Supplemental Data

Cell, Volume 133

Six-Microns-Under Acts Upstream of Draper in the Glial Phagocytosis of Apoptotic Neurons Estee Kurant, Sofia Axelrod, Dan Leaman, and Ulrike Gaul

Figure S1 Expression of apoptosis/phagocytosis markers in embryos lacking apoptosis. Ventral view of stage 16 CNS in confocal stacks, showing glia labeled with cytoplasmic GFP (simu-cytGFP, green) and expression of the three markers AnnexinV, CM1 and lysotracker (red) in wt (A)-(C) and in embryos deficient for H99, which lack the three pro-apoptotic genes hid, grim and rpr and are nearly devoid of apoptosis (Brennecke et al., 2003; Leaman et al., 2005; Rogulja-Ortmann et al., 2007; White et al., 1994; White and Steller, 1995) (D)-(F). (A, D) AnnexinV, the earliest marker, binds phosphatidylserine (PS) with high affinity, and in wt labels many particles touched or engulfed by glia, as well as numerous untouched cells. In H99 deficient embryos, only unengulfed AnnexinV-positive particles remain, suggesting that in the absence of the pro-apoptotic factors, PS still accumulates on the surface of some cells, but the subsequent steps of apoptosis, fragmentation and phagocytosis no longer take place. (B, E) As expected, H99 deficient embryos show no Caspase 3 activation as revealed by CM1 staining. (C, F) Lysotracker labels lysosomes (small vesicles) as well as phago-lysosomes (large vesicles). While in wt embryos lysotracker is found almost exclusively in large vesicles within glia and macrophages, we find it labeling small spots throughout the nerve cord in H99-deficient embryos. These findings suggest that the amount of injected lysotracker is limiting and thus in wt, where phagocytosis is high, only glial and macrophage phago-lysosomes are labeled. By contrast, when apoptosis/phagocytosis is absent as in the H99 mutant, the lysosomal content of all cells is revealed.

