To identify new proteins controlling synapse development, proteins specifically accumulating at developing NMJs of Drosophila larvae were sought. A protein-trap screen was performed in which ten lines were discovered exhibiting GFP expression at the larval NMJ; focus was placed on three independent lines showing strong GFP accumulation at larval NMJs. In these lines, a strong GFP signal is also observed in different neuropil structures of the larval brain (Besse, 2007).
To check if the distribution of tagged Bsg reflects that of the endogenous protein, wild-type larvae were stained with anti-Bsg antibodies. Endogenous Bsg shows a localization pattern identical to that of the GFP fusion, and both precisely colocalize with Discs large (Dlg), a transmembrane protein present both pre- and postsynaptically, but mainly accumulating in stacks of postsynaptic membranes named subsynaptic reticulum (SSR). Like Dlg, Bsg accumulates to higher levels at type Ib than at smaller type Is boutons. To exclusively visualize the presynaptic expression of Bsg, a GFP-tagged Bsg fusion ws specifically expressed in the presynaptic compartment, and a robust GFP signal was observed at NMJs. Consistent with an accumulation of Bsg at the presynaptic membrane, the inner aspect of both endogenous Bsg and GFP-Bsg protein-trap fusion labels partially overlap with the presynaptic membrane marker HRP (Besse, 2007).
Further analysis revealed that Bsg is not homogenously distributed at the membrane but is excluded from active zones labeled with anti-Bruchpilot (BRP) NC82 antibodies (Wagh, 2006). Therefore, similar to other transmembrane proteins involved in the structural control of synaptic terminals, such as Dlg or Fasciclin II (Sone, 2000), Bsg localizes to periactive zones (Besse, 2007).
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, or with anti-tubulin antibody to visualize microtubules. 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. These cells invariably showed a nuclear concentration of tubulin and were spherical (not flattened to the dish). The second class of cells showed elaborated microfilaments and microtubules throughout the cytoplasm. These cells appeared flattened to the dish in light microscopy (Curtin, 2005).
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 and a round morphology with a nuclear concentration of tubulin, 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 and microtubules 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 (Curtin, 2005).
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. D-basigin-V5 expression was found in three patterns. First, it was found in a fine granular pattern throughout the cell membrane. Second, D-basigin was expressed in a punctate fashion, visible as bright spots 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 and near cell edges. 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 (Curtin, 2005).
Integrins can promote cell attachment and cause cells to spread out in culture. Therefore whether D-basigin-mediated changes in cell architecture depended on integrin binding was tested. Because many integrins bind to ECM molecules, such as collagen and fibronectin, at an Arg, Gly, Asp (RGD) target sequence, the peptide GRGDS is commonly used as a competitive inhibitor for such integrin binding. When D-basigin-expressing cells were cultured in the presence of a GRGDS peptide, 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 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. Cells incubated with GRGES showed that 65% of the cells spread compared to 80% of control cells (Curtin, 2005).
Previous work indicated that basigin colocalizes with some integrins at cell-cell contacts (Berditchevski, 1997). To examine if D-basigin and integrin colocalize within the cell, antibody was generated to a peptide in the extracellular domain of D-basigin. This antibody did not label control High Five cells, but did label D-basigin-V5-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. Clear labeling of Drosophila S2 cells was seen 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 (Curtin, 2005).
It was not possible to 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. Therefore genetically altered S2 cells were used, that were permanently transfected with genes for αPS1 and ßPS integrins expressed under control of a heat-shock promoter. These cells were induced to express integrins and then double-labeled with anti-D-basigin and a mixture of monoclonal antibodies against both αPS1 and ßPS integrins. D-basigin and integrin showed partial colocalization in the cell, 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 (Curtin, 2005).
Colocalization between D-basigin and integrin in the Drosophila visual system was examined because bsg was initially identified in a visual system screen. Adult head sections were double-labeled with anti-D-basigin and monoclonal antibodies against ßPS integrin, which are expressed in the retina (Curtin, 2005).
D-basigin antibody revealed lines of immunofluorescent puncta in the retina. Labeling the same sections with anti-αPS1 integrin antibodies or anti-ßPS antibodies revealed multiple points of colocalization at these puncta, the positions of which did not correspond to ECM and were therefore probably points of cell-cell contact. Integrin-specific antibodies also showed a clear line of expression at the basement membrane whereas D-basigin antibody did not strongly label the membrane 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. D-basigin was not expressed in these pigment cells (Curtin, 2005).
The above labeling did not allow the identification of the specific retinal cell types that express D-basigin protein. To identify these, expression was examined from an enhancer trap line in the gene for D-basigin, bsg. Two P-element insertions in bsg (P1096 and P1478) 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. 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 (Curtin, 2005).
To address the in vivo requirement for Bsg at the larval NMJ, P element insertions near the transcription starts of the longer transcripts were sought and five belonging to the same lethal complementation group were found. Three of these (l[2]k13638, l[2]k14308, and NP3198), when placed in trans over a deficiency covering the locus (Df[2L]Exel7034, hereafter referred to as Df), cause an embryonic/early larval lethality that can be rescued by ubiquitous expression of a bsg transgene, and represent strong hypomorphic alleles (Curtin, 2005). Two other insertions, NP6293 and l(2)SH1217, behave genetically as weaker hypomorphic alleles, as, respectively, 30% and 50% of the corresponding hemizygotes reach third larval instar. This semilethality is reverted after precise excision of NP6293. Consistent with these data, Western blot analysis shows that Bsg expression levels are greatly reduced in Df/NP6293 and Df/l(2)SH1217 mutant larvae but restored to normal levels after precise excision of NP6293. The amount of Bsg specifically accumulating at the NMJ is also significantly reduced in Df/NP6293 larval fillets, compared with wild type. Together, these results show that NP6293 and l(2)SH1217 are bsg mutant alleles suitable for analysis of larval NMJ development and maturation. They were therefore renamed bsg6293 and bsg1217 and used for subsequent studies (Besse, 2007).
To determine whether bsg mutants exhibit defects in their motoneuron connection pattern and/or NMJ morphology, synaptic boutons and motoneuron membranes of both Df/bsg6293 and Df/bsg1217 third instar larvae were examined. Axonal targeting is not altered to a visible degree in these animals. However, the growth of synaptic boutons is strongly altered, as revealed by the considerable increase in their size. In particular, the proportion of very large boutons (>12 µm2) is greatly increased in bsg mutants compared with controls. The observed increase in bouton size is associated with a concomitant reduction of both NMJ branching and bouton number, keeping the overall NMJ size close to normal. Moreover, defects in bouton size and number are already observed in second instar animals and revert after precise excision of NP6293 (Besse, 2007).
