InteractiveFly: GeneBrief

Amalgam : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

Amalgam (Ama), a secreted member of the immunoglobulin superfamily, has been identified as a ligand for the Neurotactin (Nrt) receptor. Nrt is a member of the serine esterase-like family of membrane proteins. Ama is necessary for Nrt-expressing cells both to aggregate with one another and to associate with embryonic primary culture cells. Aggregation assays performed with truncated Nrt molecules reveal that the integrity of the cholinesterase-like extracellular domain is not required either for Ama binding or for adhesion, with only amino acids 347-482 of the extracellular domain being necessary for both activities. Moreover, the Nrt cytoplasmic domain is required for Nrt-mediated adhesion, although not for Ama binding. Using an ama-deficient stock, it has been found that ama function is not essential for viability. Pupae deficient for ama do exhibit defasciculation defects of the ocellar nerves similar to those found in nrt mutants. Although the specific roles of Nrt and Ama during Drosophila development remain quite obscure, different aspects of the puzzle are beginning to fall into place. A link has been established between these two enigmatic proteins. One of the critical missing pieces is what lies downstream of the Nrt receptor and how Ama binding to Nrt affects these downstream components. Addressing this issue and identifying orthologs in other organisms are probable key steps in a further understanding of this intriguing receptor-ligand interaction (Fremion, 2000).

Nrt functions as a heterophilic cell adhesion protein in various cell culture assays (Barthalay, 1990), suggesting that its cholinesterase domain functions as a part of a recognition/signaling transmembrane receptor-like protein. Further studies have delineated a specific region of the cholinesterase-like domain located near the membrane that is involved in this recognition process (Darboux, 1996). Ama is able to promote the aggregation of Nrt-expressing S2 cells; Ama interacts specifically with Nrt, and Ama is necessary for the interaction of Nrt-expressing S2 cells with dissociated embryonic primary culture cells, the original assay that established the heterophilic adhesion properties of Nrt. The nearly identical patterns of Ama and Nrt protein accumulation throughout embryogenesis are consistent with a receptor-ligand relationship in vivo. Furthermore, the finding that Ama protein fails to exhibit its normal pattern of accumulation in an nrt mutant is compatible with Nrt acting as the key receptor for Ama during embryogenesis. These are intriguing observations: that during embryogenesis, cells that accumulate Ama on their surface are sometimes different from the cells that are expressing ama transcripts. Certainly these differences can be explained easily for a secreted protein such as Ama. However, the functional significance, if any, of these disparities between spatial patterns of protein expression versus protein accumulation is unknown (Fremion, 2000).

How does Ama facilitate Nrt-mediated adhesion? Observations that an artificially generated membrane-anchored form of Ama can mediate homophilic aggregation in the absence of Nrt indicate that Ama can interact with itself. This may suggest one mechanism whereby Ama could function as a linker between Nrt molecules on opposing cell surfaces. Nrt would bind Ama, which would bind to a second Ama molecule that could interact with Nrt on the surface of a second cell. Thus, Ama would serve as a linker protein between Nrt-expressing cells. Alternatively, Ama could alter the Nrt protein upon binding, such that it could now recognize in a homophilic manner other Nrt molecules (Fremion, 2000).

The binding of Ama to Nrt alone is not sufficient for cell aggregation. The finding that the Nrt cytoplasmic domain is essential for adhesion indicates that important events are taking place in the cytoplasm that are critical for this adhesion process. The amino acid sequence of the Nrt cytoplasmic domain does not reveal any clear motifs such as kinase or phosphatase domains. However, it does encode several putative phosphorylation sites, and Nrt is found to be phosphorylated on serine and threonine residues. Interestingly, Nrt is enriched at sites of cellular contacts in neuronal and epithelial tissues and within Nrt transfectant aggregates (Barthalay, 1990). This recruitment to specific regions of the membrane suggests that Nrt may bind to cytoskeleton components. Nrt-mediated cell adhesion might be considered as a two-step process: (1) binding of the ligand (Ama) to the extracellular domain, a step that can occur without the Nrt cytoplasmic domain; and (2) stabilization and strengthening of the interaction through clustering of Nrt to sites of cell contact and interactions dependent upon the Nrt cytoplasmic domain. This latter step was not achieved with transfectants lacking the Nrt cytoplasmic domain, demonstrating the critical role of the Nrt cytoplasmic domain in this process (Fremion, 2000).

