InteractiveFly: GeneBrief

basigin: Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - Basigin

Synonyms -

Cytological map position- 28E3-28E5

Function - receptor

Keywords - cell adhesion, synapse, amnioserosa, neuromuscular junction

Symbol - Bsg

FlyBase ID: FBgn0261822

Genetic map position - 2L: 8,083,422..8,110,561 [+]

Classification - Ig domain superfamily

Cellular location - surface transmembrane



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

Synapses can undergo rapid changes in size as well as in their vesicle release function during both plasticity processes and development. This fundamental property of neuronal cells requires the coordinated rearrangement of synaptic membranes and their associated cytoskeleton, yet remarkably little is known of how this coupling is achieved. In a GFP exon-trap screen, Drosophila Basigin (Bsg) was identified as an immunoglobulin domain-containing transmembrane protein accumulating at periactive zones of neuromuscular junctions. Bsg is required pre- and post-synaptically to restrict synaptic bouton size, its juxtamembrane cytoplasmic residues being important for that function. Bsg controls different aspects of synaptic structure, including distribution of synaptic vesicles and organization of the presynaptic cortical actin cytoskeleton. Strikingly, bsg function is also required specifically within the presynaptic terminal to inhibit nonsynchronized evoked vesicle release. It is thus proposed that Bsg is part of a transsynaptic complex regulating synaptic compartmentalization and strength, and coordinating plasma membrane and cortical organization (Besse, 2007).

Synapses are highly specialized and asymmetric intercellular junctions organized into morphologically, biochemically, and physiologically distinct subdomains. At the presynaptic terminal membrane, active zones mediate Ca2+-dependent synaptic vesicle fusion, whereas the surrounding periactive zones are essential for synaptic vesicle endocytosis and the control of synaptic terminal growth. Definition of distinct synaptic subdomains is not restricted to the plasma membrane but is also clearly visible within the presynaptic terminal cytoplasm. Notably, synaptic vesicles are clustered at the cell cortex, in the vicinity of active zones. In addition, they seem organized into functional subpools displaying distinct release and recycling properties. Such an organization requires the precise trafficking and targeting of vesicles to their appropriate location and the specific recruitment and release of subsets of vesicles, depending on the stimulation conditions. One of the main challenges synapses have to face is maintaining such a highly organized structure while constantly adapting their morphology and strength in response to developmental programs and/or external stimuli. Indeed, synaptic terminals can adjust their size. the number, size, and composition of their pre- and post-synaptic membrane specializations; and the availability and release competence of cytoplasmic synaptic vesicles. These dynamic changes require the maintenance of precise physical and functional connections between pre- and post-synaptic compartments, as well as between cytoplasmic and plasma membrane subdomains (Besse, 2007).

To date, the mechanisms allowing such a dynamic reorganization are still poorly understood. However, using the Drosophila neuromuscular junction (NMJ) as a genetic model, different components of periactive zones, including transmembrane proteins and adaptor molecules, have been implicated in the control of terminal outgrowth. Cell adhesion molecules (CAMs) of the Ig superfamily seem particularly important in maintaining the integrity of synaptic terminals but also in transmitting signals to the cell interior, thereby promoting differentiation of pre- and postsynaptic specializations and regulating synaptic structure and function. Moreover, the actin-rich presynaptic cytoskeleton is important for rearranging synaptic domains and for controlling synaptic vesicle distribution and release ability. How the linkage between cortical cytoskeleton, cytoplasmic vesicle pools, and specialized membrane domains is mediated and, more generally, how plasma membrane and cytoplasmic membranes are spatially and functionally connected largely remain to be elucidated (Besse, 2007).

This study identified the transmembrane Ig CAM Basigin (Bsg) as a new component of periactive zones at D. melanogaster NMJ synapses. Bsg is the only Drosophila member of the Basigin/Embigin/Neuroplastin family of glycoproteins, of which mammalian Bsg has been shown to have multiple functions, including in tumor progression (Nabeshima, 2006). It seems to regulate cell architecture and cell-cell recognition (Fadool, 1993; Curtin, 2005), act in signaling (Guo, 1997; Tang, 2006), and act as a chaperone for transmembrane proteins (Kirk, 2000; Zhou, 2005). By analogy to other mammalian cell surface glycoproteins, and in particular to the CD44 transmembrane protein family (Ponta, 2003), Bsg may be essential for establishment of transmembrane complexes and for organization of cell structure and signal transduction cascades. Interestingly, mammalian Bsg and Neuroplastin have been suggested to play a role in memory functions and long-term potentiation (Naruhashi, 1997; Smalla, 2000), respectively, although their precise function has not been determined (Besse, 2007).

Drosophila Bsg is required in both pre- and postsynaptic compartments to control formation and growth of synaptic varicosities (or boutons) at larval NMJs. Bsg is a bona fide Ig CAM because (1) it can promote cell-cell adhesion and (2) its transmembrane and/or juxtamembrane cytoplasmic domains are critical for its function in vivo. Furthermore, down-regulation of bsg affects the size of postsynaptic receptor fields, as well as the distribution of synaptic vesicles within neuronal terminals. These defects are associated with alterations of the actin/Spectrin network, suggesting that Bsg accumulation at the plasma membrane regulates synaptic compartmentalization and architecture. Strikingly, it was found that Bsg function is also essential within the presynaptic compartment for the restriction of neurotransmitter release. Based on these in vivo data, it is proposed that Bsg may be part of a transsynaptic complex surrounding active zones and involved in the coordinated development of pre- and post-synaptic membranes, as well as in the functional coupling of plasma membrane and cortical subdomains (Besse, 2007).

The transmembrane Ig CAM Bsg has been identified as a new component of perisynaptic zones of Drosophila NMJs. Bsg function is required in pre- and post-synaptic compartments for the formation and growth of synaptic boutons, and Bsg controls different aspects of synapse structure, including distribution of synaptic vesicles and organization of the presynaptic terminal cortical actin network. Bsg behaves as a canonical Ig CAM, since it promotes cell-cell adhesion and has a conserved motif in its cytoplasmic tail essential for its function in vivo. It is proposed that Bsg is part of a transsynaptic complex regulating synaptic growth and structural organization. Moreover, and very originally for an Ig CAM, it was found that Bsg is essential for inhibiting transmitter release, and this function is restricted to the presynaptic compartment (Besse, 2007).

