Plasmids for the expression of the recombinant globular tail domain of MVI fused with GST (glutathione S-transferase) in E. coli was constructed by subcloning a fragment of the rat MVI nucleotide sequence (Majewski et al., 2012) (gene ID D4A5I9) corresponding to the MVI globular tail (aa 1046-1285) into the pGEX-4T1 vector (from GE Healthcare, Cat. No 28-9545-49). Glutathione Sepharose 4B was also obtained from GE Healthcare (Cat. No 17-0756-01).

The antibodies were used as follows: rabbit polyclonal antibody to MVI (Proteus, Cat. No 25-6791), mouse monoclonal antibody to β-actin (Sigma-Aldrich, Cat. No A3854), mouse monoclonal antibody to B23 (Abcam, Cat. No ab 10530), rabbit polyclonal antibody to GRP78 (Abcam, Cat. No 21685), mouse monoclonal antibody to fibrillarin (Thermo Fisher Scientific, Cat. No MA3-16771), mouse monoclonal antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Millipore, Cat. No MAB 274), mouse monoclonal antibody to RPA 194 (Pol1) (Santa Cruz Biotechnology, Cat. No sc-48385), mouse monoclonal antibody to UBF (Santa Cruz Biotechnology, Cat. No sc-13125), goat polyclonal antibody to lamin B (Santa Cruz Biotechnology, Cat. No sc-6217), mouse monoclonal antibody to p-S6 (Cell Signaling, Cat. No 62016), mouse monoclonal antibody to S6 (Cell Signaling, Cat. No 2317), rabbit monoclonal antibody to p-p70S6K (Cell Signaling, Cat. No 9234), rabbit monoclonal antibody to p70S6K (Cell Signaling, Cat. No 2708), goat anti-mouse IgG antibody, HRP conjugate (Millipore, Cat. No AP308P), goat anti-rabbit IgG antibody, HRP conjugate (Millipore, Cat. No AP307P), and donkey anti-goat IgG antibody, HRP conjugate (Santa Cruz Biotechnology, Cat. No sc-2020).

VECTASHIELD PLUS Antifade Mounting Medium with DAPI was obtained from Vector Laboratories (Cat. No H2000). For immunofluorescence studies, the following secondary antibodies were used: goat anti-rabbit IgG labeled with Alexa Fluor 488 (Invitrogen, Cat. No A11008) and goat anti-mouse IgG labeled with Alexa Fluor 546 (Invitrogen, Cat. No A11003).

The in situ proximity ligation assay (PLA) kit was purchased from Sigma-Aldrich (kit components: Duolink In Situ PLA Probe Anti-Mouse MINUS, Cat. No DUO 92004; Duolink In Situ PLA Probe Anti-Rabbit PLUS, Cat. No DUO 92002; Duolink In Situ Detection Reagents Red, Cat. No DUO 92008; Duolink In Situ Wash Buffers, Cat. No DUO 82049; and Duolink In Situ Mounting Medium with DAPI, Cat. No DUO 92006).

The non-adherent variant of PC12 cells (American Type Cell Culture Collection, ATCC, Cat. No CRL-1721) was cultured in RPMI media (Gibco, Cat. No 52400025) containing 2 mM L-glutamine, 4.5 g/L glucose supplemented with 10% heat-inactivated horse serum (Gibco, Cat. No 26050088), 5% heat-inactivated fetal bovine serum (Gibco, Cat. No 10270106), and antibiotics: 1% penicillin/streptomycin (Gibco, Cat. No 15140-122) at 37°C in humidified air containing 5% CO₂.

In addition, stable MVI knockdown (MVI-KD) and a control scrambled cell lines (control), both obtained earlier by Dr. Ł. Majewski (Majewski et al., 2011), were used. These lines were prepared using a plasmid encoding shRNA directed against the MVI mRNA and a plasmid encoding a control shRNA not recognizing any known mammalian mRNA sequences (Majewski et al., 2011). MVI-KD and control PC12 cells were cultured in F12K (Kaighn’s Modification of Ham’s F-12) (ATCC, Cat. No 30-2004) containing 2 mM L-glutamine, 1.5 g/L sodium bicarbonate supplemented with 2.5% heat-inactivated fetal bovine serum (Gibco, Cat. No 10270106), 15% heat-inactivated horse serum (Gibco, Cat. No 26050088), antibiotics: 1% penicillin/streptomycin (Gibco, Cat. No 15140-122), and hygromycin B as a selective antibiotic (250 ng/mL) at 37°C in humidified air containing 5% CO₂.