The isolation and characterization of nrt mutants have demonstrated that Nrt is required for outgrowth, fasciculation and guidance of axons (Speicher, 1998). Mutations in nrt produce quite subtle and variable defects in axon guidance within the embryonic CNS. These phenotypes are greatly enhanced when nrt mutations are combined with mutations in other cell adhesion or signaling molecules such as nrg. These results have led to the suggestion that nrt plays a cooperative or partially redundant role in axon guidance. One of the most robust phenotypes of nrt mutants alone is disruption of ocellar nerve axon pathfinding in pupae. In nrt mutants, misrouting, stalling and defasciculation of the ocellar pioneer nerves are observed at high frequencies (Fremion, 2000).

One might expect that if Ama and Nrt indeed interact in vivo as indicated by the co-immunoprecipitation experiment, mutations in these genes would lead to similar phenotypes. Since the ocellar pioneer nerve disruptions are the most consistent phenotype for nrt null mutants, a determination was made whether a loss-of-function ama mutant displays comparable phenotypes. Similar to nrt mutations, a deficiency for ama results in highly penetrant defasciculation phenotypes of the ocellar pioneer nerves in developing pupae. However, there are significant differences in the expressivity of ocellar nerve defects. Stalling or misrouting of these axons as is found in nrt mutants is not observed in ama mutant pupae. This result suggests that rather than having an instructive role in pathfinding, Ama maintains proper cohesion between these axons and contributes to the organization of these fascicles (Fremion, 2000).

The role of Ama during embryonic axon guidance cannot be addressed due to the lack of specific ama mutations. The deficiency that was used in these studies [Df(3R)ama] is also deleted for the embryonic dorsal-ventral patterning gene zerknullt. The lethality associated with zerknullt mutations could not be rescued with a wild-type zerknullt transgene in order to generate an adult viable strain that is deficient for Ama. The difficulty is that partial rescue of zerknullt embryonic functions leads to variable defects in germ band extension and retraction. These disruptions in morphogenetic movements make it impossible to assay reliably for subtle defects in formation of axon pathways in the embryonic CNS. Therefore, it is possible that loss-of-function mutations in ama could result in embryonic axon pathfinding defects that are similar to nrt loss-of-function mutations (Fremion, 2000).

Amalgam (Ama), a secreted member of the immunoglobulin superfamily, has been identified as a ligand for the Neurotactin (Nrt) receptor. Nrt is a member of the serine esterase-like family of membrane proteins. Ama is necessary for Nrt-expressing cells both to aggregate with one another and to associate with embryonic primary culture cells. Aggregation assays performed with truncated Nrt molecules reveal that the integrity of the cholinesterase-like extracellular domain is not required either for Ama binding or for adhesion, with only amino acids 347-482 of the extracellular domain being necessary for both activities. Moreover, the Nrt cytoplasmic domain is required for Nrt-mediated adhesion, although not for Ama binding. Using an ama-deficient stock, it has been found that ama function is not essential for viability. Pupae deficient for ama do exhibit defasciculation defects of the ocellar nerves similar to those found in nrt mutants. Although the specific roles of Nrt and Ama during Drosophila development remain quite obscure, different aspects of the puzzle are beginning to fall into place. A link has been established between these two enigmatic proteins. One of the critical missing pieces is what lies downstream of the Nrt receptor and how Ama binding to Nrt affects these downstream components. Addressing this issue and identifying orthologs in other organisms are probable key steps in a further understanding of this intriguing receptor-ligand interaction (Fremion, 2000).

Nrt functions as a heterophilic cell adhesion protein in various cell culture assays (Barthalay, 1990), suggesting that its cholinesterase domain functions as a part of a recognition/signaling transmembrane receptor-like protein. Further studies have delineated a specific region of the cholinesterase-like domain located near the membrane that is involved in this recognition process (Darboux, 1996). Ama is able to promote the aggregation of Nrt-expressing S2 cells; Ama interacts specifically with Nrt, and Ama is necessary for the interaction of Nrt-expressing S2 cells with dissociated embryonic primary culture cells, the original assay that established the heterophilic adhesion properties of Nrt. The nearly identical patterns of Ama and Nrt protein accumulation throughout embryogenesis are consistent with a receptor-ligand relationship in vivo. Furthermore, the finding that Ama protein fails to exhibit its normal pattern of accumulation in an nrt mutant is compatible with Nrt acting as the key receptor for Ama during embryogenesis. These are intriguing observations: that during embryogenesis, cells that accumulate Ama on their surface are sometimes different from the cells that are expressing ama transcripts. Certainly these differences can be explained easily for a secreted protein such as Ama. However, the functional significance, if any, of these disparities between spatial patterns of protein expression versus protein accumulation is unknown (Fremion, 2000).

