Two pathways converge at CED-10 to mediate actin rearrangement and corpse removal in C. elegans

The removal of apoptotic cells is essential for the physiological well being of the organism. In Caenorhabditis elegans, two conserved, partially redundant genetic pathways regulate this process. In the first pathway, the proteins CED-2, CED-5 and CED-12 (mammalian homologues CrkII, Dock180 and ELMO, respectively) function to activate CED-10 (Rac1). In the second group, the candidate receptor CED-1 (CD91/LRP/SREC) probably recognizes an unknown ligand on the apoptotic cell and signals via its cytoplasmic tail to the adaptor protein CED-6 (hCED-6/GULP), whereas CED-7 (ABCA1) is thought to play a role in membrane dynamics. Molecular understanding of how the second pathway promotes engulfment of the apoptotic cell is lacking. Here, we show that CED-1, CED-6 and CED-7 are required for actin reorganization around the apoptotic cell corpse, and that CED-1 and CED-6 colocalize with each other and with actin around the dead cell. Furthermore, we find that the CED-10(Rac) GTPase acts genetically downstream of these proteins to mediate corpse removal, functionally linking the two engulfment pathways and identifying the CED-1, -6 and -7 signalling module as upstream regulators of Rac activation.     

Pannexin 1 channels mediate 'find-me' signal release and membrane permeability during apoptosis

Apoptotic cells release 'find-me' signals at the earliest stages of death to recruit phagocytes. The nucleotides ATP and UTP represent one class of find-me signals, but their mechanism of release is not known. Here, we identify the plasma membrane channel pannexin 1 (PANX1) as a mediator of find-me signal/nucleotide release from apoptotic cells. Pharmacological inhibition and siRNA-mediated knockdown of PANX1 led to decreased nucleotide release and monocyte recruitment by apoptotic cells. Conversely, PANX1 overexpression enhanced nucleotide release from apoptotic cells and phagocyte recruitment. Patch-clamp recordings showed that PANX1 was basally inactive, and that induction of PANX1 currents occurred only during apoptosis. Mechanistically, PANX1 itself was a target of effector caspases (caspases 3 and 7), and a specific caspase-cleavage site within PANX1 was essential for PANX1 function during apoptosis. Expression of truncated PANX1 (at the putative caspase cleavage site) resulted in a constitutively open channel. PANX1 was also important for the 'selective' plasma membrane permeability of early apoptotic cells to specific dyes. Collectively, these data identify PANX1 as a plasma membrane channel mediating the regulated release of find-me signals and selective plasma membrane permeability during apoptosis, and a new mechanism of PANX1 activation by caspases.     

Continued clearance of apoptotic cells critically depends on the phagocyte Ucp2 protein

Rapid and efficient removal of apoptotic cells by phagocytes is important during development, tissue homeostasis and in immune responses. Efficient clearance depends on the capacity of a single phagocyte to ingest multiple apoptotic cells successively, and to process the corpse-derived cellular material. However, the factors that influence continued clearance by phagocytes are not known. Here we show that the mitochondrial membrane potential of the phagocyte critically controls engulfment capacity, with lower potential enhancing engulfment and vice versa. The mitochondrial membrane protein Ucp2, which acts to lower the mitochondrial membrane potential, was upregulated in phagocytes engulfing apoptotic cells. Loss of Ucp2 reduced phagocytic capacity, whereas Ucp2 overexpression enhanced engulfment. Mutational and pharmacological studies indicated a direct role for Ucp2-mediated mitochondrial function in phagocytosis. Macrophages from Ucp2-deficient mice were impaired in phagocytosis in vitro, and Ucp2-deficient mice showed profound in vivo defects in clearing dying cells in the thymus and testes. Collectively, these data indicate that mitochondrial membrane potential and Ucp2 are key molecular determinants of apoptotic cell clearance. As Ucp2 is linked to metabolic diseases and atherosclerosis, this newly discovered role for Ucp2 in apoptotic cell clearance has implications for the complex aetiology and pathogenesis of these diseases.