To explicitly determine whether these growth defects could be rescued and whether they reflected pre- and/or postsynaptic function of bsg at the NMJ, a wild-type copy of bsg was expressed in specific compartments of Df/bsg6293 larvae. Expression of wild-type Bsg solely in muscles (using mhc-Gal4 or 24B-Gal4) or in neurons (using elav-Gal4), partially, but significantly, rescued both the increase in bouton size and the reduction of bouton number observed in mutant larvae. Near-complete rescue of bouton size and junction growth was obtained only when expressing wild-type Bsg both pre- and postsynaptically (Besse, 2007).
Collectively, it is concluded that Bsg is needed for efficient outgrowth of larval NMJs and that its function is required in both pre- and postsynaptic compartments to define boutons of proper size. Such a dual requirement is documented for the Ig CAM Fas II and is thought to reflect the establishment of transsynaptic homophilic interactions. Thus whether Bsg might also promote cell-cell adhesion was tested. S2 cells transfected with a GFP-Bsg construct strongly adhere to each other, whereas S2 cells transfected with a control GFP construct do not. Thus, Bsg promotes cell aggregation, consistent with the idea that Bsg could regulate the addition and growth of synaptic boutons through modulation of cell adhesion (Besse, 2007).
Depending on the cell type and/or the protein partners, different domains of mammalian Bsg are required for its activity (Guo, 1997; Kirk, 2000; Sun, 2001). Thus, to determine which domains of Drosophila Bsg are required for its function at the larval NMJ, GFP-tagged truncated variants were generated and their capacity to rescue Df/bsg6293 morphological defects was assayed (Besse, 2007).
Bsg lacking the most C-terminal part of the intracellular domain (Δintra) rescues defects in bouton size and number similarly to the full-length tagged form (fl) when expressed presynaptically. In contrast, forms composed of the two Ig domains only (Extra) or of the two Ig domains of Bsg fused to the transmembrane and intracellular domains of rat CD2 (Bsg-CD2) do not significantly rescue the decrease in bouton number observed in bsg mutants and only poorly rescue bouton growth defects. Thus, Bsg transmembrane and/or juxtamembrane cytoplasmic domains appear crucial for regulation of NMJ bouton growth and budding by Bsg (Besse, 2007).
The cytoplasmic juxtamembrane region of Bsg contains a conserved cluster of positively charged residues (KRR). When KRR is substituted with NGG, the mutated protein only poorly rescues the reduced bouton number and only to a low extent the increased bouton size of bsg larvae. Moreover, ubiquitous expression of the KRR-->NGG mutated protein does not significantly rescue the early lethality of the strong mutant combination Df/l(2)k13638, whereas full-length Bsg does, further indicating a crucial and previously unknown role of this motif for Bsg function (Besse, 2007).
Given that bsg mutants exhibit defective NMJ morphology, whether the assembly and/or maintenance of pre- and postsynaptic specializations might also be altered was tested. The overall distribution and complementary accumulation of markers specific to perisynaptic zones and PSDs seems to be normal at bsg junctions. Moreover, no alteration of SSR integrity could be detected at the light microscopy level or at the ultrastructural level (Besse, 2007).
Next, the distribution of receptor fields and active zones was assayed, using antibodies recognizing the glutamate receptor subunit GluRIID in combination with anti-BRP NC82 antibodies (Wagh, 2006). The distribution of these two markers is normal at bsg junctions: BRP and GluRIID remain concentrated in individual puncta of normal intensity and distribution. Moreover, as described for wild-type animals, BRP+ release sites are reproducibly found in direct apposition to postsynaptic glutamate receptor clusters in bsg larvae. Consistent with these observations, transmission EM showed that active zones are found at normal frequency and that their characteristic electron-dense specializations (T-bars) are of normal morphology. Quantification, however, indicated a slight increase in the electron-dense PSD diameter, which correlates with a slight, but significant, increase in the mean size of GluRIID clusters observed using light microscopy. Thus, these results suggest that, although Bsg is involved in definition of receptor field size, its function is not essential for specifying active and periactive zone domains (Besse, 2007).
Given that Drosophila Bsg has been suggested to regulate cell architecture, possibly by modulating the cell cytoskeleton (Curtin, 2005), the integrity of the actin-based cytoskeleton was examined at bsg NMJs. α-Spectrin (α-Spec) closely associates with the NMJ juxtamembrane actin-rich cytoskeleton. Although it is mainly enriched in the postsynaptic peribouton area, α-Spec is also found at the inner presynaptic bouton cortex. In bsg larvae, even though no major alterations of the postsynaptic Spectrin cytoskeleton are observed, α-Spec aggregates are detected within the bouton lumen in ~38% of NMJ branches. These aggregates are ~0.5 µm large and contain other α-Spec-associated proteins, such as ß-and ßH-Spectrin, as well as the actin-associated protein Wasp. In contrast, no enrichment of microtubule-associated proteins was observed in these aggregates. To more directly and specifically visualize the presynaptic F-actin network, the F-actin-binding domain of Moesin fused to GFP (GFP-GMA) was expressed exclusively in neurons. GFP-GMA accumulates at the cortex of wild-type synaptic boutons. In bsg mutants, although a cortical actin network is still clearly detected at the periphery of boutons, clusters of F-actin filaments are also frequently present within them. Altogether, these observations indicate that the organization of the presynaptic F-actin network is altered at bsg NMJs (Besse, 2007).
In the course of ultrastructural analysis, it was observed that abnormally large vesicles (diameter of up to ~300 nm) are present in Df/bsg6293 boutons but are only rarely observed after presynaptic reexpression of Bsg in this background. The exact nature of these vesicles remains unclear, since no concomitant alteration in the distribution and/or size of the FYVE-GFP+ endosomal compartment was observed at the light microscopy level (Besse, 2007).
To determine whether these defects could be associated with an alteration of the synaptic vesicle compartment, synaptic vesicle distribution was analyzed using specific vesicle markers. In wild-type boutons, synaptic vesicles are clearly enriched at the cortex but are largely excluded from their central core. In contrast, in bsg larvae, preferential association of vesicles with the bouton cortex is lost in ~60% of NMJ branches, and CSP+ (cysteine string protein) vesicles fill parts of or even the entire lumen of the bouton. CSP staining is also abnormally strong in axonal tracts connecting boutons and appears more granular than in control animals. An essentially identical mislocalization was observed using two other independent markers of synaptic vesicles, Synaptotagmin and Synapsin. These defects do not indirectly result from the increase in bouton size observed in bsg mutants, since synaptic vesicle localization appears normal in fase76 hemizygous larvae, which also form abnormally large boutons. Together with the fact that such a diffuse distribution can be observed upon tracking of freshly endocytosed synaptic vesicles, the data suggest that Bsg specifically regulates the spatial distribution of synaptic vesicles and, in particular, their proper anchoring to the cortex of synaptic boutons (Besse, 2007).
