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A quantitative ultrastructural timeline of nuclear autophagy reveals a role for dynamin-like protein 1 at the nuclear envelope

Nature Cell Biology volume 27, pages 464–476 (2025)Cite this article

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Abstract

Autophagic mechanisms that maintain nuclear envelope homoeostasis are bulwarks to ageing and disease. Here we define a quantitative and ultrastructural timeline of nuclear macroautophagy (nucleophagy) in yeast by leveraging four-dimensional lattice light sheet microscopy and correlative light and electron tomography. Nucleophagy begins with a rapid accumulation of the selective autophagy receptor Atg39 at the nuclear envelope and finishes in ~300 s with Atg39-cargo delivery to the vacuole. Although there are several routes to the vacuole, at least one pathway incorporates two consecutive membrane fission steps: inner nuclear membrane (INM) fission to generate an INM-derived vesicle in the perinuclear space and outer nuclear membrane fission to liberate a double-membraned vesicle to the cytosol. Outer nuclear membrane fission occurs independently of phagophore engagement and instead relies surprisingly on dynamin-like protein 1 (Dnm1), which is recruited to sites of Atg39 accumulation by Atg11. Loss of Dnm1 compromises nucleophagic flux by stalling nucleophagy after INM fission. Our findings reveal how nuclear and INM cargo are removed from an intact nucleus without compromising its integrity, achieved in part by a non-canonical role for Dnm1 in nuclear envelope remodelling.

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Fig. 1: Dnm1 is required for nucleophagic flux of an Atg39 reporter.
Fig. 2: Dnm1 is recruited to Atg39 during nucleophagy.
Fig. 3: Dnm1 impacts Atg39 dynamics, copy number and robust delivery to vacuole.
Fig. 4: Nucleophagy proceeds through a lumenal vesicle intermediate.
Fig. 5: Dnm1 is required for ONM fission.

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Data availability

All other data supporting the findings presented in this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

The MATLAB and Jupyter Notebook Python scripts are available with no access restrictions via GitHub at https://github.com/LusKingLab/.

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Acknowledgements

We thank the Center for Cellular and Molecular Imaging for assistance with electron microscopy, particularly M. Graham, Z. Zuo and X. Liu. We are grateful to C. Kraft, M. Graef and L. Lackner for yeast strains. We thank K. Li for technical support. This work was funded by the following grants from National Institutes of Health: R01 AG090465, R56 AG071201 and R21 AG058033 to C.P.L. and T.J.M.; R01 GM105672 to C.P.L.; F32 GM139285 to N.R.A.; and F31 AG069490 to P.J.M.

Author information

Authors and Affiliations

  1. Department of Cell Biology, Yale School of Medicine, New Haven, CT, USA

    Philip J. Mannino, Andrew Perun, Ivan V. Surovtsev, Nicholas R. Ader, Lin Shao, Elisa C. Rodriguez, Thomas J. Melia, Megan C. King & C. Patrick Lusk

  2. Department of Physics, Yale University, New Haven, CT, USA

    Ivan V. Surovtsev

  3. Department of Molecular Cellular and Developmental Biology, Yale University, New Haven, CT, USA

    Megan C. King

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  1. Philip J. Mannino
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Contributions

Conceptualization: P.J.M. and C.P.L. Methodology: P.J.M., A.P., I.V.S., N.R.A., L.S. and E.C.R. Investigation: P.J.M., A.P., I.V.S. and N.R.A. Validation: P.J.M. and A.P. Data curation: P.J.M., A.P., I.V.S. and N.R.A. Formal analysis: P.J.M., A.P., I.V.S. and N.R.A. Funding acquisition: P.J.M., T.J.M. and C.P.L. Visualization: P.J.M. and A.P. Project administration: P.J.M., M.C.K. and C.P.L. Resources: L.S. and C.P.L. Software: A.P., I.V.S. and L.S. Supervision: T.J.M., M.C.K. and C.P.L. Writing—original draft: P.J.M. and C.P.L. Writing—review and editing: all authors.

Corresponding author

Correspondence to C. Patrick Lusk.