0 200

+10

CED1

DRPR-PB

MEGF10

NG3

Fibrillin1

MAMNephro-nectin

SREC-I-1

NimC1

NimB4

Eater

SIMU

EMI(-like) C...(C)...(C)...CCxGY...C

NIM-type EGF

EGF-CA

other EGF

CED1-type EGF

TB

signal peptidetransmembrane

CED1-type EGF, truncated

PxCxxxCxNGxCxxPxxCxCxxGYx4-8Cx

CxCxxgxCxpxxGxCxCxpGWxGxxC

CpxgxyGxxCxxxCxCxngxxCxpxxGxCxCxpGWxGxxC

DxdECxxxxxxCxxgxCxnTxGSYxCxCxxGYx6-8C

6(4) Cys

6 Cys

8 Cys

400 600 800 1000 aa

2871 aa

A

422651312526

3514

2926

21

28

1324689955618

16633

2738

51

56

CED1DRPRNimA

MEGF10MEGF11

PEAR1

NG3NimC2

NimC3NimC1

SIMU

MEGF7

NimB2NimB3NimB4NimB5NimB1

Eater

LTBP2Nephronectin

SREC-IEGFL6

SREC-II

Fibrillin 1

147128157135129131

141111

133124

128

134

21411717317614191

21886

8691

109

111

B

Ce Dm Hs

DHVCTVKTIVDDY--ELKKVIHTVVYNDTEQCLNPLTG-FQCTVEKRGQKASYQRQL-VKKEKYVKQCCDGY---YQTKDH-FC-LPDCN----PPC-KKG-KCIEPGKCECDPGY-------GGK--YC-PNICKRRELYNVD--VVYTELQSFQERGSTWCVT-FPP--RCSTYRIKHRVVNKTKT-IAKNRIVRDCCDGY---IA-SAG-EC-VPHCS----EPC-QHG-RCISPEKCKCDHGY-------GGP--AC-GNICIREEPYVEH--VQVPEMQPVRVRTSSWCME-IPP--RCATFKTEMREVMRVQK-LNKTRTVRFCCQGYEGNLSDSQA-TC-KPICR----GGC-GRG-SCVMPDICSCEEGY-------IGK--HC-PNVCSHWESYSVT--VQESYPHPFDQIYYTSCTDILNW-FKCTRHRVSYRTAYRHGE-KTMYRRKSQCCPGF---YE-SGE-MC-VPHCA----DKC-VHG-RCIAPNTCQCEPGW-------GGT--NC-PNVCSHWESYAVT--VQESYAHPFDQIYYTRCTDILNW-FKCTRHRISYKTAYRRGL-RTMYRRRSQCCPGY---YE-NGD-FC-IPLCT----EEC-VHG-RCVSPDTCHCEPGW-------GGP--DC-PNTCSFWESFTTT--TKESHSRPFSLLPSEPCERPWEGPHTCPQPTVVYRTVYRQVV-KTDHRQRLQCCHGF---YE-SRG-FC-VPLCA----QEC-VHG-RCVAPNQCQCVPGW-------RGD--DC-RRVCAVRAHGDPV---SESFVQRVYQPFLTTCDG--HR--ACSTYRTIYRTAYRRSPGLAPARPRYACCPGWKRTSGLPGA--CGAAICQ----PPC-RNGGSCVQPGRCRCPAGW-------RGD--TC-QGVCSKQTLVVPLH-YNESYSQPVYKPYLTLCAG--RR--ICSTYRTMYRVMWR-EVRREVQQTHAVCCQGWKKRH--PGALTC-EAICA----KPC-LNGGVCVRPDQCECAPGW-------GGK--HC-VQGCVKQAQVVKM---RGTLVTMRKHQNSANCTT------NCGP--LVGRT--RTETYL---GFTDVCCDGY---IRDENN-EC-VPLCN----D-CGASG-KCLLPNVCLCGKGY--VSRKDHG---HCDLNVCNRSQVVTYK--RYVERSRVIPYQHRSF---WSGWQ---TKYRTEYYTDEETAY-RTVMRP--SCCEGY---EG-SVE-NC-KPVCR----QQCPQHG-FCSSPNTCSCNAGY-------GGI--DC-NGHCQKNISVKYQVPVAKTRMAGAGPPNASH--PID------LDSYVVYEE--RVRW-----DNIQVCCPGY---RTILFG-FC-EPVCQ----EACPAHS-YCAEPDRCHCQRGYEPSHHHTTGHQLIC-DNYCERNETIRAT--VPVTKQRIIVKQPSKW--KIWKKT---EKITEIYDS-EEEQV---THRLVRECCPGY---LQVESG-LC-EPICS----RGCPAHA-SCAAPDRCECISGYVSARNHQDGSH-YC-SGVCYKEVPTASL--LRNSRDQFVG-NGTT-----------------PDMS------------RIQVCCDGYE--RNPHIYRRC-EPICA----DDC-RNG-ICTAPNTCVCIPGHV---RTAEGK---C-SGICYRTLTVETI--NPNSRNRQF-----------------------------------------SYCCDGYV-NKGTSQNLKC-EPICS----EDC-SNG-LCLAPEECECAPGY-----YRSNK--RC-PDKCRQEVPAVFF--QYDKEVKIVG-NSST----------------NPYMNV------------IEVCCKGWR-RYEYDWS-QC-VPDCG----ERCQENG-FCVAGGKCVCFTDFV------LNYRNNC-QHKCRIWVPPDTV--EKYSYPSVIQTDQAN----------------RLS--------------LIEVCCTGY-SASRLMGVTVC-RAQC------GC-QNG-SCKIPGECECYDGFV---RNDNGD---C-QHLCHREVPSVFF---QTERDSPVRGNGST-----------------IYFH------------RIEVCCAGY---RRDPYANEC-VPDCSASSPDNC-RNG-FCRSPGVCECFAEFV---RNEHGA---C-AQICTVNVT-RNI---KGTAVN-VQ----T-----------------------------------RDCCKGYK-KVR-SSALRC-LAQCK----VNC-GSG-FCTKPNVCTCKKGYV---NLNNDPSNRC-PNVCG-------------SRYN-------------------------------------------AYCCPGW---KTLPGGNQCIVPICR----HSC-GDG-FCSRPNMCTCPSGQ-------IAP--SC-RNVCG------------------------------------------------------------GQCCPGW---TTANSTNHCIKPVCE----PPCQNRG-SCSRPQLCVCRSGF-------RGA--RC-IGLCR-----------YGGR---------------------------------------------IDCCWGW---ARQSWG-QC-QPVCQ----PRC-KHG-ECIGPNKCKCHPGY-------AGK--TC-PGVCH-----------YGTK---------------------------------------------LACCYGW---RRNSKG-VC-EATCE----PGC-KFG-ECVGPNKCRCFPGY-------TGK--TC-QHVCV--------ASSP-SAE--------------------------------------------LQCCAGW----RQKDQ-ECTIPICE--GPDAC-QKDEVCVKPGLCRCKPGF-------FGA--HC-RNVCR----------APGSQV--------------------------------------------PTCCAGW----RQQGD-ECGIAVCE--GNSTCSENE-VCVRPGECRCRHGY-------FGA--NC- NVC S T CC GY G C VPIC C G CV P C C GY G C

