Nuclear envelope budding is a response to cellular stress

Significance A defining feature of eukaryotes is the nuclear envelope, a double lipid bilayer that serves to isolate and protect the cell’s genetic material. Transport of large molecules over this barrier is believed to occur almost exclusively via the nuclear pores. However, herpes virions and mega-ribonucleoproteins (megaRNPs) use an alternative means of transport—via nuclear envelope budding (NEB). Here, we show NEB is a ubiquitous eukaryotic phenomenon and increases when exposed to various forms of cellular stress. NEB frequency was maximal when the cell was challenged with a drug that induces protein misfolding, indicating this transport pathway plays a role in protein quality control. These results imply that NEB is an underappreciated yet potentially fundamental means of nuclear transport.

The localization of the gold particles for the ubiquitin assay, was characterized as 'membrane binding' or 'cargo binding' in accordance with their distance from the respective structures. (a, b) Labeling density of the 'cargo binding area', the 'membrane binding area' and lipid droplets (negative control). (c, d) Definition of the 'cargo binding area' and the 'membrane binding area'. As the antibody sandwich that was used for this assay has a length of approximately 30 nm, the total area of the cargo including 30 nm of the surrounding area, was considered as the 'cargo binding area'. In a similar manner, the area stretching 30 nm inwards and outwards of the cargo's membrane was considered as the 'membrane binding area'. (e) A graphical representation of the antibody sandwich and its approximate length. All graphs were generated using GraphPad Prism (version 8.1.2 (332)) software with the error bars representing the standard deviation. *P<.05, ****P<.0001 vs. lipid droplets (negative control).

Caenorhabditis elegans
The C. elegans wild type reference strain was the Bristol N2 variety. The worms were cultured on normal growing media plates (NGM plates) and the E. coli strain OP50 was used as a food source. The worms were maintained at the optimal temperature of 25C (9, 10). Adult worms were used for electron microscopy.

Saccharomyces cerevisiae
Wild type cells of S. cerevisiae (BY4741) were cultured in YPD media at 30C (11). A strain with endogenous HSP104 C-terminally tagged with GFP (12) was used for old cell isolation, heat shock and sodium arsenite experiments. The deletion mutants hsp104 and rpn4 are from the YKO collection (EUROSCARF, Frankfurt, Germany). Strains used for investigating involvement of the ESCRTpathway in NEB and the pdr5 strain are from this study (see yeast strains list). Transformations were performed following standard procedures (13), and gene deletions and endogenous tags were integrated via homologous recombination (14). For the hydrogen peroxide experiment, cells were grown in synthetic complete media (with yeast nitrogen base, without amino acids, pH 5.5, complete supplement mixture of amino acids and 2% glucose).

High-pressure freezing for electron microscopy and tomography
All samples used in this study have been prepared using high-pressure freezing followed by freeze substitution. HMC-1 cells were loaded into membrane carriers and were high-pressure frozen in a high-pressure frozen and fixed through freeze substitution (long protocol) with anhydrous acetone containing 0.25% UA, 0.1% dehydrated glutaraldehyde and 0.01% osmium tetroxide (OsO4) (7,8,16,17). Washing steps after each staining were performed in dH2O.

Immuno-electron microscopy
For the immuno-labeling experiments, the same high-pressure frozen samples embedded in HM20 resin were used, a benefit of that short FS protocol (15). For a summary of these experiments, see table 2. Grids with 70 nm thick sections were fixed in 1% paraformaldehyde (PFA) in PBS for 10 minutes. After three PBS washes of 1 minute each, samples were blocked with 0.1% fish skin gelatin and 0.8% BSA in PBS for 1 hour. For detection of NPC proteins, grids were then incubated in a 1:50 dilution of mAb414 (BioLegend, San Diego, USA) for two hours, followed by a 1:150 dilution of rabbit anti-mouse immunoglobulins (Agilent/Dako, Glostrup, Denmark) for an hour, and then a 1:70 dilution of 10 nm gold-conjugated protein A (CMC UMC Utrecht, The Netherlands) for 30 minutes. For labeling of ubiquitin, grids were incubated in a 1:20 dilution of antibody ab19247 (Abcam, Cambridge, UK) for 2 hours. Detection of GFP was performed by using a 1:5, 1:10, or 1:30 dilution of ab6556 (abcam, Camebridge, UK) and detection of Hsp104 by using a 1:100 dilution of ab69549 (abcam, Camebridge, UK) incubated overnight. Goat-anti-Rabbit IgG 10 nm gold (Electron Microscopy Sciences, Hatfield PA, USA) was then used at a 1:20 dilution for an hour. All incubations were performed at room temperature, except for the primary antibody which was kept at 4°C. Three washing steps (20 min in PBS) were carried out after incubations with each antibody. 2.5% glutaraldehyde was applied to sections for 1 hour followed by three washes (1 min in dH2O). Sections were then contrast stained in 2% UA for 5 minutes (wash 3x 2 min in dH2O) and 1 minute in Reynold's lead citrate (washed 5x 1 min in dH2O).