Cells were lysed in an ice-cold buffer that contained 50 mM Tris–HCl pH 7.5 (Sigma-Aldrich, Cat. No T6687), 150 mM NaCl (Chempure, Cat. No 117941206), 0.1% Triton X-100 (Sigma-Aldrich, Cat. No SLCJ7494), 2 mM EGTA (Sigma-Aldrich, Cat. No E3889), 1 mM DTT (Sigma-Aldrich, Cat. No 10197777001), 1 mM PMSF (Sigma-Aldrich, Cat. No P7626), cOmplete™ Protease Inhibitor Cocktail (Roche, Cat. No 04693132001), and phosphatase inhibitor PhosSTOP™ (Roche, Cat. No 4906845001).

To obtain the cytoplasmic, nuclear, and nucleolar fractions, PC12 cells were subjected to fractionation according to the Hacot protocol (Hacot et al., 2010) with several modifications. Briefly, cells were washed with PBS, harvested, and centrifuged at 200 g for 3 min at RT. The pellet was resuspended in a hypotonic buffer consisting of 10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, and 0.5 mM DTT and kept on ice for 15 min to induce osmotic shock, causing cell membrane disruption. The suspension was homogenized using a Dounce-type glass tissue homogenizer and centrifuged at 1,200 g for 5 min at 4°C. The resultant supernatant contained the cytoplasmic protein fraction, while the pellet contained both cell debris and cell nuclei. The pellet was then subjected to centrifugation in the sucrose gradient: it was resuspended in S1 buffer (0.25 M sucrose and 10 mM MgCl₂), and the suspension was placed in centrifuge tubes containing S2 buffer (0.88 M sucrose and 0.5 mM MgCl₂) and centrifuged at 1,200 g for 5 min at 4°C. The supernatant was discarded, and the pellet at the bottom of the tube (representing the purified fraction of cell nuclei) was resuspended in buffer S3 (0.35 M sucrose and 0.5 mM MgCl₂). Next, the nucleolus fraction was obtained by sonication of the purified nuclei fraction using a S-250D sonicator (Branson Ultrasonic S.A.) with a 1/8'' (3.2 mm) microtip at 30% power of the device in three cycles/sequences (10-s sonication and 10-s pause). The suspension that resulted upon sonication was applied to the surface of the S2 buffer and centrifuged at 2,000 g for 20 min at 4°C. The supernatant contained the nucleoplasmic fraction, and the pellet contained the nucleolar fraction, which was suspended in S3 buffer. All the obtained fractions were subjected to SDS-PAGE, followed by immunoblotting analysis for the presence of MVI and other marker proteins (GAPDH for the cytoplasm, and fibrillarin for the nucleoplasm and nucleolus) used as the internal loading control and indicators of fraction purity. The protein concentration was determined using the standard Bradford method.

To induce secretion, PC12 cells were cultured as described above and stimulated essentially according to Vitale et al. (1992) and Trifaró and Lee (1980). Treatment with 56 mM KCl is generally accepted as a method for in vitro PC12 cell stimulation as high concentrations of external KCl cause PC12 cell plasma membrane depolarization and evoke catecholamine release. Briefly, cells were washed with Locke’s solution containing 2.6 mM KCl, 154 mM NaCl, 2.2 mM CaCl₂, 0.5 mM KH₂PO₄, 1.25 mM K₂HPO₄, 1.2 mM MgCl₂, and 10 mM glucose. Then, they were incubated in Locke’s solution with elevated K⁺ concentration (56 mM KCl, 103.6 mM NaCl, 2.2 mM CaCl₂, 0.5 mM KH₂PO₄, 1.25 mM K₂HPO₄, 1.2 mM MgCl₂, and 10 mM glucose) to stimulate secretion or in calcium-free Locke’s solution (2.6 mM KCl, 154 mM NaCl, 0.5 mM KH₂PO₄, 1.25 mM K₂HPO₄, 1.2 mM MgCl₂, and 10 mM glucose) to block the secretion. Cells were further processed for post-embedding immunogold MVI localization.