How does Ama facilitate Nrt-mediated adhesion? Observations that an artificially generated membrane-anchored form of Ama can mediate homophilic aggregation in the absence of Nrt indicate that Ama can interact with itself. This may suggest one mechanism whereby Ama could function as a linker between Nrt molecules on opposing cell surfaces. Nrt would bind Ama, which would bind to a second Ama molecule that could interact with Nrt on the surface of a second cell. Thus, Ama would serve as a linker protein between Nrt-expressing cells. Alternatively, Ama could alter the Nrt protein upon binding, such that it could now recognize in a homophilic manner other Nrt molecules (Fremion, 2000).

The binding of Ama to Nrt alone is not sufficient for cell aggregation. The finding that the Nrt cytoplasmic domain is essential for adhesion indicates that important events are taking place in the cytoplasm that are critical for this adhesion process. The amino acid sequence of the Nrt cytoplasmic domain does not reveal any clear motifs such as kinase or phosphatase domains. However, it does encode several putative phosphorylation sites, and Nrt is found to be phosphorylated on serine and threonine residues. Interestingly, Nrt is enriched at sites of cellular contacts in neuronal and epithelial tissues and within Nrt transfectant aggregates (Barthalay, 1990). This recruitment to specific regions of the membrane suggests that Nrt may bind to cytoskeleton components. Nrt-mediated cell adhesion might be considered as a two-step process: (1) binding of the ligand (Ama) to the extracellular domain, a step that can occur without the Nrt cytoplasmic domain; and (2) stabilization and strengthening of the interaction through clustering of Nrt to sites of cell contact and interactions dependent upon the Nrt cytoplasmic domain. This latter step was not achieved with transfectants lacking the Nrt cytoplasmic domain, demonstrating the critical role of the Nrt cytoplasmic domain in this process (Fremion, 2000).

The isolation and characterization of nrt mutants have demonstrated that Nrt is required for outgrowth, fasciculation and guidance of axons (Speicher, 1998). Mutations in nrt produce quite subtle and variable defects in axon guidance within the embryonic CNS. These phenotypes are greatly enhanced when nrt mutations are combined with mutations in other cell adhesion or signaling molecules such as nrg. These results have led to the suggestion that nrt plays a cooperative or partially redundant role in axon guidance. One of the most robust phenotypes of nrt mutants alone is disruption of ocellar nerve axon pathfinding in pupae. In nrt mutants, misrouting, stalling and defasciculation of the ocellar pioneer nerves are observed at high frequencies (Fremion, 2000).

One might expect that if Ama and Nrt indeed interact in vivo as indicated by the co-immunoprecipitation experiment, mutations in these genes would lead to similar phenotypes. Since the ocellar pioneer nerve disruptions are the most consistent phenotype for nrt null mutants, a determination was made whether a loss-of-function ama mutant displays comparable phenotypes. Similar to nrt mutations, a deficiency for ama results in highly penetrant defasciculation phenotypes of the ocellar pioneer nerves in developing pupae. However, there are significant differences in the expressivity of ocellar nerve defects. Stalling or misrouting of these axons as is found in nrt mutants is not observed in ama mutant pupae. This result suggests that rather than having an instructive role in pathfinding, Ama maintains proper cohesion between these axons and contributes to the organization of these fascicles (Fremion, 2000).

The role of Ama during embryonic axon guidance cannot be addressed due to the lack of specific ama mutations. The deficiency that was used in these studies [Df(3R)ama] is also deleted for the embryonic dorsal-ventral patterning gene zerknullt. The lethality associated with zerknullt mutations could not be rescued with a wild-type zerknullt transgene in order to generate an adult viable strain that is deficient for Ama. The difficulty is that partial rescue of zerknullt embryonic functions leads to variable defects in germ band extension and retraction. These disruptions in morphogenetic movements make it impossible to assay reliably for subtle defects in formation of axon pathways in the embryonic CNS. Therefore, it is possible that loss-of-function mutations in ama could result in embryonic axon pathfinding defects that are similar to nrt loss-of-function mutations (Fremion, 2000).

