody showed no staining as expected. labeled P in Fig. 1A, are located 1145 bp from the start of We could not look for colocalization of D-basigin and transcription for the D-basigin 265 protein isoform (Fig. 1A). integrin in High Five cells because antibodies to High Five Homozygous mutant animals from both lines died after the integrins are not available. Moreover, normal Drosophila S2 second larval instar with only 3% of mutant larvae living to the Journal of Cell Science cells do not express integrins. We therefore used genetically third instar. The insertions failed to complement each other. altered S2 cells that were permanently transfected with genes Because this P-insertion did not interrupt the coding portion of for αPS1 and βPS integrins expressed under control of a heat- the gene, animals carrying this mutation may have produced shock promoter (Gotwals et al., 1994). These cells were some functioning protein. To generate a more severe allele, the induced to express integrins and then double-labeled with anti- P-element (P1478) was mobilized; such mobilization D-basigin and a mixture of monoclonal antibodies against both occasionally caused loss of genetic material near the insertion αPS1 and βPS integrins. D-basigin and integrin showed partial site. We established 200 excision lines in which the P-element colocalization in the cell (Fig. 4G,H), although there was was missing; 182 were viable, indicating a clean excision of consistently more basigin expression around the cell body. This the P-element, whereas 18 were homozygous lethal and failed suggests that basigin and integrin can at least partially to complement the original P-element allele. By DNA blot δ colocalize if expressed together. analysis, two excision lines, bsg 265 and excision number 64, were shown to be missing ~4 kb, including the first coding exon for the D-basigin 265 protein. Both lines showed high D-basigin partially colocalizes with integrin in the retina embryonic lethality with 75-80% of the animals dying as We next looked for colocalization between D-basigin and embryos. Those embryos that did hatch died within the first integrin in the Drosophila visual system because we had day and were small, lethargic and uncoordinated. originally identified bsg in a visual system screen. Adult head sections were double-labeled with anti-D-basigin (Fig. 5B) and monoclonal antibodies against βPS integrin (Fig. 5C), which D-basigin affects the subcellular structure of are expressed in the retina (Brower et al., 1995). photoreceptor neurons D-basigin antibody (Fig. 5B) revealed lines of Given that D-basigin affects cell architecture in culture, we immunofluorescent puncta in the retina. Labeling the same were interested to know if it affected cell structure in the sections with anti-αPS1 integrin antibodies (Fig. 5C) or anti- animal. To address this, we looked for the effects of D-basigin βPS antibodies (not shown) revealed multiple points of on placement of internal cellular organelles in photoreceptors. colocalization at these puncta, the positions of which did not Because the mutations are embryonic lethal, we made mosaic correspond to ECM and were therefore probably points of cell- animals in which D-basigin protein expression was missing 2654 Journal of Cell Science 118 (12) only in the eye and invariably missing from photoreceptor photoreceptor nuclei lie in tight rows across the eye (e.g. Fig. neurons. We generated such mosaics by the method of Stowers 6C), so that any mislocalization is readily detected. The nuclei and Schwarz (Stowers and Schwarz, 1999) in which FLP of the R1-R6 photoreceptors lie in the apical region of the recombinase is expressed from the eye-specific promoter of the retina (Fig. 6C). The nuclei of the R7 photoreceptors are just eyeless gene (ey). Eyeless-FLP mediates recombination in the proximal to those of R1-R6 and the R8 nuclei lie near the eye between arms bearing engineered copies of basement membrane of the retina (Fig. 6C). the FLP binding sites (FRTs) near their centromeres. We Photoreceptor nuclei of mosaic flies mutant in the eye for recombined a chromosome arm bearing a bsg mutation with a the hypomorphic P1096 allele, which encodes a nuclear β-gal, chromosome arm bearing the cell death gene hid expressed were visualized with anti-β-gal (Fig. 5A). Most nuclei were specifically in all photoreceptors. After recombination and properly located, although a few nuclei were misplaced (Fig. chromosome segregation, only photoreceptors that inherit two 6A). We saw similar results for these mosaics with anti-elav copies of mutant bsg survive to repopulate the eye; bsg eyes (not shown). In mosaics that are mutant in the eye for the were almost normal in size. bsgδ265 excision allele, elav immunolabeling revealed that 16- Photoreceptor nuclei were visualized with an antibody 50% of photoreceptor nuclei were mislocalized (Fig. 6B). against