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An ESCRT grommet cooperates with a diffusion barrier to maintain nuclear integrity

Nature Cell Biology volume 25, pages 1465–1477 (2023)Cite this article

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Abstract

The molecular mechanisms by which the endosomal sorting complexes required for transport (ESCRT) proteins contribute to the integrity of the nuclear envelope (NE) barrier are not fully defined. We leveraged the single NE hole generated by mitotic extrusion of the Schizosaccharomyces pombe spindle pole body to reveal two modes of ESCRT function executed by distinct complements of ESCRT-III proteins, both dependent on CHMP7/Cmp7. A grommet-like function is required to restrict the NE hole in anaphase B, whereas replacement of Cmp7 by a sealing module ultimately closes the NE in interphase. Without Cmp7, nucleocytoplasmic compartmentalization remains intact despite NE discontinuities of up to 540 nm, suggesting mechanisms to limit diffusion through these holes. We implicate spindle pole body proteins as key components of a diffusion barrier acting with Cmp7 in anaphase B. Thus, NE remodelling mechanisms cooperate with proteinaceous diffusion barriers beyond nuclear pore complexes to maintain the nuclear compartment.

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Fig. 1: ESCRT proteins are recruited to the SPB extrusion site in three temporal waves.
Fig. 2: Average copy numbers of ESCRTs at the SPB extrusion site.
Fig. 3: Cmp7 is required for ESCRT-III recruitment to the NE and is replaced by Wave 3 ESCRTs.
Fig. 4: Cmp7 is required to restrict the size of the NE hole in anaphase and seal the NE during G1/S.
Fig. 5: Did4 is required to remove Cmp7 to seal the NE.
Fig. 6: Cmp7 and a 1,6-HD-sensitive diffusion barrier ensure nucleocytoplasmic compartmentalization during mitosis.
Fig. 7: CMP7 is required for robust fitness and nucleocytoplasmic compartmentalization in the context of perturbed SPB protein function.

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Data availability

Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.

Code availability

The MATLAB script for quantification of ESCRT subunits is available with no access restrictions at https://github.com/LusKingLab/Find_n_Fit_Spots_3D.

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Acknowledgements

We thank the Center for Cellular and Molecular Imaging, Electron Microscopy Facility at Yale School of Medicine EM for assistance, particularly M. Graham and X. Liu. We thank L.-L. Du, A. Frost, I. Hagan, T. Pollard and NBRP (YGRC), Japan (and the investigators who have deposited their strains at this resource) for yeast strains. This work was supported by the National Institutes of Health (grant nos R01 GM105672 to C.P.L. and F32 GM139285 to N.R.A.) and an Allen Distinguished Investigator Award, a Paul G. Allen Frontiers Group advised grant of the Paul G. Allen Family Foundation (M.C.K.).

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Authors and Affiliations

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

    Nicholas R. Ader, Linda Chen, Ivan V. Surovtsev, William L. Chadwick, Elisa C. Rodriguez, Megan C. King & C. Patrick Lusk

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

    Ivan V. Surovtsev

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

    Megan C. King

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  1. Nicholas R. Ader
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Contributions

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

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Correspondence to Megan C. King or C. Patrick Lusk.

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Extended data

Extended Data Fig. 1 Did2 is not detected at the SPB extrusion site, whereas Ist1 is recruited after Cmp7.

a, Fluorescence micrographs of Did2–GFP, mCherry–Atb2 and Pcp1–mCherry in a representative cell (MKSP3342) in anaphase B. Did2–GFP (left), sum projection of mCherry–Atb2/Pcp1–mCherry (middle) and magnifications (right) of ROI (top to bottom) green, magenta, merge. b, Plot of the percentage of nuclei with Did2–GFP (MKSP3342) co-localized with SPB marker at the indicated phases of the cell cycle. Points and error bars represent the mean and range, respectively, across at least three biological replicates with at least 50 nuclei/Did2–GFP/replicate, n = 372 nuclei. c, Time-lapse fluorescence microscopy of Cmp7–GFP (green) and Ist1–mCherry (magenta) in a late anaphase B cell (MKSP3521). Cell representative of population from two biological replicates. White boxes in overview micrographs indicate ROI magnified below images as single channel and composite images. Scale bars, 1 µm (overview) and 250 nm (ROI). Source numerical data are provided.

