Nucleolar Architecture Is Modulated by a Small Molecule, the Inositol Pyrophosphate 5-InsP7

Inositol pyrophosphates (PP-InsPs); are a functionally diverse family of eukaryotic molecules that deploy a highly-specialized array of phosphate groups as a combinatorial cell-signaling code. One reductive strategy to derive a molecular-level understanding of the many actions of PP-InsPs is to individually characterize the proteins that bind them. Here, we describe an alternate approach that seeks a single, collective rationalization for PP-InsP binding to an entire group of proteins, i.e., the multiple nucleolar proteins previously reported to bind 5-InsP7 (5-diphospho-inositol-1,2,3,4,6-pentakisphosphate). Quantitative confocal imaging of the outer nucleolar granular region revealed its expansion when cellular 5-InsP7 levels were elevated by either (a) reducing the 5-InsP7 metabolism by a CRISPR-based knockout (KO) of either NUDT3 or PPIP5Ks; or (b), the heterologous expression of wild-type inositol hexakisphosphate kinase, i.e., IP6K2; separate expression of a kinase-dead IP6K2 mutant did not affect granular volume. Conversely, the nucleolar granular region in PPIP5K KO cells shrank back to the wild-type volume upon attenuating 5-InsP7 synthesis using either a pan-IP6K inhibitor or the siRNA-induced knockdown of IP6K1+IP6K2. Significantly, the inner fibrillar volume of the nucleolus was unaffected by 5-InsP7. We posit that 5-InsP7 acts as an ‘electrostatic glue’ that binds together positively charged surfaces on separate proteins, overcoming mutual protein–protein electrostatic repulsion the latter phenomenon is a known requirement for the assembly of a non-membranous biomolecular condensate.