elav, a neuron-specific nuclear protein. Normally, Nuclei were counted as misplaced only if they were obviously located between the normal position for R7 and the normal position for R8, in the region of the eye where no nuclei are usually located (see Fig. 6C). Thus nuclei that were slightly displaced were not counted. Sections from a total of 18 animals were counted (10,250 nuclei). Although the range of nuclear misplacements per fly was 16-50%, most animals fell within the lower end of this range, the average number of misplaced nuclei, pooling data from all animals, being 22%. The nuclear placement defect was rescued by expressing D- basigin 265 (Fig. 6C). We counted nuclear placement in 12 animals that were mutant in the eye for bsgδ265, but also contained a bsgδ265 transgene that expressed D-basigin 265 in photoreceptors and found only 1% of misplaced nuclei. Expression of the mouse basigin gene in photoreceptors also rescued the nuclear misplacement (Fig. 6D) with only 1.5% of nuclei misplaced in a total of 12 animals counted (7300 nuclei counted). Thus despite limited sequence homology, mouse basigin can promote the formation of normal cell architecture in flies. Photoreceptors R1-R6 terminate in the lamina, or first Journal of Cell Science optic neuropile. We examined laminas in which only the photoreceptors are mutant for bsgδ265 (i.e. the postsynaptic lamina neurons and glia are wild type). Rough endoplasmic reticulum (rER) was found misplaced into the mutant

Fig. 4. D-basigin partially colocalizes with actin and integrin. (A-F) High Five cells expressing D-basigin with a C-terminal V5 tag. (A and C) Cells labeled with anti-V5 antibody and visualized with Alexa 488-conjugated secondary antibody. (B,D-F) Cells labeled with Alexa 568-phalloidin to visualize F-actin. (A) D-basigin is expressed diffusely and in numerous vesicles within the cell. There is strong D-basigin expression at cell-cell contacts (arrow). (B) Actin colocalizes with D-basigin at cell-cell contacts (arrow). (C,D) High magnification view of the edge of a basigin-expressing cell (ca. 15 µm total width). There is significant colocalization of D-basigin (C) and actin filaments (D) as shown for marked filaments (asterisks). Some actin filaments do not colocalize with D-basigin (red arrowhead in D). D-basigin-mediated cell spreading requires integrin activity. (E) D-basigin-expressing High Five cells cultured under normal conditions. (F) Cells cultured in the presence of 200 µg/ml GRGDS peptide which competes for integrin binding sites. (G) S2 cell expressing Drosophila αPS1βPS integrins labeled with antibody against D-basigin and visualized with Alexa 568-conjugated secondary. (H) The same cell as in G labeled with a mixture of antibodies to αPS1 and βPS integrin and visualized with Alexa 488- conjugated secondary antibody. D-basigin and integrin colocalize at many sites. Bar, 20 µm (A,B); 60 µm (E,F); 10 µm (G,H). Basigin and integrin affect cell structure 2655

photoreceptor axon terminals. Normally rER, which is To look for genetic interactions between bsgδ265 and integrin continuous with the nuclear membrane, is confined distally to genes, we made double mutants by creating males that carried the photoreceptor cell body in the overlying retina. Its more the mysb45 allele (coding a mutant βPS integrin), but were also proximal displacement into the photoreceptor terminal in the homozygous mutant only in the retina for the P1096 bsg allele. lamina accords with the more proximal location of many R1- These animals showed obvious misplacement of nuclei (Fig. R6 nuclei (Fig. 7D). In addition to misplaced nuclei, 6F). The average number of misplaced photoreceptor nuclei mitochondria were also misplaced. The mitochondria per head section, after examining at least 12 animals of each accumulated in excessive numbers in the distal portion of the genotype, was three times higher in the double mutants than photoreceptor terminals (Fig. 7B), but were absent from the that predicted from the summed effect of the two single proximal portion of the terminals, where they are also mutations. Mosaics doubly mutant for mysb45 and bsgδ265 also normally found (Fig. 7C). In addition to misplaced organelles, showed a more severe photoreceptor nuclear misplacement δ bsg 265 mutant photoreceptors showed a clear increase in axon phenotype than the sum of the two single mutations would terminal size, with profiles that were >80% larger in cross- predict; 80% of nuclei were misplaced (not shown) compared sectional area (compare Fig. 7A,B) compared to the control, with an average of 24% for bsgδ265 and 1-2% for mysb45. a difference that was significant (P<0.0006; Student’s t-test; Some integrin gene allelic combinations also showed mean of means of three flies per group). None of these nuclear misplacement. Animals heterozygous for mewM6, a defects was seen in control animals in