1 Department of Molecular, Cellular and Developmental Biology, Yale University, 266 Whitney Avenue, New Haven, CT 06511, USA
2 Life Sciences Centre, Dalhousie University, 1355 Oxford Street, Nova Scotia, B3H 4J1, Canada
3 Department of Biological Sciences, Fulbright College of Arts & Sciences, University of Arkansas, Fayetteville, AR 72701, USA
* Author for correspondence (e-mail: kcurtin{at}uark.edu)
Accepted 29 March 2005
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Summary |
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Key words: Basigin, EMMPRIN, CD147, Integrin, Cell structure, Drosophila, Gelded
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Introduction |
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Several lines of evidence suggest a role for basigin in metastasis. First, basigin expression and metastasis correlate in human melanoma (Kanekura et al., 2002). Second, basigin stimulates MMPs from fibroblasts adjacent to tumors (Zucker et al., 2001
) and MMPs remodel the ECM, allowing tumor invasion (Nabeshima et al., 2002
). Third, co-culture of basigin-expressing melanoma cells with fibroblasts results in MMP induction and migration of tumor cells through a reconstituted basement membrane; anti-basigin antibodies block both of these activities (Kanekura et al., 2002
). Fourth, expression of basigin in slow-growing breast cancer cells lines that are then injected into mouse mammary tissue, leads to larger and more invasive tumors than controls (Zucker et al., 2001
). Lastly, basigin promotes adhesion-independent cell growth and this may contribute to secondary tumor formation (Marieb et al, 2004
).
Basigin is one of a three-member family in mammals that includes embigin and neuroplastin (SDR1, gp55/gp65). In vertebrates, basigin is expressed in a variety of tissues including the developing retina, blood-brain barrier, CNS, thymus, epithelial tissues and a variety of immune cells (Fadool and Linser, 1994; Fan et al., 1998b
). Embigin is expressed in mouse embryos and many tissues in the adult (Huang et al., 1990
; Fan et al., 1998a
). Neuroplastin is expressed in the nervous system in the cortex, cerebellum and hippocampus (Langnaese et al., 1997
), and in some non-neural tissues.
In mammals, the basigin gene encodes two nearly identical protein isoforms both with two IgG-C2 domains (Kanekura et al., 1991), as well as an isoform with three IgG-C2 domains (Ochrietor et al., 2003
). The neuroplastin gene also encodes two isoforms, a two-IgG protein (gp55) and a three-IgG protein (gp65). Both forms are expressed in the brain, but the gp65 protein is brain specific (Langnaese et al., 1997
). The gp55 form is expressed along the axon whereas the gp65 protein is concentrated at postsynaptic densities and may play a role in long-term potentiation (Smalla et al., 2000
).
Here we identify previously unknown functions for basigin family proteins by examining the Drosophila homologue, D-basigin. In particular, we find that D-basigin has dramatic effects on internal cell architecture, both in culture and in vivo, and that it mediates these effects through interactions with integrins. This function appears to be independent of MMPs.
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Materials and Methods |
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Cell culture and labeling
S2 cells were grown in Schneider's medium with 10% FBS (Invitrogen). High Five cells were grown in HyQ serum-free media supplemented with 10% calf serum (Invitrogen). Between 2x105 and 5x105 cells were plated on 25-mm-square coverslips pre-treated with 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 permeabilized for 10-20 minutes with PBS plus 0.3% Triton X-100. Cells were blocked for 15 minutes in blocking buffer (PBS containing 0.3% Triton X-100 and 2% BSA, Sigma). Primary and secondary antibodies were diluted in blocking buffer and applied for 1 hour each. Each antibody incubation was followed by three washes with 0.3% Triton X-100 in PBS. Coverslips were dried and mounted in glycerol gelatin (Sigma) with 1 mg/ml p-phenylene diamine (Sigma).
S2 cells with and without integrin genes were obtained from Daniel L. Brower (University of Arizona). High Five cells and the pIZT expression vector with the V5 tag were obtained from Invitrogen and the bsg265 transgene from Research Genetics. Cells were transfected with Cellfectin (Invitrogen).