Several biological activities have been attributed to the PP-InsPs. A few examples serve to illustrate this functional diversity: 1,5-InsP8 supervises the inorganic phosphate (Pi) balance in both plants [5] and animals [6]; this is metabolically critical not only because Pi is a fundamental component of the cell's "energy currency" (i.e., ATP), but also because Pi is one of the most pervasive regulators of basic cellular metabolism [7]. Other studies have demonstrated that 1,5-InsP8 exerts control over cellular nucleotide synthesis, with sometimes dramatic consequences for cellular proliferation [8]. As for 5-InsP7, it regulates PolI-mediated rRNA transcription by Saccharomyces cerevisiae [9]. In mammals, 5-InsP7 regulates insulin secretion from pancreatic beta-cells [10,11], licenses high-fat diet-induced obesity [12], modulates the degradation of the plasma membrane sodium-potassium pump [13], and it inhibits mRNA decapping and promotes the accumulation of Processing (P-) bodies [14,15].  6 and PP-InsPs. The graphic shows the proposed cyclical pathway for their interconversion in mammalian cells [3]. Mammalian cells express three isoforms of IP6Ks (types 1,2, and 3) and two PPIP5Ks (types 1 and 2). The latter each possess a kinase domain that phosphorylates 5-InsP 7 to InsP 8 and a separate phosphatase domain that dephosphorylates InsP 8 back to 5-InsP 7 . NUDT3 (also known as DIPP1) is one of five mammalian NUDT isoforms in mammals that removes β-phosphates from both 5-InsP 7 and InsP 8 . Note that little 1-InsP 7 accumulates in HCT116 cells [4]. (B). Structure of metabolically resistant 5-PCP-InsP 7 .
Several biological activities have been attributed to the PP-InsPs. A few examples serve to illustrate this functional diversity: 1,5-InsP 8 supervises the inorganic phosphate (Pi) balance in both plants [5] and animals [6]; this is metabolically critical not only because Pi is a fundamental component of the cell's "energy currency" (i.e., ATP), but also because Pi is one of the most pervasive regulators of basic cellular metabolism [7]. Other studies have demonstrated that 1,5-InsP 8 exerts control over cellular nucleotide synthesis, with sometimes dramatic consequences for cellular proliferation [8]. As for 5-InsP 7 , it regulates PolI-mediated rRNA transcription by Saccharomyces cerevisiae [9]. In mammals, 5-InsP 7 regulates insulin secretion from pancreatic beta-cells [10,11], licenses high-fat diet-induced obesity [12], modulates the degradation of the plasma membrane sodiumpotassium pump [13], and it inhibits mRNA decapping and promotes the accumulation of Processing (P-) bodies [14,15].
The molecular basis for some of the actions of PP-InsPs involves the modification of target protein functions by the donation of the molecules' β-phosphoryl groups in a non-enzymic reaction known as protein pyrophosphorylation [16][17][18]. However, in part, due to the synthesis and application of metabolically resistant PP-InsPs such as 5-PCP-InsP 7 (5-methylene-diphosphonate inositol 1,2,3,4,6-pentakisphosphate; Figure 1B), it is known that their non-covalent interactions with proteins expand the repertoire of PP-InsP signaling mechanisms [19]. Two examples of such mechanisms have emerged to date. First, the binding of PP-InsPs to certain PH domains can regulate the intracellular distribution of the host proteins [20][21][22]. The only other characterized PP-InsP-binding module is the SPX domain, but this is thought to be present in just one mammalian protein: XPR1 [6,23,24]. The latter is itself only known to have one function, namely, the regulation of the cellular efflux of Pi [6,23,25]. Consequently, it is thought that the rationalization of the functional diversity of this cell-signaling family depends upon the identification of other PP-InsP binding proteins; therefore there are ongoing efforts to catalog the entire PP-InsP interactome [19,26]. This approach has uncovered a bewilderingly large number of proteins that can associate with PP-InsPs, either directly or indirectly [19,26]. This situation is unprecedented for any other small-molecule intracellular signal, and its significance has not previously been rationalized.
Our goal has been to resolve this paradox in the current study. As an alternative to the reductive approach of attaching functional significance to the binding of a PP-InsP to a single protein, we have asked a question from a systems biology perspective: could there be a single, collective response to PP-InsP binding for an entire group of proteins? To find a potential answer, we have focused on the nucleolus, which contains a particularly high concentration of a variety of 5-InsP 7 -binding proteins, including components of RNA polymerase I complex, RNA polymerase III complex, and small nucleolar ribonucleoproteins [19,26,27]. Indeed, Arg-and Lys-rich sequences recur in nucleolar targeting motifs [28,29], thereby rendering all such proteins as candidates for ionic interactions with 5-InsP 7 , which has nine negative charges at a physiological pH. It has further been hypothesized that PP-InsPs can act as an 'electrostatic glue' that binds together positively charged surfaces on two separate proteins [2]. On a global scale, such a phenomenon could overcome protein-protein electrostatic repulsion and strongly elevate local protein concentrations, both of which are requirements for promoting the assembly of non-membranous biomolecular condensates [30]. Since the nucleolus is an important example of just such a phase-separated structure, we have hypothesized that the nucleolar volume might be increased by experimental procedures that elevate cellular 5-InsP 7 levels. Herein we describe experiments that confirm that proposal.

Loading of 5-PCP-InsP 7 into Cells Using Liposomes
Liposomes were prepared as 8 mg of lipid film inside a round-bottom glass flask, exactly as previously described [31]. As needed, the lipid film was hydrated (with vortexing) by the addition of 2 mL of 5 mM HEPES (pH 7.2 with NaOH) plus 0.25 µM 5-PCP-InsP 7 . A corresponding lipid film was prepared without the addition of 5-PCP-InsP 7 (control liposomes). The liposomal dispersion was subjected to 5-10 freeze/thaw cycles: freezing at −80 • C for 30 min and thawing at 45 • C for 5 min. The liposomes were then sequentially extruded through two membrane filters, with pore sizes of 0.45 um and then 0.2 µm. The liposomes were stored in aliquots at 4 • C for up to 2 weeks. Cells (0.6 to 0.7 × 10 6 /well, plated on coverslips in 6-well MatTek dishes) were treated for 4 h with 2 µL liposomes in Pi-free DMEM plus 10% FBS.