To induce nucleolar stress, PC12 cells were treated with the Pol1 transcription inhibitor, actinomycin D (ActD) (Sigma-Aldrich, Cat. No A1410). Briefly, examined cells were incubated at 37°C for 3 h in the culture medium in the presence or absence of 0.05 μg/mL ActD and subjected to further analyses.

PC12 cell lysates and subcellular fractions were separated using 10% polyacrylamide SDS gels and then transferred to a nitrocellulose membrane (Bio-Rad, Cat. No 1620115). After the transfer, the membrane was blocked for 1 h at room temperature in TBS containing 5% non-fat milk powder or 5% BSA (Sigma-Aldrich, Cat. No A7906-150G) and 0.2% Triton X-100 followed by overnight incubation with appropriate dilutions (from 1:100 to 1:5,000) of different primary antibodies. The primary antibodies were detected using 1:10,000 dilutions of anti-rabbit (Millipore, Cat. No AP307P), anti-mouse (Millipore, Cat. No AP308P), or anti-donkey (Cat. No sc-2020, Santa Cruz Biotechnology) secondary antibodies conjugated with horse radish peroxidase. The reaction was developed using the ECL detection kit (Pierce, Cat. No 34095 and Millipore, Cat. No P90720). Usually, 10–20 μg of protein was loaded onto the gel. Band densitometry quantification was performed using the Fiji distribution of ImageJ 1.52a software (National Institutes of Health and the University of Wisconsin, Madison, WI, United States).

The distribution of MVI and other examined proteins in PC12 cells was evaluated by indirect immunocytochemistry. Cells on coverslips were fixed in 4% paraformaldehyde for 15 min, washed three times with phosphate-buffered saline (PBS) for 5 min, and blocked in a solution that contained 2% horse serum and 0.02% Triton X-100 in PBS for 1 h at room temperature. Coverslips were then incubated overnight at 4°C with rabbit polyclonal antibody to MVI, mouse monoclonal antibody to B23, rabbit polyclonal antibody to GRP78, mouse monoclonal antibody to fibrillarin, mouse monoclonal antibody to RPA 194 (Pol1), mouse monoclonal antibody to UBF, or mouse monoclonal antibody to S6 in a blocking solution and washed three times in PBS with 0.02% Triton X-100. This was followed by incubation with Alexa Fluor 488-conjugated anti-rabbit secondary antibody or Alexa Fluor 546-conjugated secondary anti-mouse antibody in a blocking solution for 60 min.

Finally, cells were washed three times in PBS with 0.02% Triton X-100 and mounted using VECTASHIELD PLUS Antifade Mounting Medium with DAPI. The specimens were visualized using a Zeiss LSM780 spectral confocal microscope equipped with a Plan-Apochromat 63x/1.40 Oil DIC M27 lens. In double immunostaining, special care was taken to control for any possible cross-reactivity (cross-bleeding) of the detection systems. We carefully adjusted the spectral ranges of detectors and always scanned the images sequentially. For negative controls, the primary antibody was omitted.

ER was visualized by staining with the ER-specific dye, ER Tracker™ Blue/White DPX (Thermo Fisher Scientific, Cat. No E12353), which is retained within the ER lumen, thus labeling the ER tubular network, according to the manufacturer’s instructions. Briefly, cells were seeded on glass coverslips and cultured for 24 h and then incubated for 30 min at 37°C and 5% CO₂ with 1 μM ER tracker diluted in the culture medium. Then, the stained cells were fixed with 4% formaldehyde for 10 min, washed in PBS, and mounted using VECTASHIELD PLUS Antifade Mounting Medium without DAPI. Images were collected with the Zeiss LSM780, inverted Axio Observer Z.1 equipped with the 63x/1.4 Oil Plan-Apochromat DIC objective. A diode laser of 405 nm was used to excite fluorescence. Optical sections (2048 pixels × 2048 pixels × 8 Bit/pixel) were collected. The images were processed using ZEN Blue 2.1 software.