In Drosophila, the final pattern of larval motoneuron connections and the establishment of synapses are complete by the end of embryogenesis, yet NMJs are highly dynamic during larval development, expanding through sprouting of new branches and addition of new synaptic boutons. This study has shown that down-regulation of Bsg levels at the Drosophila NMJ strongly affects bouton growth and budding, resulting in a decrease in bouton number. This effect is probably independent of the increased transmitter release observed in bsg larvae because (1) it is already observed in early second instar larvae and (2), in contrast to the increased neurotransmission phenotype, it corresponds to a requirement for bsg function in both pre- and postsynaptic compartments. This may reflect a role of Bsg in regulating adhesion between pre- and postsynaptic membranes, as described for the Ig CAM Fas II. Bsg function is, however, not restricted to modulation of synaptic membrane adhesiveness; mutant forms lacking the transmembrane and/or juxtamembrane cytoplasmic domains can promote cell-cell aggregation but function poorly in vivo. Bsg may thus also signal toward the cell cytoplasm and/or regulate the actin cytoskeleton (Besse, 2007).

In addition, Bsg controls synaptic architecture: it modulates the size of postsynaptic glutamate receptor fields and, more strikingly, is required for the anchoring of synaptic vesicles to the presynaptic terminal cortex. This suggests that Bsg could be a key component coupling organization of the plasma membrane and cytoplasmic vesicular compartments. Consistent with such a role, defects were observed in 'plasma membrane versus internal membrane' sorting of presynaptic transmembrane components. It is thus proposed that Bsg may be part of a transsynaptic complex surrounding active zones and involved in the coordinated development of pre- and postsynaptic membranes, as well as in the functional coupling of plasma membrane and cytoplasmic vesicles. Notably, Bsg recently has been identified (Takamori, 2006) within synaptic vesicle preparations (Besse, 2007).

Bsg might act directly, or through interaction with transmembrane and cytoplasmic partners. Consistent with this latter hypothesis, it has been shown that conserved amino acids found in the cytoplasmic tail of Bsg are crucial for the function of the protein in vivo and that they may thus mediate transduction of a signal toward the cell cytoplasm and/or interaction with the cortical cytoskeleton (Besse, 2007).

The F-actin/Spectrin cytoskeleton underlying pre- and postsynaptic membranes seems essential for different aspects of synaptic terminal growth and plasticity, including terminal expansion, organization of presynaptic vesicle pools, and postsynaptic receptor clustering. This study has shown that Bsg modulates the organization of the presynaptic actin cytoskeleton, as revealed by the presence of ectopic aggregates of F-actin and actin-associated proteins within the lumen of bsg synaptic boutons. Although no obvious alterations of the postsynaptic actin cytoskeleton, which is intermingled with the dense membraneous network of the SSR, was detected it is nonetheless possible that Bsg also regulates this cytoskeleton. These observations further support previous reports (Schlosshauer, 1995; Curtin, 2005) showing that Bsg colocalizes with the actin cytoskeleton specifically at cell-cell contacts and that expression of Bsg in cultured cells results in reorganization of the F-actin network and consequent formation of lamellipodia (Besse, 2007).

At the NMJ, Bsg may modulate actin cytoskeleton organization indirectly, by interacting with integrin subunits at the plasma membrane. Indeed, mammalian Bsg has been found in a complex with ß1-integrin (Berditchevski, 1997; Xu, 2005), and both α- and ß-integrin subunits colocalize with Drosophila Bsg at larval NMJs. Furthermore, although no genetic interaction was observed between bsg and myspheroid (mys, which encodes the main Drosophila ß-integrin subunit) during larval NMJ development, several mys missense mutations were observed displaying a junction undergrowth phenotype very similar to that of bsg mutants. Another attractive hypothesis is that Bsg, through its juxtamembrane cytoplasmic motif, recruits Spectrin or other actin-associated proteins and thereby directly participates in organizing a cortical actin network. Interestingly, different members of the FERM domain protein family have been shown to link cell surface glycoproteins and the actin cytoskeleton by directly binding to both the intracellular region of transmembrane proteins and to actin or Spectrin. In particular, Moesin directly interacts with the intracytoplasmic domains of mammalian CD43, CD44, and intercellular adhesion molecule 2, through a positively charged amino acid cluster found in the juxtamembrane region of these proteins. The striking conservation and functional importance of the KRR juxtamembrane motif of Bsg suggests that such cytoplasmic proteins may physically link cell-surface Bsg to the underlying F-actin network and mediate organization of cortical domains at the NMJ (Besse, 2007).

Down-regulation of bsg at Drosophila NMJ terminals causes a dramatic increase in transmitter release, which, is unique among Ig CAM mutants. This phenotype corresponds to a specific presynaptic function of Bsg and may be explained by (1) an alteration of the excitability of the synaptic terminal or (2) an altered definition of the different functional synaptic vesicle pools (Besse, 2007).

Mammalian Bsg has been shown to promote translocation of transporter proteins to the plasma membrane, as well as regulate the activity of multiprotein transmembrane complexes (Kirk, 2000; Zhou, 2005). At the Drosophila NMJ, Bsg may thus be required for the proper distribution and/or clustering of ion channels regulating Ca2+ dynamics. In this context, it was recently demonstrated that the presynaptic scaffolding protein BRP is required for the clustering of Ca2+ channels and for their spatial coupling with synaptic vesicles at the Drosophila active zone. This process appears to be required for the rapid evoked component of synaptic vesicle release but not for spontaneous release (Kittel, 2006). Therefore, both the additional spontaneous and delayed evoked component of transmitter release in bsg mutants might correspond to the fusion of vesicles lacking a tight association with Ca2+ channels. An elevated contribution of asynchronous release has also been reported to occur naturally at particular synapses of the mammalian central nervous system and is thought to reflect long-lasting Ca2+ transients and a loose coupling between Ca2+ sources and vesicles. It is thus conceivable that down-regulation of Bsg alters Ca2+ dynamics, leading to an abnormal recruitment of vesicles distant from Ca2+ sources (Besse, 2007).

