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EFF-1 fusogen promotes phagosome sealing during cell process clearance in Caenorhabditis elegans

Nature Cell Biologyvolume 20pages393399 (2018) | Download Citation


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.

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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.

Author information

Author notes

  1. These authors contributed equally: Alina Rashid and Peter Insley.


  1. Laboratory of Developmental Genetics, The Rockefeller University, New York, NY, USA

    • Piya Ghose
    • , Alina Rashid
    • , Peter Insley
    • , Meera Trivedi
    • , Anupriya Singhal
    • , Yun Lu
    •  & Shai Shaham
  2. Developmental Biology Program, Sloan Kettering Institute, New York, NY, USA

    • Pavak Shah
    •  & Zhirong Bao


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P.G. and S.S. wrote the manuscript and analysed the results. A.R. and P.I. performed the iSPIM imaging. A.S. and A.R. performed the CEM imaging. M.T. helped map the mutants. P.S. and Z.B. performed the TSC ablations. Y.L. performed the electron microscopy. P.G. performed all other experiments.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Shai Shaham.

Integrated supplementary information

  1. Supplementary Figure 1 The TSC extends a microtubule-laden process required for tail morphogenesis.

    (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. (fi) 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.

  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.

  3. Supplementary Figure 3 Some phagosome markers do not localize around the TSC.

    (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.

  4. 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.

    (ad) 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. (gi) A sand-1(or552);cup-2(ar506) mutant. 5 seconds before photobleaching, (jl) time 0 seconds, (mo) and (pr) 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 information

  1. Supplementary Information

    Supplementary Figures 1–4, Supplementary Table and Supplementary Video legends.

  2. Life Sciences Reporting Summary

  3. Supplementary Table 1

    Mutants tested for tail-spike cell (TSC) persistence.

  4. Supplementary Table 2

    Statistics source data.

  5. Supplementary Table 3

    Primer sequences and plasmid construction for this study.

  6. Supplementary Table 4

    Transgenes used in this study.

  7. Supplementary Table 5

    Mutagenesis screen results for this study.


  1. Supplementary Video 1

    iSPIM movie of TSCpro::myrGFP in a wild-type animal.

  2. Supplementary Video 2

    EM serial sections of a dying TSC cell at process beading stage.

  3. Supplementary Video 3

    Movie of CEM degeneration.

  4. Supplementary Video 4

    iSPIM movie of TSC of sand-1(or552).

  5. Supplementary Video 5

    iSPIM movie of TSC of eff-1(ns634).

  6. Supplementary Video 6

    Stacks of eff-1(ns634); TSCpro::myrGFP; hyp10pro::mKate2.

  7. Supplementary Video 7

    Stacks of eff-1(ns634); TSCpro::myrGFP; hyp10pro::mKate2PH.

  8. Supplementary Video 8

    Stacks of GFP::2xFYVE; TSCp::mKate2PH in eff-1(ns634).

  9. Supplementary Video 9

    Stacks of GFP::RAB-7; TSCp::mKate2PH in eff-1(ns634).

  10. Supplementary Video 10

    Stacks of LAAT-1::mCherry; TSCp::myrGFP in eff-1(ns634).

  11. Supplementary Video 11

    Stacks of ssGFP; hyp10pro::iBlueberry in eff-1(ns634);cup-2(ar506).

  12. Supplementary Video 12

    FRAP movie for a fast-recovering eff-1(ns634) animal.

  13. Supplementary Video 13

    FRAP movie for a fast-recovering eff-1(ns634) animal.

  14. Supplementary Video 14

    FRAP movie of sand-1(or552) animal.

  15. Supplementary Video 15

    Stacks of movie of EFF-1 localization with TSCpro::mKate2PH and hyp10p::iBlueberry in eff-1(ns634).

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