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Extended data

Extended Data Fig. 1 Models of nucleophagy and characterization of Atg39 expression levels.

a, b) Schematic of two models of nucleophagy incorporating either sequential (Model 1) or simultaneous (Model 2) INM and ONM fission events. Model 1 would also lead to the formation of a lumenal vesicle/INMDV intermediate. ONM, outer nuclear membrane; INM, inner nuclear membrane; PNS, perinuclear space; INMDV, inner nuclear membrane-derived vesicle; NDV, nucleus-derived vesicle. c) RT-qPCR on indicated autophagy gene transcripts was performed in WT cells either treated with rapamycin for 2 h or cultured in medium lacking nitrogen for 2 h. Results displayed as a bar graph comparing the mean fold change of the transcripts between the rapamycin and -Nitrogen condition. Mean and standard deviation (error bars) of three biological replicates (black dots). Unpaired two-sided t tests.**** is P < 0.0001; ** is P = 0.0019; ns, not significant (P > 0.05). d) Protein levels of Atg39-3xHA fusions derived from transcripts including either the ADH1 3′UTR or the endogenous ATG39 3′UTR were assessed by western blot with anti-HA antibody directly coupled to HRP (α-HA-HRP) and ECL. Positions of molecular weight (MW) standards (kD) at left. At bottom, membranes were stained with Ponceau to evaluate total protein loads. e) Fluorescence micrographs of Atg39-GFP produced from transcripts with 3′UTRs from the ADH1 or ATG39 genes after 4 h in medium lacking nitrogen. Scale bar is 5 µm. Data are from one experiment. f) Western blot of whole protein extracts from cells expressing Atg39-pHn-mCh with fall out fragment (‘pHn) in the indicated genetic backgrounds and PMSF treatment after 24 h in sd-N. Position ‘pHn indicated on the right. An anti-GFP antibody and anti-mouse-HRP secondary followed by ECL was used to detect proteins. Positions of molecular weight (MW) standards (kD) at left. * is a non-specific band. # is the expected position of Atg39-pHn-mCh, which is undetectable in most backgrounds due to its low abundance and lack of sensitivity of the anti-GFP antibody. Numerical source data and unprocessed blots are provided.

Source data

Extended Data Fig. 2 Dnm1 colocalizes with Atg39 in the absence of Fis1.

a–c) Inverted fluorescence micrographs of a timelapse series of fis1Δ cells expressing Atg39-GFP and Dnm1-mCh with and without either an extra copy of DNM1 or the dominant-negative dnm1-K41A allele under conditions of nitrogen starvation. Green and red channels shown with merge at indicated times (T) with T = 0 s being the first detection of a Atg39-GFP focus (arrow). Arrowheads point to colocalization between Atg39-GFP and Dnm1-mCh. Numbers in merge are the z-position (in μm) of the image shown in reference to the midplane. Scale bars are 5 µm. Data shown are representative of three independent experiments.

Extended Data Fig. 3 Atg11 recruits Dnm1 to the NE.

a) Fluorescence micrographs of fis1Δ cells and fis1Δatg11Δ cells expressing Atg39-pHn, Dnm1-mCh with and without an extra copy of DNM1 or the dnm1-K41A allele under conditions of nitrogen starvation. Green and red channels shown with merge. Scale bar is 5 µm. Data shown are representative of three independent experiments. b) Plot of the mean percentage of Atg39-pHn foci that colocalize with Dnm1-mCh from experiments in A. Mean and standard deviation (error bars) from three biological replicates (black dots). For fis1Δ cells, n = 79 Atg39-pHn foci. For fis1Δ cells expressing DNM1, n = 104 Atg39-pHn foci. For fis1Δ cells expressing dnm-K41A, n = 166 Atg39-pHn foci. For fis1Δatg11Δ cells, n = 78 Atg39-pHn foci. For fis1Δatg11Δ cells expressing DNM1, n = 96 Atg39-pHn foci. For fis1Δatg11Δ cells expressing dnm1-K41A, n = 165 Atg39-pHn foci. Unpaired two-sided t test. *** is P = 0.0003. ns, not significant (P > 0.05). c) Schematic of BiFC. VN and VC are the N- and C- terminal fragments of Venus, respectively. An interaction between Dnm1-VN and VC-Atg11 leads to the reconstitution of Venus and its fluorescence. d) Fluorescence micrographs of cells expressing Atg39-ymTurquoise2 (Atg39-ymTq2), VC-Atg11, and Dnm1-VN under conditions of nitrogen starvation. Yellow (magenta) and blue (green) channels shown with merge. The arrow points out colocalization between the Venus and Atg39-ymTq2 foci. Scale bar is 5 µm. Data shown are representative of three independent experiments. e) Plot of the mean percentage of Atg39-ymTq2 foci that colocalize with Venus foci from experiments in d. Mean and standard deviation (error bars) from three biological replicates (black dots). n = 84 Atg39-ymTq2 foci. Numerical source data are provided.

