Abstract
Lysosomes are degradation and signalling centres crucial for homeostasis, development and ageing1. To meet diverse cellular demands, lysosomes remodel their morphology and function through constant fusion and fission2,3. Little is known about the molecular basis of fission. Here we identify HPO-27, a conserved HEAT repeat protein, as a lysosome scission factor in Caenorhabditis elegans. Loss of HPO-27 impairs lysosome fission and leads to an excessive tubular network that ultimately collapses. HPO-27 and its human homologue MROH1 are recruited to lysosomes by RAB-7 and enriched at scission sites. Super-resolution imaging, negative-staining electron microscopy and in vitro reconstitution assays reveal that HPO-27 and MROH1 self-assemble to mediate the constriction and scission of lysosomal tubules in worms and mammalian cells, respectively, and assemble to sever supported membrane tubes in vitro. Loss of HPO-27 affects lysosomal morphology, integrity and degradation activity, which impairs animal development and longevity. Thus, HPO-27 and MROH1 act as self-assembling scission factors to maintain lysosomal homeostasis and function.
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Data availability
All data are available in the main text and supplementary materials. Structural prediction was performed using RoseTTAFold (https://robetta.bakerlab.org) and illustrations were prepared using the program PyMOL (https://pymol.org). Images were processed using ZEN Imaging software (v.2.3, Carl Zeiss), ImageJ (v.2.1.4.7, NIH), Volocity software (v.6.3, PerkinElmer), RELION (v.3.1.3, Sjors Scheres Laboratory, MRC Laboratory of Molecular Biology), WoRx (v.6.1.1, GE Healthcare) and Matlab (v.R2017b, MathWorks). Statistical analyses were performed using GraphPad Prism (v.8.2.1, Dotmatics). Full versions of all gels and blots are provided in Supplementary Fig. 1. Source data are provided with this paper.
Code availability
No custom code was generated or applied in this study.
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Acknowledgements
We thank S. Mitani (Tokyo Women’s Medical University) and staff at the Caenorhabditis Genetics Center for C. elegans strains used in this study; K. Zhang (Yunnan University) for Drechmeria coniospora fungi; Y. Li, X. Guo, M. Wang, X. Ren, X. Su, H. Wang (Institute of Biophysics, Chinese Academy of Sciences), X. Jia and S. Li (Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences), Y. Guo and X. Wu (Institutional Center for Shared Technologies and Facilities, Kunming Institute of Zoology, Chinese Academy of Sciences), Q. Yin and X. Wang (School of Life Sciences, Yunnan University) for technical assistance; and I. Hanson for editing services. This study was equally supported by grants to X.W. from the National Natural Science Foundation of China and National Key R&D Program of China (92254303, 2021YFA1300303, 32293202 and 32130028); and grants to W.F. from the National Natural Science Foundation of China (32071191) and Strategic Priority Research Program of CAS (XDB37020302).
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X.W. supervised the study. L.L. performed most of the experiments. X.L., Y.W. and S.H. performed genetic and cell biology experiments. W.F. supervised, and S.Y. and W.W. performed, protein characterization and negative-staining EM experiments. C.Y. supervised, and M.L. performed, TEM analysis. D.L. supervised, and A.J. and X.M. performed, GI-SIM experiments. Junjie Hu analysed structure prediction data and supervised in vitro reconstitution experiments with X.W. J.Z. and Junyan Hu generated COS7 KO and stable expression cell lines and contributed to the COS7 cell experiments. Q. Zhang, Q. Zhao and Y.L. contributed to the experiments and materials. L.L., X.L., W.F. and X.W. prepared the manuscript with discussion among all authors.
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Extended data figures and tables
Extended Data Fig. 1 hpo-27 mutants contain extensive lysosomal tubules.