TVKTIVDDY--ELKKVIHTVVYNDTEQCLNPLTG-FQCTVEKRGQKASYQRQL-VKKEKYVKQCKRRELYNVD--VVYTELQSFQERGSTWCVT-FPP--RCSTYRIKHRVVNKTKT-IAKNRIVRDCIREEPYVEH--VQVPEMQPVRVRTSSWCME-IPP--RCATFKTEMREVMRVQK-LNKTRTVRFCSHWESYSVT--VQESYPHPFDQIYYTSCTDILNW-FKCTRHRVSYRTAYRHGE-KTMYRRKSQCSHWESYAVT--VQESYAHPFDQIYYTRCTDILNW-FKCTRHRISYKTAYRRGL-RTMYRRRSQCSFWESFTTT--TKESHSRPFSLLPSEPCERPWEGPHTCPQPTVVYRTVYRQVV-KTDHRQRLQCAVRAHGDPV---SESFVQRVYQPFLTTCDG--HR--ACSTYRTIYRTAYRRSPGLAPARPRYACSKQTLVVPLH-YNESYSQPVYKPYLTLCAG--RR--ICSTYRTMYRVMWR-EVRREVQQTHAVCVKQAQVVKM---RGTLVTMRKHQNSANCTT------NCGP--LVGRT--RTETYLGFTDV---CNRSQVVTYK--RYVERSRVIPYQHRSF---WSGWQ---TKYRTEYYTDEETAY-RTVMRP--SCQKNISVKYQVPVAKTRMAGAGPPNASH--PID------LDSYVVYEE--RVRW--DNIQV---CERNETIRAT--VPVTKQRIIVKQPSKW--KIWKKT---EKITEIYDS-EEEQV---THRLVREC