which non-mutant null allele for αPS1 integrin (Brower et al., 1995) showed were recombined (Fig. 7A). On the whole, these normal placement of photoreceptor nuclei. Animals defects, misplaced internal organelles and enlarged terminals, heterozygous for mysb45, a βPS1 allele, showed normal δ suggest global disruptions in cell structure in bsg 265 mutant nuclear placement, similar to the mysb45 hemizygous males cells (see Discussion), probably due to alterations in the (Fig. 6E) just discussed. However, animals heterozygous for cytoskeleton. both mewM6 and mysb45 showed 3% misplaced nuclei (Fig. 6G; >600 nuclei from three different animals counted). D-basigin and integrin interact genetically to affect retinal cell structure MMP2 is not required in the eye for photoreceptor We found colocalization of integrin and D-basigin in the architecture retina, as previously described. In addition, previous studies Because mammalian basigin stimulates secretion of MMPs, of integrin gene mutants reported that the R8 nuclei are we examined the role of MMPs in the fly visual system. sometimes misplaced, descending beneath the basement Drosophila has two MMP genes, Mmp1 and Mmp2, both membrane (Longley and Ready, 1995). We sometimes saw required for viability. Only Mmp2 is expressed in the δ this in bsg 265 mosaics and therefore looked for genetic developing eye (Llano et al., 2000; Llano et al., 2002; Page- δ interactions between bsg 265 and integrin genes with respect McCaw et al., 2003). If D-basigin were acting primarily to nuclear placement. through MMP-2, then flies lacking MMP-2 in the retina The integrin proteins expressed in the eye, αPS1 and βPS should have the same phenotypes as those found in bsgδ265 Journal of Cell Science (Zusman et al., 1993; Roote and Zusman, 1996), are encoded mutant retina. Using the same method previously described to by genes located on the X chromosome, mew codes αPS1 make bsgδ265 eye mosaics, we made flies that were mutant in integrin and mys codes βPS integrin. Mysb45 is a viable allele the eye for a null Mmp2 allele, Mmp2w307* (Page-McCaw et (Jannuzi et al., 2004) and males carrying this mutation showed al., 2003). We saw no misplaced photoreceptor cell nuclei normal placement of photoreceptor nuclei (Fig. 6E). Mutant (Fig. 6H). In case MMP1 functionally replaces MMP-2, we flies homozygous in the retina for a weak P-allele (P1096) of made mosaics that were mutant in the eye for both genes. basigin showed occasional nuclear misplacement (Fig. 6A). These also showed no misplaced nuclei (not shown). Finally,

Fig. 5. D-basigin partially colocalizes with integrin in the visual system. (A) Frozen section of retina (re) and lamina (la, bracket) of fly heterozygous for P1096 enhancer trap. This expresses nuclear β-gal and is labeled with anti-β-gal and visualized with Vector ABC kit. Expression was seen in the nuclei of photoreceptor neurons R1-6 (downward pointing arrow; these nuclei occur in a tight distal cluster in each ommatidium) and R7 (upward pointing arrow) and in the migratory retinal basal glia (RBG, arrowhead) (Choi and Benzer, 1994). The RBG lie just beneath the retinal basement membrane. (B) White-eyed animal labeled with anti-D-basigin and visualized with Alexa 568-conjugated secondary antibody. (C) The same section labeled with anti-βPS1 integrin monoclonal antibody, visualized with Alexa 488-conjugated secondary antibody. D-basigin and integrin colocalize to several places, see arrowheads. However, integrin is expressed in a thin line at the basement membrane, which lacks D-basigin expression (arrows in C). Bar, 25 µm. 2656 Journal of Cell Science 118 (12) we saw no effect on nuclear placement when we drove Discussion expression of Drosophila TIMP (tissue specific inhibitors of Basigin is found on a wide variety of metastatic tumors and MMPs) in the eye (not shown), even though this TIMP gene has been shown to enhance tumor growth and invasiveness has previously been reported to block biological activity of (Muraoka et. al., 1993; Polette et al., 1997; Bordador et al., Drosophila MMPs (Page-McCaw et al., 2003). 2000; Kanekura et al., 2002; Zucker et al., 2001). In mammalian systems, basigin stimulates secretion of MMPs (Li et al., 2001; Kanekura et al., 2002). MMPs, in turn, promote tumor cell invasion by breaking down the ECM (Kataoka et al., 1993; Li et al., 2001; Sun and Hemler, 2001). Thus basigin has been thought to act outside the cell, primarily though MMPs, even though basigin has also been shown to interact with several different molecules suggesting that at a molecular level it may be multifunctional (reviewed by Toole, 2003). In this study we identify previously uncharacterized roles for basigin. We show that D-basigin affects the intracellular architecture of the cells in which it is expressed, possibly by complexing with integrins and actin. This function appears not to require MMPs.

