DOI: 10.5061/dryad.sj3tx96df
title: Data from: Phagolysosomes break down the membrane of a non-apoptotic corpse independent of macroautophagy

description: Cell corpses must be cleared in an efficient manner to maintain tissue
 homeostasis and regulate immune responses. Ubiquitin-like Atg8/LC3 family
 proteins promote the degradation of membranes and internal cargo during
 both macroautophagy and corpse clearance, raising the question how
 macroautophagy contributes to corpse clearance. Studying the clearance of
 non-apoptotic dying polar bodies in Caenorhabditis elegans embryos, we
 show that the LC3 ortholog LGG-2 is enriched inside the polar body
 phagolysosome independent of autophagosome formation. We
 demonstrate that ATG-16.1 and ATG-16.2, which promote membrane association
 of lipidated Atg8/LC3 proteins, redundantly promote polar body membrane
 breakdown in phagolysosomes independent of their role in macroautophagy.
 We also show that the lipid scramblase ATG-9 is needed for autophagosome
 formation in early embryos but is dispensable for timely polar body
 membrane breakdown or protein cargo degradation. These findings
 demonstrate that macroautophagy is not required to promote polar body
 degradation, in contrast to recent findings with apoptotic corpse
 clearance in C. elegans embryos. Determining how factors
 regulating Atg8/LC3 promote the breakdown of different types of
 cell corpses in distinct cell types or metabolic states is likely to give
 insights into the mechanisms of immunoregulation during normal
 development, physiology, and disease.
description: Gravid C. elegans hermaphrodites were dissected in egg salts (94
 mM NaCl, 32 mM KCl, 2.8 mM MgCl<sub>2</sub>, 2.8 mM
 CaCl<sub>2</sub>, 2 mM HEPES, pH 7.5) to isolate embryos and
 mounted on a 4% agarose pad for live imaging. Z-stacks were collected
 using a Zeiss Axio Observer 7 inverted microscope with Plan-APO 40X 1.4 NA
 oil objective with Excelitas Technologies X-Cite 120LED Boost
 illumination, and Hamamatsu ORCA-Fusion sCMOS camera controlled by 3i
 SlideBook6 software. Time-lapse imaging was acquired sequentially for
 mCherry and GFP every minute. Data is provided in the SlideBook format
 (.sld).
description: # Data from: Phagolysosomes break down the membrane of a non-apoptotic
 corpse independent of macroautophagy
 [https://doi.org/10.5061/dryad.sj3tx96df](https://doi.org/10.5061/dryad.sj3tx96df) ## Description of the data and file structure In the study by Kolli, Kline et al. entitled "Phagolysosomes break down the membrane of a non-apoptotic corpse independent of macroautophagy", we confirm that ATG-9 and ATG-16.2 are required for normal autophagosome biogenesis in *C. elegans* early embryos. In the absence of autophagosomes, we find no delay in corpse membrane breakdown, no disruption in LGG-2 localization inside the polar body phagolysosome, and no delay in degradation of a corpse cargo protein. These data suggest that autophagosomes are not required for phagolysosome maturation in early embryos. We also find that ATG-16.1 and ATG-16.2 redundantly promote polar body membrane breakdown. In combination with the published role for the lipidation factor ATG-7 (Fazeli et al., Cell Rep 2016), these data suggest that the association of lipidated Atg8/LC3 with a non-autophagic membrane is important for the phagocytic