isoform SREC-I-2

0 200

+10 +10

CED1

DRPR-PB

MEGF10

NG3

Fibrillin1

MAMNephro-nectin

SREC-I-1

NimC1

NimB4

Eater

SIMU

EMI(-like) C...(C)...(C)...CCxGY...C

NIM-type EGF

EGF-CA

other EGF

CED1-type EGF

TB

signal peptidetransmembrane

CED1-type EGF, truncated

PxCxxxCxNGxCxxPxxCxCxxGYx4-8Cx

CxCxxgxCxpxxGxCxCxpGWxGxxC

CpxgxyGxxCxxxCxCxngxxCxpxxGxCxCxpGWxGxxC

DxdECxxxxxxCxxgxCxnTxGSYxCxCxxGYx6-8C

6(4) Cys

6 Cys

8 Cys

400 600 800 1000 aa

2871 aa

A

422651312526

3514

2926

21

28

1324689955618

16633

2738

51

56

422651312526

3514

2926

21

28

1324689955618

16633

2738

51

56

CED1DRPRNimA

MEGF10MEGF11

PEAR1

NG3NimC2

NimC3NimC1

SIMU

MEGF7

NimB2NimB3NimB4NimB5NimB1

Eater

LTBP2Nephronectin

SREC-IEGFL6

SREC-II

Fibrillin 1

147128157135129131

141111

133124

128

134

21411717317614191

21886

8691

109

111

147128157135129131

141111

133124

128

134

21411717317614191

21886

8691

109

111

B

Ce Dm Hs

DHVCTVKTIVDDY--ELKKVIHTVVYNDTEQCLNPLTG-FQCTVEKRGQKASYQRQL-VKKEKYVKQCCDGY---YQTKDH-FC-LPDCN----PPC-KKG-KCIEPGKCECDPGY-------GGK--YC-PNICKRRELYNVD--VVYTELQSFQERGSTWCVT-FPP--RCSTYRIKHRVVNKTKT-IAKNRIVRDCCDGY---IA-SAG-EC-VPHCS----EPC-QHG-RCISPEKCKCDHGY-------GGP--AC-GNICIREEPYVEH--VQVPEMQPVRVRTSSWCME-IPP--RCATFKTEMREVMRVQK-LNKTRTVRFCCQGYEGNLSDSQA-TC-KPICR----GGC-GRG-SCVMPDICSCEEGY-------IGK--HC-PNVCSHWESYSVT--VQESYPHPFDQIYYTSCTDILNW-FKCTRHRVSYRTAYRHGE-KTMYRRKSQCCPGF---YE-SGE-MC-VPHCA----DKC-VHG-RCIAPNTCQCEPGW-------GGT--NC-PNVCSHWESYAVT--VQESYAHPFDQIYYTRCTDILNW-FKCTRHRISYKTAYRRGL-RTMYRRRSQCCPGY---YE-NGD-FC-IPLCT----EEC-VHG-RCVSPDTCHCEPGW-------GGP--DC-PNTCSFWESFTTT--TKESHSRPFSLLPSEPCERPWEGPHTCPQPTVVYRTVYRQVV-KTDHRQRLQCCHGF---YE-SRG-FC-VPLCA----QEC-VHG-RCVAPNQCQCVPGW-------RGD--DC-RRVCAVRAHGDPV---SESFVQRVYQPFLTTCDG--HR--ACSTYRTIYRTAYRRSPGLAPARPRYACCPGWKRTSGLPGA--CGAAICQ----PPC-RNGGSCVQPGRCRCPAGW-------RGD--TC-QGVCSKQTLVVPLH-YNESYSQPVYKPYLTLCAG--RR--ICSTYRTMYRVMWR-EVRREVQQTHAVCCQGWKKRH--PGALTC-EAICA----KPC-LNGGVCVRPDQCECAPGW-------GGK--HC-VQGCVKQAQVVKM---RGTLVTMRKHQNSANCTT------NCGP--LVGRT--RTETYL---GFTDVCCDGY---IRDENN-EC-VPLCN----D-CGASG-KCLLPNVCLCGKGY--VSRKDHG---HCDLNVCNRSQVVTYK--RYVERSRVIPYQHRSF---WSGWQ---TKYRTEYYTDEETAY-RTVMRP--SCCEGY---EG-SVE-NC-KPVCR----QQCPQHG-FCSSPNTCSCNAGY-------GGI--DC-NGHCQKNISVKYQVPVAKTRMAGAGPPNASH--PID------LDSYVVYEE--RVRW-----DNIQVCCPGY---RTILFG-FC-EPVCQ----EACPAHS-YCAEPDRCHCQRGYEPSHHHTTGHQLIC-DNYCERNETIRAT--VPVTKQRIIVKQPSKW--KIWKKT---EKITEIYDS-EEEQV---THRLVRECCPGY---LQVESG-LC-EPICS----RGCPAHA-SCAAPDRCECISGYVSARNHQDGSH-YC-SGVCYKEVPTASL--LRNSRDQFVG-NGTT-----------------PDMS------------RIQVCCDGYE--RNPHIYRRC-EPICA----DDC-RNG-ICTAPNTCVCIPGHV---RTAEGK---C-SGICYRTLTVETI--NPNSRNRQF-----------------------------------------SYCCDGYV-NKGTSQNLKC-EPICS----EDC-SNG-LCLAPEECECAPGY-----YRSNK--RC-PDKCRQEVPAVFF--QYDKEVKIVG-NSST----------------NPYMNV------------IEVCCKGWR-RYEYDWS-QC-VPDCG----ERCQENG-FCVAGGKCVCFTDFV------LNYRNNC-QHKCRIWVPPDTV--EKYSYPSVIQTDQAN----------------RLS--------------LIEVCCTGY-SASRLMGVTVC-RAQC------GC-QNG-SCKIPGECECYDGFV---RNDNGD---C-QHLCHREVPSVFF---QTERDSPVRGNGST-----------------IYFH------------RIEVCCAGY---RRDPYANEC-VPDCSASSPDNC-RNG-FCRSPGVCECFAEFV---RNEHGA---C-AQICTVNVT-RNI---KGTAVN-VQ----T-----------------------------------RDCCKGYK-KVR-SSALRC-LAQCK----VNC-GSG-FCTKPNVCTCKKGYV---NLNNDPSNRC-PNVCG-------------SRYN-------------------------------------------AYCCPGW---KTLPGGNQCIVPICR----HSC-GDG-FCSRPNMCTCPSGQ-------IAP--SC-RNVCG------------------------------------------------------------GQCCPGW---TTANSTNHCIKPVCE----PPCQNRG-SCSRPQLCVCRSGF-------RGA--RC-IGLCR-----------YGGR---------------------------------------------IDCCWGW---ARQSWG-QC-QPVCQ----PRC-KHG-ECIGPNKCKCHPGY-------AGK--TC-PGVCH-----------YGTK---------------------------------------------LACCYGW---RRNSKG-VC-EATCE----PGC-KFG-ECVGPNKCRCFPGY-------TGK--TC-QHVCV--------ASSP-SAE--------------------------------------------LQCCAGW----RQKDQ-ECTIPICE--GPDAC-QKDEVCVKPGLCRCKPGF-------FGA--HC-RNVCR----------APGSQV--------------------------------------------PTCCAGW----RQQGD-ECGIAVCE--GNSTCSENE-VCVRPGECRCRHGY-------FGA--NC- NVC S T CC GY G C VPIC C G CV P C C GY G C