Lentiviral Transduction of HCT116 Cells
The pCDH-CMV-MCS-EF1a-CopGFP-T2A-Puro vector was purchased from System Biosciences (Catalog # CD513B-1). This vector was reengineered to form a pCDH-CMV-EF1a-MCS-P2A-CopGFP-T2A-Puro vector that enabled the insertion of genes under the control of the EF1a promoter. This vector was created by replacing the Xba I/Pst I fragment of the vector with a synthetic fragment from Genewiz Inc. that hosts EF1a-MCS-P2A-CopGFP. The full-length wild-type IP6K2 or kinase-dead IP6K2 K222A [32] was PCR amplified and cloned into the XhoI/BamHI site in the multiple cloning site of pCDH-CMV-EF1a-MCS-P2A-CopGFP-T2A-Puro.
All lentivirus were packaged and titered in HEK293T/17 cells (ATCC # CRL-11268) according to the published methods [33]. Briefly, 293T cells were transiently transfected with pMD2G, psPAX2, and a transfer vector containing the desired gene using Lipofectamine 2000. The supernatant was collected 48 h post-transfection and concentrated by centrifugation at 50,000× g for 2 h. Pellets were resuspended in PBS, aliquoted, and stored at −70 • C. All titers were determined by performing a digital droplet PCR to measure the number of lentiviral particles that integrated into the host genome. This titration was confirmed by using flow cytometry to monitor the co-expressed fluorescent moieties.
Lentiviral aliquots (MOI = 60) were added to HCT116 cells that had been plated at 0.1 × 10 6 cells/well in a 6-well plate and cultured for 48 h, then were transferred into a virus-free medium for a further 48 h. Transduced cells were selected with puromycin (Thermofisher catalog number: A1113803, 2.5 µg/mL) and further confirmed by GFP fluorescence, i.e., images were obtained as 8x8 TILE SCANNING using a Zeiss Colibri epifluorescence microscope, and then further stitched using the Zeiss Zen STITCH function.

Western Blotting and Analysis
Cells were harvested and rinsed with ice-cold PBS; then, a protein extract was prepared using a RIPA Lysis and Extraction Buffer (ThermoFisher Scientific catalog number: 89901), supplemented with 1% (vol/vol) protease-phosphatase inhibitor cocktail (ThermoFisher Scientific catalog number: 78442). After quantification with a Pierce BCA protein assay kit (ThermoFisher Scientific catalog number: 23221), 25 µg of each protein extract was resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with either rabbit anti-NUDT3 antibody (Thermofisher PA5-30435) or mouse anti-β-actin antibody (SantaCruz sc-47778, 1:5000). The secondary antibodies were either anti-rabbit IgG (Invitrogen 31460, 1:5000) or anti-mouse IgG (Cell Signaling Technology, 7076S, 1:5000). The immunoblots were developed using a SuperSignal™ West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific catalog number: 34580) for HRP-conjugated secondary antibodies. All the blots were scanned using a Li-Cor Odyssey ® Fc imaging system and software (Li-Cor Biosciences). Densitometric analysis of the protein bands was performed using ImageJ (v 1.51j8).