Antibodies and labels
D-basigin peptide antibody was raised in chickens (Alpha Diagnostics) to LIADENKFIIDKTDTNDDGKYSC, a peptide uniquely found in D-basigin. Other antibodies were obtained from the following sources: Anti-ß-gal, Promega (#Z378A) used 1:1000; anti-V5 (Invitrogen) used 1:500, anti-PS1 (monoclonal DK.1A4), anti-ßPS integrin (monoclonal CF.6G11) and anti-
PS2 (CF.2C7) were obtained from Daniel L. Brower (University of Arizona); anti-elav (mouse and rat monoclonal antibodies), anti-repo and 24B10 from the Developmental Studies Hybridoma Bank (University of Iowa); anti-tubulin (Sigma T4026, used according to the manufacturer's instructions); biotinylated secondary antibodies were from Vector Labs. Alexa-568 anti-chicken, Alexa-488 anti-mouse and Alexa-568 phalloidin were all from Molecular Probes.
Flies and mosaics
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 were EGUF/hid lines for FRT40A. The bsg
265 excision allele was created by crossing P1478 to a fly line containing transposase and selecting for a loss of the eye color marker encoded within the engineered P-element. Integrin alleles and integrin monoclonal antibodies were obtained from Daniel L. Brower (University of Arizona).
Electron microscopy
The lamina, innervated by either control or bsg265 mutant photoreceptors, was prepared for electron microscopy (EM) using previously reported methods (Meinertzhagen, 1996
; Meinertzhagen and O'Neil, 1991
). Single sections containing cartridge profiles cut in cross-section were examined and digital montages collected from images obtained with a Philips Tecnai 12 operated at 80 kV, using a Kodak Megaview II camera with software (AnalySIS, Soft Imaging System, Münster).
Northern blots
Total mRNA was isolated from animals at different developmental stages using a kit (Qiagen) and RNA was quantified by running 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 by isolating the basigin gene from an agarose gel and using the gene as a template in a random primer reaction made using a random primer kit (New England Biolabs). The probe was labeled with [32P]dCTP and used to probe the mRNA, as described (Sambrook and Russell, 2000
).
BLAST analysis
BLAST analyses of mammalian genes using the Drosophila genome as a database for comparison were carried out at the Berkeley Drosophila Genome Project (http://www.fruitfly.org/blast/blast_form.html.) using full protein sequences for neuroplastin, basigin and embigin. Default settings for the site were used. An amino acid-based search was chosen and the database chosen included all predicted proteins for the genome. Predicted proteins that had basigin homology were examined by following available links to see if these proteins were of similar length to basigin and had characteristic features of the basigin protein family, including transmembrane domains, IgG domains and high sequence homology in or near the transmembrane domain.
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Results |
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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 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
The bsg265 transgene that codes for D-basigin 265 was introduced permanently into insect High Five cells. These cells are derived from the embryo of the cabbage looper (Trichoplusia ni) and used as a baculovirus expression system. High Five cells permanently transfected either with empty vector or with bsg265 transgene were labeled with Alexa 568-phalloidin to visualize actin microfilaments (Fig. 3A), or with anti-tubulin antibody to visualize microtubules (Fig. 3B). Two classes of cells were seen showing two clearly distinct cytoskeletal arrangements. One class of cells showed actin filaments in an almost exclusively cortical pattern (Fig. 3A, Fig. 4F). These cells invariably showed a nuclear concentration of tubulin (Fig. 3B) and were spherical (not flattened to the dish). The second class of cells showed elaborated microfilaments (Fig. 3C, Fig. 4F) and microtubules (Fig. 3B) throughout the cytoplasm. These cells appeared flattened to the dish in light microscopy.
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When D-basigin protein was expressed in these cells, the number of each cell type changed noticeably. About 85% of control High Five cells showed cortical actin microfilaments (Fig. 3C and similar to Fig. 4F) and a round morphology with a nuclear concentration of tubulin (Fig. 3B), whereas only 15% of cells showed elaborate microfilaments and microtubules and a flattened appearance by light microscopy. By contrast 80% of D-basigin-expressing cells showed an elaboration of microfilaments (Fig. 3A, Fig. 4E) and microtubules (Fig. 3B) whereas only 20% showed a rounded morphology with cortical actin and a nuclear concentration of tubulin. Thus basigin expression in High Five cells led to a fivefold increase in the number of cells showing elaborated microfilaments and microtubules and a flattened appearance. This change in cytoskeletal rearrangement seemed to result from the cell-autonomous expression of D-basigin. First, these changes were independent of cell contact, as physically isolated basigin-expressing cells were just as likely to show the altered cytoskeletal arrangement as cells that were touching. Second, these changes in cell architecture were not due solely to secretion of a soluble factor by D-basigin-expressing cells, as medium conditioned by such cells did not induce nontransfected High Five cells to spread out and elaborate microtubules and microfilaments.