PC12 cells were cultured on Thermanox™ coverslips (Electron Microscopy Sciences, Cat. No 72274) coated with poly-L-lysine (PLL, Electron Microscopy Sciences, Cat. No 19320) in RPMI-1640 medium supplemented with 10% HS, 5% FBS, and antibiotics: 1% penicillin/streptomycin or F12K medium supplemented with 2.5% FBS, 15% HS, and antibiotics: 1% penicillin/streptomycin, depending on the cell type. Cells were washed three times for 30 s each in PBS and fixed with 2% glutaraldehyde (GA) solution in PBS for 1 h at room temperature. Next, the fixed cells were washed three times for 10 min each in PBS, followed by post-fixation in 1% osmium tetroxide (OsO₄) for 30 min at room temperature (OsO₄ not only fixes but also provides contrast to lipid membranes). Sections were then rinsed twice for 10 min in PBS and twice for 5 min in deionized water, followed by dehydration in ethanol solutions of increasing concentrations in a so-called dehydration series, starting with 50% alcohol, followed by 70%, 80%, 90%, 96%, (5 min each) and anhydrous (99.8% absolute), twice for 15 min each, and then embedded in Spurr resin (Sigma-Aldrich, Cat. No EM0300) according to the standard protocol. The resin-submerged sections were cut into ultra-thin sections (60–70 nm thick) by using a diamond knife (Micro Star Technologies) and a Leica UTC ultramicrotome and collected on copper microscope grids (Electron Microscopy Sciences, Cat. No EMS400CU). The sections were stained with 2.5% uranyl acetate and 0.4% lead citrate and then examined by using a Joel EM 100 transmission electron microscope.

PC12 cells were grown on Thermanox™ coverslips, as described earlier. The cells were gently rinsed with PBS and fixed with 4% (v/v) formaldehyde and 0.25% (v/v) GA in the same PBS buffer for 1 h at room temperature. Fixed cells were washed three times with PBS, dehydrated in graded ethanol concentrations, and embedded in LR White resin (Electron Microscopy Sciences, Cat. No 14380) according to the standard protocol. Ultrathin sections were cut by using a diamond knife (Micro Star Technologies) and a Leica UTC ultramicrotome and collected on Formvar film-coated nickel grids (Electron Microscopy Sciences, Cat. No FCF400-Ni). The sections were then pretreated with 50 mM glycine in PBS for 10 min and incubated with a blocking solution containing 3% (w/v) bovine serum albumin (BSA) in PBS for 5 min at room temperature. Next, sections were placed in 1:50 dilution of a primary MVI antibody in PBS supplemented with 0.3% BSA for 2 h, followed by incubation with a gold-conjugated anti-rabbit IgG 15-nm secondary antibody (BB International, Cat. No R14003) at 1:100 dilution in PBS with 0.1% BSA for 30 min. Both incubations were carried out at room temperature. For the negative control, the primary antibody was omitted. Finally, the sections were stained with 2.5% uranyl acetate and examined on a JEOL JEM 1010 transmission electron microscope.

The fusion proteins composed of GST and MVI C-terminal globular tail domain (GST-MVI-GD) as well as GST alone were purified, as described by Majewski et al. (2012). For the lysate, cells were lysed in an ice-cold buffer that contained 50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, 1 mM DTT, 2 mM EGTA, 50 mM NaF, 1 mM Na₃VO₄, and 1 mM PMSF and supplemented with the cOmplete™ Protease Inhibitor Cocktail and phosphatase inhibitor PhosSTOP™. The assay was performed as described by Majewski et al. (2012). Briefly, the lysates were precleared with GST-bound Glutathione Sepharose 4B beads for 2 h at 4°C to remove proteins non-specifically binding to Glutathione Sepharose 4B and/or GST and subsequently incubated with Glutathione Sepharose 4B beads bound to GST-MVI-GD or GST alone for 4 h at 4°C. The beads were exhaustively washed in the ice-cold lysate buffer, described above, and subjected to SDS–PAGE electrophoresis followed by immunoblotting.

PC12 cells after fixation were blocked in the Duolink blocking solution in a humidity chamber for 30 min at 37°C and incubated with the following primary antibodies: rabbit polyclonal anti-MVI and mouse monoclonal antibodies anti-B23, anti-fibrillarin, anti-UBF, anti-Pol1, anti-p-S6, and anti-S6, diluted in Duolink Antibody diluent solution for 3 h at 37°C. Cells were next washed two times in a wash buffer for 5 min at room temperature. Next, secondary antibodies conjugated with oligonucleotides, PLA probe anti-mouse MINUS, and PLA probe anti-rabbit PLUS were applied in the Duolink antibody diluent solution for 1 h at 37°C and then washed twice for 5 min. The Duolink assay was further performed strictly according to the manufacturer’s instructions. For negative controls, the primary antibodies were omitted.