To address whether the observed changes in the distribution of synaptic vesicles might be linked to functional changes in transmitter release, postsynaptic currents were recorded at larval NMJs. The amplitude of the postsynaptic response to the fusion of single vesicles (minis) is increased above wild-type levels in bsg mutants. This effect is most likely related to the observed enlargement of the postsynaptic glutamate receptor clusters, given that no increase in the size of synaptic vesicles was found in bsg mutants compared with w controls. Notably, the frequency of spontaneous release events is strongly elevated in bsg mutants, and these events often occur clustered in 'exocytotic bursts' (Besse, 2007).
The mean amplitude of nerve-evoked excitatory junctional currents (eEJCs) is also increased at bsg NMJs, which largely correlates with the observed enlargement of minis. However, the temporal profile of bsg mutant eEJCs is strikingly lengthened, reflecting a dramatic and atypically delayed release of vesicles. Indeed, although the charge carried by bsg mutant minis is only moderately increased (1.5-fold increase), a near eightfold elevation of the charge transferred to the postsynapse after exocytosis occurs upon initial nerve stimulation. Notably, this value decreases progressively after further low-frequency stimulation, which may result from the exhaustion of the abnormally recruited pool of vesicles responsible for the atypically delayed release component. Averaging the charge transferred over 15 consecutive sweeps nonetheless reveals a near fivefold increase in bsg mutants; therefore, a more than threefold elevation of the number of vesicles released per action potential (quantal content) is estimated to occur (Besse, 2007).
These defects reflect a requirement for Bsg within the presynaptic terminal, since sole presynaptic expression of wild-type Bsg in the mutant background rescues both the asynchronous evoked release and the high frequency of spontaneous release, whereas its sole postsynaptic reexpression does not. Interestingly, the presynaptic reexpression of Bsg even decreases the amplitude of eEJCs and the frequency of minis below control levels, indicating a dose-dependent role of presynaptic Bsg in restricting vesicle release (Besse, 2007).
Two extraembryonic tissues form early in Drosophila development. One, the amnioserosa, has been implicated in the morphogenetic processes of germ band retraction and dorsal closure. The developmental role of the other, the yolk sac, is obscure. By using live-imaging techniques, intimate interactions are reported between the amnioserosa and the yolk sac during germ band retraction and dorsal closure. These tissue interactions fail in a subset of myospheroid (mys: ßPS integrin) mutant embryos, leading to failure of germ band retraction and dorsal closure. The Drosophila homolog of mammalian basigin (EMMPRIN , CD147) -- an integrin-associated transmembrane glycoprotein -- is highly enriched in the extraembryonic tissues. Strong dominant genetic interactions between basigin and mys mutations cause severe defects in dorsal closure, consistent with basigin functioning together with ßPS integrin in extraembryonic membrane apposition. During normal development, JNK signaling is upregulated in the amnioserosa, as midgut closure disrupts contact with the yolk sac. Subsequently, the amnioserosal epithelium degenerates in a process that is independent of the reaper, hid, and grim cell death genes. In mys mutants that fail to establish contact between the extraembryonic membranes, the amnioserosa undergoes premature disintegration and death. It is concluded that intimate apposition of the amnioserosa and yolk sac prevents anoikis of the amnioserosa. Survival of the amnioserosa is essential for germ band retraction and dorsal closure. It is hypothesized that during normal development, loss of integrin-dependent contact between the extraembryonic tissues results in JNK-dependent amnioserosal disintegration and death, thus representing an example of developmentally programmed anoikis (Reed, 2004).
In Drosophila, the role of extraembryonic tissues in regulating embryonic development has only recently begun to be appreciated . Two cell types that arise at the Drosophila cellular blastoderm stage are extraembryonic (i.e., do not contribute to the mature embryo). The first, the amnioserosa, is an epithelium derived from the dorsalmost region of the blastoderm. The second, the yolk sac, originates during cellularization of the blastoderm: membrane fusion basal to the blastoderm nuclei forms both the basal membrane of each somatic cell and a single continuous plasma membrane -- the yolk sac membrane -- that envelops the yolk. Within the yolk syncytium, there are some 200 nuclei; thus, the yolk sac is a large, membrane bound, multinucleate cell (Reed, 2004).
The amnioserosa plays a key role in germ band retraction and dorsal closure. It is likely to function both in cell signaling and in generating the forces that drive these morphogenetic processes. The role of the yolk sac during development has remained obscure. The expression of several genes in the yolk nuclei, including serpent, sisterlessA, D-ret, forkhead, and those encoding imaginal disc growth factors (IDGFs), suggests that the yolk sac may play important roles in processes other than nutrition. The developmental defects produced by loss-of-function alleles of sisterlessA, which is expressed exclusively in the yolk nuclei from blastoderm stages on, have led to speculation that the yolk may play a role in morphogenesis. However, the functions of the yolk sac in morphogenesis, if any, are unknown (Reed, 2004).
Physical interaction of the amnioserosa and yolk sac has been shown to play a crucial role in both germ band retraction and dorsal closure of the embryo. βPS integrin mediates extraembryonic membrane interactions that are required for survival of the amnioserosa. Anoikis of the amnioserosa occurs during normal development after closure of the midgut disrupts integrin-dependent apposition of the amnioserosa and yolk sac. In mys mutants, failure to establish apposition of extraembryonic membranes leads to premature anoikis of the amnioserosa. A possible role for JNK signaling and the reaper/hid/grim cell death genes in amnioserosal anoikis during normal development was investigated (Reed, 2004).
In fixed, sectioned material it can be seen that as germ band retraction commences, there is a gap between the amnioserosa and the yolk sac membrane. Membrane projections from both the basal side of the amnioserosa and the dorsal region of the yolk sac can be seen to penetrate this space. This space is enriched in glycoconjugates as assayed by ruthenium red staining. Since the bulk of the extracellular matrix is not laid down at this developmental stage, these polysaccharides may be associated with transmembrane glycoproteins rather than an elaborate extracellular matrix (ECM) per se (Reed, 2004).