Source data

Extended Data Fig. 2 Use of the kinetochore protein Fta3 as a standard for ratiometric determination of ESCRT protein copy number at the NE.

a, Fluorescence micrographs of representative mixture of a GFP–ESCRT strain (Ist1–GFP; MKSP3365) and one expressing Fta3–GFP (MKSP3186). Overview micrograph: top left, strain expressing Ist1–GFP (green), mCherry–Atb2 (magenta), and Sad1–mCherry (magenta); bottom right, Fta3–GFP strain (green). Dotted white lines indicate the cell outline. The blue and orange boxes in the overview micrograph outline the ROIs magnified to the right of image as single channel images for Ist1–GFP and Fta3–GFP, as indicated. b, Fluorescence micrographs of representative Fta3–GFP expressing cells (MKSP3186) at indicated cell-cycle stage (top). The black boxes in overview micrographs outline the ROI magnified below. Schematic representation of Fta3–GFP appearance in the different cell-cycle stages (bottom). Number of molecules based on Lawrimore and colleagues27. c, x–y plot of Fta3–GFP focus fluorescence intensity against the expected number of molecules per focus in MKSP3186 based on Lawrimore and colleagues27. Points represent median of all values collected for that stage of the cell cycle (see b); error bars represent the 95% confidence interval. The red line represents a simple linear regression with the R2 shown; n = 366 foci across three biological replicates with at least 50 foci per replicate. Scale bars, 1 µm (overview) and 250 nm (ROI). Source numerical data are provided.

Source data

Extended Data Fig. 3 MGM4 and MGM2 reporters—the AID system allows for rapid depletion of Cmp7.

a, Micrographs of MGM4 (top left; MKSP3345) and MGM2 (bottom left; MKSP3960). Histogram of nucleoplasmic-to-cytoplasmic GFP fluorescence (N/C ratio) for individual cells are shown (right). Size between bins, 0.05. Dotted line at 0.75 bin, representing the cut-off we used to gauge robust nucleocytoplasmic compartmentalization. Quantification was performed for three biological replicates. MGM4 data replotted from Fig. 6a. MGM2, n = 178 nuclei, with at least 58 nuclei per biological replicate. Scale bars, 1 µm. b, Cmp7–AID was efficiently degraded within 15 min of treatment with 5IAA. Representative western blot (WB) of Cmp7–AID levels (detected with an antibody to AID) in total protein extracts from cells (MKSP3517) cultured in the presence of 5IAA for the indicated times. Ponceau stain is shown (bottom) to assess relative total protein loads. The relative amount of Cmp7–AID protein, normalized to T0, is shown below the WB as the mean and s.d. of three biological replicates. c, Depletion of Cmp7–AID recapitulates phenotypes observed in cmp7Δ cells, including abolishing recruitment of Ist1–GFP to the NE. Plots of the percentage of nuclei with Ist1–GFP co-localized with SPB marker at the indicated phases of the cell cycle in cells (MKSP3625) expressing Cmp7–AID and OsTIR1F74A. The cells were treated with DMSO (grey) or 5IAA (green) for 1 h. Points and error bars represent the mean and range, respectively, across at least three biological replicates with at least 50 nuclei per GFP–ESCRT per replicate. DMSO, n = 273 nuclei; and 5IAA, n = 296 nuclei. Source numerical data and unprocessed blots are provided.

Source data

Extended Data Fig. 4 Genes encoding constitutive SPB proteins and mitotically associated SPB proteins genetically interact with CMP7.

