henhaobio
Sign in

The Journal of Cell BiologyCell BiologyPeer-reviewed journal article

Components of an ESCRT-independent nuclear envelope assembly pathway

Emma M SydiriD, M Humam FarraiD, Abigail L WhitfordiD, Shea HinojosaiD, Pei-Yi KaoiD, João A. PauloiD, Sharan SwarupiD, C. Patrick LuskiD, J. Wade HarperiD, I‐Ju LeeiD, David S. PellmaniD

PDFDOIPreprint
In-depth full-text digest available · Jump to digest ↓

Abstract

In many cells, the nuclear envelope (NE) must be reassembled after mitosis and holes in the nuclear membrane must be "sealed." During NE assembly, the NE-specific adaptor, Cmp7, recruits/activates ESCRT-III proteins to mediate NE sealing. However, recent evidence suggests the presence of additional mechanisms. In a screen using the fission yeast, S. japonicus, we recently implicated the ESCRT adaptor, Alx1, and a conserved, but little studied protein, Vid27, in Cmp7-independent NE assembly. Here, we provide direct evidence that Alx1 functions in a Cmp7- and ESCRT-independent NE assembly pathway via positive regulation of Vid27. Consistent with a role in membrane remodeling, Vid27 localizes to sites of postmitotic NE sealing and is essential in S. japonicus. Alx1 and Vid27 interact and mutations disrupting the predicted interaction interface abolish Alx1s enhancement of Vid27 function at the NE. These findings define components of a new Cmp7- and ESCRT-independent NE assembly pathway, advancing our understanding of mechanisms that maintain the integrity of the nucleus.

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (27 Jul 2026) · CC BY 4.0

In S. japonicus, the ESCRT adaptor Alx1 activates the essential PH-domain protein Vid27 through interaction, driving postmitotic nuclear envelope sealing independently of Cmp7 and ESCRT-III.

Cmp7 is the only known nuclear envelope adaptor for ESCRT-III, yet losing it or ESCRT-III is only partially detrimental in fission yeast and human cells, implying other mechanisms. This work identifies Alx1 and Vid27 as components of a Cmp7- and ESCRT-independent assembly pathway.

Key results

  1. ShownVid27 is essential in S. japonicus. Degron-mediated depletion slows postmitotic nuclear envelope reassembly and increases large membrane gaps seen by electron microscopy.

    Mean reassembly time 4.1 min (control), 9.8 min (degron strain, DMSO), 17.1 min (degron, IAA+ASV); permutation p=0.0007, 0.0068, <0.0001; n=31-37 nuclei. Cells with gaps ≥150 nm rose about 4-fold (n>90 cells, Welch P≤0.05); imaging cut off at 30.8 min. · S. japonicus Vid27 is essential and depletion of…; Figure legends

  2. ShownVid27 concentrates at mitotic tails, the main sites of postmitotic nuclear envelope sealing, and this does not depend on Cmp7. It is not concentrated at spindle pole bodies.

    99% (511/516) of mitotic tails accumulated Vid27-INTmNG. Only 2.63% of 86,416 Vid27 puncta overlapped the spindle pole body marker Pcp1. Localization was unchanged by cmp7Δ or vid27-G67R. · Vid27 localizes to the major sites of…; Microscopy and image analysis

  3. ShownThe Alx1-E308V suppressor works without ESCRT-III: it suppresses vps32Δ and mostly loses ESCRT-III binding. Vid27-G67R still bypasses cmp7Δ without Alx1, and the Vid27-Vps32 co-IP depends on Alx1, suggesting Alx1 bridges them.

    alx1-E308V suppressed the nuclear integrity defect of vps32Δ cells (n=200-205 cells per strain, 3 experiments, P≤0.01). Vid27-Vps32 co-IP was lost in alx1Δ and alx1-E308V. cmp7Δ alx1Δ vid27-G67R grew better than cmp7Δ alx1Δ. · alx1-E308V establishes the existence of an…; Vid27 functions with Alx1 but independently of…; The ESCRT-III-independent function of Alx1 at…

  4. ShownAlphaFold2 predicts the Alx1 C-terminal proline-rich tail binds the Vid27 PH2 domain. Charge-swap mutants on either side strongly disrupt binding, and they eliminate the Alx1-E308V bypass of cmp7Δ.