Sucrose Density Gradient Fractionation of Ribosome Subunits
Cells were grown on two 150mm tissue culture dishes at approximately 80 % confluence. All growth media was then removed by aspiration, and the cells were rinsed with ice-cold PBS. After rinsing, PBS supplemented with cycloheximide at a concentration of 100 µg/mL was added to the dishes, which were then incubated at 37 • C for 15 min. The cells were harvested by scraping adherent cells from the plate and transferring them to a centrifuge tube which was then placed on the ice. Cells were spun down at 2000 rpm for 5 min. The cell pellet was then transferred to a 1.5 mL centrifuge tube. Cells were again pelleted, and all remaining PBS was then removed. The cell pellet was then resuspended in the Polysome Extraction Buffer (20 mM Tris-HCl pH 7.4, 60 mM KCl, 10 mM MgCl2, 1 mM DTT, 1% [v/v] Triton X-100, 0.1 mg/mL cycloheximide, 0.2 mg/mL heparin) supplemented with RNAse Inhibitor. Cells were nutated at 4 • C for one hour and then spun at 13,000 rpm at 4 • C for 30 min. RNA from cell lysate was quantitated using a Qubit fluorometer (Invitrogen) following the manufacturer's protocol. A total of 150 ug of RNA was loaded onto a 7-47% sucrose gradient. Sucrose gradients were spun at 260,110× g at 4 • C for 2.5 h. The gradients were then fractionated using the Brandel BR-188 Density Gradient Fractionation System with a UV absorbance monitored at 254 nm.

Total RNA Analysis by Automated Electrophoresis
Cells were counted upon harvesting to ensure an equal number of cells per sample for the total RNA extraction as described above, and each sample was resuspended in 40 µl. RNA stocks were stored at −80 • C before use. Total ribosomal RNA was estimated by the quantification of the two largest mature rRNAs [36], 18S and 28S, in mammals, using a Bioanalyzer Automated Electrophoresis system (Agilent Technologies) with an RNA 6000 Nano Chip. Briefly, aliquots of the total RNA stocks were diluted with equal ratios to bring the sample concentration into the quantitative range of the Nano Chip (25-500 ng/uL). Nano Chip gel was prepared per instructions, stored at 4 • C, and used within 4 weeks of preparation. Gel-dye was added to the gel per instructions and immediately loaded into the Nano Chip before the loading sample. Diluted RNA samples were heat denatured at 70 • C for 2 min and immediately returned to ice before loading 1 uL of diluted sample into the Nano chip. Each sample was loaded on the chip in quadruplets for every biological sample; throughout the entire study, just one technical replicate exhibited a RIN number < 9.0, and it was discarded. The RNA assay data were manually imported into Excel for further analysis.

Nucleolar Granular Volume Is Elevated in PPIP5K KO HCT116 Cells
It has previously been reported that the CRISPR-based knockout of the PPIP5Ks interrupts the PP-InsP metabolic cycle ( Figure 1A), leading to an elevation in 5-InsP 7 levels in HCT116 cells; there is no off-target impact on the separate metabolic pathway to InsP 6 synthesis [14,37]. Thus, we investigated the impact of the PPIP5K KO on nucleolar architecture. We first recorded the nucleolar granular volume in the intact cells. Previous studies have quantified the size of this nucleolar compartment by staining protein markers such as nucleostemin (NSM) and nucleophosmin [38][39][40]. The analysis of endogenously expressed proteins avoids potential artifacts that might arise from the heterologous expression of fluorescently tagged nucleolar proteins. We recruited endogenous NSM to record the nucleolar granular volume in wild-type and PPIP5K KO HCT116 cells.

The KO of PPIP5Ks Does Not Affect rRNA Synthesis
There are a considerable number of published studies that testify to an inexorable link between nucleolar morphology and ribosome biogenesis (e.g., [41]). The nucleolar fibrillar compartment is the site of rRNA transcription. The volume of this compartment was not affected by the PPIP5K KO, as measured by the staining of the RNA polymerase upstream binding factor, UBF ( Figure 2D-F). Furthermore, the PPIP5K KO was not accompanied by any significant change in the cellular rRNA content, as determined by automated electrophoresis ( Figure 3A). The PPIP5K KO also did not affect either 28S:18S rRNA ratios ( Figure 3B) or 60S:40S ribosomal subunit ratios ( Figure 3C). Thus, we con- To analyze the morphological consequences for the nucleolus in the PPIP5K KO cells, we combined confocal laser scanning microscopy with Airyscan to acquire two-and threedimensional images, respectively (Figure 2A,B). In two independent clones of PPIP5K KO cells (KO1 and KO2), the total granular volume (as a ratio to the nuclear volume) was calculated to be 85% larger in both KO1 and KO2 lines ( Figure 2C). By contrast, the DAPI-stained nuclear volume was very similar to that of the wild-type cells (see the legend in Figure 2).