a-r, Confocal fluorescence images of the hypodermis in day 2 adult worms carrying different hpo-27 mutant alleles (a-p), or treated with hpo-27 RNAi (q, r). The worms were expressing NUC-1::CHERRY and AJM-1::GFP to label lysosomes and lateral seam cells, respectively. hpo-27 mutants contain extensive lysosomal tubules and greatly diminished NUC-1::CHERRY fluorescence in hyp7 cells. The percentage of worms with the representative lysosomal pattern is shown at the lower left corner in each panel. s, qPCR analysis of hpo-27 gene expression in control RNAi and hpo-27 RNAi worms. Three independent experiments were performed, and data are shown as mean ± SD. Student’s two-tailed unpaired t-test was performed to compare hpo-27 RNAi with control RNAi. P values are indicated. t-v”, Confocal fluorescence images of the hypodermis in wild type (WT) (t-t”) and hpo-27(tm5336) mutant adults (u-v”) carrying NUC-1::CHERRY and Psemo-1GFP (n = 15 worms in each strain). Lysosomes labeled by NUC-1::CHERRY are seen in both hyp7 cells positive for GFP and lateral seam cells that lack GFP expression (arrows). In hpo-27 mutants, extensive lysosomal tubules are present in hyp7 and seam cells, and NUC-1::CHERRY fluorescence is diminished in hyp7 but not seam cells. w, Cloning of hpo-27. The hpo-27 gene structure is shown with filled boxes representing exons, thin lines indicating introns, and gray bars designating 3’ and 5’ UTRs. The arrow delineates the direction of transcription. x, Schematic diagram showing the organization of the HPO-27 protein. The mutation sites identified in all the hpo-27 alleles and the region deleted in the tm5336 allele are indicated. Gray boxes indicate the HEAT motifs. Scale bars: 5 µm.
Extended Data Fig. 2 HPO-27 is homologous to human MROH1.
a, Sequence alignment of C. elegans (c.e) HPO-27 and human (h.s) MROH1. Identical residues are shaded in black. The red box indicates the disordered region and blue boxes designate the HEAT motifs. b, The percentage of worms with diminished NUC-1::CHERRY fluorescence was quantified in the indicated strains at adult day 3 (n = 50 worms in each of 3 biological repeats for each strain). Data are shown as mean ± SD. One-way ANOVA with Tukey’s multiple comparison test was performed to compare mutant datasets with WT or datasets that are linked by lines. P values are indicated.
Extended Data Fig. 3 Loss of HPO-27 affects lysosome morphology in intestine and body wall muscle cells.
a-d, Confocal fluorescence images of the intestine and body wall muscle cells in WT and hpo-27(tm5336) adult worms expressing LAAT-1::GFP. Arrowheads indicate tubular lysosomes. Quantifications are shown in e, f, h (n = 14 worms per strain) and g (n = 15 worms per strain). Data are shown as mean ± SD. Two-way ANOVA with Bonferroni post-test (e, f, h) or one-way ANOVA with Tukey’s multiple comparison test (g) was performed to compare mutant datasets with WT. P values are indicated. i-q, Confocal fluorescence images of the hypodermis in WT and hpo-27(tm5336) worms at adult day 2 expressing AJM-1::GFP (which stains the seam cell boundary) and RNST-2::CHERRY (i-k) or SCAV-3::tagRFPt (l-n), or carrying Pcol-12LMP-1::RFP which is expressed in hyp7 but not seam cells (o-q). The percentage of worms with the representative lysosomal pattern is shown at the lower left corner in each panel. In (q), the DIC image is merged to show a hyp7 cell with invisible LMP-1::RFP. r-v’, Confocal fluorescence time-lapse images of the hypodermis in adult hpo-27(tm5336) worms co-expressing RNST-2::CHERRY and SCAV-3::GFP. The lysosomal tubular networks are clearly observed at “0 min”, but they gradually collapse. White arrowheads indicate the lysosomal tubules that gradually disappear. The lysosomal membrane protein SCAV-3::GFP labels tubular structures, which collapse to form small vesicles (yellow arrowhead) and eventually became diffuse. Scale bars: 5 µm.
Extended Data Fig. 4 Loss of HPO-27 affects lysosomal integrity and causes abnormalities of the ER and mitochondria.