    Vid27-E3K, Vid27-DE6K, Alx1-KR4E and Alx1-KR7E disrupted co-IP. With vid27-DE6K or vid27-E3K, alx1-E308V no longer suppressed cmp7Δ growth. At 19°C, alx1-E308V-KR4E/KR7E cmp7Δ grew like cmp7Δ. · Vid27 and Alx1 are predicted to form a complex; The ESCRT-III-independent function of Alx1 at…

  5. SuggestedThe N-terminal half of Vid27, with its two PH-like domains, appears to carry the essential function. Loss of the C-terminal β-propeller is tolerated alone but is synthetically lethal with cmp7Δ. Lem2 clustering did not track with nuclear envelope defects across vid27 mutants.

    vid27ΔN was inviable, and vid27ΔPH1 was worse than vid27ΔPH2 for growth and nuclear integrity. vid27ΔC grew only slightly slower but was synthetically lethal with cmp7Δ. vid27ΔC formed Lem2 clusters but vid27ΔPH1 did not. · The essential function of Vid27 is contained…; Vid27 and Alx1 promote NE assembly independent…

Methods, data and reproducibility

Genetic suppressor and colony-size analysis, auxin/Asunaprevir degron depletion, GFP-NLS compartmentalization imaging, electron microscopy, TMT immunoprecipitation mass spectrometry, co-IP blots, and AlphaFold2-guided charge-swap mutagenesis.

  • S. japonicus strains with deletions, point mutations, truncations and tagged alleles of vid27, alx1, cmp7, vps32, lem2 and les1; colony-size analysis after ascus dissection · S. japonicus (fission yeast) · n = 20-45 asci analyzed per cross
  • GFP-NLS nuclear/cytoplasmic ratio imaging in postmitotic binucleated cells · S. japonicus (fission yeast) · n = 609-1280 nuclei measured per strain/condition
  • Time-lapse imaging of nuclear envelope reassembly after Vid27 depletion · S. japonicus (fission yeast) · n = 31-37 nuclei per condition, 3 experiments
  • Electron microscopy of nuclear envelope gaps in Vid27-depleted versus control cells · S. japonicus (fission yeast) · n = more than 90 cells, 3 experiments
  • TMT immunoprecipitation mass spectrometry of Vid27, Vid27-G67R, Vid27ΔC, Alx1 and Alx1-E308V · S. japonicus (fission yeast) · n = 3 replicates (Vid27 variants); 4 replicates (Alx1 variants)
Methods & tools
AlphaFold2, mAID-SMASh degron (IAA and Asunaprevir), TMTpro labeling, Orbitrap Exploris480 and Orbitrap Fusion Lumos mass spectrometers, Comet database search with linear discriminant analysis, MSconvert, ImageJ/FIJI, CellProfiler, GraphPad Prism, UniProt S. japonicum reference database
Reagents & environment
Mass spectrometry searches used the S. japonicum UniProt reference database (downloaded February 2023); analysis in ImageJ/FIJI, CellProfiler and GraphPad Prism (no versions given).
Reproducibility gaps
No code or data repository URLs or deposition accessions printed; mass spectrometry results and strain lists are in supplementary tables (S1-S4) not included in the text.
Limitations (6)
  • Direct Alx1-Vid27 binding is supported by AlphaFold2 and co-IP only; reconstitution with purified proteins is still needed to characterize the complex. · stated by the authors
  • Few cmp7Δ les1Δ vid27-G67R triple mutants could be obtained because of genetic linkage, which limits sensitivity to detect a Les1 relationship. · stated by the authors
  • Imaging stopped at 30.8 min, so reassembly times in depleted cells are underestimates. The C-terminal mNG fusion also impaired Vid27 function, so localization relied on an internal mNG insertion. · stated by the authors
  • Vid27ΔC is expressed at higher levels than wild-type Vid27, which complicates interpreting its genetic interaction with cmp7Δ as a simple loss of function. · stated by the authors
  • All functional evidence comes from one species, S. japonicus. Conservation of the pathway in other organisms is argued from homology and localization reports rather than tested here. · noted in this summary
  • Several mass spectrometry interactors, including some Alx1-E308V partners, missed the significance threshold because of replicate variability and rely on directed co-IP blots for validation. · noted in this summary

Read this if you study nuclear envelope sealing, ESCRT-independent membrane remodeling, or yeast genetics and proteomics approaches to finding parallel membrane-repair pathways.

Citations
2
FWCI
4.10
Field percentile
top 8%

Reproducibility ledger

Nobody has reported trying to reproduce this paper yet. Did you replicate an experiment or run the code? Your report saves the next lab a week.

Sign in to file a reproducibility report.

Related papers

Similarity Map
SelectedSame fieldCross-field
THISGatta '20Ader '22Webster '16Gu '16Thaller '19Carlton '16
74% semantic matchCell Biology
A. T. Gatta, Y. Olmos, C. L. Stoten+3 more·bioRxiv·Apr 20, 2020