D-basigin colocalizes to the actin cytoskeleton
The D-basigin protein expressed in High Five cells had a V5 epitope tag at its C-terminus. Antibody to this tag was used to assess the subcellular distribution of D-basigin (Fig. 4A,C). D-basigin-V5 expression was found in three patterns. First, it was found in a fine granular pattern throughout the cell membrane (Fig. 4A,C). Second, D-basigin was expressed in a punctate fashion, visible as bright spots (Fig. 4A) seen to be vesicles by phase-contrast microscopy. High Five cells normally contain many vesicles even when D-basigin is not expressed. Lastly, a 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 colocalization in isolated cells varied. However, in cells that were in physical contact, D-basigin-actin colocalization at cell-cell contacts was invariable (Fig. 4A,B).
D-basigin-mediated changes in cellular architecture require integrin binding
Integrins can promote cell attachment and cause cells to spread out in culture. We therefore tested whether D-basigin-mediated changes in cell architecture depended on integrin binding. Because many integrins bind to ECM molecules, such as collagen and fibronectin, at an Arg, Gly, Asp (RGD) target 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 were cultured in the presence of a GRGDS peptide (Fig. 4F), the cells looked indistinguishable from control High Five cells, showing a rounded morphology with cortical actin filaments. By contrast, D-basigin-expressing cells grown without peptide (Fig. 4E) had elaborated microfilaments and a flattened appearance. D-basigin-expressing cells were much less affected by a control peptide, GRGES at the same concentration of 200 µg/ml (not shown). Cells incubated with GRGES showed that 65% of the cells spread compared to 80% of control cells.
D-basigin partially colocalizes with integrin in integrin-transfected S2 cells
Previous work indicated that basigin colocalizes with some 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 of D-basigin. This antibody did not label control High Five cells, but did label D-basiginV5-expressing High Five cells. When these latter cells were double-labeled with both the peptide antibody and the V5 antibody, nearly identical patterns of labeling were seen, suggesting that this antibody indeed 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 cells express D-basigin. Control staining of S2 cells with anti-integrin antibody showed no staining as expected.
We could not look for colocalization of D-basigin and integrin in High Five cells because antibodies to High Five integrins are not available. Moreover, normal Drosophila S2 cells do not express integrins. We therefore used genetically altered S2 cells that were permanently transfected with genes for PS1 and ßPS integrins expressed under control of a heat-shock promoter (Gotwals et al., 1994
). These cells were induced to express integrins and then double-labeled with anti-D-basigin and a mixture of monoclonal antibodies against both
PS1 and ßPS integrins. D-basigin and integrin showed partial colocalization in the cell (Fig. 4G,H), although there was consistently more basigin expression around the cell body. This suggests that basigin and integrin can at least partially colocalize if expressed together.
D-basigin partially colocalizes with integrin in the retina
We next looked for colocalization between D-basigin and integrin in the Drosophila visual system because we had originally identified bsg in a visual system screen. Adult head sections were double-labeled with anti-D-basigin (Fig. 5B) and monoclonal antibodies against ßPS integrin (Fig. 5C), which are expressed in the retina (Brower et al., 1995).
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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 obtained from the Bloomington Drosophila Stock Center. Both contain a bacterial lacZ gene encoding a nuclear form of ß-galactosidase. This lacZ gene contains no regulatory sequences and thus the bsg regulatory elements should drive expression (i.e. it should act as an `enhancer trap'). Anti-ß-gal revealed expression in photoreceptors and basal glia in adult head-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 either of these cell types at this stage.