Amalgam regulates the receptor tyrosine kinase pathway through Sprouty in glial cell development

The receptor tyrosine kinase (RTK) pathway plays an essential role in development and disease by controlling cell proliferation and differentiation. This study profiled the Drosophila larval brain by single cell RNA-sequencing and identified Amalgam (Ama), encoding a cell adhesion protein of the immunoglobulin IgLON family, that regulates the RTK pathway activity during glial cell development. Depletion of Ama reduces cell proliferation, affects glial cell type composition and disrupts the blood-brain barrier (BBB) that leads to hemocyte infiltration and neuronal death. Ama depletion lowers RTK activity by upregulating Sprouty (Sty), a negative regulator of RTK pathway. Knockdown of Ama blocks oncogenic RTK signaling activation in the Drosophila glioma model and halts malignant transformation. Finally, knockdown of a human ortholog of Ama, LSAMP, results in upregulation of SPOUTY2 in glioblastoma cell lines suggesting that the relationship between Ama and Sty is conserved (Ariss, 2020).

The RTK signaling pathway is critical in a plethora of glial cell functions, such as migration, differentiation, proliferation, neurodegeneration and locomotion. This study identified Ama as a regulator of RTK signaling in glial cells and uncover an essential function of Ama in the maintenance of the BBB. This study underscores the power of scRNA-seq profiling to explore a knockdown phenotype and led to the identification of Sty as a major Ama target during regulation of RTK signaling (Ariss, 2020).

Profiling cells by scRNA-seq identified distinct changes in gene expression profiles across multiple glial cell types in Ama-depleted brain that otherwise would have not been possible using conventional approaches. First, it was found that Ama depletion affects gene expression in surface glia, which in turn leads to a disruption in the BBB. Second, the increase in hemocyte numbers in repo>Ama^RNAi scRNA-seq dataset enabled the discovery of infiltrating blood cells in Ama-depleted brains. Third, the increase in Sty and decrease in PntP1 levels following Ama knockdown in glia encouraged exploration of the impact of Ama in RTK signaling (Ariss, 2020).

scRNA-seq identifies and clusters cells based on similarity in gene expression profile to uncover the cellular heterogeneity in a tissue. In this study, scRNA-seq of the normal fly brains identified all the glial cell types in addition to a Fas cluster that appeared to encompass multiple glial cell types. This cell cluster displayed high expression levels of cell adhesion proteins, which is a hallmark of glial cells, while also supporting the observation that scRNA-seq not only clusters cells by type but also by similar biological features. This underlies the robustness of scRNA-seq, since it uncovers complex cellular dynamics related to certain stimuli and continuous temporal differentiation processes, as well as the spatial arrangement of cells. Notably, the cellular perturbations in clustering that were observed following Ama depletion were validated experimentally by genetic analysis, immunofluorescence and other assays. This type of single-cell data validation is essential as it addresses the concern of a batch effect between scRNA-seq samples. Thus, this work highlights the power of scRNA-seq to profile a knockdown or mutant phenotype (Ariss, 2020).

The results revealed that Ama is critical for maintaining the BBB, as its depletion results in discontinuous surface glia (SG) membranes, suggesting a lack of tight junctions or organization that leads to disruption of the barrier. This in turn exposes the larval brains to the high potassium content of the hemolymph, which damages neurons. Ama knockdown decreases overall glial cell proliferation, which can affect SG cells and the BBB in two additional ways. First, lack of proliferation in PG cells can potentially affect the secretion of metabolites, which is important to prevent neurodegeneration. Second, Ama depletion can alter subperineurial glia (SPG) growth by reducing endoreplication and endomitosis, as evident by reduced expression of cell cycle genes in repo>Ama^RNAi, and, therefore, hinder the ability of SPG to accommodate the growing brain during late larval stages (Ariss, 2020).

The conventional way to determine the intactness of the BBB function is by labeling brains with a fluorescent dextran dye. If the barrier is permissible to large molecules, such as the dye, then the BBB is considered to be broken. This study presents an alternative approach to monitor a disruption in the BBB by measuring the increase in infiltrating hemocytes in larval brains both by scRNA-seq and through staining with a hemocyte-specific antibody. Although SG protect neurons from the hemolymph, penetration of hemocytes into the brain through the damaged BBB has not been previously reported. Whether infiltrating hemocytes have a role in inflicting the damage in the brain is unknown but raises the possibility that they might have a function in that context (Ariss, 2020).

