SNS: adhesive properties, localization requirements and ectodomain dependence in S2 cells and embryonic myoblasts

Abstract

The body wall muscles in the Drosophila larva arise from interactions between Duf/Kirre and Irregular chiasm C-roughest (IrreC-rst)-expressing founder myoblasts and sticks-and-stones (SNS)-expressing fusion competent myoblasts in the embryo. Herein, we demonstrate that SNS mediates heterotypic adhesion of S2 cells with Duf/Kirre and IrreC-rst-expressing S2 cells, and colocalizes with these proteins at points of cell contact. These properties are independent of their transmembrane and cytoplasmic domains, and are observed quite readily with GPI-anchored forms of the ectodomains. Heterotypic interactions between Duf/Kirre and SNS-expressing S2 cells occur more rapidly and to a greater extent than homotypic interactions with other Duf/Kirre-expressing cells. In addition, Duf/Kirre and SNS are present in an immunoprecipitable complex from S2 cells. In the embryo, Duf/Kirre and SNS are present at points of contact between founder and fusion competent cells. Moreover, SNS clustering on the cell surface is dependent on Duf/Kirre and/or IrreC-rst. Finally, although the cytoplasmic and transmembrane domains of SNS are expendable for interactions in culture, they are essential for fusion of embryonic myoblasts.

Analysis of the Cell Adhesion Molecule Sticks-and-Stones Reveals Multiple Redundant Functional Domains, Protein-Interaction Motifs and Phosphorylated Tyrosines That Direct Myoblast Fusion in Drosophila melanogaster

“…Cloning and constructs: The full-length sns cDNA isolated by Bour et al (2000) was subcloned into pRmHa3 (Bunch et al 1988), using the EcoRI linker from the bacteriophage library at the 59 end and an NheI site at position 5612 of the cDNA sequence (Galletta et al 2004). A single hemagglutinin (HA) tag was added immediately following the last amino acid of the SNS coding sequence and cloned into pUAST to generate UAS-sns-HA.…”

“…For the HA-tagged membrane proximal deletions, the corresponding region in pUAST-sns-HA was replaced using XhoI and XbaI restriction sites. The untagged membrane distal deletion (UAS-snsD1279-1482) was similarly generated by PCR and replaced into pUAST-sns (Galletta et al 2004), using AflII and KpnI. To make UAS-sns5xPDZ-HA, UAS-sns2xPXXP, UAS-snsA17-HA, and UAS-snsF14-HA, site-directed mutagenesis was employed and the region amplified by PCR using mismatch oligonucleotides, in several rounds until all the desired sites were mutagenized.…”

“…Homozygous mutant embryos were identified by the absence of b-galactosidase (b-gal) activity and verified to be 25% of the population. Primary antibodies included a monoclonal antibody to myosin heavy chain (MHC) (1:1000, D. Kiehart), rabbit polyclonal antib-gal (1:1000, MP Biomedicals), and affinity-purified rabbit anti-SNS antisera at 1:100 (Galletta et al 2004). Colorimetric detection was performed using the Vectastain ABC elite kit (Vector Laboratories, Burlingame, CA) according to manufacturer's instructions with biotinyl-tyramide enhancement for anti-SNS.…”

“…They function as ligand-receptor pairs that become localized to points of cell-cell contact (Galletta et al 2004), whereupon they recruit and/or activate downstream components that lead to myoblast fusion. Duf/Kirre is thought to act upstream of the monomeric guanosine triphosphatase (GTPase) Rac1 in the FM.…”

FuRMAS: triggering myoblast fusion in Drosophila

“…Like Duf/Kirre, Rst/IrreC is a member of the Immunoglobulin Superfamily (IgSF) and can also attract myoblasts (Strü nkelnberg et al, 2001); however, in contrast to Duf/ Kirre, Rst/IrreC is expressed in both myoblast types. In aggregation experiments with S2 cells, the fcm-specific IgSF protein Sticks and Stones (SNS) was observed to mediate heterotypic adhesion with Duf/Kirreand Rst/Irre C-expressing cells (Bour et al, 2000;Galletta et al, 2004). Hibris (Hbs), the paralogue of SNS, is also expressed in fcms (Artero et al, 2001;Dworak et al, 2001), but interestingly, Hbs seems not to be absolutely essential for myoblast fusion since its loss of function does not disturb myoblast fusion (Artero et al, 2001;Dworak et al, 2001).…”