Live imaging of germ band retraction and dorsal closure has revealed that contacts between the yolk sac membrane and the amnioserosa initiate at the beginning of germ band retraction and are remarkably dynamic. Imaging was carried out by using combinations of three different GFP fusion proteins that serve as markers of the F actin-based cytoskeleton (actin-GFP); the amnioserosal and yolk sac membranes (DE-cadherin-GFP), and G289, a homozygous viable PTT line that reports basigin expression as a basigin-GFP fusion protein (Reed, 2004).
The initial, transient contacts between the amnioserosa and the yolk sac membrane, referred to here as phase I interactions, occur as germ band retraction initiates and are accomplished by two classes of cellular extensions: filopodia that emanate from the amnioserosa and contact the yolk sac membrane, and membrane bound projections emanating from the yolk sac, which contact the amnioserosa (marked by basigin-GFP). Their lack of stable association with their target cells and their highly dynamic character suggest that neither the amnioserosal nor the yolk sac projections generate the mechanical forces that drive morphogenesis. Instead these projections may facilitate a chemosensory or signaling function between the amnioserosa and yolk sac membrane (Reed, 2004).
The intimate and persistent interaction between the amnioserosa and yolk sac -- phase II -- initiates in the dorsal-anterior region of the amnioserosa. This contact is maintained and further contact is established in an anterior-to-posterior direction as retraction progresses. Close apposition of the amnioserosa and yolk sac membranes persists during dorsal closure (Reed, 2004).
In mammals, basigin has been reported to be expressed and to function in extraembryonic tissues during early development, when it is required for embryo implantation. Basigin also functions in retinal epithelial morphogenesis. Since Drosophila Basigin is highly enriched on the extraembryonic membranes prior to and during their close apposition, attention was directed to the structure and function of Drosophila Basigin (Reed, 2004).
The Drosophila basigin transcription unit (CG31605, FBgn0051605) encodes multiple transcript variants. The transcripts encode two distinct protein isoforms: a long, 298 amino acid (aa) isoform and a short, 265 aa isoform. The long and short isoforms differ only at their amino and carboxy termini: the first 50 aa of the long form are substituted by 25 aa in the short form; the long form also has an 8 aa carboxy-terminal extension. The distinct N-terminal regions each contain their own unique transmembrane domains and signal peptide cleavage sites. The long isoform's N-terminal region is glycine rich. Database searches show that long and short isoforms also exist for human basigin (Reed, 2004).
Drosophila and mammalian basigin exhibit strong conservation of immunoglobulin (Ig) domain organization, location of predicted O linked glycosylation sites, as well as extracellular and cytoplasmic tail length. Both mammalian and Drosophila basigin have two extracellular Ig domains, the C-terminal of which appears to be representative of a 'primordial' Ig domain. There is an additional, more C-terminal 50 amino acid stretch of conservation, which will be referred to as the 'basibox' and which includes the predicted transmembrane domain. One of the defining features of the basibox is a glutamic acid residue in the middle of the transmembrane domain. The basibox is 52%-54% identical between Drosophila and vertebrates; the central 27 amino acids show 78%-81% identity (Reed, 2004).
There are multiple P element inserts in or near the basigin gene. One, the NP6293 GAL4 P element insertion, is in the 5'UTR of a predicted basigin transcript. This insertion causes leaky postembryonic lethality when homozygous and is referred to here as bsgNP6293. Homozygous bsgNP6293 embryos show no defects in germ band retraction and dorsal closure. A P element insert that causes male sterility has been referred to as gelded (Castrillon; 1993; Reed, 2004).
Basigin and integrins associate physically in mammals, possibly through direct contacts between basigin and the β1 integrin subunit. In Drosophila there is a single β integrin, called βPS integrin, which is encoded by the myospheroid (mys) gene. mys1 mutants show germ band retraction and dorsal closure defects (Reed, 2004).
Basigin and βPS integrin mutants show striking dominant genetic interactions: while bsgNP6293 mutants show no defects in dorsal closure and mys1 mutant embryos show only weak dorsal closure defects -- evidenced by a small dorsal hole -- mys1 mutant embryos from females in which the dose of the basigin gene is reduced by 50% show a striking increase in the size of the dorsal hole, while double mutant embryos show an even greater increase in dorsal hole size. The dominant genetic interaction of bsg and mys mutants is consistent with the possibility that basigin and integrin proteins interact physically in Drosophila (Reed, 2004).
Live imaging shows that those mys1 mutant embryos that fail germ band retraction exhibit apparently normal phase I interactions (for example, yolk sac projections are produced and contact the amnioserosa). However, phase II membrane apposition fails completely. Most striking is a failure of the dorsal-anterior region of the amnioserosa to initiate contact with the yolk sac membrane. In those mys1 mutant embryos that complete germ band retraction, there is failure to maintain the apposition of the amnioserosa and yolk sac membrane, with subsequent high penetrance failure of dorsal closure (Reed, 2004).
In summary, phase II membrane intimacy is compromised in mys1 mutants, implicating βPS integrin in the close apposition of amnioserosal and yolk sac membranes. The failure of both germ band retraction and dorsal closure in mys1 mutants suggests that close apposition of the extraembryonic membranes is required for these morphogenetic processes. The strong enhancement of mys1 dorsal closure defects by bsgNP6293 mutants suggests that Basigin functions together with βPS integrin in these morphogenetic processes. Anterior-to-posterior 'zipping up' of the membranes may generate forces that help push the germ band posteriorly. Alternatively, the role of integrin-dependent membrane apposition may be indirect, promoting survival of the amnioserosa, which in turn directs retraction and closure via signaling and/or physical contacts (Reed, 2004).
In wild-type embryos, the concomitant closure of the dorsal epidermis and midgut abrogate apposition of the amnioserosa and yolk sac. It was therefore asked when during normal development the amnioserosa loses integrity and dies. It has been shown, by using live imaging, that a small subset of the amnioserosal cells drop out of the epithelium prior to completion of closure. However, live imaging of the majority of amnioserosal cells (which remain in the epithelium) after dorsal closure has not been attempted previously (Reed, 2004).
Therefore, embryos in which amnioserosal cells had been specifically labeled were live-imaged, thus definitively addressing the fate of the amnioserosa after dorsal closure: the amnioserosa invaginates to form a tube-like structure with its perimeter cells aligning on the dorsal side of the tube, beneath the dorsal midline of the embryo. Over a period of 2-3 hr, individual nonperimeter cells round up and are extruded from the tube. Finally, the amnioserosal perimeter cells also dissociate. As amnioserosal cells are extruded, they are rapidly engulfed by hemocytes, which thus become GFP positive. These results are fully consistent with those inferred from analysis of fixed sectioned embryos (Reed, 2004).
