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. 2022 Dec;13(1):144-154.
doi: 10.1080/19491034.2022.2047289.

Dictyostelium spastin is involved in nuclear envelope dynamics during semi-closed mitosis

Ulrike Schweigel  1 , Petros Batsios  1 , Annette Müller-Taubenberger  2 , Ralph Gräf  1 , Marianne Grafe  1
Affiliations

Dictyostelium spastin is involved in nuclear envelope dynamics during semi-closed mitosis

Ulrike Schweigel et al. Nucleus. 2022 Dec.

Abstract

Dictyostelium amoebae perform a semi-closed mitosis, in which the nuclear envelope is fenestrated at the insertion sites of the mitotic centrosomes and around the central spindle during karyokinesis. During late telophase the centrosome relocates to the cytoplasmic side of the nucleus, the central spindle disassembles and the nuclear fenestrae become closed. Our data indicate that Dictyostelium spastin (DdSpastin) is a microtubule-binding and severing type I membrane protein that plays a role in this process. Its mitotic localization is in agreement with a requirement for the removal of microtubules that would hinder closure of the fenestrae. Furthermore, DdSpastin interacts with the HeH/ LEM-family protein Src1 in BioID analyses as well as the inner nuclear membrane protein Sun1, and shows subcellular co-localizations with Src1, Sun1, the ESCRT component CHMP7 and the IST1-like protein filactin, suggesting that the principal pathway of mitotic nuclear envelope remodeling is conserved between animals and Dictyostelium amoebae.

Keywords: ESCRT; LEM-domain; Spastin; dictyostelium; mitosis; nuclear envelope; sun1.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Domain conservation and membrane orientation of DdSpastin. (a) Schematic of DdSpastin domains and membrane orientation by motif predictions using ELM , . See text for further descriptions; PNS, perinuclear space. (b) Immunoprecipitation using GFP-Trap Agarose beads showing tubulin-binding of DdSpastin-GFP. Proteins in the supernatant (lysate; corresponding to ~106 cells) and the GFP-Trap eluate (corresponding to 1 × 107 cells) were separated by SDS-PAGE, and stained with Coomassie or evaluated by immunoblot staining with anti-β-tubulin; *, this particular band was analyzed by mass spectrometry resulting in a hit for α-tubulin (see table S1). (c) In vitro microtubule severing assay. Polymerized porcine brain tubulin and DdSpastin-GFP (green) were incubated with and without 1 mM ATP. The reaction mixture was fixed with formaldehyde on poly-L-lysine coated coverslips and stained with anti-α-tubulin (red). Green spots most likely represent DdSpastin-GFP clusters that have formed via hydrophobic interactions of the transmembrane domains. Bar, 5 µm. (d) Verification of membrane orientation using isolated nuclei from DdSpastin-GFP overexpression cells. Nuclei were fixed with and without Triton X-100 permeabilization. Merged images of three examples each and corresponding single channel images are shown. Bar, 2 μ
Figure 2.
Figure 2.
Localization of DdSpastin-NEON (knock-in). Cells were fixed with glutaraldehyde and stained with DAPI(blue), and anti-α-tubulin (red). DdSpastin-NEON (green) accumulated at spindle Poles beginning in early telophase and in late telophase at the central spindle. The DdSpastin-NEON channel alone is shown below the merged images. A quantitative evaluation of all investigated cells is given in Table S2. Bar, 5 μm.
Figure 3.
Figure 3.
DdSpastin interactions and co-localizations. (a) Immunoblot of whole cell extracts of AX2 control cells, BirA*-DdSpastin cells and DdSpastin-BirA* cells stained with anti-BirA* antibodies. Fusion protein bands and BirA* with signal peptide are labeled with an asterisk. (b) BioID with nuclear extracts of DdSpastin-BirA* (lane 1–6) and negative control BirA* cells (lane 7). Western blots were stained with alkaline phosphate conjugated to the antibodies/protein stated on top. The interactors Src1 and Sun1 are labeled with red asterisks, DdSpastin-BirA* is labeled with a blue asterisk (lane 2). Lane 1 control w/o biotin incubation and lane 7 BirA* control show no specific bands at these positions. (c-h) Fluorescence microscopy of the strains stated in the figures. Cells were fixed with either glutaraldehyde (c-g) or methanol (h), and additionally labeled with DAPI (blue). Close-ups show co-localization (d, e, g, h). GFP-CHMP7 labeling is shown in a single channel because this protein has not been previously published (f). Bar, 5 μm.
Figure 4.
Figure 4.
Schematic illustration of Dictyostelium cell in late mitosis (a) and corresponding nuclear envelope fenestrae in late telophase. Centrosomes are still inserted in the NE in late anaphase and leave the NE in late telophase. (b) Enlargement with putative proteins involved in closure of the membrane fenestrae at the spindle Poles (1) and central spindle (2) in Dictyostelium. Inspired by Sundquist& Ullman (2015) [9]. See text for further details.
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References

    1. Sazer S, Lynch M, Needleman D.. Deciphering the evolutionary history of open and closed mitosis. Curr Biol. 2014;24(22):R1099–103. - PMC - PubMed
    1. Dey G, Culley S, Curran S, et al. Closed mitosis requires local disassembly of the nuclear envelope. Nature. 2020;585(7823):119–123. - PMC - PubMed
    1. Batsios P, Gräf R, Koonce MP, et al. Nuclear envelope organization in Dictyostelium discoideum. Int J Dev Biol. 2019;63(8–9–10):509–519. - PubMed
    1. McIntosh JR, Roos UP, Neighbors B, et al. Architecture of the microtubule component of mitotic spindles from Dictyostelium discoideum. J Cell Sci. 1985;75(1):93–129. - PubMed
    1. Meyer I, Peter T, Batsios P, et al. CP39, CP75 and CP91 are major structural components of the Dictyostelium centrosome’s core structure. Eur J Cell Biol. 2017;96(2):119–130. - PubMed

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This work was supported by the Deutsche Forschungsgemeinschaft [GR1642/9-1].

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