Alternatively, the observed transmitter release phenotype may not be associated with an alteration of Ca2+ signals, but rather reflects a role of Bsg in organizing synaptic vesicle populations. It has been suggested that synaptic vesicles are organized into functionally distinct pools (readily releasable pool, recycling pool, and reserve pool) with specific recycling and mobilization properties. This study has shown that down-regulation of bsg leads to an abnormal distribution of vesicles in resting terminals, as well as an aberrant trafficking of vesicles to the center of boutons (where reserve pool vesicles are thought to reside) under conditions where synaptic vesicle recycling is normally restricted to the periphery (and to the recycling pool). These data suggest that the definition of different synaptic vesicle populations may be altered in bsg mutants, which in turn may explain the observed defects in precise recruitment and release of vesicles. This is of particular interest given that mammalian Bsg has been suggested to physically associate with synaptic vesicles (Takamori, 2006). Additionally, presynaptic actin filaments have been proposed to provide a physical barrier impeding vesicle dispersion and, in particular, to regulate the availability of the reserve pool. They have also been suggested to participate in a mechanism restraining fusion of synaptic vesicles in cultured hippocampal neurons. An attractive possibility is therefore that Bsg controls synaptic vesicle organization and retention through its effect on the cortical actin cytoskeleton (Besse, 2007).

Sphingolipids regulate neuromuscular synapse structure and function in Drosophila

Sphingolipids are found in abundance at synapses and have been implicated in regulation of synapse structure, function and degeneration. Serine Palmitoyl-transferase (SPT) is the first enzymatic step for synthesis of sphingolipids. Analysis of the Drosophila larval neuromuscular junction revealed mutations in the SPT enzyme subunit, lace/SPTLC2 resulted in deficits in synaptic structure and function. Although NMJ length is normal in lace mutants, the number of boutons per NMJ is reduced to approximately 50% of the wild type number. Synaptic boutons in lace mutants are much larger but show little perturbation to the general ultrastructure. Electrophysiological analysis of lace mutant synapses revealed strong synaptic transmission coupled with predominance of depression over facilitation. The structural and functional phenotypes of lace mirrored aspects of Basigin (Bsg), a small Ig-domain adhesion molecule also known to regulate synaptic structure and function. Mutant combinations of lace and Bsg generated large synaptic boutons, while lace mutants showed abnormal accumulation of Bsg at synapses, suggesting that Bsg requires sphingolipid to regulate structure of the synapse. This data points to a role for sphingolipids in the regulation and fine-tuning of synaptic structure and function while sphingolipid regulation of synaptic structure may be mediated via the activity of Bsg (West, 2018).

A prominent enrichment of glycosphingolipids has been demonstrated within synaptic structures in the mammalian brain. To date understanding of the role of these enigmatic lipids in synapse structure and function has yet to be fully elucidated. Sphingolipids are major lipid components of the plasma and endomembrane system and have been implicated in many forms of neuropathy and neurodegeneration. Sphingolipids are proposed to generate structure in membranes due to their rigidity and association with cholesterol. They are also known to be potent signalling molecules regulating processes such as apoptosis, proliferation, migration and responses to oxidative stress (West, 2018).

Numerous neurological and neurodegenerative conditions are directly attributable to the inability to synthesise or catabolise sphingolipids. The failure to synthesise all or particular sphingolipids gives rise to a number of neurological conditions such as infant-onset symptomatic epilepsy (loss of GM3 ganglioside synthesis, bovine spinal muscular atrophy (loss of 3-ketohydrosphingosine reductase and hereditary sensory and autonomic neuropathy type 1 (HSAN1; recessive and dominant mutations in serine palmitoyl transferase subunit 1 (SPTLC1). Conversely failure to catabolise sphingolipids in the lysosome generates a subset of lysosomal storage diseases/disorders (LSD's) known as sphingolipidoses, of which there are approximately 14 identified separate genetic conditions. Sphingolipids are now suggested to have a prominent role in the onset and progression of Alzheimer's disease while the production after bacterial infection of autoimmune antibodies to gangliosides present at the neuromuscular synapse is likely to cause the dramatic and often lethal paralysis seen in Guillain-Barrê and Miller-Fisher syndromes. The presence of sphingolipids at the synapse is further attested by the ability of tetanus and botulinal toxins to effect their entry to synapses via co-attachment to synaptic glycosphingolipids (West, 2018).

While the presence of sphingolipids (in particular, glycosphingolipids) at the synapse is well established, little is known about their functional or structural role in the operational life of the synapse. Some in vitro studies have addressed the role of sphingolipids at synapses in the context of sphingolipid/cholesterol microdomains and indicate roles in the function and localisation of neurotransmitter receptors and synaptic exocytosis. The prominence of sphingolipids in neurological disease suggests that absence or accumulation of sphingolipids can exert an influence in synaptic function and indicates an inappropriately large gap in knowledge regarding the actions of these lipids at the synapse. In the above outlined context, roles for sphingolipids in synapse structure and function remain to be determined. To this end, this study has undertaken an analysis of sphingolipid function at a model synapse, the third instar neuromuscular junction of Drosophila. Mutations were analyzed in SPT2/SPTLC2 (Serine Palmitoyltransferase, Long Chain Base Subunit 2), which encodes an essential subunit of the Serine Palmitoyltransferase (SPT) heterodimer necessary for the initial step in sphingolipid synthesis, for defects in neuromuscular synapse structure. Evidence is presented to suggest that sphingolipids are essential for synaptic structure and function, and structural regulation may be mediated partially through function of the Ig domain adhesion protein Basigin/CD147 (Bsg) (West, 2018).