It is possible to visualize a subset of the amnioserosal cells as acridine orange positive either before they leave the tube or shortly thereafter. Both acridine orange staining and engulfment by hemocytes are hallmarks of dying cells. To determine whether death of amnioserosal cells might be reaper dependent, it was asked whether reaper expression could be visualized in the amnioserosal cells prior to or after extrusion. No reaper-expressing cells were detected. To further test whether amnioserosal cell death might be reaper dependent, the H99 deficiency [Df(3L)H99] was used; this deficiency removes the reaper, head involution defective (hid), and grim genes, and the amnioserosa with anti-HNT antibody was visualized. If amnioserosal death were reaper dependent, one would expect HNT-positive cells to persist in H99 mutants when compared with wild-type. Such persistence does not occur. While it is conceivable that HNT expression is downregulated in a persistent amnioserosa, the simplest interpretation of these data is that death of the amnioserosa is reaper independent. This conclusion is consistent with the recent suggestion that Drosophila embryos have a caspase-independent cell engulfment system, which is still operative in H99 mutants (Reed, 2004).
It has been shown that loss of integrin-dependent contact between cells and the extracellular matrix leads to cell death, a process referred to as anoikis. Anoikis is promoted by the Jun amino-terminal kinase (JNK) pathway. Previous analyses have shown that JNK signaling in the amnioserosa is downregulated prior to dorsal closure. In those analyses, puckered-lacZ expression was used as a read-out of JNK signaling, and it was shown that relocation of JUN and FOS proteins from the nucleus to the cytoplasm of amnioserosal cells correlates with downregulation of JNK signaling. While JNK signaling is downregulated in the amnioserosa prior to dorsal closure, JNK signaling is upregulated in this tissue as dorsal closure approaches completion. Thus, reactivation of JNK signaling in the amnioserosa follows loss of integrin-dependent apposition of the amnioserosa and yolk sac membrane and precedes amnioserosal disintegration and death. These data are consistent with the hypothesis that midgut closure disrupts integrin-dependent apposition of the amnioserosa and yolk sac, thus inducing JNK signaling in the amnioserosa and its subsequent anoikis (Reed, 2004).
Therefore, in the Drosophila embryo, intimate apposition of the extraembryonic membranes is integrin dependent and promotes the integrity and survival of the amnioserosa. During normal development, closure of the midgut abrogates contact between the amnioserosa and yolk sac. JNK signaling is then upregulated in the amnioserosa, which subsquently disintegrates and dies, consistent with this being an example of developmentally programmed anoikis. In a subset of mys (βPS integrin) mutant embryos, apposition of the extraembryonic membranes never occurs, and the amnioserosa undergoes premature anoikis. The strong genetic interaction of mys and basigin mutants is consistent with the known physical interaction of these molecules in mammals (Berditchevski, 1997) and suggests that basigin might act together with integrin to promote extraembryonic membrane interaction and to prevent anoikis of the amnioserosa. Failure of germ band retraction and dorsal closure occurs in integrin mutants and is greatly enhanced when basigin levels are reduced. Together, these results suggest that extraembryonic membrane interaction promotes survival of the amnioserosa, which in turn directs germ band retraction and dorsal closure through physical contacts and/or intercellular signaling (Reed, 2004).
The hypothesis that amnioserosal anoikis is triggered during normal development by loss of integrin-mediated contact with the yolk sac membrane allows several testable predictions: (1) that in mutants in which the amnioserosa undergoes premature apoptosis prior to germ band retraction (e.g., hindsight), phase II apposition of the amnioserosa and yolk sac membrane may fail; (2) that premature amnioserosal apoptosis in these mutants is a consequence, rather than a cause of loss of amnioserosal epithelial integrity; (3) that the amnioserosa may persist in mutants lacking a midgut or in those defective for midgut closure (Reed, 2004).
It remains to be determined whether disintegration and death of the amnioserosa during normal development is caused solely by loss of contact with the yolk sac (i.e., is nonautonomously induced) versus whether signals from cell types other than the yolk -- or even an amnioserosa-autonomous program -- also play a role. For example, it is possible that upregulation of JNK signaling in the amnioserosa is independent of loss of contact with the yolk sac. Analysis of mutants lacking a midgut provide a test of this possibility: if disintegration and death of the amnioserosa occur even when apposition with the yolk sac is maintained, signals from other cell types or amnioserosa-autonomous processes would be implicated (Reed, 2004).
The specific role of JNK signaling in amnioserosal anoikis is difficult to assess because downregulation of JNK signaling in the amnioserosa and up-regulation of JNK signaling in the leading edge of the epidermis are required for dorsal closure. Thus JNK pathway mutants stall morphogenesis prior to dorsal closure, making it impossible to assess a possible later role. Expression of dominant-negative JNK specifically in the amnioserosa only later in development, when closure is almost complete, will be necessary to rigorously test the role of JNK activation in amnioserosal anoikis (Reed, 2004).
All of the data presented above support the hypothesis that phase II amnioserosa-yolk sac membrane association is necessary for maintenance of the amnioserosal epithelium and, thus, the morphogenetic processes of germ band retraction and dorsal closure. However, the role of the transient phase I interaction is less clear. It is unlikely that the phase I interactions play a role in generation of the forces that lead to close apposition of these extraembryonic membranes. It seems more likely that the transient interactions play a role in communication between the yolk sac and the amnioserosa. The ecdysone receptor and active ecdysteroids are reported to be present in the amnioserosa and required for germ band retraction. Expression of a dominant-negative form of the ecdysone receptor worsens germ band retraction defects in mys (βPS integrin) mutants. Furthermore, it has been speculated that enzymes residing in the yolk might participate in conversion of ecdysone to its active forms. Dynamic invaginations of the yolk sac membrane, which dive into the yolk mass and transiently contact the yolk spheres, have been observed. Thus, one tantalizing possibility is that these invaginations transport active forms of ecdysone -- as well as other key signaling molecules -- from the yolk spheres to the yolk sac membrane. Phase I amnioserosa-yolk membrane contacts and/or phase II intimate membrane apposition might subsequently bring these molecules to the amnioserosa (Reed, 2004).
It is concluded that the extraembryonic tissues of Drosophila play a crucial role in directing embryonic morphogenesis. Close apposition of the yolk sac membrane and the basal cell membranes of the amnioserosa is dependent on βPS integrin. This intimate membrane association is required to promote survival and to prevent anoikis of the amnioserosa. The amnioserosa then directs germ band retraction and dorsal closure through physical contacts and/or signaling. Disintegration and death of the amnioserosa after closure of the epidermis and midgut correlates with upregulation of JNK signaling in the amnioserosa, is independent of reaper/hid/grim function, and is likely to represent the first example of developmentally programmed anoikis in Drosophila (Reed, 2004).