a-i, Transmission electron micrographs of the hypodermis in WT and hpo-27 RNAi worms expressing SCAV-3::APEX. Both “membrane whorl”-containing compartments and tubules are stained by diaminobenzidine (DAB) (arrowheads). j-n, Transmission electron micrographs of the hypodermis in hpo-27(tm5336) at adult day 3. Few lysosomes (green arrowhead) are observed, and they are surrounded by ribosome-containing ER membranes (yellow arrowheads in k-n). Red arrowheads indicate membranes of autophagosomes (AP) enclosing a mitochondrion or an ER microsome (yellow asterisks). The pink arrowhead indicates a piece of broken ER. “M” indicates a fragmented and dilated mitochondrion. Quantification of the area of hypodermal lysosomes is shown in o (left to right: n = 4, 3, 4, 6 worms). Data are shown as mean ± SEM. Student’s two-tailed unpaired t-test was performed to compare mutant datasets with WT. P values are indicated. p-aa, Super-resolution images of the hypodermis in the indicated strains expressing the ER marker GFP::TRAM-1 (p-u) or the mitochondrial marker Mito::GFP (v-aa) at adult day 1 and day 3. Arrowheads indicate abnormal ring-like ER structures. The percentage of worms with the representative ER or mitochondrial pattern is shown at the lower left corner in each panel. ab-ae, Transmission electron micrographs of the hypodermis in WT and hpo-27(tm5336) at adult day 1 and day 3. Pink arrowheads indicate normal ER and yellow arrowheads indicate microsomes probably generated by structural damage of the ER. “M” indicates a mitochondrion, and yellow asterisks designate abnormal mitochondria. af, ag, Confocal fluorescence images of the hypodermis in WT expressing HPO-27::mKate2 and Mito::GFP or GFP::TRAM-1. The boxed region is magnified in the zoom images. Magenta and white arrowheads indicate HPO-27::mKate2 puncta and mitochondrial or ER structures, respectively. Quantification is shown in ah (n = 10 worms per strain). Data are shown as mean ± SD. Scale bars represent 0.5 µm in (a-n, ab-ae) and 5 µm in (p-aa, af, ag).
Extended Data Fig. 5 Loss of hpo-27 affects lysosomal degradation activity.
a, Relative activity of cathepsin B and L in lysosomes purified from WT and hpo-27(tm5336) mutants. The amount of lysosomes in each strain was determined by western blot with an anti-ASP-4 antibody. b, Western blot analysis of NUC-1::CHERRY cleavage in WT and hpo-27(tm5336) at day 1. Quantification is shown in c. Four (a) and five (c) independent experiments. d-r, Confocal fluorescence images of WT, epg-5(tm3425), and hpo-27(tm5336) animals expressing GFP::LGG-1. Comma and 4-fold are embryonic stages; day 1 and day 3 are adult stages. LGG-1 puncta are indicated by white arrowheads. Quantifications are shown in s (n = 14 worms per strain). t-w, DIC images of the embryo (t, v) and germline (u, w) in WT and hpo-27(tm5336) mutants. Cell corpses are indicated by arrowheads. Quantifications are shown in x (n = 10 worms per strain), y (n = 15 worms per strain). Comma, 1.5 F, 2 F, 2.5 F, 3 F and 4 F (4-fold) are different embryonic stages. L4 is larval stage 4. In (a, c, s, x, y), data are shown as mean ± SD. Two-way ANOVA with Bonferroni post-test (s, x) or Student’s two-tailed unpaired t-test (a, b, y) was performed to compare mutant datasets with WT. P values are indicated. Scale bars: 5 µm. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 6 dyn-1 mutants exhibit a weak lysosomal tubule phenotype.
a-b’, DIC and confocal fluorescence images of the gonads in WT (a, a’) and hpo-27(tm5336) (b, b’) stained by Lysosensor green. White and yellow arrowheads indicate cell corpses positive and negative for Lysosensor green, respectively. Quantification is shown in c (n = 15 worms per strain). d, Confocal time-lapse images of WT and hpo-27(tm5336) embryos expressing HIS-24::GFP and GFP::RAB-7. Arrowheads indicate phagolysosomes positive for GFP::RAB-7. The time point just prior to the appearance of GFP::RAB-7 on phagosomes was set as “0 min”. Quantification is shown in e, with average duration (±SD) shown in parentheses. 40 cell corpses were analyzed in each strain. f-i and q-w, Confocal fluorescence images of the adult hypodermis in the indicated strains expressing NUC-1::CHERRY without or with expression of DYN-1(WT)::GFP or DYN-1(T67A)::GFP. Arrowheads indicate tubular lysosomes. Quantifications are shown in j (left to right: n = 20, 13, 13, 15, 13 worms), k (left to right: n = 12, 10, 15, 10 worms), x (left to right: n = 18, 17, 21 worms), y (left to right: n = 18, 16, 17, 15 worms). l-m”, 3D reconstruction images of lysosomes in WT and dyn-1(ky51) adults co-expressing HPO-27::GFP and NUC-1::CHERRY. Arrowheads indicate association of HPO-27 with lysosomes. Quantification is shown in n (n = 15 worms per strain). o, Confocal fluorescence image of the hypodermis in a WT adult co-expressing DYN-1::GFP and NUC-1::CHERRY. The boxed region is magnified in the zoom image. White and magenta arrowheads indicate a DYN-1 punctum and a lysosome, respectively. Quantification is shown in p (n = 10 per test). NUC-1::CHERRY and DYN-1::GFP puncta were analyzed separately to yield Manders overlap coefficient M1 (DYN-1/NUC-1) and M2 (NUC-1/DYN-1), respectively. The experiments were performed at 25 °C in (f-m”), which is the non-permissive temperature of the dyn-1(ky51) allele. In c, j, k, n, p, x, y, data are shown as mean ± SD. One-way ANOVA with Tukey’s multiple comparison test (k, x, y) or Student’s two-tailed unpaired t-test (c, e, j, n) was performed to compare mutant datasets with WT (c, e, j, k, n), datasets without and with DYN-1(WT) or DYN-1(T67A) expression (x), all other datasets with hpo-27 RNAi (y) or datasets that are linked by lines. P values are indicated. Scale bars: 2.5 µm in (d), 5 µm in (a-b’, f-i, l-m”, o, q-w).