The enrichment of sphingolipids at synapses has been long known. Assigning functions for these enigmatic lipids at the synapse has remained problematic. Ablation of gangliosides in mouse has identified subtle defects in neurotransmission while loss of G3- ganglioside synthesis results in an infantile onset epilepsy, the mechanism for which remains obscure. A specific role for sphingosine has been identified in promoting SNARE protein fusion and synaptic exocytosis (West, 2018).

Many sphingolipid species present in the outer leaflet of the plasma membrane are found in association with cholesterol as 'lipid rafts'. Neurons receive supplementary cholesterol from glia which is essential for supporting synapse maturation and additional synaptogenesis suggesting cholesterol, and potentially lipid rafts, are rate limiting for these processes. Depletion of both cholesterol and sphingolipids together has been shown to reduce and enlarge dendritic spines with eventual loss of synapses in hippocampal neurons in culture possibly due to reduced association with lipid rafts of synapse structure promoting proteins such as Post-Synaptic Density protein 95 (PSD95). This study reduced synthesis of sphingolipids with a mutation in SPTLC2, and examined the development of neuromuscular synapses in the Drosophila larval preparation. This approach has allowed study of the genetic depletion of sphingolipids at an identified synapse in vivo and investigate a role for sphingolipids in the regulation of synaptic structure and activity. As part of this study, a potential role was identified for the Ig domain cell adhesion protein Bsg in sphingolipid dependent regulation of synaptic structure (West, 2018).

The enrichment of sphingolipids at synapses has been long known. Assigning functions for these enigmatic lipids at the synapse has remained problematic. Ablation of gangliosides in mouse has identified subtle defects in neurotransmission while loss of G3-ganglioside synthesis results in an infantile onset epilepsy, the mechanism for which remains obscure. A specific role for sphingosine has been identified in promoting SNARE protein fusion and synaptic exocytosis. Many sphingolipid species present in the outer leaflet of the plasma membrane are found in association with cholesterol as 'lipid rafts'. Neurons receive supplementary cholesterol from glia which is essential for supporting synapse maturation and additional synaptogenesis suggesting cholesterol, and potentially lipid rafts, are rate limiting for these processes. Depletion of both cholesterol and sphingolipids together has been shown to reduce and enlarge dendritic spines with eventual loss of synapses in hippocampal neurons in culture possibly due to reduced association with lipid rafts of synapse structure promoting proteins such as Post-Synaptic Density protein 95 (PSD95). This study has reduced synthesis of sphingolipids with a mutation in SPTLC2, and examined the development of neuromuscular synapses in the Drosophila larval preparation. This approach has allowed study of the genetic depletion of sphingolipids at an identified synapse in vivo and investigate a role for sphingolipids in the regulation of synaptic structure and activity. As part of this study, a potential role has been identified for the Ig domain cell adhesion protein Bsg in sphingolipid dependent regulation of synaptic structure (West, 2018).

On examination of sphingolipid deficient synapses, a disruption to the normal synaptic structure was observed. Synaptic boutons were enlarged and the overall numbers of boutons reduced by ~50% while the length of the neuromuscular synapse remained indistinguishable from wildtype. This phenotype is highly reminiscent of the reduction of synapse number, but increase in synapse size observed in hippocampal neurons in culture depleted for lipid rafts. Nevertheless, beyond the structural deficit of the synapse, the ultrastructure of the synapse was remarkably intact, suggesting a role in fine-tuning of synaptic properties (West, 2018).

Synapses depleted for sphingolipids were capable of greater growth when combined with the synaptic overgrowth mutation highwire (hiw). The data suggests the mutations in lace, encoding a pyridoxal phosphate-dependent transferase, and sphingolipid depletion decouples bouton structure from normal synaptic length. Large boutons are observed in mutants of mothers against dpp (mad), thick veins (tkv), saxophone (sax) medea (med), and glass-bottom-boat (gbb), components of the TGF-β pathway that is known to regulate synaptic growth. However these mutations reduce synaptic length by ~50% and ultrastructural synaptic defects such as non-plasma membrane attached active zones (T-bars), large endosomal vesicles and ripples in pre-synaptic peri-active membranes are observed. One obvious ultrastructural defect that is present in sphingolipid depleted synapses is enlarged mitochondria. Enlarged mitochondria are observed in a number of sensory neuropathies and it is of interest that dominant mutations in SPTLC1 and SPTLC2 that generate aberrant sphingolipids give rise to Hereditary and Sensory Neuropathy Type 1 (HSAN1) where enlarged mitochondria are often observed. This may be attributable to a recognised role for sphingolipids in mitochondrial fission (West, 2018).

To dissect the spatial requirement for sphingolipid regulation of synapse structure, the lace mutant was rescued with a rescue transgene, expressed globally, pre- or post-synaptically. It was possible to rescue synaptic bouton size and number with a global expression of the rescue transgene, but no aspects of the phenotype could be rescued with a pre-synaptic expression. Perturbed NMJ morphology could also be rescued by glial or post-synaptic expression of lace, however post-synaptic muscle expression generated a partial rescue, with an excess of 'satellite' boutons, a phenotype normally associated with integrin dysfunction or endocytic defects. Previous data feeding lace mutant larvae with sphingosine, the product of the SPT enzyme, partially rescued lace mutant associated phenotypes. Taken together with the current analysis, there is a strong suggestion that sphingolipid precursors such as sphingosine may be able to act non-cell autonomously, and traffic between cells to support synapse structure and function, but not when supplied from the nervous system (West, 2018).