Ama and Lachesin are part of the IgLON family, with Lachesin also shown to be required for the BBB maintenance. Since IgLONs are also expressed in the BBB in mammals, this suggests that the immunoglobin superfamily may indeed also have an evolutionarily conserved BBB function in humans. These findings highlight an important role of IgLONs in neurodegenerative diseases that result in BBB breakdown (Ariss, 2020).

Through scRNA-seq and measuring P-ERK and PntP1 levels, this study found a drastic reduction in the level of RTK signaling in Ama-depleted brains. It is suggested that Ama regulates the RTK pathway since its depletion increases Sty levels, a general inhibitor of the pathway, and, conversely, overexpression of Ama has an opposite effect. Sty in repo>Ama^RNAi brains predominantly localizes to the nuclear and plasma membranes. Intriguingly, in mammalian cells, SPRY2 localization to the membrane has been shown to be crucial for its phosphorylation and inhibitory effect. The results of this study therefore suggest that, while depletion of Ama increases Sty levels, there might additionally be an effect in the cellular localization or post-translational modification of Sty. Genetic experiments indicate that Sty is the key target of Ama and that activated ERK can partially rescue the repo>Ama^RNAi phenotype, whereas activated EGFR in the glioma fly model cannot. These epistatic interactions support the model that Ama, acts through Sty, and is downstream of EGFR but upstream of ERK in the RTK signaling pathway (Ariss, 2020).

Although knockdown of Sty in Ama-depleted brains partially rescues the glial cell numbers and brain size, it fails to suppress the neuronal apoptosis in repo>Ama^RNAi brains. Since Ama can act non-cell-autonomously, it cannot be excluded that Ama might also affect neurons non-cell-autonomously and, therefore, the expression of Sty in glial cells fails to rescue neurons. This is noteworthy since LSAMP, the human counterpart of Ama, was reported to control neurite growth (Akeel, 2011). The precise mechanism of how Ama affects Sty is unknown and requires further investigation. However, SPRY2 and EGFR in human cell lines have been shown to compete with c-Cbl (a ubiquitin ligase) binding. In this context, the ubiquitin ligase attenuates the inhibitory effect of SPRY2 and vice versa. Since Ama and Sty localize to cell membranes, one possible explanation is that they may interact with each other, whereby loss of Ama releases Sty to bind to RTK receptors and promotes inhibition of the signaling pathway (Ariss, 2020).

Finally, this study shows that the role of Ama in controlling Sty levels is conserved in human glioblastoma (GBM) cell lines. Additionally, GBM patients with EGFR mutations display a significant increase in survival when they have low expression levels of LSAMP, suggesting a potential role for the IgLON family member in this cancer. Previous work has shown that LSAMP either promotes growth or acts as a tumor suppressor (Kresse, 2009). Intriguingly, SPRY2 inhibits or activates RTK signaling based on the context and cell type and also acts either as an oncogene or tumor suppressor (Masoumi-Moghaddam, 2014). These results suggest a potential connection between LSAMP and SPRY2 that may help to explain the role of LSAMP in tumorigenesis and, thus, point to LSAMP as a potential therapeutic target (Ariss, 2020).

REGULATION

Transcriptional Regulation

In an attempt to identify gene targets of ash2, an expression analysis was performed by using cDNA microarrays. Genes involved in cell cycle, cell proliferation, and cell adhesion are among these targets, and some of them are validated by functional and expression studies. Genes involved in cell adhesion and/or development of the neural system (i.e., FasII, mfas, Ama, Lac, and shg) are two of the main classes regulated by ash2. Even though trithorax proteins act by modulating chromatin structure at particular chromosomal locations, evidence of physical aggregation of ash2-regulated genes has not been found. This work represents the first microarray analysis of a trithorax-group gene (Beltran, 2003).

Protein Interactions

Neurotactin is a transmembrane protein whose extracellular domain is able to bind a ligand(s). Heterotypic binding assays utilizing embryonic cells obtained from gastrula stage embryos or transfected S2 cells expressing Nrt protein has indicated that an Nrt ligand(s) is present on the surface of embryonic cells. This Nrt ligand is also found as a soluble form, since auto-aggregation of Nrt-expressing S2 cells can be induced with a 100,000 g supernatant prepared from embryonic extracts (Fremion, 2000 and references therein).