Source data

Extended Data Fig. 4 Atg8 recruitment, copy number, and Atg39 dynamics during nucleophagy.

a) Fluorescence micrographs of cells expressing Atg39-GFP and 2xmCh-Atg8 imaged every 10 seconds under conditions of nitrogen starvation. Green and red channels shown with merge. Numbers in merge are z-position (in μm) of the displayed image in reference to the midplane. T (s) indicates the time since the first detection of the Atg39-GFP focus (indicated above, arrowhead). Arrows point to colocalization between Atg39-GFP and 2xmCh-Atg8. * indicates fluorescence from the vacuole in the red channel. Scale bar is 3 µm. Data shown are representative of three independent experiments. b) Histogram of the number of colocalization instances at the indicated time after the appearance of an Atg39-GFP focus. Data are from the experiments in A; n = 73 cells, 74 tracks, three biological replicates. c) To calculate copy number of Atg39-GFP within individual foci, we imaged budding yeast expressing Atg39-GFP next to S. pombe expressing Fta3-GFP, a kinetochore protein which has 111 molecules/focus in anaphase B61. By directly relating the fluorescence intensities, we calculated Atg39-GFP copy number/focus as presented in Fig. 3d. Scale bar is 5 µm for the large image and 500 nm for the insets. Data shown are representative of three independent experiments. d) Inverted fluorescence micrographs of WT cells expressing Atg39-GFP and Vph1-mCh imaged every two seconds under conditions of nitrogen starvation. Green and red channels shown with merge. Numbers in merge are z-position (in μm) of the displayed image in reference to the midplane. T (s) indicates the time in seconds since the first detection of the Atg39-GFP focus (green arrowhead). Another less intense focus of Atg39-GFP is visible merging with the initial particle (pink arrowhead). Scale bar is 3 µm. Data is from one experiment. Numerical source data are provided.

Source data

Extended Data Fig. 5 Atg11 dependence during nucleophagy.

a) Fluorescence micrographs of a time series of atg11Δ cells expressing Atg39-GFP under conditions of nitrogen starvation. Numbers in merge are z-position (in μm) of the displayed image in reference to the midplane. T (s) indicates the time in seconds since the first detection of the Atg39-GFP focus (indicated above, arrowhead). Cells shown are representative examples of two classifications of the Atg39-GFP foci dynamics that either disappear prior to internalization in the vacuole (aborted) or don’t (not aborted). Scale bar is 3 µm. Data shown are representative of three independent experiments. b) Plot of the mean number of focal accumulations divided by the total number of cells divided by the total time of the experiment (h) for the indicated genotype. Data are from three biological replicates (black dots) where n = 294, n = 557 and n = 522 cells were assessed for WT, dnm1Δ and atg11Δ, respectively. Ordinary one-way ANOVA with multiple comparisons. ** is P = 0.0032; ns, not significant (P > 0.05). c) Fluorescence intensity of Atg39-GFP foci in atg11Δ cells plotted versus time after their first detection. Data are split into two groups, aborted (red, squares) and not aborted (blue, circles) from three biological replicates. The ‘aborted’ group comprised n = 40 cells, 42 tracks and the ‘not aborted’ group, n = 37 cells, 38 tracks. Numerical source data are provided.

Source data

Extended Data Fig. 6 Characteristics of Atg39-GFP mobility and cargo capture.

a) Fluorescence micrographs of WT cells expressing Atg39-GFP and a model INM cargo comprising the INM targeting and transmembrane domain of Heh1 fused to two tandem mCh fluorophores (H1-2xmCh) under conditions of nitrogen starvation. Green and red channels shown with merge. Scale bar is 3 µm. b) Inverted fluorescence micrographs of a time series of WT cells expressing Atg39-GFP and H1-2xmCh under conditions of nitrogen starvation. Time (T) 0 is the first detected point of Atg39-GFP accumulation. Green and red channels shown with merge. Arrowheads point to colocalization between Atg39-GFP and H1-2xmCh. Cell boundaries are outlined. Numbers in merge are z-position (in μm) of the image in reference to the midplane. Scale bar is 3 µm. Data shown are representative of two independent experiments. c) Scatter plot of the elapsed time from the first detection of an Atg39-GFP focus and the first instance of colocalization with H1-2xmCh from experiments as in B. The median is the black bar. Data are from n = 36 cells in which Atg39-GFP and H1-2xmCh were colocalized outside of the nucleus out of n = 42 total cells from two biological replicates. d) Bar graph of the percentage of Atg39-GFP foci tracked from first appearance that do not enter the vacuole over a 20-minute timelapse experiment as noted in Fig. 3f for the indicated genotype. Mean and standard deviation (error bars) from 5 (WT cells) or 4 (dnm1∆ cells) biological replicates (black dots). n = 114 tracks, 99 cells for WT cells and n = 95 tracks, 82 cells for dnm1∆ cells. Unpaired two-sided t test. *** is P = 0.0008. e) Plot of the percentage of time that Atg39-GFP foci spend in each phase as per color code in Fig. 3e and g in WT and dnm1Δ cells. The latter are also segregated based on those that enter the vacuole (unperturbed) and those that don’t (stalled). Numerical source data are provided.