Basigin, an IgG family glycoprotein found on the surface of human metastatic tumors, stimulates fibroblasts to secrete matrix metalloproteases that remodel the extracellular matrix. Using Drosophila melanogaster, intracellular, matrix metalloprotease-independent, roles for basigin have been identified. Specifically, Basigin, interacting with integrin, is required for normal cell architecture in some cell types. Basigin promotes cytoskeletal rearrangements and the formation of lamellipodia in cultured insect cells. Loss of basigin from photoreceptors leads to misplaced nuclei, rough ER and mitochondria, as well as to swollen axon terminals. These changes in intracellular structure suggest cytoskeletal disruptions. These defects can be rescued by either fly or mouse Basigin. Basigin and integrin colocalize to cultured cells and to the visual system. Basigin-mediated changes in the architecture of cultured cells require integrin binding activity. Basigin and integrin interact genetically to affect cell structure in the animal, possibly by forming complexes at cell contacts that help organize internal cell structure (Curtin, 2005).
The two P-element insertions in bsg are located 1145 bp from the start of transcription for the D-basigin 265 protein isoform. 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. Two hundred excision lines were establised 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, bsgΔ265 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 (Curtin, 2005).
Effects of D-basigin on placement of internal cellular organelles in photoreceptors werre examined. Because the mutations are embryonic lethal, mosaic animals were made in which D-basigin protein expression was missing only in the eye and invariably missing from photoreceptor neurons. Such mosaics were generated by expression of FLP recombinase 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. A chromosome arm bearing a bsg mutation was recombined 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 (Curtin, 2005).
Photoreceptor nuclei were visualized with an antibody against Elav, a neuron-specific nuclear protein. Normally, photoreceptor nuclei lie in tight rows across the eye, so that any mislocalization is readily detected. The nuclei of the R1-R6 photoreceptors lie in the apical region of the retina. 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 (Curtin, 2005).
Photoreceptor nuclei of mosaic flies mutant in the eye for the hypomorphic P1096 allele, which encodes a nuclear ß-gal, were visualized with anti-ß-gal. Most nuclei were properly located, although a few nuclei were misplaced. Similar results were seen for these mosaics with anti-elav. In mosaics that are mutant in the eye for the bsgΔ265 excision allele, Elav immunolabeling revealed that 16-50% of photoreceptor nuclei were mislocalized. 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. 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% (Curtin, 2005).
The nuclear placement defect was rescued by expressing D-basigin 265. Nuclear placement was counted 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 only 1% of misplaced nuclei were found. Expression of the mouse basigin gene in photoreceptors also rescued the nuclear misplacement with only 1.5% of nuclei misplaced in a total of 12 animals counted. Thus despite limited sequence homology, mouse basigin can promote the formation of normal cell architecture in flies (Curtin, 2005).
Photoreceptors R1-R6 terminate in the lamina, or first optic neuropile. Laminas were examined 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 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. In addition to misplaced nuclei, mitochondria were also misplaced. The mitochondria accumulated in excessive numbers in the distal portion of the photoreceptor terminals, but were absent from the proximal portion of the terminals, where they are also normally found. In addition to misplaced organelles, bsgΔ265 mutant photoreceptors showed a clear increase in axon terminal size, with profiles that were >80% larger in cross-sectional area compared to the control, a difference that was significant. None of these defects was seen in control animals in which non-mutant chromosomes were recombined. On the whole, these defects, misplaced internal organelles and enlarged terminals, suggest global disruptions in cell structure in bsgΔ265 mutant cells, probably due to alterations in the cytoskeleton (Curtin, 2005).
Colocalization of integrin and D-basigin was found in the retina. In addition, studies of integrin gene mutants have reported that the R8 nuclei are sometimes misplaced, descending beneath the basement membrane. This was somtimes seen in bsgΔ265 mosaics and therefore genetic interactions were examined between bsgΔ265 and integrin genes with respect to nuclear placement (Curtin, 2005).
The integrin proteins expressed in the eye, αPS1 and ßPS, are encoded by genes located on the X chromosome, mew codes αPS1 integrin and mys codes ßPS integrin. Mysb45 is a viable allele and males carrying this mutation showed normal placement of photoreceptor nuclei. Mutant flies homozygous in the retina for a weak P-allele (P1096) of basigin showed occasional nuclear misplacement. To look for genetic interactions between bsgΔ265 and integrin genes, double mutants were maed 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. 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 bsgΔ265 also showed a more severe photoreceptor nuclear misplacement phenotype than the sum of the two single mutations would predict; 80% of nuclei were misplaced compared with an average of 24% for bsgΔ265 and 1-2% for mysb45 (Curtin, 2005).
Some integrin gene allelic combinations also showed nuclear misplacement. Animals heterozygous for mewM6, a null allele for αPS1 integrin showed normal placement of photoreceptor nuclei. Animals heterozygous for mysb45, a ßPS1 allele, showed normal nuclear placement, similar to the mysb45 hemizygous males. However, animals heterozygous for both mewM6 and mysb45 showed 3% misplaced nuclei (Curtin, 2005).
Because mammalian basigin stimulates secretion of MMPs, the role of MMPs in the fly visual system was examined. Drosophila has two MMP genes, Mmp1 and Mmp2, both required for viability. Only Mmp2 is expressed in the developing eye (Llano, 2000; Llano, 2002; Page-McCaw, 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 bsgΔ265 mutant retina. Using the same method previously described to make bsgΔ265 eye mosaics, flies were made that were mutant in the eye for a null Mmp2 allele, Mmp2w307* (Page-McCaw, 2003). No misplaced photoreceptor cell nuclei were seen. In case MMP1 functionally replaces MMP-2, mosaics were made that were mutant in the eye for both genes. These also showed no misplaced nuclei. Finally, no effect was seen on nuclear placement when expression of Drosophila TIMP (tissue specific inhibitors of MMPs) was driven in the eye, even though this TIMP gene has previously been reported (Page-McCaw, 2003) to block biological activity of Drosophila MMPs (Curtin, 2005).