Analysis of EJP and miniEJP characteristics at the 3rd instar larval NMJ reveals mutations in lace produce, at the Ca2+/Mg2+ concentrations that were used, a small but significant increase in synaptic strength, accompanied by a change in short-term plasticity, with synaptic depression predominating over synaptic facilitation. NMJs with high-quantal content EJPs normally show synaptic depression during paired or short-train repetitive stimulation, while those with a low basal quantal content show synaptic facilitation (Lnenicka, 2000; Lnenicka, 2006). Further analysis is required, for instance using a range of Ca2+ concentrations, to establish whether this apparent change in synaptic plasticity is commensurate with a greater basal synaptic strength in the lace mutant larvae, or whether it represents a specific effect of the mutation, disrupting the normal link between mechanisms that couple basal quantal content to short-term synaptic plasticity. In vitro and in vivo analysis has suggested a role for sphingolipids in synaptic vesicle endocytosis and exocytosis in addition to a role in neurotransmitter distribution. No evident defects were observed in neurotransmitter receptor distribution. Interestingly, ablation of major subsets of gangliosides and subsequent synaptic function at the NMJ in a mouse model reveals a more pronounced run-down of neurotransmitter release upon sustained stimulation, consistent with the data presented in this study. It is not possible, however, to directly attribute the apparent deficit in synaptic facilitation observed in lace mutants to exoF or endocytosis, at this point (West, 2018).

A phenotypic similarity at the larval neuromuscular synapse is noted between the lace mutants and mutations in the small Ig domain adhesion protein Basigin/CD147. Bsg is a glycoprotein localised in the plasma membrane that is known to genetically interact with integrins during development of the Drosophila eye. In Bsg mutants, synaptic boutons at the larval neuromuscular junction are enlarged in size and reduced in number with a modest reduction in synaptic span. Bsg has previously been localised to sphingolipid enriched lipid rafts in invading epithelial breast cells, and this study observed that Bsg is abundant in the lipid raft associated membrane fraction, co-sedimenting with syntaxin, a known component of lipid rafts. It cannot be said at this juncture if Bsg function is directly regulated by sphingolipids. Indeed, recruitment of Bsg to lipid rafts can be critical for the recruitment of other protein factors such as claudin-5 in retinal vascular epithelial cells. However, given the genetic interaction between Bsg and lace, with bsg;lace transheterozygous double mutants phenocopying both lace and bsg mutants, the data suggests Bsg and sphingolipids genetically interact to regulate synaptic structure. This interaction is interpreted as indirect; the loss of sphingolipid generated in the lace mutant affecting Bsg function to regulate synapse structure and function (West, 2018).

Synaptic sphingolipids have previously been implicated in synaptic vesicle release, endocytosis, neurotransmitter receptor localisation and maintenance of synaptic activity. However other roles at the synapse for these enigmatic lipids remain elusive. Two potential functions for sphingolipid at the synapse are suggested by this study. Mitochondrial uptake of Ca2+ shapes Ca2+ dependent responses. The enlarged mitochondria observed in lace mutants may impinge on Ca2+ uptake to affect synaptic facilitation. A further deficit in Ca2+ handling at the synapse is suggested by the recent finding that Bsg is an obligatory subunit of plasma membrane Ca2+-ATPases (PMCAs). PMCAs extrude Ca2+ to the extracellular space, and knock-out of Bsg considerably affects Ca2+ handling by PMCAs. Sphingolipid deficient synapses in the lace mutant have deficits in Bsg function which may in turn have an effect on Ca2+ dynamics via PMCA function (West, 2018).

Ablation of sphingolipid synthesis at a Drosophila model synapse supports a role for sphingolipids in maintenance of synaptic activity and regulation of synaptic structure. The analysis also points to sphingolipid dependent regulation of synaptic structure via function of the small Ig-domain protein Bsg. The precise regulation of synapse structure and function is a potent mechanism underlying synaptic plasticity and it is suggested that the presence of sphingolipids at synapse may partially reflect this function (West, 2018).


REGULATION


DEVELOPMENTAL BIOLOGY

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).

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, 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).

Basigin expression in cultured cells and in vivo

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).


EFFECTS OF MUTATION

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).

Integrin-dependent apposition of Drosophila extraembryonic membranes promotes morphogenesis and prevents anoikis

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 interacts with integrin to affect cellular architecture

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/EMMPRIN/CD147 mediates neuron-glia interactions in the optic lamina of Drosophila

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).

Mutational analysis of Drosophila basigin function in the visual system

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).


EVOLUTIONARY HOMOLOGS

Interactions of mammalian Basigin is reviewed in the following article, which is freely available online:

Yurchenko, V., Constant, S. and Bukrinsky, M. (2006). Dealing with the family: CD147 interactions with cyclophilins. Immunology 117(3): 301-9. Medline abstract: 16476049

Cell surface expression of CD147/EMMPRIN is regulated by cyclophilin 60

CD147, also known as extracellular matrix metalloproteinase inducer, is a regulator of matrix metalloproteinase production and also serves as a signaling receptor for extracellular cyclophilins. Cell surface expression of CD147 is sensitive to cyclophilin-binding drug cyclosporin A, suggesting involvement of a cyclophilin in the regulation of intracellular transport of CD147. This report identifies this cyclophilin as cyclophilin 60 (Cyp60), a distinct member of the cyclophilin family of proteins. CD147 co-immunoprecipitates with Cyp60, and confocal immunofluorescent microscopy revealed intracellular co-localization of Cyp60 and CD147. This interaction with Cyp60 involves proline 211 of CD147, which was shown previously to be critical for interaction between CD147 and another cyclophilin, cyclophilin A, in solution. Mutation of this proline residue abrogates co-immunoprecipitation of CD147 and Cyp60 and reduces surface expression of CD147 on the plasma membrane. Suppression of Cyp60 expression using RNA interference had an effect similar to that of cyclosporin A: reduction of cell surface expression of CD147. These results suggest that Cyp60 plays an important role in the translocation of CD147 to the cell surface. Therefore, Cyp60 may present a novel target for therapeutic interventions in diseases where CD147 functions as a pathogenic factor, such as cancer, human immunodeficiency virus infection, or rheumatoid arthritis (Pushkarsky, 2005).