D-basigin alters cell structure In High Five cells, D-basigin expression promotes the rearrangement of both the actin and tubulin cytoskeleton with consequent formation of lamellipodia. Likewise, in Drosophila photoreceptors, D-basigin is required for normal cell architecture. In mosaics in which D-basigin expression is missing from the photoreceptors, the nuclei, rough endoplasmic reticulum and mitochondria in these cells are all misplaced. Photoreceptor terminals mutant for bsgδ265 are also larger than wild-type terminals. All these defects suggest disruption of the cytoskeleton.

D-basigin acts through integrins Journal of Cell Science There are many reasons to believe that D-basigin affects cell structure by interacting with integrins. First, D-basigin- mediated cell spreading is blocked by peptides that block integrin binding sites. Second, D-basigin and integrin Fig. 6. D-basigin expression in the retinal photoreceptor neurons is colocalize in the Drosophila retina. Third, D-basigin and necessary for the proper placement of their nuclei. Frozen sections integrin colocalize to sites within the cell in integrin-expressing through the retina (brackets in B and C); la, lamina. The normal Drosophila S2, as well as in human cells (Berditchevski et al., locations of the R1-8 nuclei are marked in C. (A) Section labeled 1997), in which the two proteins also co-immunoprecipitate. with anti-β-gal expressed in R1-R6 nuclei. (B-G) Sections labeled This suggests they may form a complex in the membrane. with antibody to the neural nuclear protein, elav. (A) Mutant for the Fourth, allelic combinations of integrin gene mutations show hypomorphic bsg allele, P1096. A few photoreceptor nuclei are δ265 disruption of retinal cell structure, e.g. misplaced nuclei, displaced (arrow). (B) Mutant for bsg . Nuclei are scattered δ265 δ265 throughout the retina (arrows). (C) Mutant for bsgδ265 rescued by a similar to those in bsg mosaics. Fifth, bsg and integrin GMR-bsg265 transgene that drives expression of D-basigin 265 in all gene mutations interact to affect nuclear placement. Sixth, bsg photoreceptor neurons. The nuclei of the R7 photoreceptors are just and integrin genes have been shown to interact genetically to proximal to those of R1-R6 and the R8 nuclei lie near the basement affect dorsal closure and germ band retraction in the membrane of the retina. (D) Mutant for bsgδ265 rescued by a GMR- Drosophila embryo (Reed et al., 2004). mouse basigin transgene (Bsg). D-basigin and integrin interact to Two additional findings support the idea that D-basigin and affect nuclear placement. (E) Male carrying a viable allele of δ intregin interact to affect cell structure. First, antibodies against integrin, βPS1 (mysb45). (F) integrin bsg 265 double mutant male D-basigin can block integrin-mediated adhesion of T cells to b45 carrying mys also mutant in the eye for the P1096 bsg. Arrows ECM (Allain et al., 2002). Second, expression of embigin, a indicate some of the misplaced nuclei. The number of misplaced basigin family member, causes normally non-adherent mouse nuclei is much greater than the sum of the two mutations independently (A and E). (G) Fly heterozygous for both the αPS1 L cells to spread in an integrin-mediated fashion (Huang et al., integrin allele, mewM6 and the βPS integrin allele mysb45. Examples 1993), similar to what we see with D-basigin. Given that of misplaced nuclei are marked with arrows. (H) Fly mutant in the basigin does not localize to focal adhesions (Berditchevski et eye for Mmp2w307, which shows no abnormality in nuclear al., 1997), the mechanism by which it mediates integrin- placement. Bar, 30 µm. mediated cell attachment is not clear. Basigin and integrin affect cell structure 2657 Journal of Cell Science