Basigin gene mutations
The two P-element insertions in bsg mentioned previously, labeled P in Fig. 1A, are located 1145 bp from the start of transcription for the D-basigin 265 protein isoform (Fig. 1A). Homozygous mutant animals from both lines died after the second larval instar with only 3% of mutant larvae living to the third instar. The insertions failed to complement each other. Because this P-insertion did not interrupt the coding portion of the gene, animals carrying this mutation may have produced some functioning protein. To generate a more severe allele, the P-element (P1478) was mobilized; such mobilization occasionally caused loss of genetic material near the insertion site. We established 200 excision lines in which the P-element was missing; 182 were viable, indicating a clean excision of the P-element, whereas 18 were homozygous lethal and failed to complement the original P-element allele. By DNA blot analysis, two excision lines, bsg265 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 embryonic lethality with 75-80% of the animals dying as embryos. Those embryos that did hatch died within the first day and were small, lethargic and uncoordinated.
D-basigin affects the subcellular structure of photoreceptor neurons
Given that D-basigin affects cell architecture in culture, we were interested to know if it affected cell structure in the animal. To address this, we looked for the effects of D-basigin on placement of internal cellular organelles in photoreceptors. Because the mutations are embryonic lethal, we made mosaic animals in which D-basigin protein expression was missing only in the eye and invariably missing from photoreceptor neurons. We generated such mosaics by the method of Stowers and Schwarz (Stowers and Schwarz, 1999) in which FLP recombinase is expressed from the eye-specific promoter of the eyeless gene (ey). Eyeless-FLP mediates recombination in the eye between chromosome arms bearing engineered copies of the FLP binding sites (FRTs) near their centromeres. We recombined a chromosome arm bearing a bsg mutation with a chromosome arm bearing the cell death gene hid expressed specifically in all photoreceptors. After recombination and chromosome segregation, only photoreceptors that inherit two copies of mutant bsg survive to repopulate the eye; bsg eyes were almost normal in size.
Photoreceptor nuclei were visualized with an antibody against elav, a neuron-specific nuclear protein. Normally, photoreceptor nuclei lie in tight rows across the eye (e.g. Fig. 6C), so that any mislocalization is readily detected. The nuclei of the R1-R6 photoreceptors lie in the apical region of the retina (Fig. 6C). The nuclei of the R7 photoreceptors are just proximal to those of R1-R6 and the R8 nuclei lie near the basement membrane of the retina (Fig. 6C).
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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 bsg265, but also contained a bsg
265 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 optic neuropile. We examined laminas in which only the photoreceptors are mutant for bsg265 (i.e. the postsynaptic lamina neurons and glia are wild type). Rough endoplasmic reticulum (rER) was found misplaced into the mutant photoreceptor axon terminals. Normally rER, which is continuous with the nuclear membrane, is confined distally to the photoreceptor cell body in the overlying retina. Its more proximal displacement into the photoreceptor terminal in the lamina accords with the more proximal location of many R1-R6 nuclei (Fig. 7D). In addition to misplaced nuclei, mitochondria were also misplaced. The mitochondria accumulated in excessive numbers in the distal portion of the photoreceptor terminals (Fig. 7B), but were absent from the proximal portion of the terminals, where they are also normally found (Fig. 7C). In addition to misplaced organelles, bsg
265 mutant photoreceptors showed a clear increase in axon terminal size, with profiles that were >80% larger in cross-sectional area (compare Fig. 7A,B) compared to the control, a difference that was significant (P<0.0006; Student's t-test; mean of means of three flies per group). None of these defects was seen in control animals in which non-mutant chromosomes were recombined (Fig. 7A). On the whole, these defects, misplaced internal organelles and enlarged terminals, suggest global disruptions in cell structure in bsg
265 mutant cells (see Discussion), probably due to alterations in the cytoskeleton.
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The integrin proteins expressed in the eye, PS1 and ßPS (Zusman et al., 1993
; Roote and Zusman, 1996
), are encoded by genes located on the X chromosome, mew codes
PS1 integrin and mys codes ßPS integrin. Mysb45 is a viable allele (Jannuzi et al., 2004
) and males carrying this mutation showed normal placement of photoreceptor nuclei (Fig. 6E). Mutant flies homozygous in the retina for a weak P-allele (P1096) of basigin showed occasional nuclear misplacement (Fig. 6A). To look for genetic interactions between bsg
265 and integrin genes, we made double mutants by creating males that carried the mysb45 allele (coding a mutant ßPS integrin), but were also homozygous mutant only in the retina for the P1096 bsg allele. These animals showed obvious misplacement of nuclei (Fig. 6F). The average number of misplaced photoreceptor nuclei per head section, after examining at least 12 animals of each genotype, was three times higher in the double mutants than that predicted from the summed effect of the two single mutations. Mosaics doubly mutant for mysb45 and bsg
265 also showed a more severe photoreceptor nuclear misplacement phenotype than the sum of the two single mutations would predict; 80% of nuclei were misplaced (not shown) compared with an average of 24% for bsg
265 and 1-2% for mysb45.