Regulation of CD147 cell surface expression: involvement of the proline residue in the CD147 transmembrane domain

CD147, also known as extracellular matrix metalloproteinase inducer, is a regulator of matrix metalloproteinase production and serves as a signaling receptor for extracellular cyclophilins. The cell surface expression of CD147 is regulated by cyclophilins via the transmembrane domain of CD147. Solution binding experiments demonstrated that the transmembrane domain was both necessary and sufficient for CD147 binding to cyclophilin A (CypA). Treatment with cyclosporin A significantly reduces surface expression of CD147 and of CD8-CD147 fusion protein carrying the extracellular domain of CD8 fused to the transmembrane and cytoplasmic domains of CD147, but does not affect expression of CD8. Peptide binding studies demonstrated specific interaction between CypA and the proline-containing peptide from the CD147 transmembrane domain. Mutation of this proline residue reduces binding of CD147-derived peptides to CypA and also diminishes transport of CD147 to the plasma membrane without reducing the total level of CD147 expression. These results suggest involvement of a cyclophilin-related protein in CD147 cell surface expression and provide molecular details for regulation of CD147 trafficking by cyclophilins (Yurchenko, 2005).

The basolateral targeting signal of CD147 consists of a single leucine and is not recognized by retinal pigment epithelium

CD147, a type I integral membrane protein of the immunoglobulin superfamily, exhibits reversed polarity in retinal pigment epithelium (RPE). CD147 is apical in RPE in contrast to its basolateral localization in extraocular epithelia. This stimulated an interest in understanding the basolateral sorting signals of CD147 in prototypic Madin-Darby canine kidney (MDCK) cells. The cytoplasmic domain of CD147 has basolateral sorting information but is devoid of well-characterized basolateral signals, such as tyrosine and di-leucine motifs. Hence, systematic site-directed mutagenesis was carried out to delineate basolateral targeting information in CD147. This detailed analysis identified a single leucine (252) as the basolateral targeting motif in the cytoplasmic tail of CD147. Four amino acids (243-246) N-terminal to leucine 252 are also critical basolateral determinants of CD147, because deletion of these amino acids leads to mistargeting of CD147 to the apical membranes. The involvement of adaptor complex 1B (AP1B) in the basolateral trafficking of CD147 was ruled out, because LLC-PK1 cells lacking AP1B, target CD147 basolaterally. At variance with MDCK cells, the human RPE cell line ARPE-19 does not distinguish between CD147 (WT) and CD147 with leucine 252 mutated to alanine and targets both proteins apically. Thus, this study identifies an atypical basolateral motif of CD147, which comprises a single leucine and is not recognized by RPE cells. This unusual basolateral sorting signal will be useful in unraveling the specialized sorting machinery of RPE cells (Deora, 2004).

Mechanisms regulating tissue-specific polarity of monocarboxylate transporters and their chaperone CD147 in kidney and retinal epithelia

Proton-coupled monocarboxylate transporters (MCT) MCT1, MCT3, and MCT4 form heterodimeric complexes with the cell surface glycoprotein CD147 and exhibit tissue-specific polarized distributions that are essential for maintaining lactate and pH homeostasis. In the parenchymal epithelia of kidney, thyroid, and liver, MCT/CD147 heterocomplexes are localized in the basolateral membrane where they transport lactate out of or into the cell depending on metabolic conditions. A unique distribution of lactate transporters is found in the retinal pigment epithelium (RPE), which regulates lactate levels of the outer retina. In RPE, MCT1/CD147 is polarized to the apical membrane and MCT3/CD147 to the basolateral membrane. The mechanisms responsible for tissue-specific polarized distribution of MCTs are unknown. This study demonstrates that CD147 carries sorting information for polarized targeting of the MCT1/CD147 hetero-complexes in kidney and RPE cells. In contrast, MCT3 and MCT4 harbor dominant sorting information that cotargets CD147 to the basolateral membrane in both epithelia. RNA interference experiments show that MCT1 promotes CD147 maturation. These results open a unique paradigm to study the molecular basis of tissue-specific polarity (Deora, 2005).

Links between CD147 function, glycosylation, and caveolin-1

Cell surface CD147 shows remarkable variations in size (31-65 kDa) because of heterogeneous N-glycosylation, with the most highly glycosylated forms functioning to induce matrix metalloproteinase (MMP) production. All three CD147 N-glycosylation sites make similar contributions to both high and low glycoforms (HG- and LG-CD147). l-Phytohemagglutinin lectin binding and swainsonine inhibition experiments indicated that HG-CD147 contains N-acetylglucosaminyltransferase V-catalyzed, beta1,6-branched, polylactosamine-type sugars, which account for its excess size. Therefore, CD147, which is itself elevated on invasive tumor cells, may make a major contribution to the abundance of beta1,6-branched polylactosamine sugars that appear on invasive tumor cells. Caveolin-1 associates with CD147, thus inhibiting CD147 self-aggregation and MMP induction. This study shows that caveolin-1 associates with LG-CD147 and restricts the biosynthetic conversion of LG-CD147 to HG-CD147. In addition, HG-CD147 (but not LG-CD147) was preferentially captured as a multimer after treatment of cells with a homobifunctional cross-linking agent and was exclusively recognized by monoclonal antibody AAA6, a reagent that selectively recognizes self-associated CD147 and inhibits CD147-mediated MMP induction. In conclusion, this study has (1) determined the biochemical basis for the unusual size variation in CD147, (2) established that CD147 is a major carrier of beta1,6-branched polylactosamine sugars on tumor cells, and (3) determined that caveolin-1 can inhibit the conversion of LG-CD147 to HG-CD147. Because it is HG-CD147 that self-aggregates and stimulates MMP induction, a mechanism has been found to explain how caveolin-1 inhibits these processes. These results help explain the previously established tumor suppressor functions of caveolin-1 (Tang, 2004).