Extended Data Fig. 7 HPO-27 is widely expressed and associates with lysosomes.
a-i’, DIC and confocal fluorescence images of WT animals at different developmental stages carrying the HPO-27::mKate2 knock-in reporter. The muscle cell, vulva, and spermatheca are indicated by green asterisks. j-j”, Confocal fluorescence images of the hypodermis in WT expressing HPO-27::mKate2 and Psemo-1GFP. HPO-27::mKate2 puncta are present in the hyp7 cell (GFP-positive; white arrowheads) and in the lateral seam cell (GFP-negative; yellow arrowhead). k-l”, 3D reconstruction images of lysosomes in the hypodermis of a WT adult expressing both HPO-27::mKate2 and SCAV-3::GFP (k-k”’) or both MROH1::GFP and NUC-1::CHERRY (l-l”). The boxed region is magnified in k”’. Arrowheads indicate association of HPO-27 or MROH1 with lysosomes. m, n, Confocal fluorescence images of the hypodermis in WT adults co-expressing NUC-1::CHERRY and HPO-27(ΔN)::GFP or HPO-27(ΔC)::GFP. Both HPO-27 truncations fail to associate with lysosomes. In a-n, 15 animals were examined in each strain. Scale bars: 5 µm.
Extended Data Fig. 8 Loss of MROH1 affects acidity and proteolytic activity of lysosomes.
a-a”, Super-resolution images of a WT COS7 cell stably expressing GFP-MROH1 and containing chased fluorescent Dextran A555. The boxed region is magnified in the zoom images. Arrows and arrowheads indicate association of MROH1 with vesicular and tubular lysosomes labeled by Dextran. b, CRISPR-mediated generation of the MROH1 knockout COS7 cell line, and verification by sequencing. The knockout clone contains a 28-bp deletion, which causes a frameshift and results in a premature stop codon. c, qPCR analysis of MROH1 expression in the indicated cells. Three independent experiments were performed. Data are shown as mean±SD. d-o, Confocal fluorescence images of WT (d-f), MROH1 KO (g-i), siControl (j-l), and siMROH1 (m-o) COS7 cells stained by Lysotracker Red (d, g, j, m), Magic Red (e, h, k, n), or DQ-BSA (f, i, l, o). Cell boundaries are outlined by dashed circles. Quantifications are shown in p (n = 20 cells in each of 4 biological repeats), q (n = 20 cells in each of 3 biological repeats). Data are shown as mean±SD. r-s”’, Confocal fluorescence images of Lysotracker Blue-stained COS7 cells co-expressing GFP-MROH1 and CHERRY-RAB7A(WT) (r-r”’) or CHERRY-RAB7A(T22N) (s-s”’). Arrowheads indicate enrichment of MROH1 on RAB7-positive vesicles. In c, p, q, Student’s two-tailed unpaired t-test was performed to compare MROH1 mutant or siRNA datasets with WT or siControl. P values are indicated. Scale bars: represent 5 μm.