The indicated strains were serially diluted tenfold, plated on YE5S plates and incubated at the indicated temperatures. The plates were imaged after 2 d (30, 32 and 36 °C) or 4 d (22 °C). WT (MKSP399), cmp7Δ (MKSP3107), alp4-1891 (MKSP4067), cmp7Δalp4-1891 (MKSP4163), alp6-719 (MKSP3475), cmp7Δalp6-719 (MKSP4031), alp14Δ (MKSP4063), cmp7Δalp14Δ (MKSP4165), brr6.ts8 (MKSP3461), cmp7Δbrr6.ts8 (MKSP3528), cam1-116F (MKSP4061), cmp7Δcam1-116F (MKSP4167), cdc11-123 (MKSP4060), cmp7Δcdc11-123 (MKSP4169), cut11-1 (MKSP3469), cmp7Δcut11-1 (MKSP4040), gtb1-93 (MKSP4062), cmp7Δgtb1-93 (MKSP4174), kms2 DAmP (MKSP1474), cmp7Δkms2 DAmP (MKSP3683), mto1Δ (MKSP1303), cmp7Δmto1Δ (MKSP4027), mto2Δ (MKSP1305), cmp7Δmto2Δ (MKSP4151), mzt1-T27W (MKSP3476), cmp7Δmzt1-T27W (MKSP4045), plo1-24C (MKSP1461), cmp7Δplo1-24C (MKSP4048), sad1-1 (MKSP3468), cmp7Δsad1-1 (MKSP4033), sid4-SA1 (MKSP4065) and cmp7Δsid4-SA1 (MKSP4171).

Extended Data Fig. 5 Conditional depletion of Pcp1 and Ppc89 impairs nucleocytoplasmic compartmentalization without impacting mitosis progression.

a, WB of Pcp1–AID from cells expressing OsTir1F74A treated with 5IAA as indicated. Ponceau to assess relative total protein. −OsTir1F74A (MKSP4112); +OsTir1F74A (MKSP4113); −OsTir1F74A, cmp7Δ (MKSP4114); +OsTir1F74A, cmp7Δ (MKSP4115). b, Nuclei of pcp1–AID (MKSP3773) and cmp7Δpcp1–AID (MKSP3775) strains expressing MGM4, Cut11–mCherry, mCherry–Atb2 and OsTir1F74A: top, MGM4 (green); middle, Cut11–mCherry/mCherry–Atb2 (magenta); bottom, merge. Cells were treated with DMSO/5IAA as indicated. c, Percentage of pcp1–AID (grey bars, MKSP3773) and cmp7Δpcp1–AID (teal bars, MKSP3775) cells in b with N/C fluorescence ratios >0.75 reflecting a loss of robust nucleocytoplasmic compartmentalization. Points are the mean from each biological replicate; bars and error bars represent the total mean and s.d., respectively. Points average of at least 18 nuclei per cell-cycle phase/strain/replicate, n = 649 nuclei. d, WB of Ppc89–AID levels as in a. −OsTir1F74A (MKSP4175); +OsTir1F74A (MKSP4136); −OsTir1F74A, cmp7Δ (MKSP4176); +OsTir1F74A, cmp7Δ (MKSP4135). e, Nuclei of ppc89–AID (MKSP4136) and cmp7Δppc89–AID (MKSP4135) strains expressing MGM4, Cut11–mCherry and OsTir1F74A shown as in b. f, Percentage of ppc89–AID (grey bars, MKSP4136) and cmp7Δppc89–AID (teal bars, MKSP4135) cells shown in e with N/C fluorescence ratios >0.75 reflecting a loss of robust nucleocytoplasmic compartmentalization. Points are the mean from each biological replicate; bars and error bars represent the total mean and s.d., respectively. Points represent the average of at least 11 nuclei/cell cycle phase/strain/replicate, n = 504 nuclei. g, Time-lapse of mCherry–Atb2 and Cut11–mCherry in a mitotic pcp1–AID (MKSP3773) cell and a cmp7Δpcp1–AID cell (MKSP3775) expressing OsTir1F74A. Imaging began after 1 h of treatment with DMSO/5IAA as indicated. h, Time-lapse of Cut11–mCherry in a mitotic ppc89–AID (MKSP4136) cell and a cmp7Δppc89–AID cell (MKSP4135) expressing OsTir1F74A. Imaging began after 1 h treatment with DMSO/5IAA as indicated. i, pcp1–AID cell (MKSP3773) expressing OsTir1F74A containing a monopolar spindle arranged horizontally with MGM4 (green; left), Cut11–mCherry (magenta) and mCherry–Atb2 (magenta) in middle, and merge (right) 3 h after treatment with 5IAA. Scale bars, 1 µm. Source numerical data and unprocessed blots are provided.