TVKTIVDDY--ELKKVIHTVVYNDTEQCLNPLTG-FQCTVEKRGQKASYQRQL-VKKEKYVKQCKRRELYNVD--VVYTELQSFQERGSTWCVT-FPP--RCSTYRIKHRVVNKTKT-IAKNRIVRDCIREEPYVEH--VQVPEMQPVRVRTSSWCME-IPP--RCATFKTEMREVMRVQK-LNKTRTVRFCSHWESYSVT--VQESYPHPFDQIYYTSCTDILNW-FKCTRHRVSYRTAYRHGE-KTMYRRKSQCSHWESYAVT--VQESYAHPFDQIYYTRCTDILNW-FKCTRHRISYKTAYRRGL-RTMYRRRSQCSFWESFTTT--TKESHSRPFSLLPSEPCERPWEGPHTCPQPTVVYRTVYRQVV-KTDHRQRLQCAVRAHGDPV---SESFVQRVYQPFLTTCDG--HR--ACSTYRTIYRTAYRRSPGLAPARPRYACSKQTLVVPLH-YNESYSQPVYKPYLTLCAG--RR--ICSTYRTMYRVMWR-EVRREVQQTHAVCVKQAQVVKM---RGTLVTMRKHQNSANCTT------NCGP--LVGRT--RTETYLGFTDV---CNRSQVVTYK--RYVERSRVIPYQHRSF---WSGWQ---TKYRTEYYTDEETAY-RTVMRP--SCQKNISVKYQVPVAKTRMAGAGPPNASH--PID------LDSYVVYEE--RVRW--DNIQV---CERNETIRAT--VPVTKQRIIVKQPSKW--KIWKKT---EKITEIYDS-EEEQV---THRLVREC

isoform SREC-I-2

Figure S2 Comparison of EMI(-like)+NIM domain proteins.