Extended Data Fig. 9 HPO-27 and MROH1 interact with RAB-7 and self-interact in a “head-to-tail” mode.
a, Schematic illustration of the tripartite split-GFP assay. b-g’, DIC and confocal fluorescence images of the hypodermis in WT adults co-expressing GFP1-9, NUC-1::CHERRY, WT or mutant forms of GFP10::RAB-7, and HPO27::GFP11 (b-d’) or MROH1::GFP11 (e-g’). Arrowheads indicate co-localization of reconstituted GFP and lysosomes. h-s’ DIC and confocal fluorescence images of the hypodermis in WT adults co-expressing GFP1-9, HPO-27::mKate2 (h-k’, p-s’) or NUC-1::CHERRY (l-o’), and the indicated HPO-27 (h-k’), MROH1 (l-o’), HPO-27(ΔN) (p-q’), or HPO-27(ΔC) (r-s’) fusion proteins. Arrowheads indicate co-localization of reconstituted GFP with HPO-27::mKate2 or NUC-1::CHERRY. In b-s’, 15 animals were examined in each strain. Scale bars: 5 µm.
Extended Data Fig. 10 HPO-27 tends to oligomerize.
a, Coomassie blue staining of indicated purified recombinant proteins. Data are representative of six independent experiments. b-e, Confocal fluorescence time-lapse images of supported membrane tubes (SMrTs) labeled by Texas red DHPE without (b) or with recombinant EGFP (c), HPO-27-EGFP (d) or MROH1-EGFP (e) proteins. “-PA” indicates absence of phosphatidic acid (PA) in Texas red-labeled SMrTs. Three independent experiments were performed. f-h, Tertiary structures of HPO-27 and human MROH1 predicated by RoseTTAFold. Comparison and illustrations were prepared using the program PyMOL (https://pymol.org). Both proteins contain 37 HEAT motifs, each of which is composed of two α-helices (A- and B- helices) colored in green and brown, respectively. The first (1st) and the last (37th) HEAT motifs are removed in HPO-27(ΔN) and HPO-27(ΔC), respectively. Superimposition of the predicated tertiary structures of HPO-27 (green) and MROH1 (brown) is shown in (g). Negatively charged (red) and positively charged (blue) areas on the surface of an HPO-27 molecule are shown in a top (upper) or bottom (lower) view in (h). i, Negative-staining EM showing monomeric and oligomeric states of HPO-27. Corresponding cartoon illustrations are shown underneath. j-r, Photo-bleaching and single-molecule imaging assay of EGFP (j-l), HPO-27-EGFP (m-o) and HPO-27(ΔC)-EGFP (p-r). TIRF images are shown in (j, m, p). Representative traces show one-step photobleaching of EGFP and HPO-27(ΔC)-EGFP, and two-step photobleaching of HPO-27-EGFP (arrows; k, n, q). A summary of the number of photobleaching steps is shown in (l, o, r; n = 200 foci in each of 3 biological repeats). Data are shown as mean ± SD. Scale bars represent 5 µm in (b-e, j, m, p) and 20 nm in (i). s, Proposed model of HPO-27 function in lysosomal fission. For gel source data, see Supplementary Fig. 1.
Supplementary information
Supplementary Fig. 1
Raw images of western blots, lipid dot-blots and SDS–PAGE data shown in Figs. 4 and 5 and Extended Data Figs. 5 and 10. The boxed region was cropped for the final figure. In Extended Data Fig. 5b, tubulin was run on the same gel as a sample processing control. The blot was stripped and rehybridized using anti-tubulin antibodies.
Supplementary Data
C. elegans strains (Data 1), expression vectors (Data 2) and primers (Data 3) used in this study.
Supplementary Video 1
Tubular lysosomal networks collapse in hpo-27 mutants.
Supplementary Video 2
Dynamic changes of lysosomes in the hypodermis.
Supplementary Video 3
HPO-27 mediates lysosome fission.
Supplementary Video 4
HPO-27 mediates lysosome fission.
Supplementary Video 5
MROH1 mediates lysosome fission.
Supplementary Video 6
MROH1 mediates lysosome fission.
Supplementary Video 7
HPO-27 assembles on and severs SMrTs.
Supplementary Video 8
MROH1 assembles on and severs SMrTs.
Source data
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Li, L., Liu, X., Yang, S. et al. The HEAT repeat protein HPO-27 is a lysosome fission factor. Nature 628, 630–638 (2024). https://doi.org/10.1038/s41586-024-07249-8
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DOI: https://doi.org/10.1038/s41586-024-07249-8
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