The KO of PPIP5Ks Does Not Affect rRNA Synthesis
There are a considerable number of published studies that testify to an inexorable link between nucleolar morphology and ribosome biogenesis (e.g., [41]). The nucleolar fibrillar compartment is the site of rRNA transcription. The volume of this compartment was not affected by the PPIP5K KO, as measured by the staining of the RNA polymerase upstream binding factor, UBF ( Figure 2D-F). Furthermore, the PPIP5K KO was not accompanied by any significant change in the cellular rRNA content, as determined by automated electrophoresis ( Figure 3A). The PPIP5K KO also did not affect either 28S:18S rRNA ratios ( Figure 3B) or 60S:40S ribosomal subunit ratios ( Figure 3C). Thus, we conclude that elevated 5-InsP 7 levels do not significantly alter ribosome biogenesis in HCT116 cells.

The Impact of NUDT3 KO upon Nucleolar Granular Volume
Mammalian cells express five isoforms of the NUDT/DIPP phosphatases ( Figure 1A); we are not aware of any published studies with mammalian cells that have assayed the impact upon PP-IP levels for knocking out any of these phosphatases. However, kinetic studies with recombinant enzymes indicate that NUDT3/DIPP1 has the highest catalytic efficiency [42], and so we reasoned that cells in which the corresponding gene knocked out would show elevated levels of 5-InsP7, thereby yielding another method to study the impact of this PP-InsP upon the nucleolus.
We have used CRISPR to create two independent clones of HCT116 NUDT3 KO cells (namely KOα and KOβ; Figure 4A). The HPLC analysis of [ 3 H]inositol-labeled cells showed that both clonal lines contained 40% higher levels of 5-InsP7 compared to WT cells ( Figure 4B,C). This has no significant impact on InsP6, which is not surprising as its levels are approximately 20-fold higher than those of 5-InsP7 ( Figure 4B). This is why we followed a usual practice in the field, which is to consider InsP6 as an internal standard to which levels of 5-InsP7 and InsP8 can be normalized. Significantly, in NUDT3 KO cells, the nucleolar granular volume was about two-fold higher than that of wild-type cells ( Figure  4E,F). This KO did not alter InsP8 levels ( Figure 4B,D), which is consistent with the previous kinetic analysis of PPIP5Ks' separate kinase and phosphatase activities that predict cellular InsP8 levels to be relatively insensitive to changes in 5-InsP7 concentration [43].

The Impact of NUDT3 KO upon Nucleolar Granular Volume
Mammalian cells express five isoforms of the NUDT/DIPP phosphatases ( Figure 1A); we are not aware of any published studies with mammalian cells that have assayed the impact upon PP-IP levels for knocking out any of these phosphatases. However, kinetic studies with recombinant enzymes indicate that NUDT3/DIPP1 has the highest catalytic efficiency [42], and so we reasoned that cells in which the corresponding gene knocked out would show elevated levels of 5-InsP 7 , thereby yielding another method to study the impact of this PP-InsP upon the nucleolus.
We have used CRISPR to create two independent clones of HCT116 NUDT3 KO cells (namely KOα and KOβ; Figure 4A). The HPLC analysis of [ 3 H]inositol-labeled cells showed that both clonal lines contained 40% higher levels of 5-InsP 7 compared to WT cells ( Figure 4B,C). This has no significant impact on InsP 6 , which is not surprising as its levels are approximately 20-fold higher than those of 5-InsP 7 ( Figure 4B). This is why we followed a usual practice in the field, which is to consider InsP 6 as an internal standard to which levels of 5-InsP 7 and InsP 8 can be normalized. Significantly, in NUDT3 KO cells, the nucleolar granular volume was about two-fold higher than that of wild-type cells ( Figure 4E,F). This KO did not alter InsP 8 levels ( Figure 4B,D), which is consistent with the previous kinetic analysis of PPIP5Ks' separate kinase and phosphatase activities that predict cellular InsP 8 levels to be relatively insensitive to changes in 5-InsP 7 concentration [43].