2

Typical domain organizations of CED-1, its homologs, and other proteins containing an N-terminal EMI(-like)+NIM domain from worm, fly, and human; names of proteins with a demonstrated role in phagocytosis are highlighted in grey. Note that in most insect proteins the common EMI(-like)+NIM core is followed by additional NIM domains, while the vertebrate proteins contain tandem arrays of EGF-like repeats similar to those found in CED1 or other EGF-type domains. We follow Kurucz et al. (2007) and Callebaut et al. (2003) for the identification and domain structure of the proteins shown, but verified using Psi-Blast and manual inspection; we also shifted the delineation of the NIM domain by one Cysteine relative to the consensus reported by Kurucz et al. (2007), consistent with the standard parsing of EGF-type repeats. (B) Sequence alignment of the EMI(-like)+NIM core for all known proteins containing this signature from worm, fly, and human. Proteins with similar domain organization as represented in (A) are grouped and bracketed; identical residues are boxed in black, similar residues in grey. The alignment was constructed using the ClustalW algorithm as implemented in VectorNTI 9.0, with manual adjustments. Note that the EMI(-like) domains all share a highly conserved CCxGY motif at the C-terminal end of the domain, with invertebrate and vertebrate proteins showing different forms of internal truncation relative to the canonical EMI domain as represented by CED-1. A separate superimposed alignment (identical residues boxed in dark blue, similar residues in light blue) reveals the stronger similarity of the Drosophila NimC proteins, including SIMU, with the canonical EMI domain; although the two internal Cysteines are missing in the NimC proteins, other motifs are preserved. Callebaut et al. (2003) in their analysis in fact classified the N-terminal domains of NimC1 (=CG8942), NimC3 (=CG16880), and NimC4 (=SIMU, CG16876) as typical EMI-domains.

Figure S3 3D analysis of engulfment of apoptotic particles (CM1, red) by glia (repo::CD8GFP, green) in the CNS. Shown are single-label (glia) and merged images of local projections of 4-5 mm depth in three orthogonal planes (XY, YZ, XZ), centered around similar large CM1-positive particles near the midline for all genotypes, see Experimental Procedures. Note that in wt (A) and drpr (C), glial processes envelop these particles on all sides, while in simu (B) and in the simu; drpr double mutant (D) the particles are merely touched. References

Brennecke, J., Hipfner, D. R., Stark, A., Russell, R. B., and Cohen, S. M. (2003). bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell 113, 25-36.

Callebaut, I., Mignotte, V., Souchet, M., and Mornon, J. P. (2003). EMI domains are widespread and reveal the probable orthologs of the Caenorhabditis elegans CED-1 protein. Biochem Biophys Res Commun 300, 619-623.

Kurucz, E., Markus, R., Zsamboki, J., Folkl-Medzihradszky, K., Darula, Z., Vilmos, P., Udvardy, A., Krausz, I., Lukacsovich, T., Gateff, E., et al. (2007). Nimrod, a putative phagocytosis receptor with EGF repeats in Drosophila plasmatocytes. Curr Biol 17, 649-654.

Leaman, D., Chen, P. Y., Fak, J., Yalcin, A., Pearce, M., Unnerstall, U., Marks, D. S., Sander, C., Tuschl, T., and Gaul, U. (2005). Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development. Cell 121, 1097-1108.

Rogulja-Ortmann, A., Luer, K., Seibert, J., Rickert, C., and Technau, G. M. (2007). Programmed cell death in the embryonic central nervous system of Drosophila melanogaster. Development 134, 105-116.

White, K., Grether, M. E., Abrams, J. M., Young, L., Farrell, K., and Steller, H. (1994). Genetic control of programmed cell death in Drosophila. Science 264, 677-683.

White, K., and Steller, H. (1995). The control of apoptosis in Drosophila. Trends Cell Biol 5, 74-78.

3