Source data

Extended Data Fig. 6 An ESCRT grommet and SPB-dependent diffusion barrier maintain nucleocytoplasmic compartmentalization.

A model of NE sealing and maintenance of mitotic nucleocytoplasmic compartmentalization by the ESCRT machinery and the SPB in S. pombe anaphase B. (1) At mitotic entry, the SPB (dark magenta rounded rectangle) is inserted into the NE to organize the mitotic spindle (light magenta rectangles). (2) Once the SPB is extruded to the cytosol, Cmp7 is recruited by Heh1/Lem2 (not shown). Concurrently, the SPB contributes to the formation of a proteinaceous diffusion barrier (translucent magenta) sensitive to 2.5% 1,6-HD to prevent free diffusion through the unsealed NE. (3) A second wave of ESCRT recruitment comprising Vps32 and Ist1 (multiple circles represent copy number ratio to Cmp7) occurs in late anaphase B, which generates a grommet that restricts the size of the NE hole and supports the SPB-dependent diffusion barrier. The Ist1 circle at partial opacity represents copy number increase during mitosis and into G1/S. (4) Finally, in the G1/S of the following cell cycle, the third wave of ESCRTs, Did4 and Vps24 (multiple circles represent copy number ratio to Cmp7), is recruited, displacing Cmp7 and sealing the NE. N, nucleoplasm (blue); C, cytoplasm (beige).

Supplementary information

Reporting Summary

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Supplementary Video 1

CLEM and electron tomography of a WT cell in early anaphase B. Electron tomogram of an early anaphase B cell (MKSP3180) with segmentation. NE, blue; SPB, dark orange; microtubules, yellow. Scale bar, 100 nm.

Supplementary Video 2

CLEM and electron tomography of a WT cell in late anaphase B. Electron tomogram of a late anaphase B cell (MKSP3180) with segmentation. NE, blue; SPB, dark orange; microtubules, yellow. Annotations at end of the video show a NPC and SPB extrusion hole. Scale bar, 100 nm.

Supplementary Video 3

CLEM and electron tomography of a cmp7∆ cell in early anaphase B. Electron tomogram of an early anaphase B cell (MKSP3243) with segmentation. NE, blue; SPB, dark orange; microtubules, yellow. Scale bar, 100 nm.

Supplementary Video 4

CLEM and electron tomography of a cmp7∆ cell in late anaphase B. Electron tomogram of a late anaphase B cell (MKSP3243) with segmentation. NE, blue; SPB, dark orange; microtubules, yellow. Scale bar, 100 nm.

Supplementary Table 1

GFP tagging of key ESCRT machinery. ESCRT protein in S. pombe is shown (left). Orthologues in S. cerevisiae and Homo sapiens are shown (centre). Tagging approach to ensure function is shown (right).

Supplementary Table 2

List of S. pombe strains used in this study.

Supplementary Table 3

List of plasmids used in this study.

Supplementary Table 4

List of primers used in this study.

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Ader, N.R., Chen, L., Surovtsev, I.V. et al. An ESCRT grommet cooperates with a diffusion barrier to maintain nuclear integrity. Nat Cell Biol 25, 1465–1477 (2023). https://doi.org/10.1038/s41556-023-01235-4

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