Extracellular matrix metalloproteinase inducer stimulates tumor angiogenesis by elevating vascular endothelial cell growth factor and matrix metalloproteinases

Matrix metalloproteinases (MMPs) are endopeptidases that play pivotal roles in promoting tumor disease progression, including tumor angiogenesis. In many solid tumors, MMP expression can be attributed to tumor stromal cells and is partially regulated by tumor-stroma interactions via tumor cell-associated extracellular matrix metalloproteinase inducer (EMMPRIN). The role of EMMPRIN during tumor angiogenesis and growth was explored by modulating EMMPRIN expression and activity using recombinant DNA engineering and neutralizing antibodies. In human breast cancer cells, changes in EMMPRIN expression influences vascular endothelial growth factor (VEGF) production at both RNA and protein levels. In coculture of tumor cells and fibroblasts mimicking tumor-stroma interactions, VEGF expression is induced in an EMMPRIN- and MMP-dependent fashion, and is further enhanced by overexpressing EMMPRIN. Conversely, VEGF expression is inhibited by suppressing EMMPRIN expression in tumor cells, by neutralizing EMMPRIN activity, or by inhibiting MMPs. In vivo, EMMPRIN overexpression stimulates tumor angiogenesis and growth; both are significantly inhibited by antisense suppression of EMMPRIN. Expression of both human and mouse VEGF and MMP, derived from tumor and host cells, respectively, is regulated by EMMPRIN. These results suggest a novel tumor angiogenesis mechanism in which tumor-associated EMMPRIN functionally mediates tumor-stroma interactions and directly contributes to tumor angiogenesis and growth by stimulating VEGF and MMP expression (Tang, 2005; full text of article).

Membrane type 1 matrix metalloproteinase (MT1-MMP/MMP-14) cleaves and releases a 22-kDa extracellular matrix metalloproteinase inducer (EMMPRIN) fragment from tumor cells

Proteolytic shedding is an important step in the functional down-regulation and turnover of most membrane proteins at the cell surface. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a multifunctional glycoprotein that has two Ig-like domains in its extracellular portion and functions in cell adhesion as an inducer of matrix metalloproteinase (MMP) expression in surrounding cells. Although the shedding of EMMPRIN is reportedly because of cleavage by metalloproteinases, the responsible proteases, cleavage sites, and stimulants are not yet known. In this study, it was found that human tumor HT1080 and A431 cells shed a 22-kDa EMMPRIN fragment into the culture medium. The shedding is enhanced by phorbol 12-myristate 13-acetate and inhibited by TIMP-2 but not by TIMP-1, suggesting the involvement of membrane-type MMPs (MT-MMPs). Indeed, down-regulation of the MT1-MMP expression in A431 cells using small interfering RNA inhibits the shedding. The 22-kDa fragment was purified, and the C-terminal amino acid was determined. A synthetic peptide spanning the cutting site was cleaved by MT1-MMP in vitro. The cleavage site is located in the linker region connecting the two Ig-like domains. The N-terminal Ig-like domain is important for the MMP inducing activity of EMMPRIN and for cell-cell interactions, presumably through its ability to engage in homophilic interactions, and the 22-kDa fragment retained the ability to augment MMP-2 expression in human fibroblasts. Thus, the MT1-MMP-dependent cleavage eliminates the functional N-terminal domain of EMMPRIN from the cell surface, which is expected to down-regulate its function. At the same time, the released 22-kDa fragment may mediate the expression of MMPs in tumor tissues (Egawa, 2006).

CD147 is a regulatory subunit of the gamma-secretase complex in Alzheimer's disease amyloid beta-peptide production

gamma-Secretase is a membrane protein complex that cleaves the beta-amyloid precursor protein (APP) within the transmembrane region, after prior processing by beta-secretase, producing amyloid beta-peptides Abeta(40) and Abeta(42). Errant production of Abeta-peptides that substantially increases Abeta(42) production has been associated with the formation of amyloid plaques in Alzheimer's disease patients. Biophysical and genetic studies indicate that presenilin-1, which contains the proteolytic active site, and three other membrane proteins [nicastrin, anterior pharynx defective-1 (APH-1), and presenilin enhancer-2 (PEN-2)] are required to form the core of the active gamma-secretase complex. This study reports the purification of the native gamma-secretase complexes from HeLa cell membranes and the identification of an additional gamma-secretase complex subunit, CD147, a transmembrane glycoprotein with two Ig-like domains. The presence of this subunit as an integral part of the complex itself was confirmed through coimmunoprecipitation studies of the purified protein from HeLa cells and of solubilized complexes from other cell lines such as neural cell HCN-1A and HEK293. Depletion of CD147 by RNA interference was found to increase the production of Abeta peptides without changing the expression level of the other gamma-secretase components or APP substrates whereas CD147 overexpression has no statistically significant effect on Abeta-peptide production, other gamma-secretase components or APP substrates, indicating that the presence of the CD147 subunit within the gamma-secretase complex down-modulates the production of Abeta-peptides (Zhou, 2005; full text of article).

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)

Translocation of monocarboxylate transporters MCT1 and MCT4 to the plasma membrane requires CD147 (basigin) with which they remain tightly associated. However, the importance of CD147 for MCT activity is unclear. MCT1 and MCT4 are both inhibited by the cell-impermeant organomercurial reagent p-chloromercuribenzene sulfonate (pCMBS). This study demonstrates by site-directed mutagenesis that removal of all accessible cysteine residues on MCT4 does not prevent this inhibition. pCMBS treatment of cells abolishes co-immunoprecipitation of MCT1 and MCT4 with CD147 and enhances labeling of CD147 with a biotinylated-thiol reagent. This suggested that CD147 might be the target of pCMBS, and further evidence for this was obtained by treatment of cells with the bifunctional organomercurial reagent fluorescein dimercury acetate that caused oligomerization of CD147. Site-directed mutagenesis of CD147 implicated the disulfide bridge in the Ig-like C2 domain of CD147 as the target of pCMBS attack. MCT2, which is pCMBS-insensitive, co-immunoprecipitates with gp70 rather than CD147. The interaction between gp70 and MCT2 was confirmed using fluorescence resonance energy transfer between the cyan fluorescent protein- and yellow fluorescent protein-tagged MCT2 and gp70. pCMBS strongly inhibits lactate transport into rabbit erythrocytes, where MCT1 interacts with CD147, but not into rat erythrocytes where it interacts with gp70. These data imply that inhibition of MCT1 and MCT4 activity by pCMBS is mediated through its binding to CD147, whereas MCT2, which associates with gp70, is insensitive to pCMBS. It is concluded that ancillary proteins are required to maintain the catalytic activity of MCTs as well as for their translocation to the plasma membrane (Wilson, 2005; full text of article).