Image acquisition and electron tomography
All thin sections were imaged at 120 kV either on a LEO 912 OMEGA (Zeiss, Krefeldt, Germany) equipped with a 2k x 2k VELETA Olympus CCD camera or on a Tecnai T12 transmission electron microscope equipped with a Ceta CMOS 16M camera (FEI Co., Eindhoven, The Netherlands). Double axis tilt series of serial sections of T. brucei samples were acquired every degree using the serialEM software (19) on a Tecnai TF30 300 kV IVEM microscope (FEI Co., The Netherlands) equipped with a Ultrascan 785 4k x 4k camera binned to 2k x 2k (pixel size 1 nm). For S. pombe, digital images (Gatan Ultrascan 890 or 895 camera, pixel size 1.5 nm) single axis tilt series were taken every 1.5° over a ±60°-65° range operating a Tecnai TF20 electron microscope (FEI Co., Eindhoven, The Netherlands) (7). For S. cerevisiae, double axis tilt series of serial sections were acquired on a Tecnai TF30 300 kV microscope (FEI Co., The Netherlands) equipped with a Gatan One View camera (pixel size 1.6 nm, increment 1.5° over a ±60° range). Tomograms were acquired with the use of serialEM (19). The tomographic reconstruction was performed using of the IMOD software package (20).

Isolation of old yeast cells
Isolation of old yeast cells was performed according to Smeal et al. with some modifications in order to reduce mechanical stress and improve the cell morphology for electron microscopy (21). In brief, the cell surface of exponentially growing cells was labeled with biotin by incubating the cells with 0.5 mg/ml Sulfo-NHS-LC biotin (#21335, Thermo Fisher scientific) for 20 min at room temperature. Cells were grown in YPD, harvested prior saturation of culture, and washed in PBS + 0.5% glucose. Biotinylated cells were labeled with 17.5 ug/ml Streptavidin magnetic beads (#21344, Thermo Fisher scientific) for 1.5 hours followed by 3 x 15 min magnetic sorting with PBS + 0.5% glucose washes in between.
Another two rounds of growth, streptavidin-labeling, and sorting was performed. The old cells and their unbound daughters were recovered in YPD for 4 hours before high-pressure freezing.

Stress treatments of S. cerevisiae
Cells were grown at 30C to mid exponential phase. For mild heat shock, the culture was shifted to 38C and samples were collected after 0, 5, 15, 30, 45, and 90 minutes (22). The more severe heat shock was performed at 42C for 30 min. For hydrogen peroxide, sodium arsenite, and AZC treatments the culture was split into two, one kept as unstressed control and one treated with the stressor. Cells were exposed to 0.6 mM hydrogen peroxide for 90 minutes, 0.5 mM sodium arsenite for 60 minutes, or 1 mg/mL AZC for 30 and 90 minutes (corresponding control culture was grown for 60 minutes after mid exponential phase). Cells were harvested by filtration followed by high-pressure freezing.

Proteasome inhibition
The rpn4 strain and a wild type control was grown to mid exponential phase and prepared for highpressure freezing. The pdr5 strain was grown to mid exponential phase prior addition of either 50 M MG132 (#C2211, Sigma-Aldrich) dissolved in DMSO or an equivalent volume of DMSO. A 60 min incubation at 30C followed before cells were prepared for high-pressure freezing.