To perform co-immunoprecipitation, PC12 cells (CRL-1721) were lysed in a buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EGTA, 0.1% Triton X-100, 2 mM MgCl₂, 2 mM MgATP, 50 mM NaF, and 1 mM Na₃VO_4, supplemented with the cOmplete™ Protease Inhibitor Cocktail and phosphatase inhibitor PhosSTOP™. The lysates were pre-cleared with A/G agarose beads (Santa Cruz Biotechnology, Cat. No sc-2003) for 30 min at 4°C and subsequently incubated for 4 h at 4°C with 10 µg of the anti-MVI antibody (Proteus, Cat. No 25-6791) or non-immunized rabbit IgG (Santa Cruz Biotechnology, Cat. No sc-2027) as a control, followed by overnight incubation with the aforementioned agarose beads. Next, the beads were washed with the lysis buffer and then subjected to SDS–PAGE electrophoresis followed by immunoblotting with antibodies of interest to detect the co-immunoprecipitated complexes.

RNA was isolated from 5 × 10⁶ PC12 cells (scrambled and MVI-KD) using the RNeasy Plus Universal Mini Kit (Qiagen, Cat. No 73404) according to the manufacturer’s instructions. DNA contamination from RNA samples was removed through treatment with RNase-Free DNase I (Qiagen, Cat. No 79254). First-strand cDNA synthesis was performed using 1 µg of RNA and the SuperScript™ III Reverse Transcriptase Kit (Thermo Fisher Scientific, Cat. No 18080-093) with random hexamers. Quantitative PCR was performed using the Fast SYBR Green Master Mix (Applied Biosystems, Cat. No 4385612) with an Applied Biosystems 7900HT Fast Real-Time PCR System. The oligonucleotide primer sequence used for rRNA analysis included 45S pre-rRNA—forward: 5′-TGG​GGC​AGC​TTT​ATG​ACA​AC-3´; 45S pre-rRNA—reverse: 5′-TAG​CAC​CAA​ACG​GGA​AAA​CC-3´;

18S rRNA—forward: 5′GTT​GGT​TTT​CGG​AAC​TGA​GGC3’;

18S rRNA—reverse: 5′GTC​GGC​ATC​GTT​TAT​GGT​CG3’.

Pre-rRNA and 18S rRNA levels were quantified using the ΔΔ CT method (2^−ΔΔCT). Expression values were obtained from four independent experiments run in triplicates of each cDNA sample: 45S pre-rRNA relative to 18S rRNA (from the same cDNA preparations) (Kalita et al., 2008).

To identify a nucleolar localization signal (NoLS) within the MVI heavy chain, the NoD webserver was used (http://www.compbio.dundee.ac.uk/nod) (Scott et al., 2011).

All experiments were performed at least three times in two–three technical replicates. The results were expressed as means ± SD (standard deviation). If the data were normally distributed, we performed parametric two-tailed Student’s t-test or one-way ANOVA using GraphPad Prism 8.4.3 software (San Diego, CA, United States). Data that were non-normally distributed were analyzed with a nonparametric Mann–Whitney U-test to determine the significance. Statistical significance was defined as * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and ns for no statistical significance (p > 0.05).

We started the examination with identification of structural grounds for the presence of MVI in the nucleolus. For this, we performed an analysis using a nucleolar localization sequence detector, NoD, created on the basis of the data of 46 human-confirmed nucleolar localization signals (NoLS) (Scott et al., 2011). The analysis predicted the presence of one NoLS within the MVI heavy chain with the following sequence: FHRRLKVYHAWKSKNKKRNTETEQRAPKS (Figures 1A and B). This positively charged region with the pi value of 10.99 (calculated with a protein isoelectric point calculator, http://isoelectric.org) spans residues 1114 and 1142, situated within the MVI cargo-binding domain. Furthermore, it overlaps the bipartite nuclear localization signal (NLS) and contains both the RRL motif, involved in electrostatic interaction with MVI partners, and the positively charged region (WKSKNKKRN), involved in PIP₂ binding (Tumbarello et al., 2013; Spudich et al., 2007).

