Apoptosis culminates in engulfment and degradation of the apoptotic cell

Apoptosis culminates in engulfment and degradation of the apoptotic cell. and autoimmune diseases. There is increasing evidence that the engulfment machinery can regulate the execution of apoptosis. However, the underlying molecular mechanisms are poorly understood. We show that dynein mediates cell non-autonomous cross-talk between the engulfment and apoptotic programs in the germline. Dynein is an ATP-powered microtubule-based molecular motor, built from several subunits. CP544326 (Taprenepag) Dynein has many diverse functions including transport of cargo around the cell. We show that both dynein light chain 1 (DLC-1) and dynein heavy chain 1 (DHC-1) localize to the nuclear membrane inside apoptotic germ cells in where three types of apoptosis occur7,8. During embryogenesis and larval development, 131 cells undergo apoptosis (developmental)9,10. In the germline, around half of the germ CP544326 (Taprenepag) cells are eliminated by apoptosis during adulthood (physiological)11. Finally, germ cells can undergo apoptosis if exposed to different stressors, DNA damage or checkpoint failure (damage-induced)12. The core apoptotic machinery is indispensable for all types of apoptosis and is comprised of the anti-apoptotic CED-9 (Bcl-2)13, the caspase CED-3 (caspase-9)14, and the pro-apoptotic adaptor CED-4 (Apaf1)15. Pro-apoptotic signals inhibit CED-9, which releases its inhibition of CED-4, which in turn activates CED-312,16C18. In the germline, CED-4 localizes primarily to the nuclear membrane19, but mitochondrial localization has also been reported20. Upon apoptotic stimuli, such as DNA damage, CED-4 accumulation increases at the nuclear membrane20,21. Apoptosis culminates in engulfment and degradation of the apoptotic cell. In and pathway and the and pathway23. The engulfment pathways cooperate with the apoptotic machinery to induce developmental apoptosis, since overactivation of engulfment causes more cells to die24, while a defect in engulfment causes more cells to survive under limited caspase activity25C29. However, little is known about the interaction between engulfment and the apoptotic machinery. The dynein complex is a highly conserved microtubule-based motor of 1600?kDa that consists of two heavy chains, two intermediate chains, three different light chains, and two light intermediate chains30. Dynein has a variety of functions inside the CP544326 (Taprenepag) cell; many related to transport or cell division30. In the germline of dynein light chain 1 (DLC-1) has 95% sequence homology to the human light chains DYNLL1 and DYNLL2. Loss-of-function mutations of and cause embryonic lethality32C34. In suppresses accumulation of apoptotic corpses in engulfment mutants. We show that both DLC-1 and DHC-1 localize to the nuclear membrane of apoptotic germ cells and that lack of either inhibits physiological apoptosis in mutants defective in engulfment. Furthermore, DLC-1 is required for accumulation of CED-4 at the nuclear membrane of germ cells, which is necessary for execution of apoptosis. Our study demonstrates a novel role of the dynein complex as a switch controlling live/death decisions in the germline in cooperation with the engulfment machinery. Results DLC-1 accumulates at the nuclear membrane of apoptotic germ cells DLC-1 regulates germ cell apoptosis in response to ionizing radiation via CP544326 (Taprenepag) a signal from somatic tissues37. However, DLC-1 is also expressed in the germline36C38. To establish if DLC-1 plays a role within germ cells undergoing apoptosis we expressed DLC-1::GFP in mutants, where apoptotic cells accumulate due to impaired engulfment11. Expression of the DLC-1::GFP transgene did not alter germline morphology and the fusion protein was found evenly distributed in healthy germ cells consistent with previous studies38. Strikingly, we found that DLC-1::GFP strongly localized to DIC positive apoptotic germ cells (Fig.?1a). To verify that the cells marked by DLC-1::GFP were indeed apoptotic, we expressed DLC-1::GFP in mutants, in which apoptosis is blocked7 and treated them with RNAi against to block engulfment. In mutants, we found no DIC positive corpses nor any germ cells marked with DLC-1::GFP, demonstrating that DLC-1::GFP accumulation specifically marks apoptotic cells (Fig.?1b). Physiological apoptosis also occurs in wild-type animals but fewer corpses are found because they are rapidly removed by engulfment. We found that some apoptotic cells in wild-type worms were also marked by the accumulation of DLC-1::GFP but generally to a lesser extent than in engulfment U2AF1 mutants (Fig. S1A). The expression of the single copy DLC-1::GFP transgene did not significantly alter the level of apoptosis in wild-type animals or engulfment mutants (Fig. S1B-D). The low number of apoptotic corpses in wild-type animals could perhaps mask an effect. Hence, we investigated the effect of the DLC-1::GFP transgene in mutants and worms treated with RNAi against the cytoplasmic.