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Organelle degradation in the lens by PLAAT phospholipases

Nature volume 592, pages 634–638 (2021)Cite this article

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Abstract

The eye lens of vertebrates is composed of fibre cells in which all membrane-bound organelles undergo degradation during terminal differentiation to form an organelle-free zone1. The mechanism that underlies this large-scale organelle degradation remains largely unknown, although it has previously been shown to be independent of macroautophagy2,3. Here we report that phospholipases in the PLAAT (phospholipase A/acyltransferase, also known as HRASLS) family—Plaat1 (also known as Hrasls) in zebrafish and PLAAT3 (also known as HRASLS3, PLA2G16, H-rev107 or AdPLA) in mice4,5,6—are essential for the degradation of lens organelles such as mitochondria, the endoplasmic reticulum and lysosomes. Plaat1 and PLAAT3 translocate from the cytosol to various organelles immediately before organelle degradation, in a process that requires their C-terminal transmembrane domain. The translocation of Plaat1 to organelles depends on the differentiation of fibre cells and damage to organelle membranes, both of which are mediated by Hsf4. After the translocation of Plaat1 or PLAAT3 to membranes, the phospholipase induces extensive organelle rupture that is followed by complete degradation. Organelle degradation by PLAAT-family phospholipases is essential for achieving an optimal transparency and refractive function of the lens. These findings expand our understanding of intracellular organelle degradation and provide insights into the mechanism by which vertebrates acquired transparent lenses.

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Fig. 1: Plaat1 and PLAAT3 are essential for the degradation of lens organelles in zebrafish and mice, respectively.
Fig. 2: Organelle targeting of Plaat1 is required for degradation of lens organelles in zebrafish.
Fig. 3: Translocation of PLAAT3 and Plaat1 to organellar membranes depends on membrane damage and Hsf4.
Fig. 4: plaat1-deficient zebrafish and Plaat3-deficient mice both develop cataracts and refractive defects.

Data availability

All relevant data generated and analysed in this study are included in the Article. Microarray data that support the findings of this study have been deposited in the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE168029. Uncropped gels are provided in Supplementary Fig. 1. Source data are provided with this paper.

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Acknowledgements

We thank K. Kawakami for the Tol2 transposon system; C. Kishi-Itakura and M. Koike for help with the electron microscopy; S. Shimizu and S. Honda for help with the mouse experiments; Y. Aoyama for help with zebrafish screening; N. Sato and Y. Kanda for maintaining the animals; J. Sakamaki and R. L. Knorr for help with the in vitro experiments; Y. Ishida for help with the production of schematics; K. Igarashi for help with the histological examinations; and M. Komatsu for invaluable support. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (grant number 25111005 to N.M.), a Grant-in-Aid for Research Activity Start-up (grant number 16H06730 to H.M.) and a Grant-in-Aid for Young Scientists (grant number 18K14694 to H.M.) from the Japan Society for the Promotion of Science; a grant for Exploratory Research for Advanced Technology (grant number JPMJER1702 to N.M.) from the Japan Science and Technology Agency (JST); and a grant from the Mitsubishi Foundation (grant number 6705 to N.M.).

Author information

Author notes

  1. Hideaki Morishita

    Present address: Department of Physiology, Graduate School of Medicine, Juntendo University, Tokyo, Japan

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo, Japan

    Hideaki Morishita, Tomoya Eguchi, Satoru Takahashi, Chieko Saito, Ikuko Koyama-Honda & Noboru Mizushima

  2. Laboratory Animal and Genome Sciences Section, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, Chiba, Japan

    Satoshi Tsukamoto

  3. Microscopy Research Support Unit Research Core, Tokyo Medical and Dental University, Tokyo, Japan

    Yuriko Sakamaki

  4. Department of Neurosurgery, Tokyo Medical and Dental University, Tokyo, Japan

    Satoru Takahashi

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Contributions

H.M. and N.M. conceived and designed the overall study. H.M. performed most of the in vivo experiments using zebrafish and mice. H.M. and T.E. performed live imaging experiments using zebrafish. T.E. performed most of the in vitro experiments using culture cells and liposomes. S. Tsukamoto generated Plaat3-deficient mice. Y.S., S. Takahashi, C.S. and I.K-H. performed electron microscopy. All the authors analysed the data. H.M. and N.M. wrote the manuscript with input from all the authors.

