Phagocytosis of dying cells is critical in development and immunity1,2,3. Although proteins for recognition and engulfment of cellular debris following cell death are known4,5, proteins that directly mediate phagosome sealing are uncharacterized. Furthermore, whether all phagocytic targets are cleared using the same machinery is unclear. Degeneration of morphologically complex cells, such as neurons, glia and melanocytes, produces phagocytic targets of various shapes and sizes located in different microenvironments6,7. Thus, such cells offer unique settings to explore engulfment programme mechanisms and specificity. Here, we report that dismantling and clearance of a morphologically complex Caenorhabditis elegans epithelial cell requires separate cell soma, proximal and distal process programmes. Similar compartment-specific events govern the elimination of a C. elegans neuron. Although canonical engulfment proteins drive cell soma clearance, these are not required for process removal. We find that EFF-1, a protein previously implicated in cell–cell fusion8, specifically promotes distal process phagocytosis. EFF-1 localizes to phagocyte pseudopod tips and acts exoplasmically to drive phagosome sealing. eff-1 mutations result in phagocytosis arrest with unsealed phagosomes. Our studies suggest universal mechanisms for dismantling morphologically complex cells and uncover a phagosome-sealing component that promotes cell process clearance.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Cell Regeneration Open Access 03 February 2021
Nature Communications Open Access 01 May 2018
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Lekstrom-Himes, J. A. & Gallin, J. I. Immunodeficiency diseases caused by defects in phagocytes. N. Engl. J. Med. 343, 1703–1714 (2000).
Levin, R., Grinstein, S. & Canton, J. The life cycle of phagosomes: formation, maturation, and resolution. Immunol. Rev. 273, 156–179 (2016).
Mallat, M., Marin-Teva, J. L. & Cheret, C. Phagocytosis in the developing CNS: more than clearing the corpses. Curr. Opin. Neurobiol. 15, 101–107 (2005).
Hochreiter-Hufford, A. & Ravichandran, K. S. Clearing the dead: apoptotic cell sensing, recognition, engulfment, and digestion. Cold Spring Harb. Perspect. Biol. 5, a008748 (2013).
Reddien, P. W. & Horvitz, H. R. The engulfment process of programmed cell death in Caenorhabditis elegans. Annu. Rev. Cell Dev. Biol. 20, 193–221 (2004).
Osterloh, J. M. et al. dSarm/Sarm1 is required for activation of an injury-induced axon death pathway. Science 337, 481–484 (2012).
Simon, D. J. et al. Axon degeneration gated by retrograde activation of somatic pro-apoptotic signaling. Cell 164, 1031–1045 (2016).
Mohler, W. A. et al. The type I membrane protein EFF-1 is essential for developmental cell fusion. Dev. Cell 2, 355–362 (2002).
Chiorazzi, M. et al. Related F-box proteins control cell death in Caenorhabditis elegans and human lymphoma. Proc. Natl Acad. Sci. USA 110, 3943–3948 (2013).
Maurer, C. W., Chiorazzi, M. & Shaham, S. Timing of the onset of a developmental cell death is controlled by transcriptional induction of the C. elegans ced-3 caspase-encoding gene. Development 134, 1357–1368 (2007).
Wu, Y. et al. Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 108, 17708–17713 (2011).
Nehme, R. et al. Transcriptional upregulation of both egl-1 BH3-only and ced-3 caspase is required for the death of the male-specific CEM neurons. Cell Death Differ. 17, 1266–1276 (2010).
Singhal, A. & Shaham, S. Infrared laser-induced gene expression for tracking development and function of single C. elegans embryonic neurons. Nat. Commun. 8, 14100 (2017).
Oren-Suissa, M., Hall, D. H., Treinin, M., Shemer, G. & Podbilewicz, B. The fusogen EFF-1 controls sculpting of mechanosensory dendrites. Science 328, 1285–1288 (2010).
Wu, Y. C. & Horvitz, H. R. C. elegans phagocytosis and cell-migration protein CED-5 is similar to human DOCK180. Nature 392, 501–504 (1998).
Zhou, Z., Hartwieg, E. & Horvitz, H. R. CED-1 is a transmembrane receptor that mediates cell corpse engulfment in C. elegans. Cell 104, 43–56 (2001).
Kinchen, J. M. & Ravichandran, K. S. Identification of two evolutionarily conserved genes regulating processing of engulfed apoptotic cells. Nature 464, 778–782 (2010).
Ellis, R. E., Jacobson, D. M. & Horvitz, H. R. Genes required for the engulfment of cell corpses during programmed cell death in Caenorhabditis elegans. Genetics 129, 79–94 (1991).
Koppen, M. et al. Cooperative regulation of AJM-1 controls junctional integrity in Caenorhabditis elegans epithelia. Nat. Cell Biol. 3, 983–991 (2001).