Analysis of polyubiquitination
Cells were cultured and treated as described above. Mid-exponentially grown yeast corresponding to OD600 = 20 was harvested, washed once in distilled water and resuspended in 1 ml lysis buffer (100 mM Tris pH 7.5, 100 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20 mM N-ethylmaleimide). Homogenisation was conducted via an Avestin Emulsiflex C-15, applying a homogenization pressure of 18 000 psi. Samples were cleared from cell debris and unlysed cells by centrifugation for 5 min at 3500 rcf and 4°C. 100 µl of cleared lysate was mixed with the same volume of 2x Laemmli buffer (100 mM Tris pH 6.8, 4% SDS, 20% glycerol, 0.2% bromophenol blue, 200 mM 2-mercaptoethanol) and incubated for 15 min at 95°C. The samples were then applied for SDS-PAGE and immunoblotting following standard protocols. Equal loading was controlled via Ponceau S staining directly after wet electrotransfer on PVDF membranes and blots were decorated with an anti-ubiquitin antibody (1:1000, HRP-conjugated, P4D1, sc8017, Santa Cruz). Clarity Western ECL Substrate (BIO-RAD, 1705060) and a ChemiDoc XRS+ Imaging System (BIO-RAD, 1708265) were used for detection

Analysis of cell death
Loss of membrane integrity was assessed with propidium iodide (PI) staining as previously described (23). Briefly, cells were harvested in 96-well plates after the respective stressor/mock treatment and resuspended in 250 µl of PI solution (100 µg/l PI in phosphate buffered saline PBS; 25 mM potassium phosphate, 0.9% NaCl; adjusted to pH 7.2). After 10 min incubation in the dark, cells were washed with recorded per strain and condition using InCyte software (3.1).

Confocal microscopy
For visualization of nuclei, yeast cells were harvested and resuspended in DRAQ5 staining solution (5 µM DRAQ5 in PBS). After 10 min incubation in the dark, cells were washed once with PBS and immobilized on agar slides. Specimen were analyzed with a ZEISS LSM700 microscope using ZEISS ZEN blue software control. Plan-Apochromat 63x/1.40 Oil M27 objective was employed. Appropriate filter settings were used to visualize GFP and DRAQ5. Micrographs were analyzed and processed with the open-source software Fiji (24). To reduce image noise, Gaussian filtering (σ = 1) was applied, followed by background subtraction (rolling ball radius = 100 pixels). Pictures within an experiment were captured and processed in the same way.

Phylogenetic tree
A phylogenetic tree was constructed showing all the organisms where NEB events have been observed. The tree was generated based on the NCBI taxonomy browser for scientific names and visualized by the EvolView online software (http://www.evolgenius.info/evolview/).

Statistics and reproducibility
The frequency of NEB events was achieved by counting the number of events present in approximately 100-200 thin sections of nuclei. The percentage of sections containing events in each organism was presented as a bar graph. For the NPC immuno-EM assay, different cell compartments (NPCs, NEBs and lipid droplets) were categorized as labeled or not labeled based on the presence or absence of gold particles. Similarly, the percentage of labeled compartments was presented as a bar graph. For the statistical analysis, different statistical tests were examined but a non-parametric Wilcoxon test was performed as our data represent frequencies and the numerical values are not following a normal distribution nor are continuous variants (values were considered as 0 for the absence of events and 1 for the presence of events).. The test was performed using the MATLAB multi-paradigm programming language. For further details on the examined statistic tests see Supplementary table 2. For the ubiquitin and Hsp104-GFP immuno-EM, the area and number of gold particles of different cell compartments were measured using IMOD (https://bio3d.colorado.edu/imod/). The amount of gold particles per area was presented as bar graph.
Movie S1. 1.5 nm thick tomographic slices of the budding event observed in S. pombe ( Figure   6b). The extra bilayer around the bud is becoming easily noticeable. The movie was created as a sequence of snap pictures acquired through the IMOD software, frame rate: 6 per second. Scale bar size is 200 nm.