The Impact upon Nucleolar Granular Volume of Changes in Cellular IP6K Activity
We increased the capacity of HCT116 cells to synthesize 5-InsP7 through the overexpression of IP6K2. Because IP6Ks have both catalytic and non-catalytic (scaffolding) roles, it is usual to compare the effects of wild-type enzymes versus a catalytically dead To further interrogate the relationship between cellular 5-InsP 7 levels and nucleolar granular volume, we next performed a series of independent experiments in which we directly modulated the levels of this PP-IP by using several independent protocols ( Figure 5A): the viral transduction of IP6K constructs, the pharmacological inhibition of IP6Ks, siRNA against IP6Ks, or delivery into cells of a metabolically stable 5-InsP 7 analog using lipid nanoparticles. This application of multiple protocols represents a particularly robust approach to the identification of PP-InsP functionality.

The Impact upon Nucleolar Granular Volume of Changes in Cellular IP6K Activity
We increased the capacity of HCT116 cells to synthesize 5-InsP 7 through the overexpression of IP6K2. Because IP6Ks have both catalytic and non-catalytic (scaffolding) roles, it is usual to compare the effects of wild-type enzymes versus a catalytically dead construct, along with fluorescent probes to monitor the expression [15]. Thus, in our experiments, we transduced HCT116 cells with lentivirus hosting either wild-type human IP6K2 or a catalytically dead IP6K2 K222A mutant [32] (Figure 5A,B). The co-expression of copGFP was tracked to the success of the transduction ( Figure 5B). We found that the NSM volume was substantially elevated by wild-type IP6K2 but not by the kinase-dead mutant ( Figure 5B,C), thereby demonstrating that the size of the nucleolar granular compartment correlates with the catalytic activity of this kinase (i.e., 5-InsP 7 synthesis).
Biomolecules 2023, 13, x FOR PEER REVIEW 10 of 15 construct, along with fluorescent probes to monitor the expression [15]. Thus, in our experiments, we transduced HCT116 cells with lentivirus hosting either wild-type human IP6K2 or a catalytically dead IP6K2 K222A mutant [32] ( Figure 5A,B). The co-expression of copGFP was tracked to the success of the transduction ( Figure 5B). We found that the NSM volume was substantially elevated by wild-type IP6K2 but not by the kinase-dead mutant ( Figure 5B,C), thereby demonstrating that the size of the nucleolar granular compartment correlates with the catalytic activity of this kinase (i.e., 5-InsP7 synthesis).
In separate experiments, we strongly reduced 5-InsP7 synthesis by treating wild-type and PPIP5K HCT116 KO cells for 18 hr with 10 µM of the pan-IP6K inhibitor, TNP, exactly as previously described for these cell types [14,31]. This pharmacological approach completely reversed the increase in the NSM volume that is normally observed in PPIP5K KO cells ( Figure 5D). Another important aspect of this particular experiment is that PPIP5K KO cells cannot synthesize 1-InsP7 nor InsP8 [31]. Thus, the impact of TNP upon the nucleolar volume in these cells must be independent of these two PP-InsPs. We also decreased IP6K activity in HCT116 cells by the siRNA-mediated knockdown of IP6K1 plus IP6K2 exactly as previously described for these cells [14]. This IP6K1/IP6K2 knockdown in PPIP5K KO cells shrank the NSM volume to that observed in wild-type cells ( Figure  5E).