Fig. 7. R1-R6 in bsgδ265 mutant terminals exhibit a mutant ultrastructural phenotype. (A) Cross section of a control cartridge innervated by non- mutant axons (control animals were generated by recombining a wild-type chromosome arm using the same basic procedure used to create mutant terminals as already described). This control exhibits a wild-type structure (Meinertzhagen and O’Neil, 1991) in which a ring of photoreceptor terminals (R) surrounds the axon profiles of lamina cells and L2. The entire cartridge is surrounded by lamina epithelial glia (*). Terminals contain normal mitochondria (m) and synaptic profiles composed of synaptic vesicles, capitate projections (arrowheads) and T- bar synaptic ribbon release sites (arrow). (B) Cartridge innervated by bsgδ265 photoreceptors; the terminals are of variable sizes, most are larger than controls in A, which are shown at the same magnification, although one terminal is very small (*). At this distal section plane in the lamina, close to the eye, there are more mitochondrial profiles than normal. (C) In the same lamina as B, but cut at a proximal level, R1-R6 terminals lack mitochondrial profiles. Such profiles disappear in the distal third of the lamina’s depth, with a cut-off that is sharply localized. At that level, cartridge cross-sections, as here, have some terminals with (m) and some without (*) mitochondrial profiles. (D) Individual R1-R6 terminal from C, exhibits misplaced rough ER (long arrows), never normally seen in either control or wild-type terminals and pleiomorphic profiles of synaptic vesicles (short arrow); capitate projections are mostly lacking, except those that are shallow (arrowhead), whereas T-bar ribbons are normal (double arrowhead). Bar, 1 µm. 2658 Journal of Cell Science 118 (12) D-basigin and the cytoskeleton positive stretch. Comparing basigin and gp55 from several D-basigin partially colocalizes with actin in High Five cells and species we see the following consensus: Y E K R/K R/K this was especially evident at cell-cell contacts and cell edges. R/K/N. Embigin, the most divergent family member, shows a Partial colocalization was also previously reported in chicken similar sequence, Y T H K K K (mouse). Beyond this, there is retinal pigment (Schlosshauer et al, 1995). D- little or no homology in the intracellular portion of the basigin and actin colocalization may occur as an indirect molecules. This pattern of sequence conservation between consequence of the interaction of D-basigin with integrins. The basigin from many species is consistent with the observations D-basigin/actin colocalization seen here was similar to that that the extracellular portion of basigin has biological activity seen for D-basigin/integrin colocalization in cultured human (Guo et al., 1997) and that basigin interacts with proteins in cells (Berditchevski et al., 1997), primarily at cell contacts. In the plane of the membrane. addition, integrins are linked to actin via adaptor proteins, such There is also congruence of function between basigin in flies as talin, that bind to specific sequences in the intracellular tail and mammals during development. For example, rod cells in of integrins (reviewed by Arnaout et al., 2002). Basigin family the Bsg knockout mouse retina exhibit gross morphological proteins have no known binding motifs inside the cell for actin- differences, having smaller outer segments (Ochrietor et al., binding proteins and there is little conservation between D- 2001). Mouse basigin is expressed in both retinal neurons and basigin and mouse basigin in the intracellular tail. However, Müller cell glia (Ochrietor et al., 2003). Anti-basigin can block direct interactions between D-basigin and actin-binding neuronal-glial adhesion in disassociated cultures from avian proteins cannot be ruled out. retina (Fadool and Linser, 1993). Drosophila basigin also Although D-basigin colocalizes with actin at cell contacts in affects neuron-glia interactions (our unpublished data). Flies or culture, its effects on internal cell structure may result from mice mutant for the basigin gene both have defects in olfaction. alterations in either microfilaments or, more indirectly, Thus, Bsg knockout mice are unable to respond to noxious microtubules. Although organelle anchoring has not been odors (Igakura et al., 1996); and, in flies, a P-element mutation studied in Drosophila, in many systems, nuclei are anchored in an upstream non-coding exon from the bsg class 1 and 2 in their final positions by attachment to actin (Apel et al., transcripts leads to a loss of sensitivity