Cheng, S. et al. PtdIns(4,5)P(2) and PtdIns3P coordinate to regulate phagosomal sealing for apoptotic cell clearance. J. Cell Biol. 210, 485–502 (2015).
Guo, P., Hu, T., Zhang, J., Jiang, S. & Wang, X. Sequential action of Caenorhabditis elegans Rab GTPases regulates phagolysosome formation during apoptotic cell degradation. Proc. Natl Acad. Sci. USA 107, 18016–18021 (2010).
Fares, H. & Greenwald, I. Genetic analysis of endocytosis in Caenorhabditis elegans: coelomocyte uptake defective mutants. Genetics 159, 133–145 (2001).
Kumari, S. & Mayor, S. ARF1 is directly involved in dynamin-independent endocytosis. Nat. Cell Biol. 10, 30–41 (2008).
Podbilewicz, B. et al. The C. elegans developmental fusogen EFF-1 mediates homotypic fusion in heterologous cells and in vivo. Dev. Cell 11, 471–481 (2006).
del Campo, J. J. et al. Fusogenic activity of EFF-1 is regulated via dynamic localization in fusing somatic cells of C. elegans. Curr. Biol. 15, 413–423 (2005).
Smurova, K. & Podbilewicz, B. Endocytosis regulates membrane localization and function of the fusogen EFF-1. Small GTPases 8, 177–180 (2017).
Kinet, M. J. et al. HSF-1 activates the ubiquitin proteasome system to promote non-apoptotic developmental cell death in C. elegans. eLife 5, e12821 (2016).
van der Bliek, A. M. & Meyerowitz, E. M. Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic. Nature 351, 411–414 (1991).
Simunovic, M. et al. Friction mediates scission of tubular membranes scaffolded by BAR proteins. Cell 170, 172–184.e11 (2017).
Neumann, B. et al. EFF-1-mediated regenerative axonal fusion requires components of the apoptotic pathway. Nature 517, 219–222 (2015).
Ghosh-Roy, A., Wu, Z., Goncharov, A., Jin, Y. & Chisholm, A. D. Calcium and cyclic AMP promote axonal regeneration in Caenorhabditis elegans and require DLK-1 kinase. J. Neurosci. 30, 3175–3183 (2010).
Oren-Suissa, M., Gattegno, T., Kravtsov, V. & Podbilewicz, B. Extrinsic repair of injured dendrites as a paradigm for regeneration by fusion in Caenorhabditis elegans. Genetics 206, 215–230 (2017).
Rasmussen, J. P., English, K., Tenlen, J. R. & Priess, J. R. Notch signaling and morphogenesis of single-cell tubes in the C. elegans digestive tract. Dev. Cell 14, 559–569 (2008).
Shemer, G. et al. EFF-1 is sufficient to initiate and execute tissue-specific cell fusion in C. elegans. Curr. Biol. 14, 1587–1591 (2004).
Fedry, J. et al. The ancient gamete fusogen HAP2 is a eukaryotic class II fusion protein. Cell 168, 904–915 (2017).
Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).
Mello, C. C., Kramer, J. M., Stinchcomb, D. & Ambros, V. Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959–3970 (1991).
Wicks, S. R., Yeh, R. T., Gish, W. R., Waterston, R. H. & Plasterk, R. H. Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map. Nat. Genet. 28, 160–164 (2001).
Lundquist, E. A., Reddien, P. W., Hartwieg, E., Horvitz, H. R. & Bargmann, C. I. Three C. elegans Rac proteins and several alternative Rac regulators control axon guidance, cell migration and apoptotic cell phagocytosis. Development 128, 4475–4488 (2001).
Bao, Z. & Murray, J. I. Mounting Caenorhabditis elegans embryos for live imaging of embryogenesis. Cold Spring Harb. Protoc. 2011, prot065599 (2011).
We thank members of the Shaham lab for discussions and critical comments on the manuscript. We thank F. Soulavie and M. Sundaram for sharing data. Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440) and the National Bioresource Project of Japan. The work was supported by a Rockefeller Women and Science Fellowship and the NIH grant 1F32HD089640 to P.G. and by NIH grants R01NS081490 and R01NS078703 to S.S.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
(a) Section of TSC distal process of 1.5-fold embryo. Arrows, microtubules. n = 1. Scale bar, 500 nm (b,c) The TSC results from AFF-1-dependent fusion of two precursor cells. Forked tails with bifurcating processes (arrow) from unfused TSC precursors appear in aff-1(tm2214) mutants. n = 2 biologically independent animals. (d,e) Normal and TSC-ablated L1 animals, respectively. n = 5 biologically independent experiments. Arrow, misshapen tail tip in TSC-ablated animal. Scale bars: 5 μm. (f–i) TSCpro-myristoylated mCherry expressing embryos showing degeneration sequence similar to that of GFP version .n = 2 biologically independent animals. s = soma, p = proximal process, d = distal process. Statistics source data are provided in Supplementary Table 2.