Corresponding authors

Correspondence to Hideaki Morishita or Noboru Mizushima.

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The authors declare no competing interests.

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Peer review information Nature thanks Steven Bassnett and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Live imaging of large-scale organelle degradation in the lens of zebrafish.

a, Live imaging of ER–eGFP and mito–mRFP in the lens of zebrafish. The equatorial diameter of the whole lens (black) and the mitochondria- (red) or endoplasmic-reticulum-free zone (green) was quantified in zebrafish (n = 16) at the indicated time points. Solid horizontal bars indicate medians, boxes the interquartile range (25th–75th percentiles) and whiskers 1.5× the interquartile range; outliers are plotted individually. Scale bar, 20 μm. b, Live imaging of ER–eGFP and mito–mRFP in the lens of zebrafish from 56 hpf (n = 3). Magnified time lapse-images in the indicated regions are shown in the right panels. A model of degradation of the endoplasmic reticulum and mitochondria is shown below. Scale bars, 20 μm (leftmost panels), 5 μm (all other panels). c, d, Live imaging of mito–mRFP (c) and ER–eGFP (d) in the lens of rb1cc1+/− (n = 7) and rb1cc1−/− zebrafish (n = 5). Scale bars, 20 μm.

Source data

Extended Data Fig. 2 Identification of Plaat1 as a factor that is essential for the degradation of membrane-bound organelles in the lens of zebrafish.

a, Microarray mRNA expression profiles of the lens and other tissues of zebrafish at 10 days after fertilization. b, Schematic of the screening procedure. c, Results of the founder CRISPR–Cas9-knockout analysis of lens-specific genes and other candidate genes. Relative expression levels were calculated by dividing the normalized signal intensity of the lens by that of other tissues, which were obtained by microarray analyses. d, Schematic of the Cas9-gRNA-targeted site in the plaat1 genomic locus. The targeted site is underlined. The protospacer-adjacent motif sequence is shown in red. The 67-bp insertion and 4-bp deletion in the mutated allele are shown. e, Genotyping of plaat1-mutant zebrafish by PCR. Data are representative of at least five independent experiments. Uncropped gels are provided in Supplementary Fig. 1. f, External appearance of zebrafish at 96 hpf (n = 8). Scale bar, 1 mm. g, Haematoxylin and eosin staining of transverse sections of the eye of zebrafish at 72 hpf (n = 3). Scale bar, 50 μm. h, Electron micrographs of fibre cells in the central region of the lens of zebrafish at 96 hpf (n = 3). Scale bar, 500 nm. i, Live imaging of the plasma membrane and organellar membranes stained with BODIPY FL C5-ceramide (n = 6). Scale bars, 20 μm (main panels), 2 μm (inset panels). j, Staining of nuclear DNA with Hoechst 33342 in the lens of zebrafish at 72 hpf (n = 3). Scale bar, 20 μm. k, Live imaging of histone H2B–eGFP in the lens of zebrafish (n = 8). Scale bar, 20 μm.

Extended Data Fig. 3 Plaat1 is required for degradation of partially rupturing lysosomes in the lens of zebrafish.

a, Live imaging of LAMP1–eGFP and Dnase1l1l–mRFP in the lens of wild-type zebrafish starting at 52 hpf (n = 4). Magnified time-lapse images in the indicated regions in the leftmost panels are shown in the panels to the right. Arrows and arrowheads indicate rupturing lysosomes. Signal intensities (shown as per cent of maximum intensity) of the LAMP1–eGFP- and Dnase1l1l–mRFP-positive puncta (arrows, approximately 2 μm2) were quantified (after background signals were subtracted). Scale bars, 20 μm (leftmost panels), 2 μm (all other panels). b, c, Live imaging of LAMP1–eGFP and Dnase1l1l–mRFP (n = 5) (b) and eGFP–galectin 3 and LAMP1–mRFP (n = 6) (c) in zebrafish. Box plots denote the median, 25th–75th percentiles and minimum and maximum values. Differences were statistically analysed using a two-sided Welch’s t-test. Scale bars, 20 μm (main panels), 2 μm (inset panels). d, A model of degradation of lysosomes and nuclear DNA.