PC12 cell fractionation and immunogold staining showed that MVI is present in the nucleolar fraction (Figures 1C–E). Furthermore, analysis of immunogold staining revealed that the presence of MVI within the nucleolus is increased upon cell stimulation with 56 mM KCl (Figures 1D and E). Quantification of MVI-associated gold particles revealed that MVI localizes mainly to the granular component (GC) and dense fibrillar component (DFC), but the majority was present within the GC. This distribution does not depend on stimulation, as the same fraction of MVI is visible in both sub-compartments regardless of stimulation (Figure 1E).

To show whether MVI can interact with markers of nucleolar compartments, namely, UBF (upstream binding factor, marker of FC), fibrillarin (marker of DFC), and B23 (marker of GC), we performed a series of experiments using the immunoprecipitation (IP) as well as pull-down techniques, immunofluorescence staining, and proximity ligation assay (PLA) (Figure 2; Supplementary Figures S1, S2).

The pull-down assay with the MVI cargo-binding domain (GST-MVI-GD) as a bait, followed by immunoblotting, revealed that the selected marker proteins were present in the fractions precipitated with the MVI fragment and not with GST alone (Figure 2A). In addition, the analysis of the fractions co-IPed with the anti-MVI antibody (see Figure 2B) demonstrated the presence of the above-mentioned nucleolar proteins and MVI in the precipitates obtained upon incubation of cell lysates with the antibody but not with the control non-immune serum. It should be noted that the low yield of IP and pull-down assays for fibrillarin suggests that these interactions may be weak and/or transient.

Double immunostaining for MVI and the aforementioned nucleolar proteins showed their co-localization, further confirming their interaction with MVI (Figure 2C; Supplementary Figure S1).

To check whether these interactions also exist in cellulo, we employed the proximity ligation assay (PLA), designed for in situ detection of two proteins existing within close intracellular proximity (within the 20–40 nm range) (Soderberg et al., 2006). As shown in Figure 2D and Supplementary Figure S2A, red dots representing positive PLA signals indicative of the close proximity of two examined proteins were observed within the nucleus for UBF, fibrillarin, and B23. Thus, these data confirm the interaction of MVI with these proteins in situ. Positive signals in the perinuclear region were also detected for B23. Quantification of the number of PLA foci per nucleus revealed that the interaction does not take place in all the nuclei and varies between the examined proteins (Figure 2D; Supplementary Figure S2B). The highest fraction of “foci-positive” nuclei was observed for fibrillarin (∼65%), and the lowest for UBF (∼36%). As for B23, the fraction of “foci-positive” nuclei constitutes ∼57%, but in case of these interactions, we observed the highest fraction of the nuclei, which contained ≥3 foci (∼17%).

Since MVI is known to be involved in the organization of cytoskeletal compartments, and in PC12 cells (Majewski et al., 2011), we tested whether and how depletion (by ∼90%) of this molecular motor (Figure 3A) affects the organization of the nucleolus. For this, we employed transmission electron microscopy, which showed different phenotypes, with more or less defective nucleoli in MVI-depleted cells (Figure 3B; Supplementary Figure S3). In the presented examples, the nucleoli are disorganized, with structural defects in both the DFC and GC subdomains. Furthermore, in some of the images, identification of these nucleolar compartments was not possible at all.

Since depletion of MVI causes disorganization of the nucleolus, we checked whether its presence is required for preserving the association of the examined nucleolar proteins with their locations. For this, we performed immunostaining for UBF, fibrillarin, and B23 in MVI-KD cells (Figure 4; Supplementary Figures S4–S6). Furthermore, the morphological changes observed in the nucleoli of MVI-KD cells suggest that MVI depletion may evoke conditions resembling nucleolar stress to some extent.