Supplementary Figure 2 R77K mutant is defective in fusion but not localization and its TSC clearance defect is independent of hyp10 cell-cell fusion defects.
(a) EFF-1(R77K) fails to rescue TSC process clearance in eff-1(ns634). n = biologically independent animalsfor statistics are as follows, Line 1: n = 35 (non-transgenic), n = 50 (transgenic); Line 2: n = 34 (non-transgenic), n = 50 (transgenic); Line 3: n = 45 (non-transgenic), n = 50 (transgenic). Data are mean +/− s.e.m. Statistics: two-tailed unpaired t-test. Individual p values: see Supplementary Table 2. Numbers inside bars, total animals scored per genotype. 3 independent transgenic lines were scored. (b,c) eff-1(ns634); TSCpro::mKate2PH animals expressing AJM-1::GFP to show apical junctions between unfused hyp10 cells. Yellow arrows indicate cell-cell junctions, white arrow, TSC. n = 20 biologically independent animals. In (b) TSC is absent. TSC is present as in (d). Wild-type L1 animal expressing EFF-1(R77K)::GFP showing EFF-1 localization at the cell membrane and in vesicles (yellow arrows); n = 4 biologically independent animals. Scale bar: 5 μm. Statistics source data are provided in Supplementary Table 2.
(a) TSCpro::mKate2PH, (b) ced-1p::GFP::2xFYVE, and (c) merge in eff-1(ns634). (d) TSCpro::mKate2PH, (e) eff-1p::GFP::RAB-7, and (f) merge in eff-1(ns634) (g) TSCpro::myrGFP, (h) ced-1p::LAAT-1::mCherry, and (i) merge in eff-1(ns634). n = 10 biologically independent animals per marker. Scale bar: 5 μm. Statistics source data are provided in Supplementary Table 2.
Supplementary Figure 4 Muscle-secreted ssGFP accumulates within the phagosome of eff-1(ns634);cup-2(ar506) mutants and GFP around TSC does not recover in a sand-1(or552);cup-2(ar506) FRAP experiment.
(a–d) The TSC distal process surrounded by muscle-secreted GFP (ssGFP) within an open phagosome in eff-1(ns634);cup-2(ar506) mutants. White arrow, ssGFP within phagosome; yellow arrow, phagosome opening. n = 4 biologically independent animals. (e) Open phagosome model: photobleached GFP should recover by entry of extracellular GFP into the open phagosome. (f) Closed phagosome model: photobleached GFP should not recover as phagosome is sealed. (g–i) A sand-1(or552);cup-2(ar506) mutant. 5 seconds before photobleaching, (j–l) time 0 seconds, (m–o) and (p–r) 5 seconds and 30 minutes after photobleaching, respectively. Arrow, ssGFP-containing phagosome. Note GFP signal around TSC does not return. n = 1 animal. Scale bar: 5 μm. Statistics source data are provided in Supplementary Table 2.
Supplementary Figures 1–4, Supplementary Table and Supplementary Video legends.
Mutants tested for tail-spike cell (TSC) persistence.
Statistics source data.
Primer sequences and plasmid construction for this study.
Transgenes used in this study.
Mutagenesis screen results for this study.
iSPIM movie of TSCpro::myrGFP in a wild-type animal.
EM serial sections of a dying TSC cell at process beading stage.
Movie of CEM degeneration.
iSPIM movie of TSC of sand-1(or552).
iSPIM movie of TSC of eff-1(ns634).
Stacks of eff-1(ns634); TSCpro::myrGFP; hyp10pro::mKate2.
Stacks of eff-1(ns634); TSCpro::myrGFP; hyp10pro::mKate2PH.
Stacks of GFP::2xFYVE; TSCp::mKate2PH in eff-1(ns634).
Stacks of GFP::RAB-7; TSCp::mKate2PH in eff-1(ns634).
Stacks of LAAT-1::mCherry; TSCp::myrGFP in eff-1(ns634).
Stacks of ssGFP; hyp10pro::iBlueberry in eff-1(ns634);cup-2(ar506).
FRAP movie for a fast-recovering eff-1(ns634) animal.
FRAP movie for a fast-recovering eff-1(ns634) animal.
FRAP movie of sand-1(or552) animal.
Stacks of movie of EFF-1 localization with TSCpro::mKate2PH and hyp10p::iBlueberry in eff-1(ns634).
About this article
Cite this article
Ghose, P., Rashid, A., Insley, P. et al. EFF-1 fusogen promotes phagosome sealing during cell process clearance in Caenorhabditis elegans. Nat Cell Biol 20, 393–399 (2018). https://doi.org/10.1038/s41556-018-0068-5
This article is cited by
Cell Regeneration (2021)
Nature Communications (2018)