Source data

Extended Data Fig. 4 Plaat3-deficient mice show defects in lens organelle degradation.

a, Schematic of the Cas9-gRNA-targeted site in the mouse Plaat3 genomic locus. The targeted site is underlined. The protospacer-adjacent motif sequence is shown in red. A 16-bp deletion in the mutated allele is shown. The positions of PCR primers are shown. b, Genotyping of Plaat3-mutant mice by PCR. Data are representative of at least five independent experiments. Uncropped gels are provided in Supplementary Fig. 1. c, Haematoxylin and eosin staining of transverse sections of the lens of mice at 0.5 days (n = 3). Scale bar, 200 μm. d–f, h, Immunohistochemistry of the lens of mice at post-natal day 0.5 (d, e, h) (n = 3) and at 2 months old (f) (n = 3). The regions in which the nuclei are condensed (corresponding to the organelle-free zone (OFZ)) are indicated by broken white lines. Arrows indicate colocalized signals. Partially overlapping image tiles were combined to produce a single image. The percentage of LAMP1-positive puncta with galectin 3 in the observed area (approximately 10,000 μm2) was calculated. Data are mean ± s.e.m. (n = 3) (h). Scale bars, 100 μm (main panels), 5 μm (inset panels) (d–f); 100 μm (leftmost panels), 5 μm (all other panels), 1 μm (inset panels) (h). g, i, j, Electron micrographs of fibre cells in the peripheral and central regions of the lens (g, i) and red blood cells (j) of mice at post-natal day 0.5 (n = 2). The number of organelles (>50 nm in the major axis) was quantified in at least 10 randomly selected areas in g. Arrowheads indicate the nuclear envelope. N, nucleus. Schematic of nuclear degradation is shown on the right. Scale bars, 500 nm (g), 200 nm (i), 1 μm (left panels in j), 100 nm (right panels in j). k, l, Immunohistochemistry of the tail of mice at 2 months of age (n = 2), using the indicated antibodies and Hoechst 33342. The regions in which the nuclei were absent are indicated by broken white lines. Scale bar, 5 μm.

Source data

Extended Data Fig. 5 Plaat1 and PLAAT3 localize to various organelles during lens organelle degradation.

a, Schematic of the plaat1 genomic locus and the putative promoter region. The position of the first codon (ATG) is indicated. Live imaging of the lens of zebrafish injected with mito-mRFP mRNA and pT2AL200R150-plaat1(−793/+755):eGFP-Plaat1 was performed at 60 hpf (n = 5). Signal intensities of eGFP–Plaat1- and mito–mRFP-positive puncta (arrows, approximately 2 μm2) were quantified (after background signals were subtracted). Scale bars, 20 μm (leftmost panels), 2 μm (all other panels). b, c, Live imaging of the lens of eGFP–Plaat1 transgenic zebrafish (cryaa promoter) injected with LAMP1-mRFP mRNA (b) or ER-mRFP mRNA (c) at 51 hpf (n = 3). Scale bars, 20 μm (main panel), 2 μm (inset panels) (b), 5 μm (c). d, The catalytic cysteine in human PLAAT3(C113) and its corresponding residue (C130) in zebrafish Plaat1 are shown in red on amino acid sequence alignment of human PLAAT3 and zebrafish Plaat1 (ClustalW algorithm). e–i, Immunohistochemistry of endogenous PLAAT3 in the lens of wild-type mice at embryonic day 18.5 using the indicated antibodies and Hoechst 33342 (n = 3). The regions in which the nuclei are condensed (corresponding to the organelle-free zone) are indicated by broken white lines. Arrows indicate colocalized signals. Partially overlapping image tiles were combined to produce a single image. The percentages of the indicated organelles with PLAAT3 signals in the observed area (approximately 10,000 μm2) are shown for the three regions as indicated in the bottom panel (i): (1) periphery (approximately between 50 μm and 100 μm in depth from the lens surface), (2) boundary (approximately 50 μm in width outside of the organelle-free zone), and (3) organelle-free zone (approximately 50 μm in width from the boundary of the organelle-free zone). Data are mean ± s.e.m. (n = 3) (i). Scale bars, 50 μm (main panels), 2 μm (inset panels (e–g) or bottom panels (h)).

