In search of novel genes required for phagocytosis of apoptotic cells by macrophages, we took an unbiased approach and carried out a deficiency screen of the Drosophila autosomes (NC Franc and K White, unpublished data). We also more recently developed an in vitro phagocytosis assay in Schneider S2 cells, a Drosophila cell line with macrophage properties, which we used to carry out a genome-wide RNAi screen for genes required for apoptotic cell clearance by these cells at the Drosophila RNAi Screening Center (L Cuttell and NC Franc, unpublished data).

We identified undertaker, a gene essential for efficient phagocytosis of apoptotic cells by embryonic macrophages (Cuttell et al, Cell; 135(3):524-34). The undertaker gene encodes a novel Drosophila Junctophilin-related protein. Junctophilins have been found to couple voltage-gated Ca2+ channels at the plasma membrane to those of the endoplasmic reticulum, the Ryanodine receptors. We found that, as for the mammalian Junctophilins, the Undertaker (UTA) protein (also known as Retinophilin) acts with the Drosophila Ryanodine receptor, Rya-r44F, and behaves as a Junctophilin. UTA is localized in punctae at the Endoplasmic Reticulum (ER), and can be found at or near the Plasma Membrane (PM), as well as on the phagosomal membrane where it can be seen to colocalize with CRQ. We also found a requirement for Ca2+ entry in apoptotic cell clearance and demonstrated a role for dSTIM, a sensor of Ca2+ of the ER/SR lumen (Roos et al., 2005) and CRACM1/dOrai, a Ca2+-release activated Ca2+ channel (CRAC) required for store-operated Ca2+ entry (SOCE) (Feske et al., 2006; Vig et al., 2006) in this process by acting in the same pathway as UTA and Rya-r44F in vivo. With these results, we demonstrated a direct role for calcium homeostasis during phagocytosis of apoptotic corpses in vivo.

In C. elegans, two pathways including the C. elegans death genes ced-1, -6 and -7, and ced-2, -5, -10 and -12, have been characterized that participate in apoptotic cell clearance. While the CED-2/5/10/12 pathway leads to the activation of the small GTPase CED10, which controls actin cytoskeleton rearrangement during phagocytosis, the role of the CED-1/6/7 pathway remains unclear. We found that uta and rya-r44F genetically interact with drced-6 and drpr (ced-1 homologue), and that uta, drced6 and drpr are required for SOCE in S2 cells, demonstrating that these genes act in the same pathway. uta, dstim, dorai, drced-6 and drpr are also required for efficient phagocytosis of bacteria, demonstrating a general role for this pathway in phagocytosis.

Thus, altogether our results provide a link between SOCE and phagocytosis, imply that UTA plays a similar role in macrophages to that of Junctophilins in excitable cells, and shed light on a role for the CED1/6/7 pathway in Ca2+ homeostasis during phagocytosis of apoptotic cells and in bacterial engulfment.

A role for Ca2+ in phagocytosis of various particles by mammalian phagocytes has been previously described, but the molecular mechanisms underlying Ca2+ fluxes associated with these events are not known (Dewitt and Hallett, 2002; Rubartelli et al., 1997; Tejle et al., 2002). Thus, lessons learned from our work in the fly are likely to further our understanding of phagocytosis in mammalian systems, including man. Indeed, a mutation in human Orai1 was found in some patients with Severe Combined Immune Deficiency (Feske et al., 2006).

Our goal is now to understand how Ca2+ homeostasis promotes phagocytosis. We have preliminary data suggesting that other channels are required for phagocytosis and may cooperate with the CRAC channels to promote Ca2+ homeostasis. We have also identified several candidate genes in our genome-wide RNAi screen that are required for phagocytosis and encode proteins with Ca2+ binding motifs that may provide us with some clues as to how Ca2+ may promote phagocytosis.

We will pursue this project along the following three lines:

1) Pursue the genetic dissection of the molecular mechanisms controlling Ca2+ homeostasis during phagocytosis;

2) Confirm and further characterize the candidate genes encoding Ca2+-binding motifs containing proteins and study their role in phagocytosis;

3) Study the in vivo localization of UTA and all other components of the phagocytosis machinery identified thus far to try and understand the dynamics in time and space of these molecules during phagocytosis in wild-type and phagocytosis-defective mutant macrophages.