The role of Dictyostelium discoideum annexin C1 in cellular responses

Annexins are big family of proteins which are binding to phosphplipids in a Ca2+-dependent manner. A lot is known about their distribution in the cells and tissues, their involvement in endocytosis and exocytosis, vesicular trafficking and rearrangement of the cytoskeleton. For several annexins mouse knock out models are established. However, loss of one particular annexin had relatively mild effects suggesting compensation of the lost annexin function by other annexins. This study focused on Dictyostelium discoideum annexin C1, its dynamic behavior and possible interactions with other proteins. GFP-tagged full length annexin, a mutated form of annexin and the annexin core domain were used to study involvement of annexin in cellular processes in vivo and in vitro, with emphasis on processes like pinocytosis, phagocytosis, osmoregulation and development. Dictyostelium annexin C1 localizes in the cell cytosol and nucleus and on the plasma membrane. In the cytosol annexin is associated with internal membranes. Fractionation of membranes on discontinuous sucrose gradient showed that annexin C1 was present on plasma membrane, endosomal and lysosomal membranes, as well as in Golgi membranes and in a lesser amounts in ER fraction. In addition, co-immunostaining with several protein markers revealed annexin C1 in association with endosomes and contractile vacuoles. Although its localization on contractile vacuoles suggested a role in osmoregulation, mutant lacking annexin (SYN¯) behaved similar to wild type cells (AX2) under osmotic stress conditions. Despite the presence of annexin C1 on pinosomes and phagosomes the SYN¯ mutant was not impaired in endocytosis and exocytosis when compared with AX2 and showed an increased rate of phagocytosis. In accordance with this is the decreased rate of phagocytosis observed with cells overexpressing annexin. Furthermore we observed in vivo translocation of annexin from the cytosol to the plasma membrane upon a decrease in cytosolic pH. This finding showed that the protein could bind to phospholipids in the absence of Ca2+ and was in accordance with previously published in vitro data for annexin A5 and B12. We also found that annexin C1 associated with Triton X-100 insoluble pellet only in the presence of Ca2+. A mutation in one Ca2+-binding site in the third annexin repeat affected this behavior. Considering proposed high affinity for Ca2+ of this particular Ca2+-binding site and the previously described cooperation in binding of Ca2+ we speculate that his particular substitution of aspartic acid residue at the position 293 with alanine reduced protein affinity for binding of Ca2+ and eventually affected binding of Ca2+ to other Ca2+-binding sites.Gel filtration and immunoprecipitation experiments revealed the presence of annexin C1 monomers in the cytoplasm and interactions with two small Ca2+-binding proteins, CBP2 and DdPEF-2. Furthermore, we found annexin to be phosphorylated but the physiological role of phosphorylation is needed to be further studied in detail. Taken together, annexin C1 is not crucial for the survival of Dictyostelium. In annexin minus mutant SYN¯ however development was noticeably affected and was retarded by more than four hours and not all multicellular structures finally developed into fruiting bodies consisting of spore and stalk cells. Although all fusion proteins showed same localization in slugs, stalks and spore cells, impairment in development of SYN¯ cells could only be rescued by the full length protein pointing out the crucial importance of annexin function for development.