Source data

Extended Data Fig. 6 PLAAT3 localizes to damaged organelles.

a, b, HeLa cells stably expressing eGFP–mouse PLAAT3 were treated with LLOME (1 mM) for 1 h (a) or Q-VD-OPh (control, 20 μM) alone or with Q-VD-OPh (20 μM), actinomycin D (1 μM) and ABT-737 (10 μM) for 3 h (b), and stained with the indicated antibodies. Scale bars,10 μm (main panels), 1 μm (inset panels). c, d, Semi-intact U2OS cells were generated by depleting the cytosolic factors, incubated with recombinant eGFP–PLAAT3 in the presence or absence of 50 μM cBid (c) or 20 μM melittin (d) for 30 min at 30 °C, and stained with the indicated antibodies and DRAQ5 (e). Scale bars, 10 μm (main panels), 1 μm (inset panels). e, Semi-intact U2OS cells were incubated with recombinant eGFP–PLAAT3 or eGFP–PLAAT3(ΔC) in the presence or absence of 100 μM cBid or 20 μM melittin for 30 min at 30 °C. Scale bar, 10 μm. Data are representative of at least five (a), two (c, d) and one (e) independent experiments.

Extended Data Fig. 7 Mitochondria are partially damaged independently of Plaat1 and PLAAT3 in the lens.

a, b, Live imaging of wild-type zebrafish injected with zebrafish Lactb (N-terminal region)-eGFP mRNA and mito-mRFP mRNA (n = 3). Arrows and arrowheads indicate the rupturing mitochondria. Signal intensities of the Lactb–eGFP- and Mito–mRFP-positive puncta (arrows, approximately 2 μm2) were quantified (after background signals were subtracted). Scale bars, 20 μm (a, leftmost panels in b), 2 μm (all other panels in b). c, The percentage of the CIIICI-positive mitochondria with cytochrome c in the observed area (approximately 10,000 μm2) was calculated for the periphery (approximately between 50 μm and 100 μm in depth from the lens surface) and boundary of the organelle-free zone (approximately 50 μm in width outside of the organelle-free zone) of the lens of mice at post-natal day 0.5 (n = 3). Data are mean ± s.e.m. (n = 3), two-sided Welch’s t-tests. Scale bars, 100 μm (main panels), 2 μm (inset panels).

Source data

Extended Data Fig. 8 Hsf4 is required for lens organelle degradation in zebrafish.

a, Schematic of the Cas9-gRNA-targeted site in the zebrafish hsf4 genomic locus. The targeted site is underlined. The protospacer-adjacent motif sequence is shown in red. b, Genotyping of wild-type and F0 hsf4-knockout zebrafish by PCR. Uncropped gels are provided in Supplementary Fig. 1. c–f, Live imaging of histone H2B–eGFP (c), mito–mRFP (d), and ER–eGFP (e) in the lens of zebrafish. The equatorial diameter of the whole lens and the mitochondria- or endoplasmic-reticulum-free zones was quantified at 72 hpf (n = 11) (f). Box plots denote the median, 25th–75th percentiles and minimum and maximum values, two-sided Welch’s t-tests. Scale bar, 20 μm. g, Whole mount in situ hybridization of endogenous plaat1 in wild-type and hsf4-knockout zebrafish at 48 hpf using antisense and control sense RNA probes (n = 6). Scale bar, 1 mm.

Source data

Extended Data Fig. 9 Exogenous PLAAT3 induces rupture of peroxisomes in HeLa cells.

a, Immunofluorescence images of HeLa cells transiently expressing eGFP–PLAAT3. Cells were stained with anti-PMP70 (peroxisomes), COX IV (mitochondria), GM130 (cis-Golgi), calnexin (endoplasmic reticulum) and LAMP1 (lysosomes) antibodies and DAPI. Scale bar, 10 μm. b, Time-lapse images of HeLa cells expressing eGFP–PLAAT3, mRuby–PTS1 (peroxisomal luminal protein) and HaloTag7–PEX16 (peroxisomal membrane protein). Z-stack images were acquired (0.5 μm × 7 slices) and projection images are shown. Arrows indicate peroxisomes before and after disappearance of mRuby–PTS1 signals from peroxisomes. Scale bars, 10 μm (main panels), 1 μm (inset panels). Data are representative of at least two (a) and one (b) independent experiments.