Fractionation experiments using embryo extracts show that Ama is present in soluble fractions. Western blot analysis with Ama-specific antisera indicates that extracts prepared from embryonic cells contained immunoreactive polypeptides of ~45 kDa within the membrane fraction and in the 100,000g supernatant. Higher molecular weight bands are only observed in the membrane fraction pellet and might be related to Ama molecules trapped in protein complexes. The ama gene encodes a protein with an N-terminal signal sequence and a weakly hydrophobic C-terminal domain. Immunostaining of whole-mount embryos suggests that Ama is a membrane-associated protein, although the weakly hydrophobic C-terminal domain is unlikely to tether Ama directly to the membrane (Fremion, 2000).

In order to confirm further that Ama is a secreted protein, S2 cells were transfected with a plasmid construct encoding the ama cDNA under the control of an inducible metallothionein promoter. After induction with divalent cations, products were immunodetected by Western blot analysis of whole-cell extracts. The culture medium in which Ama transfectants have grown contains soluble Ama protein. Taken together, these data indicate that Ama is a secreted, soluble protein that can associate with the cell surface (Fremion, 2000.

To determine whether Ama plays a role in Nrt-mediated heterophilic adhesion, Nrt transfectants, which are not able to aggregate by themselves, were incubated in culture medium containing secreted Ama protein. Aggregate formation, similar to that in the experiment where the 100 000 g embryonic extract supernatant was used as a soluble fraction containing ligand activity, was observed. Moreover, when a soluble fraction is prepared from embryos deleted for the ama gene, Nrt transfectants do not aggregate. To determine whether Ama interacts specifically with Nrt, an S2 cell pull-down assay was conducted. Untransfected and transfected S2 cells expressing Nrt were incubated with soluble protein fractions prepared from either wild-type or ama-deficient embryos. These S2 cells were then pelleted and total cellular proteins were analyzed by Western blot analysis with Nrt- and Ama-specific antibodies. Nrt-expressing S2 cells are able to pull-down Ama from wild-type embryonic extracts, while control S2 cells do not. Not surprisingly, no Ama-immunoreactive material is found associated with Nrt-expressing S2 cells that are incubated with soluble protein fractions prepared from ama-deficient embryos. These results show that Ama is necessary for Nrt-mediated adhesion. A molecular association between Ama and Nrt has been demonstrated by a co-immunoprecipitation assay (Fremion, 2000).

These results suggest that a membrane-anchored form of Ama might interact directly with Nrt-expressing cells and facilitate heterophilic aggregation. To test this hypothesis, a transmembrane form of Ama (Ama-TM) was generated. Ama-TM was created by fusing the entire ama open reading frame to the transmembrane and cytoplasmic domain of the Drosophila Neuroglian protein. When plated onto plastic slide flasks, the Ama-TM S2 transfectants were able to bind methylene blue-stained Nrt-expressing S2 cells. This result suggests that the Ama membrane-bound form may also bind the Nrt molecule. Ama-TM-expressing S2 cells form large aggregates in the cell aggregation assay. Thus, it would appear that Ama protein can interact with itself in addition to its interaction with Nrt (Fremion, 2000).

Nrt is a type II transmembrane protein inserted in the lipid bilayer by a single hydrophobic region composed of 22 amino acids that separates the N-terminal cytoplasmic domain (323 amino acids) from the C-terminal extracellular domain (500 amino acids). By expressing truncated Nrt proteins in S2 cells and using a soluble fraction prepared from embryonic extracts that promote cell aggregation, a region within the extracellular domain between His347 and His482 that is essential for the adhesive function of Nrt has been localized (Darboux, 1996). In order to determine if this in vitro recognition process requires only Ama, the same experiments were repeated by replacing the crude extract with culture medium containing secreted Ama protein. Consistent with previous results, Nrt molecules that were truncated downstream of residues Pro452 were found to be inactive, while truncation downstream of His482 generates a molecule that possesses the same adhesive properties as the full-length Nrt. Simultaneously with these assays, aliquots of cells or aggregates were analyzed on SDS-PAGE, and Ama binding to transfectants was evaluated by Western blot analysis. Only Delta EXT3 transfectants are able to form aggregates, and protein analysis demonstrates that these cells bind Ama while Delta EXT1 and Delta EXT2 transfectants stay as single cells and no Ama binding is detected. Among the series of truncated molecules that have been analyzed (Delta EXT1, Delta EXT2 and Delta EXT3), the presence of Ama was found to correlate with the capacity to form aggregates. This suggests that at least in vitro, Ama is the major component involved in this Nrt-mediated cell-cell recognition process (Fremion, 2000).

