Abstract
Removal of apoptotic cells is critical for the physiological well-being of the organism1 and defects in corpse removal have been linked to disease states2,3. Genes regulating corpse recognition and internalization have been identified4, but few molecules involved in the processing of internalized corpses are known. Through a combination of targeted and unbiased reverse genetic screens in Caenorhabditis elegans, and studies in mammalian cells, we have identified genes required for maturation of apoptotic-cell-containing phagosomes. We have further ordered these candidates, which include the GTPases RAB-5 and RAB-7 and the HOPS complex, into a coherent linear pathway for the maturation of apoptotic cells within phagosomes. In depth analysis of two additional candidate genes, the phosphatidylinositol 3 kinase (PI(3)K) vps-34 (A001762) and dyn-1/dynamin, showed an accumulation of internalized, but undegraded, corpses within abnormal Rab5-negative phagosomes. We ordered these candidates in our pathway, with DYN-1 functioning upstream of VPS-34 in the recruitment and/or retention of RAB-5 to the phagosome. Finally, we have also identified a previously undescribed biochemical complex containing Vps34, dynamin and Rab5GDP, thus providing a mechanism for Rab5 recruitment to the nascent phagosome.
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Acknowledgements
The authors would like to thank members of the Ravichandran and Hengartner laboratories for helpful input and suggestions. We thank Dorothy Schafer for dynamin plasmids and purified Dyn2 protein, David Castle, Jim Casanova and Heidi McBride for Rab5 expression constructs, Jae Jung for Vps34 expression constructs, Lukas Neukomm for pLN022 and pLN019 plasmids and Jan Redick and Christie Davis from the Advanced Microscopy Facility for help with confocal microscopy. Some strains used in this work were obtained from the Caenorhabditis Genetics Center (CGC). This work was supported by grants from the NIGMS/NIH to K. S. R. and from the EU Project Apoclear, the Ernst Hadorn Foundation, the University of Zurich and the Swiss National Science Foundation to M. O. H. J. M. K. is an Arthritis Foundation Postdoctoral Fellow.
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J. M. K., K. D., J. A. and L. S. contributed to the generation of nematode transgenics and fluorescence microscopy studies; K. D. and J. A. conducted the unbiased screen; J. M. K. conducted the targeted screen; J. M. K. performed the mammalian cell culture experiments and wrote the manuscript; all authors contributed to editing the manuscript; A. C. T., C. D. S., M. O. H. and K. S. R. contributed to the data analysis, project planning and writing of the manuscript.
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Supplementary Figures S1, S2, S3, S4, S5, S6, Supplementary Tables S1, S2, S3, S4, S5 and Supplementary Methods (PDF 1741 kb)
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Kinchen, J., Doukoumetzidis, K., Almendinger, J. et al. A pathway for phagosome maturation during engulfment of apoptotic cells. Nat Cell Biol 10, 556–566 (2008). https://doi.org/10.1038/ncb1718
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DOI: https://doi.org/10.1038/ncb1718
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