Extended Data Fig. 10 Targeting of PLAAT3 to mitochondria induces membrane degradation in HeLa cells and in vitro.

a, Schematic of wild-type mouse PLAAT3 and mouse PLAAT3(ΔCT). The C-terminal positively charged residues and transmembrane domain are indicated by red and brown letters, respectively. b, Immunofluorescence images of HeLa cells expressing eGFP–PLAAT3(ΔCT) (lipase-active) or its C113S mutant (lipase-dead). Scale bars, 10 μm (main panels), 2 μm (inset panels). Data are representative of at least two independent experiments. c, Correlative light and electron microscopy images of HeLa cells expressing eGFP–PLAAT3(ΔCT) (lipase-active). Arrows indicate the edges of ruptured outer mitochondrial membranes. TEM, transmission electron microscopy. Scale bars, 2 μm (top panels), 500 nm (magnified panels) and 100 nm (in panels marked 1 and 2). d, Z-series images of array tomography of the mitochondria with herniated inner mitochondrial membranes (indicated by arrowheads) in HeLa cells expressing lipase-active eGFP–PLAAT3(ΔCT). SEM, scanning electron microscopy; m, mitochondria. Scale bar, 500 nm. e, f, Purified mitochondria (stained with MITO-ID) were incubated with MBP–eGFP–PLAAT3 or its mutants (n = 3) at 37 °C (e). The amount of free fatty acids in the supernatant was quantified (f) (n = 6 for wild-type PLAAT3 and PLAAT3(ΔTMD), and n = 3 for lipase-dead PLAAT3). Data are mean ± s.e.m. (f). Scale bars, 10 μm (main panels), 5 μm (inset panels) (e).

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Supplementary information

Supplementary Figure 1

Uncropped gel images. Red boxes indicate regions of blots used in Extended Data Fig. 2e, 4b, 8b.

Reporting Summary

Video 1

Time-lapse video of the lens of zebrafish expressing ER-eGFP (left, green) and mito-mRFP (middle, red) recorded from 54 to 62 h post-fertilization (hpf). Time interval, 5 min. Scale bar, 20 μm.

Video 2

Magnification of Supplementary Video 1. Scale bar, 5 μm.

Video 3

Time-lapse video of the lens of zebrafish expressing LAMP1-eGFP (left, green) and Dnase1l1l-mRFP (middle, red) recorded from 50 to 56 hpf. Time interval, 10 min. Scale bar, 20 μm.

Video 4

Magnification of Supplementary Video 3. Scale bar, 2 μm.

Video 5

Time-lapse video of the lens of zebrafish expressing eGFP-Plaat1 (left, green) and mito-mRFP (middle, red) recorded from 56 to 72 hpf. Time interval, 5 min. Scale bar, 20 μm.

Video 6

Magnification of Supplementary Video 5, from 61.5 to 64 hpf. Scale bar, 1 μm.

Video 7

Time-lapse video of the lens of zebrafish expressing Lactb-eGFP (left, green) and mito-mRFP (middle, red) recorded from 55 to 63 hpf. Time interval, 15 min. Scale bar, 20 μm.

Video 8

Magnification of Supplementary Video 7. Scale bar, 2 μm.

Video 9

Time-lapse video of HeLa cells expressing eGFP-PLAAT3 (green), mRuby-PTS1 (red), and HaloTag7-PEX16 (blue). Time interval, 5 min. Scale bar, 10 μm.

Video 10

A z-stack montage and 3D surface reconstruction of HeLa cells expressing lipase-active eGFP-PLAAT3ΔCT (shown in Extended Data Fig. 10c). Red, a herniated inner mitochondrial membrane; green, a swollen outer mitochondrial membrane.

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Morishita, H., Eguchi, T., Tsukamoto, S. et al. Organelle degradation in the lens by PLAAT phospholipases. Nature 592, 634–638 (2021). https://doi.org/10.1038/s41586-021-03439-w

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