clearance of a non-apoptotic corpse. The imaging data in the archived .sld files are viewable for free in SlideBook Reader software or after installing the SlideBook Bio-Formats plugin in FIJI or ImageJ software (Linked below under Code/software). ### Files and variables #### File: PLoS One Figure Guide.csv **Description:** Spreadsheet detailing where the raw imaging data in the figures of the Kolli, Kline et al. manuscript are found in the following zip archives containing sld files. #### File: WEH95\_Archive.zip **Description:** Time-lapse imaging data of wild type control embryos with green membrane marker degraded in somatic cells and red histones *Genotype: xnIs390[pie-1::GFP::ZF1::PH(PLCdelta1), unc-119(+)]; unc-119(ed3) III; Is[mCherry::HistoneH2B] IV* Contains WEH95-10%.sld #### File: WEH683\_Archive.zip **Description:** Time-lapse imaging data of *atg-16.1* mutant embryos with green membrane degraded in somatic cells and red histones *Genotype: xnIs390[pie-1::GFP::ZF1::PH(PLC1delta1), unc-119(+)] II; Is[mCherry::HistoneH2B] IV; atg-16.1(gk668615[Q356Stop]) X* Contains WEH683-10%.sld and WEH683-10%_2.sld #### File: WEH684\_Archive.zip **Description:** Time-lapse imaging data of *atg-16.2* mutant embryos with green membrane degraded in somatic cells and red histones *Genotype: atg-16.2(ok3224) xnIs390[pie-1::GFP::ZF1::PH(PLC1delta1), unc-119(+)] II; Is[mCherry::HistoneH2B] IV* Contains WEH684-10%.sld,  WEH684-10%_2.sld, WEH684-10%_3.sld,  WEH684-10%_4.sld, and WEH684-10%_5.sld #### File: WEH696\_Archive.zip **Description:** Time-lapse imaging data of homozygous *atg-16.2; atg-16.1* double mutant embryos with green membrane degraded in somatic cells and red histones *Genotype: atg-16.2(ok3224) xnIs390[pie-1::GFP::ZF1::PH(PLC1delta1), unc-119(+)] II; unc-119(ed3) III; Is[mCherry::HistoneH2B] IV; atg-16.1(gk668615[Q356Stop]) X* Contains WEH696-10%.sld and WEH696-10%_2.sld #### File: WEH700\_Archive.zip **Description:** Time-lapse imaging data of wild type control embryos with green membrane and red histones degraded in somatic cells *Genotype: wurIs144[pGF13: pie-1::ZF1::mCherry::his-15; unc-119(+)] I; unc-119(ed3) ltIs38[pie-1::GFP::PH(PLC1delta1), unc-119(+)] III* Contains WEH700-10%.sld, WEH700-10%-2c.sld,  WEH700-10%-2c2.sld, WEH700-10%-2c3.sld,  WEH700-10%-2c4.sld, WEH700-10%-2c5.sld, WEH700-10%-2c6.sld, WEH700-10%-2c7.sld, WEH700-10%-3c.sld, WEH700-10%-3c2.sld, WEH700-10%-4c.sld, WEH700-10%-4c2.sld, and WEH700-10%-4c3.sld #### File: WEH708\_Archive.zip **Description:** Time-lapse imaging data of *atg-16.2* mutant embryos with green membrane and red histones degraded in somatic cells *Genotype: wurIs144[pGF13: pie-1::ZF1::mCherry::his-15; unc-119(+)] I; atg-16.2(gk145022[W253Stop]) II; unc-119(ed3) ltIs38[pie-1::GFP::PH(PLC1delta1), unc-119(+)] III* Contains WEH708-10%.sld and WEH708-10%_2.sld #### File: WEH714\_Archive.zip **Description:** Time-lapse imaging data of homozygous *atg-16.2; atg-16.1* double mutant embryos with green membrane and red histones degraded in somatic cells *Genotype: wurIs144[pGF13: pie-1::ZF1::mCherry::his-15; unc-119(+)] I; atg-16.2(gk145022[W253Stop]) II; unc-119(ed3) ltIs38[pie-1::GFP::PH(PLC1delta1), unc-119(+)] III; atg-16.1(gk668615[Q356Stop]) X* Contains WEH714-10%.sld,  WEH714-10%_2.sld, and WEH714-10%_3.sld #### File: WEH722\_Archive.zip **Description:** Time-lapse (10% fluorescence) and still (50% fluorescence) imaging data of wild type control embryos with green histones and red LC3 homolog LGG-2 *Genotype: Si[pVIG57: Pmex-5::mCherry::LGG-2::tbb-2 3'UTR, C.b. unc-119(+)] II; unc-119(ed3) ruIs32[pAZ132: pie-1::GFP::H2B, unc-119(+)] III* Contains WEH722-10%.sld,  WEH722-10% 1 cell movies.sld, and WEH722-50%.sld #### **File: WEH728\_Archive.zip** **Description:** Still images of *atg-16.2* mutant embryos with green histones and red LC3 homolog LGG-2 *Genotype: Si[pVIG57: Pmex-5::mCherry::LGG-2::tbb-2 3'UTR, C.b. unc-119(+)] atg-16.2(gk145022[W253Stop]) II; unc-119(ed3) ruIs32[pAZ132: pie-1::GFP::H2B, unc-119(+)] III* Contains WEH728-50%.sld #### File: WEH729\_Archive.zip **Description:** Still images of *atg-9* mutant embryos with green histones and red LC3 homolog LGG-2 *Genotype: Si[pVIG57: Pmex-5::mCherry::LGG-2::tbb-2 3'UTR, C.b. unc-119(+)] II; unc-119(ed3) ruIs32[pAZ132: pie-1::GFP::H2B, unc-119(+)] III; atg-9(bp564) V* Contains WEH729-50%.sld #### File: WEH731\_Archive.zip **Description:** Time-lapse imaging data of *atg-9* mutant embryos with green membrane and red histones degraded in somatic cells *Genotype: wurIs144[pGF13: pie-1::ZF1::mCherry::his-15; unc-119(+)] I; unc-119(ed3) ltIs38[pie-1::GFP::PH(PLC1delta1), unc-119(+)] III; atg-9(bp564) V* Contains WEH731-10%.sld, WEH731-10%-2.sld, WEH731-10% 2 hr.sld, WEH731-10% 2 hr2.sld, WEH731-10% 2 hr3.sld, WEH731-10% 2 hr-2c9.sld, WEH731-10% 2 hr-2c10.sld, and WEH731-10% 2 hr-3c.sld #### File: WEH734\_Archive.zip **Description:** Still images of *atg-16.1* mutant embryos with green histones and red LC3 homolog LGG-2 *Genotype: Si[pVIG57: Pmex-5::mCherry::LGG-2::tbb-2 3'UTR, C.b. unc-119(+)] II; unc-119(ed3) ruIs32[pAZ132: pie-1::GFP::H2B, unc-119(+)] III; atg-16.1(gk668615[Q356Stop*]) X Contains WEH734-50%.sld #### File: WEH739\_Archive.zip **Description:** Time-lapse (10% fluorescence) and still (50% fluorescence) imaging data of homozygous *atg-16.2; atg-16.1* double mutants with green histones and red LC3 homolog LGG-2 *Genotype: Si[pVIG57: Pmex-5::mCherry::LGG-2::tbb-2 3'UTR, C.b. unc-119(+)] atg-16.2(gk145022[W253Stop]) II; unc-119(ed3) ruIs32[pAZ132: pie-1::GFP::H2B, unc-119(+)] III; atg-16.1(gk668615[Q356Stop]) X* Contains WEH739-2 cell movies 10%.sld and WEH739-50%.sld #### File: WEH755\_Archive.zip **Description:** Still images of wild type control embryos with green histones and red LC3 homolog LGG-2 degraded in blastomeres after the first mitotic division *Genotype: wurSi2[pVIG57-CTPD: Pmex-5::CTPD::mCherry::LGG-2::tbb-2 3'UTR, C.b. unc-119(+)] II; unc-119(ed3) ruIs32[pAZ132: pie-1::GFP::H2B, unc-119(+)] III* Contains WEH755-50%-1s exposure red.sld ## Code/software SlideBook Reader software [https://go.intelligent-imaging.com/get-slidebook-reader](https://go.intelligent-imaging.com/get-slidebook-reader) SlideBook Bio-Formats plugin in FIJI or ImageJ software [https://fiji.sc](https://fiji.sc) Instructions on how to add SlideBook access to the Bio-Formats plugin can be found on the 3i website below [https://www.intelligent-imaging.com/technical-answers](https://www.intelligent-imaging.com/technical-answers) ## Access information Data was derived from the following sources: * Imaging data generated in the Wehman Lab at the University of Denver (now relocated to Texas A&M University)