The role of the Nrt cytoplasmic domain in aggregation was investigated by using a construct designated Delta CYT. The Delta CYT molecule lacks the 293 amino acid cytoplasmic domain, including five putative phosphorylation sites, leaving intact a short terminal sequence for correct initiation of translation as well as sequences near the transmembrane domain for proper membrane insertion and orientation. The Delta CYT construct was used to transfect S2 cells, and protein expression was analyzed by Western blot analysis. The apparent molecular weight of the Delta CYT molecule (60 kDa) is consistent with the predicted size of the glycosylated extracellular domain (522 amino acids). The correct translocation of the Delta CYT was investigated further by mild papain digestion of the transfectants. This treatment releases a polypeptide whose molecular weight (55 kDa) is compatible with the expected accessibility of the extracellular domain to proteases. These observations indicate that the removal of 293 amino acids from the cytoplasmic domain does not impair efficient insertion of Nrt into the membrane or the overall stability of the Nrt protein (Fremion, 2000).

Delta CYT-expressing transfectants does not form aggregates in the presence of the culture medium containing secreted Ama protein, although the controls (full-length Nrt transfectants) aggregate efficiently. This result demonstrates that the Nrt cytoplasmic domain is necessary for cells to aggregate. Interestingly, Ama binding to Delta CYT transfectants is detected. This demonstrates that Ama binding to the Nrt extracellular domain alone is not sufficient to promote aggregation and that the Nrt cytoplasmic domain is also required for Nrt-mediated aggregation (Fremion, 2000).

The quaternary structure of amalgam, a Drosophila neuronal adhesion protein, explains its dual adhesion properties

Amalgam (Ama) is a secreted neuronal adhesion protein that contains three tandem immunoglobulin domains. It has both homophilic and heterophilic cell adhesion properties, and is required for axon guidance and fasciculation during early stages of Drosophila development. This study reports its biophysical characterization; small-angle x-ray scattering was used to determine its low-resolution structure in solution. The biophysical studies revealed that Ama forms dimers in solution, and that its secondary and tertiary structures are typical for the immunoglobulin superfamily. Ab initio and rigid-body modeling by small-angle x-ray scattering revealed a distinct V-shaped dimer in which the two monomer chains are aligned parallel to each other, with the dimerization interface being formed by domain 1. These data provide a structural basis for the dual adhesion characteristics of Ama. Thus, the dimeric structure explains its homophilic adhesion properties. Its V shape suggests a mechanism for its interaction with its receptor, the single-pass transmembrane adhesion protein neurotactin, in which each 'arm' of Ama binds to the extracellular domain of neurotactin, thus promoting its clustering on the outer face of the plasma membrane (Zeev-Ben-Mordehai, 2009).

DEVELOPMENTAL BIOLOGY

Embryonic

Accumulation of Ama is first observed during early stage 8 of embryogenesis, shortly after the formation of the three germ layers during gastrulation. At this stage, Nrt is already expressed throughout the ectoderm and mesoderm. During germ band extension, Ama begins to be expressed within a row of midline cells that appear to be a subset of mesectodermal cells (Seeger, 1988); Nrt is expressed by midline cells and also more generally by the ectoderm layer. During embryonic stages 11 and 12, from the fully extended germ band through germ band shortening, neuroblasts undergo a series of asymmetric cell divisions to produce ganglion mother cells that in turn divide symmetrically, generating two neurons. Nrt is expressed ubiquitously in the ectoderm layer, outlining the epithelial cells and the developing neuroblasts and their progeny (Piovant, 1998). The Ama expression pattern is more restricted: Ama is not found on the neuroblasts; however, high levels of protein are found on their neural but not GMC progeny (Seeger, 1988). Both proteins are expressed in a subset of mesodermal derivatives including the fat body and the dorsal vessel. By stage 13 of embryogenesis, mature neurons are extending axons along stereotyped pathways forming the segmentally repeated arrays of commissural and longitudinal axon bundles or fascicles. During these stages, Ama and Nrt accumulation is seen within the fat body and throughout the central nervous system (CNS) on both neuronal cell bodies and their axons (Piovant, 1998; Seeger, 1988; de la Escalera, 1990; Hortsch, 1990). During early stages of peripheral nervous system (PNS) development, Nrt is expressed weakly on axon pathways that connect the ventral, lateral and dorsal clusters, while Ama expression is not detectable. However, in later stage embryos, both proteins concentrate on external sensory organ precursors. Overall, the patterns of Ama and Nrt accumulation during embryogenesis are strikingly similar (Fremion, 2000 and references therein).

