Grafe M et al. (2019), Supramolecular Structures of the Dictyostelium ...

XB-ART-55709

Cells 2019 Feb 16;82:. doi: 10.3390/cells8020162.

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Supramolecular Structures of the Dictyostelium Lamin NE81.

Grafe M , Batsios P , Meyer I , Lisin D , Baumann O , Goldberg MW , Gräf R .

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Nuclear lamins are nucleus-specific intermediate filaments (IF) found at the inner nuclear membrane (INM) of the nuclear envelope (NE). Together with nuclear envelope transmembrane proteins, they form the nuclear lamina and are crucial for gene regulation and mechanical robustness of the nucleus and the whole cell. Recently, we characterized Dictyostelium NE81 as an evolutionarily conserved lamin-like protein, both on the sequence and functional level. Here, we show on the structural level that the Dictyostelium NE81 is also capable of assembling into filaments, just as metazoan lamin filament assemblies. Using field-emission scanning electron microscopy, we show that NE81 expressed in Xenopous oocytes forms filamentous structures with an overall appearance highly reminiscent of Xenopus lamin B2. The in vitro assembly properties of recombinant His-tagged NE81 purified from Dictyostelium extracts are very similar to those of metazoan lamins. Super-resolution stimulated emission depletion (STED) and expansion microscopy (ExM), as well as transmission electron microscopy of negatively stained purified NE81, demonstrated its capability of forming filamentous structures under low-ionic-strength conditions. These results recommend Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope involving all relevant protein components known in animal cells.

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Species referenced: Xenopus
Genes referenced: cdk1 myc

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	Figure 1. Filamentous structures at intranuclear membrane stacks obtained upon expression of FLAG-NE81 in Xenopus oocytes. (A) TEM image showing intranuclear membrane stacks elicited through FLAG-NE81 expression. (B,C,C′) Field-emission (fe)SEM image showing Xenopus lamin B2 filaments (B) and Dictyostelium FLAG-NE81 filaments (C,C′) associated with intranuclear membrane stacks. Scale bars = 100 nm.
	Figure 2. (A,B) Transmission electron microscopy of permeabilized Dictyostelium cells showing spongy GFP-NE81ΔNLSΔCLIM clusters (Cl) studded by particles representing ribosomes. The nucleus (Nu), nucleoli (No), and mitochondria (Mi) are labeled. (B) is an enlarged view of (A). Scale bars = 1 µm.
	Figure 3. Expression of tagged NE81 in AX2 control cells or NE81 knockout cells. (A) Fluorescence deconvolution microscopy using a PlanApo 1.4/100× objective; GFP-NE81 cells (A), GFP-NE81KO cells (A′), HisMyc-NE81KO cells (A′′), and AX2 control cells (A′′′) were fixed with glutaraldehyde, stained with 4′,6-diamidino-2-phenylindole (DAPI) and, if appropriate (A′′′), with anti-Myc/anti-mouse-AlexaFluor 488 or anti-NE81/anti-rabbit-AlexaFluor 488. Scale bar = 5 µm. NE81 fluorescence is shown in green, DAPI in blue, and inverted phase contrast emphasizing the dark nucleoli is shown in red. (B,C) Immunoblots stained with alkaline phosphatase/NBT/BCIP show the absence of endogenous NE81 in the respective knockout (KO) strains and the band shift of the tagged protein compared to endogenous NE81.
	Figure 4. Formation of cytosolic HisMyc-NE81ΔNLSΔCLIM clusters requires an intact S122 target for CDK1. (A) Domain overview of HisMyc-NE81 variants used in this work. (B) Immunoblots of whole-cell extracts showing relative expression levels of endogenous NE81 compared to the tagged variants. The anti-actin staining is shown as a loading control. (C,C′) Expansion microscopy employing an LCI PlanNeo 1.3/63× objective. Cells were fixed with glutaraldehyde and stained with Hoechst33342, anti-myc/anti-mouse-AlexaFluor 488, and anti-tubulin/anti-rat-AlexaFluor 568 as indicated. Cytosolic HisMyc-NE81ΔNLSΔCLIM clusters are present only in cells with a native serine 122 (C), but not in cells carrying the phosphomimetic S122E mutation (C′). Expansion factors are 3.2 in (C) and 3.7 in (C′); scale bars = 5 µm (referring to the original size).
	Figure 5. Formation of HisMyc-NE81ΔNLSΔCLIM assemblies is salt-dependent in vitro. (A) Affinity purification of HisMyc-NE81ΔNLSΔCLIM expressed in Dictyostelium at high-salt conditions (0.5 M NaCl). Proteins were separated on an 8% SDS-PAGE. A Coomassie blue staining (left) and a Western blot stained with anti-Myc/alkaline phosphatase and NBT/BCIP color detection of the total extract and Ni-NTA-chromatography fractions are shown. Molecular masses of standard proteins are indicated on the left. (B) Soluble purified HisMyc-NE81ΔNLSΔCLIM at two starting concentrations (low concentration = 0.2 mg/mL; high concentration = 0.4 mg/mL) was dialyzed against 25 mM Tris-HCl pH = 8.0 and indicated NaCl concentrations, followed by centrifugation. Equivalent amounts of pellet (P) and supernatant (S) were loaded on SDS gels, blotted, stained with anti-myc antibodies, and detected as described above. Low concentration: protein amount in the pellet decreases with increasing NaCl concentration. (C) Chart depicting densitometric percentages of HisMyc-NE81ΔNLSΔCLIM in supernatants and pellets obtained at the indicated salt conditions (low protein concentration = 0.2 mg/mL protein). (D) Percentages of HisMyc-NE81ΔNLSΔCLIM in supernatants and pellets obtained at the indicated salt and pH conditions (low protein concentration = 0.2 mg/mL protein). Key: +++ most protein in supernatant, + around half of protein in supernatant, -- most of protein in pellet.
	Figure 6. Light microscopy reveals reticular HisMyc-NE81ΔNLSΔCLIM assemblies. Purified protein was fixed with formaldehyde and stained with anti-NE81/anti-rabbit-AlexaFluor 488. Phase-contrast (A–C; Ph) and fluorescence images (A–C′; Fl) are shown. (A,A′) At high NaCl concentration, no clear assemblies are visible, whereas the protein forms reticular assemblies at no-salt conditions and high protein concentration (B,B′). At low protein concentration (C,C′), individual filamentous structures become apparent. Scale bars = 5 µm (inset = 1 µm).
	Figure 7. Super-resolution fluorescence microscopy of HisMyc-NE81ΔNLSΔCLIM filaments. (A–A′′) stimulated emission depletion (STED) microscopy: assemblies formed at low protein concentration (5 µg/mL) were stained with anti-NE81/anti-rabbit-Atto 647N. Scale bars = 1 µm. (B,B′) Expansion microscopy with deconvolution, 3.5-fold expanded; assemblies formed at 0.3 mg/mL (B) and 5 µg/mL (B′) were stained with anti-NE81/anti-rabbit-AlexaFluor 488. Maximum-intensity projections are presented. Refer to supplemental videos 1 and 2 to get a three-dimensional impression. The scale bar (2 µm) refers to the original size of the specimen.
	Figure 8. Reticular HisMyc-NE81ΔNLSΔCLIM filament networks visualized by negative-staining transmission electron microscopy. Two representative examples are shown. Scale bar = 1 µm.

References [+] :

Adam, Regulation of nuclear lamin polymerization by importin alpha. 2008, Pubmed, Xenbase