Nucleolar Granular Volume Is Expanded by Liposomal Delivery of Metabolically Stable 5-PCP-InsP7
We recently described a procedure by which PP-InsPs could be encapsulated into liposomes for delivery into the cytoplasm of intact HCT116 cells [6,14]. It has been determined that liposomes will release their cargo into the cytoplasm following their endocytosis and delivery into tubular recycling endosomes [44]. We used this same procedure ( Figure 5A) to deliver metabolically stable 5-PCP-InsP7 [45] into wild-type HCT116 cells.
In these experiments, nucleolar granular volume was elevated approximately two-fold ( Figure 5F). This is a particularly significant result because five-PCP-InsP7 cannot support protein pyrophosphorylation, which is one molecular mechanism by which PP-IPs can modify protein function [16,18]. Thus, we conclude that 5-InsP7 may regulate nucleolar volume through non-covalent interactions with appropriate target proteins [46].

Discussion
The main accomplishment of the current study has been to demonstrate that the granular portion of the nucleolus responds to multiple orthogonal, chemical, and genetic procedures, which all have in common the ability to manipulate 5-InsP7 levels in intact cells. For example, we show that the granular, NSM-positive volume expands in response to elevations in the 5-InsP7 accumulation brought about by the CRISPR-based KO of either All data are mean values ± standard errors from three experiments. ** p < 0.01, *** p < 0.001. (G) A hypothetical electrostatic 'glue' to locally elevate protein concentration for promoting the formation of biomolecular condensates. Both 5-InsP 7 (drawn as a space-filling model) and proteins-1 and -2 are depicted as electrostatic surface plots with the intensity of blue and red coloration denoting the degree of positive and negative electrostatic potentials at physiological pH. Any similarity to real protein structures is purely coincidental.
In separate experiments, we strongly reduced 5-InsP 7 synthesis by treating wild-type and PPIP5K HCT116 KO cells for 18 hr with 10 µM of the pan-IP6K inhibitor, TNP, exactly as previously described for these cell types [14,31]. This pharmacological approach completely reversed the increase in the NSM volume that is normally observed in PPIP5K KO cells ( Figure 5D). Another important aspect of this particular experiment is that PPIP5K KO cells cannot synthesize 1-InsP 7 nor InsP 8 [31]. Thus, the impact of TNP upon the nucleolar volume in these cells must be independent of these two PP-InsPs. We also decreased IP6K activity in HCT116 cells by the siRNA-mediated knockdown of IP6K1 plus IP6K2 exactly as previously described for these cells [14]. This IP6K1/IP6K2 knockdown in PPIP5K KO cells shrank the NSM volume to that observed in wild-type cells ( Figure 5E).

Nucleolar Granular Volume Is Expanded by Liposomal Delivery of Metabolically Stable 5-PCP-InsP 7
We recently described a procedure by which PP-InsPs could be encapsulated into liposomes for delivery into the cytoplasm of intact HCT116 cells [6,14]. It has been determined that liposomes will release their cargo into the cytoplasm following their endocytosis and delivery into tubular recycling endosomes [44]. We used this same procedure ( Figure 5A) to deliver metabolically stable 5-PCP-InsP 7 [45] into wild-type HCT116 cells. In these experiments, nucleolar granular volume was elevated approximately two-fold ( Figure 5F). This is a particularly significant result because five-PCP-InsP 7 cannot support protein pyrophosphorylation, which is one molecular mechanism by which PP-IPs can modify protein function [16,18]. Thus, we conclude that 5-InsP 7 may regulate nucleolar volume through non-covalent interactions with appropriate target proteins [46].