Since the data from S2 cell transfection experiments show that Ama is secreted into the culture medium, one might expect to find differences between AMA RNA and protein expression within tissues during development. For nrt, there is a precise correspondence between patterns of nrt RNA and protein localization (de la Escalera, 1990; Hortsch, 1990). In the case of AMA, there are interesting differences between RNA and protein accumulation profiles. During early stages of embryogenesis, there is a strong correlation between patterns of AMA RNA and protein expression. For example, high levels of both AMA RNA and protein are observed in the mesoderm invagination during early stages of germ band extension. During later stages of embryogenesis, differences between the patterns of RNA and protein accumulation become apparent. By stage 14, Ama protein is found predominantly within the CNS, while in situ hybridizations show no RNA expression within this tissue. The same observation was made for the fat body, which accumulates large amounts of Ama protein. Once again, low levels of AMA RNA are detected in this tissue. By stage 14, weak AMA RNA signals are found around the gut, but never accumulated in discrete areas such as the external sensory organs in the PNS where Ama protein is concentrated. Clearly, there are regions where Ama protein accumulation does not correspond to high levels of AMA transcripts, suggesting that Ama protein turnover is low (Fremion, 2000).

The overall patterns of Ama protein accumulation during embryogenesis are interrelated with patterns of NRT RNA and protein expression (Seeger, 1988; de la Escalera, 1990; Hortsch, 1990). Differences in the patterns of AMA RNA and Nrt expression are observed. For instance, in a stage 10 embryo double stained for AMA RNA and with anti-Nrt antibody, differences are apparent. AMA transcripts, which identify cells producing the ligand, are located more apically than Nrt-expressing cells. Clearly, many cells that express high levels of AMA transcript do not express Nrt protein. The normal pattern of Ama protein accumulation is also dependent upon the presence of Nrt. In nrt mutant embryos, the pattern of Ama protein expression is clearly aberrant (Fremion, 2000).

REFERENCES

Search PubMed for articles about Drosophila Amalgam

Akeel, M., McNamee, C. J., Youssef, S. and Moss, D. (2011). DIgLONs inhibit initiation of neurite outgrowth from forebrain neurons via an IgLON-containing receptor complex. Brain Res 1374: 27-35. PubMed ID: 21167820

Ariss, M. M., Terry, A. R., Islam, A., Hay, N. and Frolov, M. V. (2020). Amalgam regulates the receptor tyrosine kinase pathway through Sprouty in glial cell development. J Cell Sci. PubMed ID: 32878945

Barthalay, Y., Hipeau-Jacquotte, R., de la Escalera, S., Jimenez, F. and Piovant, M. (1990). Drosophila Neurotactin mediates heterophilic cell adhesion. EMBO J. 9(11): 3603-3609. PubMed Citation: 2120048

Beltran, S., et al. (2003). Transcriptional network controlled by the trithorax-group gene ash2 in Drosophila melanogaster. Proc. Natl. Acad. Sci. 100(6): 3293-8. 12626737

Darboux, I., et al. (1996). The structure-function relationships in Drosophila neurotactin show that cholinesterasic domains may have adhesive properties. EMBO J. 15(18):4835-43. PubMed Citation: 8890157

de la Escalera, S., Bockamp, E. O., Moya, F., Piovant, M. and Jimenez, F. (1990). Characterization and gene cloning of Neurotactin, a Drosophila transmembrane protein related to cholinesterases. EMBO J. 9(11): 3593-3601. PubMed Citation: 2120047

Fremion, F., et al. (2000). Amalgam is a ligand for the transmembrane receptor neurotactin and is required for neurotactin-mediated cell adhesion and axon fasciculation in drosophila. EMBO J. 19(17): 4463-72. PubMed Citation: 10970840

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date revised: 20 December 2020

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