to noxious odors 2000). However, in fly photoreceptors, both nuclei (Fan and (Anholt et al., 2002). In addition, Bsg knockout mice are also Ready, 1997; Patterson et al., 2003) and mitochondria (Stowers male sterile (Igakua et al., 1998; Saxena et al., 2002). et al., 2002) require microtubules for their proper migration Intriguingly, a screen for male sterile mutants in Drosophila (Fan and Ready, 1997; Patterson et al., 2003). Our identified a fly line containing a P-element insertion in bsg mitochondrial placement defect is also similar to that seen in about 500 bp upstream from the P-element previously the motor axons of kinesin mutants (Hurd and Saxton, 1996). described (Castrillon et al., 1993). It was based on this insertion that the was originally called gelded (gel). However, our There are direct physical links between microfilaments and δ microtubules, as well as interactions between the two bsg 265 deletion allele compliments the original gel allele for (Rodriguez et al., 2003; Cao et al., 2004), so that an affect on male sterility. This makes it unclear, at present, if the male one of these cytoskeletal elements may also effect changes in sterility phenotype is really due to a mutation in bsg. Lastly, in the other. mice, a knockout that eliminates basigin remains viable, Journal of Cell Science whereas in flies bsgδ265 mutants are lethal. The most likely explanation for this difference is that mammals contain three MMP-independent functions of D-basigin basigin family members that may be required for different There are several reasons to conclude that many functions of aspects of development whereas flies contain only one. D-basigin in the fly do not depend on its putative role as an MMP inducer. When we made mosaics in which MMP This work was supported by grants from the NIH (GM57889 to function is missing in the eye, there was no effect on R.J.W. and EY-03592 to I.A.M.), and aided by ACS IRG-58-012-45 photoreceptor cell structure and no effect on the placement of (to K.D.C.). We thank Rita Kostyleva and Jane Anne Horne, both at glial cell nuclei. Likewise, when we misexpressed Drosophila Dalhousie University, for help with electron microscopy and data analysis, respectively. We thank Danny Brower (University of TIMP (tissue specific inhibitor of MMPs) (Page-McCaw, Arizona) for helpful comments and for flies, cell lines and antibodies, 2003) in photoreceptors we also saw no effect on cell structure. Paul Linser (University of Florida) for providing the mouse basigin There is an even stronger reason to believe that D-basigin δ clone and Andrea Page-McCaw (University of California, Berkeley) has MMP-independent functions in the fly. Bsg 265 mutants are for MMP-2 mutants. embryonic lethal. If D-basigin acted only through MMPs, then MMP mutants would also be embryonic lethal; this is not the case. MMP-1-null mutants survive through the second larval References instar, MMP-2-null mutants survive into the pupal stage, and Allain, F., Vanpouille, C., Carpentier, M., Slomianny, M.-C., Durieux, S. double mutants also survive well into the larval stages (Page- and Spik, G. (2002). Interaction with glycosaminoglycans is required for McCaw et al., 2003). B to trigger integrin-mediated adhesion of peripheral blood T to extracellular matrix. Proc. Natl. Acad. Sci. USA 99, 2714- 2719. Anholt, R. R., Dilda, C. L., Chaing, S., Fanara, J.-J., Kulkarni, N. H., Mouse basigin and D-basigin Ganguly, I., Rollmann, S. M., Kamdar, K. P. and Mackay, T. F. (2003). Mouse basigin can replace the function of D-basigin in the fly The genetic architecture of odor-guided behavior in Drosophila: epistasis and the transcriptome. Nat. Genet. 35, 180-184. visual system. The homology between fly and mouse basigin Apel, E. D., Lewis, R. M., Grady, R. M. and Sanes, J. R. (2000). Syne-1,a lies in the external and transmembrane domains, and in the six dytrophin- and Kalrsicht-related protein associated with synaptic nuclei at cytoplasmic residues closest to the membrane, that form a short neuromuscular junction. J Biol. Chem. 275, 31986-31995. Basigin and integrin affect cell structure 2659

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