Basigin, an IgG family glycoprotein found on the surface of human metastatic tumors, stimulates fibroblasts to secrete matrix metalloproteases (MMPs) that remodel the extracellular matrix, and is thus also known as Extracellular Matrix MetalloPRotease Inducer (EMMPRIN). Using Drosophila novel roles for basigin have been identified. Specifically, photoreceptors of flies with basigin eyes show misplaced nuclei, rough ER and mitochondria, and swollen axon terminals, suggesting cytoskeletal disruptions. This study demonstrates that basigin is required for normal neuron-glia interactions in the Drosophila visual system. Flies with basigin mutant photoreceptors have misplaced epithelial glial cells within the first optic neuropile, or lamina. In addition, epithelial glia insert finger-like projections -- capitate projections (CPs) -- sites of vesicle endocytosis and possibly neurotransmitter recycling. When basigin is missing from photoreceptors terminals, CP formation between glia and photoreceptor terminals is disrupted. Visual system function is also altered in flies with basigin mutant eyes. While photoreceptors depolarize normally to light, synaptic transmission is greatly diminished, consistent with a defect in neurotransmitter release. Basigin expression in photoreceptor neurons is required for normal structure and placement of glia cells (Curtin, 2007).
Drosophila Basigin is a cell-surface glycoprotein of the Ig superfamily and a member of a protein family that includes mammalian EMMPRIN/CD147/basigin, neuroplastin, and embigin. Drosophila basigin has shown that it is required for normal photoreceptor cell structure and normal neuron-glia interaction in the fly visual system. Specifically, the photoreceptor neurons of mosaic animals that are mutant in the eye for basigin show altered cell structure with nuclei, mitochondria and rER misplaced and variable axon diameter compared to wild-type. In addition, glia cells in the optic lamina that contact photoreceptor axons are misplaced and show altered structure. All these defects are rescued by expression of either transgenic fly basigin or transgenic mouse basigin in the photoreceptors demonstrating that mouse Basigin can functionally replace fly Basigin. To determine what regions of the Basigin protein are required for each of these functions, mutant basigin transgenes were created coding for proteins that are altered in conserved residues, these were introduced into the fly genome, and they were tested for their ability to rescue both photoreceptor cell structure defects and neuron-glia interaction defects of basigin. The results suggest that the highly conserved transmembrane domain and the extracellular domains are crucial for Basigin function in the visual system while the short intracellular tail may not play a role in these functions (Munro, 2010).
Reference names in red indicate recommended papers.
Search PubMed for articles about Drosophila Basigin
Berditchevski, F., Chang, S., Bodorova, J. and Hemler, M. (1997). Generation of monoclonal antibodies to integrin-associated proteins. Evidence that alpha3beta1 complexes with EMMPRIN/basigin/OX47/M6. J. Biol. Chem. 272 (46): 29174-80. Medline abstract: 9360995
Besse, F., Mertel, S., Kittel, R. J., Wichmann, C., Rasse, T. M., Sigrist, S. J. and Ephrussi, A. (2007). The Ig cell adhesion molecule Basigin controls compartmentalization and vesicle release at Drosophila melanogaster synapses. J. Cell Biol. 177(5): 843-55. Medline abstract: 17548512
Castrillon, D. H., et al. (1993). Toward a molecular genetic analysis of spermatogenesis in Drosophila melanogaster: characterization of male-sterile mutants generated by single P element mutagenesis. Genetics 135: 489-505. Medline abstract: 8244010
Chen, Z., et al. (2005). Function of HAb18G/CD147 in invasion of host cells by severe acute respiratory syndrome coronavirus. J. Infect. Dis. 191: 755-60. Medline abstract: 15688292
Collins, M. O., et al. (2006). Molecular characterization and comparison of the components and multiprotein complexes in the postsynaptic proteome. J. Neurochem. 97: 16-23. Medline abstract: 16635246
Curtin, K.D., Meinertzhagen, I. A. and Wyman, R. J. (2005). Basigin (EMMPRIN/CD147) interacts with integrin to affect cellular architecture. J. Cell Sci. 118: 2649-2660. Medline abstract: 15928045
Curtin, K. D., Wyman, R. J. and Meinertzhagen, I. A. (2007). Basigin/EMMPRIN/CD147 mediates neuron-glia interactions in the optic lamina of Drosophila. Glia 55(15): 1542-53. PubMed Citation: 17729283
Deora, A. A., et al. (2004). The basolateral targeting signal of CD147 (EMMPRIN) consists of a single leucine and is not recognized by retinal pigment epithelium. Mol. Biol. Cell. 15(9): 4148-65. Medline abstract: 15215314
Deora, A. A., Philp, N., Hu, J., Bok, D. and Rodriguez-Boulan, E. (2005). Mechanisms regulating tissue-specific polarity of monocarboxylate transporters and their chaperone CD147 in kidney and retinal epithelia. Proc. Natl. Acad. Sci. 102(45): 16245-50. Medline abstract: 16260747
Egawa, N., et al. (2006). Membrane type 1 matrix metalloproteinase (MT1-MMP/MMP-14) cleaves and releases a 22-kDa extracellular matrix metalloproteinase inducer (EMMPRIN) fragment from tumor cells. J. Biol. Chem. 281(49): 37576-85. Medline abstract: 17050542
Fadool, J. M., and Linser, P. J. (1993). 5A11 antigen is a cell recognition molecule which is involved in neuronal-glial interactions in avian neural retina. Dev. Dyn. 196: 252-262. Medline abstract: 8219348
Fadool, J. M. and Linser, P. J. (1996). Evidence for the formation of multimeric forms of the 5A11/HT7 antigen. Biochem. Biophys. Res. Commun. 229: 280-286. Medline abstract: 8954119
Fan, Q. W., et al. (1998). Expression of basigin, a member of the immunoglobulin superfamily, in the mouse central nervous system. Neurosci. Res. 30: 53-63. Medline abstract: 9572580
Guo, H., et al. (1997). Stimulation of matrix metalloproteinase production by recombinant extracellular matrix metalloproteinase inducer from transfected Chinese hamster ovary cells. J. Biol. Chem. 272: 24-27. Medline abstract: 8995219
Huang, R.P., et al. (1993). Embigin, a member of the immunoglobulin superfamily expressed in embryonic cells, enhances cell-substratum adhesion. Dev. Biol. 155: 307-314. Medline abstract: 8432389
