Calcium-dependent activities, transcription, and localization of the Caenorhabditis elegans annexin homolog nex-1

Annexins are a family of eukaryotic calcium-dependent phospholipid-binding proteins. There have been many localization studies and calcium-dependent activity measurements that characterize annexins and imply possible physiological functions, but none have identified a physiological function for annexins. To investigate the physiological function of annexins, the nematode Caenorhabditis elegans is being developed as a system for making genetic manipulations of expressed annexins.;After the initial identification of nex-1 by Creutz et al. (1996), the C. elegans genomic sequencing project identified two other annexin homologs, nex-2 and nex-3. RT-PCR examination verified that nex-2 and nex-3 are expressed at the mRNA level. An RT-PCR screen also determined that nex-1 mRNA and nex-3 mRNA are trans-spliced to the splice leader sequence SL-1. However, the function of the splice leaders is unknown, and thus implications for functional differences between nex-1 and nex-3 versus nex-2 are unknown. Subsequent to these investigations another annexin homolog nex-4 was identified.;The in vitro calcium-dependent activities of nex-1 were examined for comparison with previously characterized annexins. The threshold calcium concentration for nex-1 binding to membranes and aggregating granules is approximately 30 ;The cellular localization of nex-1 was examined by transforming worms with a plasmid that produces the green fluorescent protein under control of the nex-1 promoter. The GFP reporter verified that nex-1 is expressed in the gland cells, vulva muscles, and spermathecal valve. The GFP reporter also identified the hypodermal cells as a novel site of nex-1 expression. To determine whether nex-1 was expressed extracellularly, TRITC-labeled anti-nex-1 IgG was microinjected into live worms. The injected anti-nex-1 IgG failed to specifically stain any structure indicating that nex-1 is only expressed intracellularly.