Some integrin gene allelic combinations also showed nuclear misplacement. Animals heterozygous for mewM6, a null allele for PS1 integrin (Brower et al., 1995
) showed normal placement of photoreceptor nuclei. Animals heterozygous for mysb45, a ßPS1 allele, showed normal nuclear placement, similar to the mysb45 hemizygous males (Fig. 6E) just discussed. However, animals heterozygous for both mewM6 and mysb45 showed 3% misplaced nuclei (Fig. 6G; >600 nuclei from three different animals counted).
MMP2 is not required in the eye for photoreceptor architecture
Because mammalian basigin stimulates secretion of MMPs, we examined the role of MMPs in the fly visual system. Drosophila has two MMP genes, Mmp1 and Mmp2, both required for viability. Only Mmp2 is expressed in the developing eye (Llano et al., 2000; Llano et al., 2002
; Page-McCaw et al., 2003
). If D-basigin were acting primarily through MMP-2, then flies lacking MMP-2 in the retina should have the same phenotypes as those found in bsg
265 mutant retina. Using the same method previously described to make bsg
265 eye mosaics, we made flies that were mutant in the eye for a null Mmp2 allele, Mmp2w307* (Page-McCaw et al., 2003
). We saw no misplaced photoreceptor cell nuclei (Fig. 6H). In case MMP1 functionally replaces MMP-2, we made mosaics that were mutant in the eye for both genes. These also showed no misplaced nuclei (not shown). Finally, we saw no effect on nuclear placement when we drove expression of Drosophila TIMP (tissue specific inhibitors of MMPs) in the eye (not shown), even though this TIMP gene has previously been reported to block biological activity of Drosophila MMPs (Page-McCaw et al., 2003
).
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Discussion |
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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 bsg265 are also larger than wild-type terminals. All these defects suggest disruption of the cytoskeleton.
D-basigin acts through integrins
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 colocalize in the Drosophila retina. Third, D-basigin and integrin colocalize to sites within the cell in integrin-expressing Drosophila S2, as well as in human cells (Berditchevski et al., 1997), in which the two proteins also co-immunoprecipitate. This suggests they may form a complex in the membrane. Fourth, allelic combinations of integrin gene mutations show disruption of retinal cell structure, e.g. misplaced nuclei, similar to those in bsg
265 mosaics. Fifth, bsg
265 and integrin gene mutations interact to affect nuclear placement. Sixth, bsg and integrin genes have been shown to interact genetically to affect dorsal closure and germ band retraction in the Drosophila embryo (Reed et al., 2004
).
Two additional findings support the idea that D-basigin and intregin interact to affect cell structure. First, antibodies against D-basigin can block integrin-mediated adhesion of T cells to ECM (Allain et al., 2002). Second, expression of embigin, a basigin family member, causes normally non-adherent mouse L cells to spread in an integrin-mediated fashion (Huang et al., 1993
), similar to what we see with D-basigin. Given that basigin does not localize to focal adhesions (Berditchevski et al., 1997
), the mechanism by which it mediates integrin-mediated cell attachment is not clear.
D-basigin and the cytoskeleton
D-basigin partially colocalizes with actin in High Five cells and this was especially evident at cell-cell contacts and cell edges. Partial colocalization was also previously reported in chicken retinal pigment epithelium (Schlosshauer et al, 1995). D-basigin and actin colocalization may occur as an indirect consequence of the interaction of D-basigin with integrins. The D-basigin/actin colocalization seen here was similar to that seen for D-basigin/integrin colocalization in cultured human cells (Berditchevski et al., 1997
), primarily at cell contacts. In addition, integrins are linked to actin via adaptor proteins, such as talin, that bind to specific sequences in the intracellular tail of integrins (reviewed by Arnaout et al., 2002
). Basigin family proteins have no known binding motifs inside the cell for actin-binding proteins and there is little conservation between D-basigin and mouse basigin in the intracellular tail. However, direct interactions between D-basigin and actin-binding proteins cannot be ruled out.