Source data

Extended Data Fig. 7 Ultrastructure of sites of Atg39 accumulation during nucleophagy.

a–e) CLEM/tomography of sites of Atg39-hfYFP foci. From left to right: fluorescence image of WT cells expressing Atg39-hfYFP (green), Vph1-mCh and Nup170-mCh (magenta). Scale bar is 3 µm. EM tomogram acquired of boxed region in fluorescence image and tomogram virtual slice without and with annotation shown alongside a snapshot of a 3D model. Arrows point to INMDVs. ONM is green, INM is light blue, phagophore/autophagosome is orange, vacuole membrane is magenta. Scale bar is 200 nm. C, cytosol; N, nucleus; V, vacuole; ONM, outer nuclear membrane; INM, inner nuclear membrane; ER, endoplasmic reticulum; INMDV, inner nuclear membrane derived vesicle; AM, autophagic membrane. Numbers at right refer to classification scheme in Fig. 4f. Data are from one experiment.

Extended Data Fig. 8 Ultrastructure of sites of Atg39 accumulation in dnm1Δ cells.

a–e) CLEM/tomography of sites of Atg39-hfYFP foci in dnm1Δ cells. From left to right: fluorescence image of WT cells expressing Atg39-hfYFP (green), Vph1-mCh and Nup170-mCh (magenta). Scale bar is 3 µm. EM tomogram acquired of boxed region in fluorescence image and tomogram virtual slice without and with annotation shown alongside a snapshot of a 3D model. Arrows point to INMDVs. ONM is green, INM is light blue, phagophore/autophagosome is orange, vacuole membrane is magenta. Scale bar is 200 nm. C, cytosol; N, nucleus; V, vacuole; ONM, outer nuclear membrane; INM, inner nuclear membrane; ER, endoplasmic reticulum; INMDV, inner nuclear membrane derived vesicle; AM, autophagic membrane; LD, lipid droplet. Numbers at right refer to classification scheme in Fig. 4f. Data are from one experiment.

Extended Data Fig. 9 Proposed model of nucleophagy.

Illustrative model of the molecular and morphological steps of nucleophagy. Atg39 locally accumulates adjacent to the NVJ in a mechanism supported by Atg11 (blue cylinders). It is likely that this clustering coincides with cargo capture and the evagination and fission of the INM to generate an INMDV. Initial small (50-100 nm INMDVs) may grow by fusion to generate larger (200–300 nm) INMDVs that are ultimately released from the NE through a Dnm1-dependent ONM fission step, or from the cortical ER through a proposed Scs2-dependent mechanism. Engagement with the phagophore occurs at or just after ONM fission. Autophagosomes containing double membrane nuclear derived vesicles (NDVs) fuse with vacuoles. Some INMDVs may also be capable of transiting through the broader ER but their fate remains uncertain.

Supplementary information

Reporting Summary

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Supplementary Table 1

Lists of yeast strains, plasmids and primers used in this study.

Supplementary Video 1

A 5-minute time series of cells expressing Atg39–GFP and Vph1–mCh imaged every 10 s by LLSM. Vph1–mCh signal is rendered as a surface (magenta) and Atg39–GFP foci are rendered as balls (green). Scale bar, 10 µm. Frame rate is 24 frames per second.

Supplementary Video 2

Electron tomogram of a cell expressing Atg39–hfYFP, Nup170–mCh and Vph1–mCh obtained at a site of Atg39–hfYFP accumulation shown in Fig. 4d. Note that the outer membrane of the autophagosome and the vacuole extend towards each other but do not touch. However, content with similar electron density of the vacuole lumen is observed between the inner and outer membrane of the autophagosome suggesting prior fusion with the vacuole. NE, green; INMDV, blue; autophagic membranes, orange; vacuole, magenta. Scale bar, 100 nm.

Supplementary Video 3

Electron tomogram of a cell expressing Atg39–hfYFP, Nup170–mCh and Vph1–mCh in a dnm1Δ cell obtained at a region of Atg39–hfYFP accumulation shown in Fig. 5d. NE, green; INMDV, blue; autophagic membranes, orange; vacuole, magenta. Scale bar, 100 nm.

Source data

Source Data Figs. 1–4 and Extended Data Figs. 1 and 3–6

Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed western blots and Ponceau.

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Mannino, P.J., Perun, A., Surovtsev, I.V. et al. A quantitative ultrastructural timeline of nuclear autophagy reveals a role for dynamin-like protein 1 at the nuclear envelope. Nat Cell Biol 27, 464–476 (2025). https://doi.org/10.1038/s41556-025-01612-1

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