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Molecular and cellular mechanisms in phagocytosis of apoptotic cells

Final Report Summary - AC REMOVAL MECHANISM (Molecular and cellular mechanisms in phagocytosis of apoptotic cells)

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis plays a crucial role in development and tissue homeostasis of multicellular organisms. Inefficient or defective removal of apoptotic cells leads to inflammation and autoimmune diseases. Given that in vertebrates phagocytosis is a complex and highly redundant process, we are using the Drosophila model, which permits comprehensive in vivo studies and provides complement to clinical approaches in the field.
The goal of the proposed research is to elucidate the molecular and cellular mechanisms underlying recognition of apoptotic cells by phagocytes and subsequent signaling for engulfment. We previously identified and characterized a novel phagocytic receptor, named Six Microns Under (SIMU), which is required for recognition and engulfment of apoptotic cells by glia in the nervous system and by macrophages elsewhere. However, the mode of SIMU action was, like of many other phagocytic receptors, unknown.
Our objectives were (1) to gain a deeper insight into the molecular basis of SIMU function by finding its partners in phagocytosis and (2) to discover new phagocytic receptors acting in the same or parallel pathways with SIMU.
The first step in searching for SIMU partners was structure function analysis of the protein in order to characterize which domains are responsible for SIMU binding to apoptotic cells and to its partner on phagocytic surface. This analysis intriguingly showed that different SIMU domains have distinct functions, even though they belong to the same protein family (Nimrod Family).
We identified Phosphatidylserine (PS) on apoptotic cells as a ligand for SIMU and reported that PS exposure alone is not sufficient for engulfment in vivo. Moreover, we showed that SIMU specifically binds to PS on apoptotic cells through its EMI, NIM1 and NIM2 domains and its presence on phagocytic glial membranes in vivo in embryos lacking caspase activation is not sufficient for engulfment. Further analysis of caspase mutants revealed that, in addition to PS exposure, caspase dependent ligands on apoptotic cells are required for their clearance by phagocytes.
We have previously shown that embryos lacking SIMU function and Draper (an additional receptor) function are still capable to phagocytose apoptotic particles, meaning that additional receptors/pathways of apoptotic cell clearance exist in the embryo. To discover these pathways we focused on identifying and characterizing novel phagocytic receptors starting with two interesting candidates, the fly homologs of CD36 and LDL receptors, which have been implicated in mammalian phagocytosis. These receptors have a molecular domain structure distinct from SIMU and Draper, making it likely that they act by different mechanisms. We generated a knock-out mutant for the LDL receptor homolog and we are currently in process of making CD36 homolog knock-out mutant. We tested apoptotic cell clearance in embryos containing the novel LDL deletion and found no abnormality in glial phagocytosis during embryogenesis. These data indicate that LDL receptor homolog seems not to be involved in apoptotic cell clearance during Drosophila embryogenesis. We plan to test the generated CD36 homolog mutants in a few months.
Taken together the results achieved so far provide a good entry point for identifying novel players in apoptotic cell clearance and mechanistic understanding of how apoptotic particles are recognized by phagocytic receptors.

Information related to our research and project activities can be found on the PI’s laboratory Web site at: http://kurant.rappinst.com/