Therefore, we incubated MVI-KD cells with the Pol1 inhibitor, ActD, at the concentration of 0.05 μg/mL, known to inhibit the activity of this polymerase only (Reich et al., 1961). Since there is no information on the effect of ActD on the nucleoli of PC12 cells, we visualized the effects of this Pol1 inhibitor using transmission electron microscopy on the nucleoli of the examined cells (Supplementary Figure S7). As expected, the ActD treatment caused a collapse of the nucleoli with a segregation of its fibrillar and granular components (Yang et al., 2018; Lafita-Navarro and Conacci-Sorrell, 2023). In addition, we showed that ActD at this concentration does not affect the content of MVI in the cytoplasm and nuclear fractions of control cells (Supplementary Figure S8).

As presented in Figure 4A and Supplementary Figure S4, depletion of MVI did not affect the localization of UBF, as in both scrambled and MVI-KD cells, this protein was dispersed within the nucleolus. In control cells treated with ActD, UBF localized to the nucleolar caps shaped around the nucleolar remnants (Yang et al., 2018). Localization of this protein in the ActD-treated MVI-KD cells did not substantially differ from that in the control counterparts. A similar observation was made for fibrillarin (Figure 4B; Supplementary Figure S5). Thus, depletion of MVI in the presence or absence of ActD does not affect the localization of UBF and fibrillarin. Furthermore, MVI knockdown does not affect the overall level of these two proteins (Supplementary Figure S9).

However, the examination of localization of B23 revealed the evident difference between the two tested conditions (Figure 4C; Supplementary Figure S6). While in scrambled cells, this protein mostly localized to the nucleolar periphery, in MVI-KD cells, it was present within the entire nucleolus. Treatment of both cell types with ActD caused delocalization of B23 to the nucleoplasm, but in MVI-depleted cells, incubated with the inhibitor, the nucleoplasmic staining for B23 was more prominent, and the protein was still present at the edges of the nucleolar remnants (Figure 4C; Supplementary Figure S6). Immunoblotting analysis did not reveal statistically significant changes in the level of B23 in MVI-KD lysates (Supplementary Figure S9).

Next, we decided to test whether depletion of MVI could affect Pol1-based transcription (see Figure 5; Supplementary Figures S10 and S11).

As shown in Figures 5A and B and Supplementary Figure 10, Pol1 co-localizes with MVI and interacts with this motor protein in situ, as revealed by the immunofluorescence and PLA assays. Quantification of the number of PLA foci per nucleus revealed that the Pol1–MVI interaction takes place in about 50% nuclei (Figure 5B).

In addition, the depletion of MVI did not have an evident effect on Pol1 distribution (Figure 5C; Supplementary Figure 11).

To test whether MVI could be involved in Pol1-based transcription, we used the q-RT-PCR technique to examine whether the depletion of MVI affects the synthesis of 45S pre-rRNA transcript (Kalita et al., 2008). As shown in Figure 5D, there is no significant change in the amount of the transcript in MVI-KD cells with respect to control cells, suggesting that MVI does not play a significant role in Pol1 activity.

Our previous results indicate that in PC12 cells, MVI not only localizes to the endoplasmic reticulum (ER) membranes but also interacts with ribosomal protein S6 (Majewski et al., 2018), which suggests that this molecular motor might be involved in the ribosome and/or ER organization. Furthermore, our current observation that MVI is important for the maintenance of the nucleolar organization makes this suggestion plausible.

The analysis of the TEM images revealed that in MVI-KD cells, the ER membranes are not only inflated but also less decorated with the ribosomes with respect to control scrambled cells (Figure 6A). In addition, live staining for the ER membranes with the ER Tracker showed profound changes in the ER organization in MVI-KD cells (Figure 6B, upper panels).

Immunostaining for GRP78, a key protein involved in protein folding and quality control in the ER (Kleizen and Braakman 2004), confirmed the disorganization/fragmentation of this membranous compartment in MVI-KD cells (Figure 6B, middle panels). Immunostaining for S6 (Figure 6B, bottom panels) revealed a very weak signal in MVI-KD cells. This is in contrast to our observation that the overall level of S6, similarly to GRP78, was not affected by MVI depletion (Figure 6C). This could be explained in terms that depletion of MVI might impair the availability of an S6 epitope in cellulo. We also examined the level of p70S6K (and its active, phosphorylated form, p-p70S6K), a kinase involved in regulation of S6 activity. As presented in Figure 6C, the ratio of p-p70S6K to p70S6K level is not affected by MVI depletion, but the ratio of p-S6 to S6 is decreased, indicating that the activity of S6 is associated with MVI.