Kasinrerk, W., Tokrasinwit, N. and Phunpae, P. (1999). CD147 monoclonal antibodies induce homotypic cell aggregation of monocytic cell line U937 via LFA-1/ICAM-1 pathway. Immunology. 96: 184-192. Medline abstract: 10233694
Kirk, P., et al. (2000). CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression. EMBO J. 19: 3896-3904. Medline abstract: 10921872
Kittel, R.J., et al. (2006). Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release. Science 312: 1051-1054. Medline abstract: 16614170
Llano, E., et al. (2000). Dm1-MMP, a matrix metalloproteinase from Drosophila with a potential role in extracellular matrix remodelling during neural development. J. Biol. Chem. 275: 35978-35985. Medline abstract: 10964925
Llano, E., Adam, G., Pendas, A. M., Quesada, V., Sanchez, L. M., Santamaria, I., Noselli, S. and Lopez-Otin, C. (2002). Structural and enzymatic characterization of Drosophila Dm2-MMP, a membrane bound Matrix Metalloproteinase with tissue specific expression. J. Biol. Chem. 277: 23321-23329. Medline abstract: 11967260
Munro, M., Akkam, Y. and Curtin, K. D. (2010). Mutational analysis of Drosophila basigin function in the visual system. Gene 449(1-2): 50-8. PubMed Citation: 19782733
Muramatsu, T. and Miyauchi, T. (2003). Basigin (CD147): a multifunctional transmembrane protein involved in reproduction, neural function, inflammation and tumor invasion. Histol. Histopathol. 18: 981-987. Medline abstract: 12792908
Nabeshima, K., et al. (2006). Emmprin (basigin/CD147): matrix metalloproteinase modulator and multifunctional cell recognition molecule that plays a critical role in cancer progression. Pathol. Int. 56: 359-367. Medline abstract: 16792544
Naruhashi, K., et al. (1997). Abnormalities of sensory and memory functions in mice lacking Bsg gene. Biochem. Biophys. Res. Commun. 236: 733-737. Medline abstract: 9245724
Ochrietor, J. D., Moroz, T. M., Kadomatsu, K., Muramatsu, T. and Linser, P. J. (2001). Retinal degeneration following failed photoreceptor maturation in 5A11/basigin null mice. Exp. Eye Res. 72: 467-477. Medline abstract: 11273674
Ochrietor, J. D., Moroz, T. P., van Ekeris, L., Clamp, M. F., Jefferson, S. C., deCarvalho, A. C., Fadool, J. M., Wistow, G., Muramatsu, T. and Linser, P. J. (2003). Retina-specific expression of 5A11/Basigin-2, a member of the immunoglobulin gene superfamily. Invest. Ophthalmol Vis. Sci. 44: 4086-4096. Medline abstract: 12939332
Page-McCaw, A., Serano, J., Sante, J. M. and Rubin, G. M. (2003). Drosophila matrix metalloproteinases are required for tissue remodeling, but not embryonic development. Dev. Cell. 4: 95-106. Medline abstract: 12530966
Ponta, H., Sherman, L. and Herrlich, P. A. (2003). CD44: from adhesion molecules to signalling regulators. Nat. Rev. Mol. Cell Biol. 4: 33-45. Medline abstract: 12511867
Pushkarsky, T., et al. (2005). Cell surface expression of CD147/EMMPRIN is regulated by cyclophilin 60. J. Biol. Chem. 280(30): 27866-71. Medline abstract: 15946952
Reed, B. H., Wilk, R., Schock, F. and Lipshitz, H. D. (2004). Integrin-dependent apposition of Drosophila extraembryonic membranes promotes morphogenesis and prevents anoikis. Curr. Biol. 14(5): 372-80. Medline abstract: 15028211
Schlosshauer, B., Bauch, H. and Frank, R. (1995). Neurothelin: amino acid sequence, cell surface dynamics and actin colocalization. Eur. J. Cell Biol. 68: 159-166. Medline abstract: 8575462
Schreiner, A., et al. (2007). Junction protein shrew-1 influences cell invasion and interacts with invasion-promoting protein CD147. Mol. Biol. Cell 18(4): 1272-81. Medline abstract: 17267690
Smalla, K.H., et al. (2000). The synaptic glycoprotein neuroplastin is involved in long-term potentiation at hippocampal CA1 synapses. Proc. Natl. Acad. Sci. 97: 4327-4332. Medline abstract: 10759566
Sone, M., et al. (2000). Synaptic development is controlled in the periactive zones of Drosophila synapses. Development. 127: 4157-4168. Medline abstract: 10976048
Sun, J. and Hemler, M. E. (2001). Regulation of MMP-1 and MMP-2 production through CD147/extracellular matrix metalloproteinase inducer interactions. Cancer Res. 61: 2276-2281. Medline abstract: 11280798
Takamori, S., et al. (2006). Molecular anatomy of a trafficking organelle. Cell. 127: 831-846. Medline abstract: 17110340
Tang, W., Chang, S. B. and Hemler, M. E.(2004). Links between CD147 function, glycosylation, and caveolin-1. Mol. Biol. Cell 15(9): 4043-50. Medline abstract: 15201341
Tang, Y., et al. (2005). Extracellular matrix metalloproteinase inducer stimulates tumor angiogenesis by elevating vascular endothelial cell growth factor and matrix metalloproteinases. Cancer Res. 65: 3193-9. Medline abstract: 15833850
Tang, Y., et al. (2006). Regulation of vascular endothelial growth factor expression by EMMPRIN via the PI3K-Akt signaling pathway. Mol. Cancer Res. 4: 371-377. Medline abstract: 16778084
Toole, B. P. (2003). Emmprin (CD147), a cell surface regulator of matrix metalloproteinase production and function. Curr. Top. Dev. Biol. 54: 371-389. Medline abstract: 12696756
Wagh, D. A., et al. (2006). Bruchpilot, a protein with homology to ELKS/CAST, is required for structural integrity and function of synaptic active zones in Drosophila. Neuron. 49: 833-844. Medline abstract: 16543132
Wilson, M. C., et al. (2005). Basigin (CD147) is the target for organomercurial inhibition of monocarboxylate transporter isoforms 1 and 4. the ancillary protein for the insensitive MCT2 is EMBIGIN (gp70). J. Biol. Chem. 280: 27213-21. Medline abstract: 15917240
Xu, D. and Hemler, M. E. (2005). Metabolic activation-related CD147-CD98 complex. Mol. Cell. Proteomics. 4: 1061-1071. Medline abstract: 15901826
Yurchenko, V., Pushkarsky, T., Li, J. H., Dai, W. W., Sherry, B. and Bukrinsky, M. (2005). Regulation of CD147 cell surface expression: involvement of the proline residue in the CD147 transmembrane domain. J. Biol. Chem. 280(17): 17013-9. Medline abstract: 15671024
Yurchenko, V., Constant, S. and Bukrinsky, M. (2006). Dealing with the family: CD147 interactions with cyclophilins. Immunology 117(3): 301-9. Medline abstract: 16476049
Zhou, S., et al. (2005). CD147 is a regulatory subunit of the gamma-secretase complex in Alzheimer's disease amyloid beta- peptide production. Proc. Natl. Acad. Sci. 102: 7499-7504. Medline abstract: 15890777
date revised: 10 August 2010
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