Regulation of vascular endothelial growth factor expression by EMMPRIN via the PI3K-Akt signaling pathway

Extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN) is a cell surface glycoprotein overexpressed in many solid tumors. In addition to its ability to stimulate stromal MMP expression, tumor-associated EMMPRIN also induces vascular endothelial growth factor (VEGF) expression. To explore the underlying signaling pathways used by EMMPRIN, the involvement of phosphoinositide 3-kinase (PI3K)-Akt, mitogen-activated protein kinase (MAPK), JUN, and p38 kinases in EMMPRIN-mediated VEGF regulation, was studied. Overexpression of EMMPRIN in MDA-MB-231 breast cancer cells stimulates the phosphorylation of only Akt and MAPKs but not that of JUN and p38 kinases. Conversely, inhibition of EMMPRIN expression results in suppressed Akt and MAPK phosphorylation. Furthermore, the PI3K-specific inhibitor LY294002 inhibits VEGF production by EMMPRIN-overexpressing cells in a dose- and time-dependent manner. In contrast, the MAPK inhibitor U0126 does not affect VEGF production. In vivo, EMMPRIN-overexpressing tumors with elevated VEGF expression have a high level of phosphorylation of Akt and MAPK. Finally, when fibroblast cells are treated with recombinant EMMPRIN, Akt kinase but not MAPK is phosphorylated concomitant with an increase in VEGF production. Both the activation of Akt kinase and the induction of VEGF are specifically inhibited with a neutralizing antibody to EMMPRIN. These results show that in both tumor and fibroblast cells EMMPRIN regulates VEGF production via the PI3K-Akt pathway but not via the MAPK, JUN, or p38 kinase pathways (Tang, 2006; full text of article).

Metabolic activation-related CD147-CD98 complex

Cell surface CD147 protein promotes production of matrix metalloproteinases and hyaluronan, associates with monocarboxylate transporters and integrins, and is involved in reproductive, neural, inflammatory, and tumor functions. This study combined covalent cross-linking, mass spectrometric protein identification, and co-immunoprecipitation to show selective CD147 association with three major types of transporters (CD98 heavy chain (CD98hc)-L-type amino acid transporter, ASCT2, and monocarboxylate transporters) as well as a regulator of cell proliferation (epithelial cell adhesion molecule). In the assembly of these multicomponent complexes, CD147 and CD98hc play a central organizing role. RNA interference knock-down experiments established a strong connection between CD147 and CD98hc expression and a strong positive association of CD147 (and CD98hc) with cell proliferation. As the CD147-CD98hc complex and proliferation diminished, AMP-activated protein kinase (a cellular 'fuel gauge') became activated, indicating a disturbance of cellular energy metabolism. These data point to a CD147-CD98 cell surface supercomplex that plays a critical role in energy metabolism, likely by coordinating transport of lactate and amino acids. Furthermore this study showed how covalent cross-linking, together with mass spectrometry, can be used to identify closely associated transmembrane proteins. This approach should also be applicable to many other types of transmembrane proteins besides those associated with CD98hc and CD147 (Xu, 2005; full text of article).

Junction protein shrew-1 influences cell invasion and interacts with invasion-promoting protein CD147

Shrew-1 was isolated from an endometriotic cell line in a search for invasion-associated genes. It is a membrane protein that targets to the basolateral membrane of polarized epithelial cells, interacting with E-cadherin-catenin complexes of adherens junctions. Paradoxically, the existence of adherens junctions is incompatible with invasion. To investigate whether shrew-1 can indeed influence cellular invasion, it was overexpressed in HT1080 fibrosarcoma cells. This resulted in enhanced invasiveness, accompanied by an increased matrix metalloprotease (MMP)-9 level in the supernatant, raising the question about the role of shrew-1 in this process. Interaction with CD147, a known promoter of invasiveness and MMP activity, was tested. Indeed, genetics-based, biochemical, and microscopy experiments revealed shrew-1- and CD147-containing complexes in invasive endometriotic cells and an interaction in epithelial cells, which was stronger in MCF7 tumor cells, but weaker in Madin-Darby canine kidney cells. In contrast to the effect mediated by overexpression, small interfering RNA-mediated down-regulation of either shrew-1 or CD147 in HeLa cells decreased invasiveness without affecting the proliferation behavior of HeLa cells, but the knockdown cells displayed decreased motility. Altogether, these results imply that shrew-1 has a function in the regulation of cellular invasion, which may involve its interaction with CD147 (Schreiner, 2007).

Function of HAb18G/CD147 in invasion of host cells by severe acute respiratory syndrome coronavirus

To identify the function of CD147 in invasion of host cells by severe acute respiratory syndrome (SARS) coronavirus (CoV), the protein-protein interaction among HAb18G/CD147, cyclophilin A (CyPA), and SARS-CoV structural proteins were analyzed by coimmunoprecipitation and surface plasmon resonance analysis. Although none of the SARS-CoV proteins was found to directly bind CD147, the nucleocapsid (N) protein of SARS-CoV is bound to CyPA, which interacts with CD147. Further research showed that CD147 is highly expressed on 293 cells and that CyPA is integrated with SARS-CoV. CD147-antagonistic peptide (AP)-9, an AP of CD147, had a high rate of binding to 293 cells and an inhibitory effect on SARS-CoV. These results show that CD147, mediated by CyPA bound to SARS-CoV N protein, plays a functional role in facilitating invasion of host cells by SARS-CoV. These findings provide some evidence for the cytologic mechanism of invasion by SARS-CoV and provide a molecular basis for screening anti-SARS drugs (Chen, 2005; full text of article).


REFERENCES

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

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Biological Overview

date revised: 10 August 2018

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