Although D-basigin colocalizes with actin at cell contacts in culture, its effects on internal cell structure may result from alterations in either microfilaments or, more indirectly, microtubules. Although organelle anchoring has not been studied in Drosophila, in many systems, nuclei are anchored in their final positions by attachment to actin (Apel et al., 2000). However, in fly photoreceptors, both nuclei (Fan and Ready, 1997
; Patterson et al., 2003
) and mitochondria (Stowers et al., 2002
) require microtubules for their proper migration (Fan and Ready, 1997
; Patterson et al., 2003
). Our mitochondrial placement defect is also similar to that seen in the motor axons of kinesin mutants (Hurd and Saxton, 1996
). There are direct physical links between microfilaments and microtubules, as well as interactions between the two (Rodriguez et al., 2003
; Cao et al., 2004
), so that an affect on one of these cytoskeletal elements may also effect changes in the other.
MMP-independent functions of D-basigin
There are several reasons to conclude that many functions of D-basigin in the fly do not depend on its putative role as an MMP inducer. When we made mosaics in which MMP function is missing in the eye, there was no effect on photoreceptor cell structure and no effect on the placement of glial cell nuclei. Likewise, when we misexpressed Drosophila TIMP (tissue specific inhibitor of MMPs) (Page-McCaw, 2003) in photoreceptors we also saw no effect on cell structure.
There is an even stronger reason to believe that D-basigin has MMP-independent functions in the fly. Bsg265 mutants are 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 instar, MMP-2-null mutants survive into the pupal stage, and double mutants also survive well into the larval stages (Page-McCaw et al., 2003
).
Mouse basigin and D-basigin
Mouse basigin can replace the function of D-basigin in the fly visual system. The homology between fly and mouse basigin lies in the external and transmembrane domains, and in the six cytoplasmic residues closest to the membrane, that form a short positive stretch. Comparing basigin and gp55 from several species we see the following consensus: Y E K R/K R/K R/K/N. Embigin, the most divergent family member, shows a similar sequence, Y T H K K K (mouse). Beyond this, there is little or no homology in the intracellular portion of the molecules. This pattern of sequence conservation between basigin from many species is consistent with the observations that the extracellular portion of basigin has biological activity (Guo et al., 1997) and that basigin interacts with proteins in the plane of the membrane.
There is also congruence of function between basigin in flies and mammals during development. For example, rod cells in the Bsg knockout mouse retina exhibit gross morphological differences, having smaller outer segments (Ochrietor et al., 2001). Mouse basigin is expressed in both retinal neurons and Müller cell glia (Ochrietor et al., 2003
). Anti-basigin can block neuronal-glial adhesion in disassociated cultures from avian retina (Fadool and Linser, 1993
). Drosophila basigin also affects neuron-glia interactions (our unpublished data). Flies or mice mutant for the basigin gene both have defects in olfaction. Thus, Bsg knockout mice are unable to respond to noxious odors (Igakura et al., 1996
); and, in flies, a P-element mutation in an upstream non-coding exon from the bsg class 1 and 2 transcripts leads to a loss of sensitivity to noxious odors (Anholt et al., 2002). In addition, Bsg knockout mice are also male sterile (Igakua et al., 1998; Saxena et al., 2002
). Intriguingly, a screen for male sterile mutants in Drosophila identified a fly line containing a P-element insertion in bsg about 500 bp upstream from the P-element previously described (Castrillon et al., 1993
). It was based on this insertion that the locus was originally called gelded (gel). However, our bsg
265 deletion allele compliments the original gel allele for male sterility. This makes it unclear, at present, if the male sterility phenotype is really due to a mutation in bsg. Lastly, in mice, a knockout that eliminates basigin remains viable, whereas in flies bsg
265 mutants are lethal. The most likely explanation for this difference is that mammals contain three basigin family members that may be required for different aspects of development whereas flies contain only one.
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Acknowledgments |
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