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).
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).
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