Discussion
The main accomplishment of the current study has been to demonstrate that the granular portion of the nucleolus responds to multiple orthogonal, chemical, and genetic procedures, which all have in common the ability to manipulate 5-InsP 7 levels in intact cells. For example, we show that the granular, NSM-positive volume expands in response to elevations in the 5-InsP 7 accumulation brought about by the CRISPR-based KO of either NUDT3 or PPIP5Ks (Figures 2 and 4); through the heterologous expression of wild-type IP6K2 (but not through the expression of the kinase-dead mutant, Figure 5B,C) the delivery into cells of 5-PCP-InsP 7 was achieved ( Figure 5F). Conversely, the nucleolar volume can be returned to the level seen in wild-type cells upon treatment with PPIP5K KO cells with either the pan-IP6K inhibitor TNP ( Figure 5D), or by siRNA-induced knockdown of IP6K1+IP6K2 ( Figure 5E). Our observation that 5-InsP 7 influences the granular but not the fibrillar volume speak to the specificity of the action, which could be explained by functional interactions of this PP-InsP primarily with granular proteins. This is an unprecedented demonstration that nucleolar architecture is modulated by a naturally occurring messenger molecule. Proteins and other macromolecules have been the focus of previous studies into electrostatic interactions that contribute to the dynamic regulation of the assembly, function, and disassembly of biomolecular condensates [47,48]. However, the 5-InsP 7 molecule has an exceptional negative charge density and has previously been demonstrated to associate electrostatically with multiple nucleolar proteins [19,26,27]. Indeed, 5-InsP 7 may act as an 'electrostatic glue' that binds together positively charged surfaces on two separate proteins ( Figure 5G and [2]). At a nucleolar level, such a phenomenon could overcome general protein-protein electrostatic repulsion and strongly elevate local protein concentrations, both of which are requirements for promoting the assembly of non-membranous biomolecular condensates [30]. It is this process, we propose, that can explain how an increase in 5-InsP 7 levels facilitates the expansion of the nucleolar granular compartment. This hypothesis takes PP-InsP research into a new direction by its emphasis on the collective significance of multiple ligand-protein interactions, as opposed to prior studies that attach separate biological outcomes of 5-InsP 7 binding to individual proteins. This electrostatic model could, in theory, also apply to the other two mammalian PP-InsPs, 1-InsP 7, and 1,5-InsP 8 ( Figure 1). However, we deem this as an unlikely scenario, as these molecules are 10-and 50-fold less abundant than 5-InsP 7 [3]. Moreover, both 1-InsP 7 and 1,5-InsP 8 cannot be synthesized by our PPIP5K KO cell line, which is used in the experiments described in Figure 5. We also do not exclude the possibility that the unique stereochemistry of 5-InsP 7 may contribute to its regulation of nucleolar morphology.
The further biological significance of our study arises from it providing a new perspective on the impact of dynamic changes in the nucleolar volume, which have previously largely focused on the consequences for rRNA synthesis and ribosome biogenesis [41,47,49]. The rRNA is transcribed in the nucleolar fibrillar compartment; its volume was not affected by changes in the 5-InsP 7 levels ( Figure 2D,E,F). Moreover, we have shown that neither rRNA synthesis nor ribosome biogenesis is affected by elevations in the levels of 5-InsP 7 (Figure 3).
In previously reported experiments with the yeast S. cerevisiae [9], rRNA synthesis was impaired upon the elimination of all IP6K activity following the deletion of the Kcs1 gene. The latter study concluded that the Kcs1∆ yeast strain exhibited a defect in transcriptional elongation due to the loss of the 5-InsP 7 -mediated pyrophosphorylation of PolI [9]. We cannot exclude the possibility that this PP-InsP may have a similar, constitutive role that maintains rRNA synthesis in mammals, because. In our study we did not completely eliminate 5-InsP 7 synthesis from HCT116 cells. In any case, the mechanisms by which an elevation in 5-InsP 7 levels regulates the nucleolar volume in the current study must involve a separate process that is independent of protein pyrophosphorylation, since it is recapitulated by the metabolically stable analog, 5-PCP-InsP 7 ( Figure 5E).
There are indications that our findings may have human health relevance. For example, a non-canonical function for the granular compartment of the nucleolus is homeostatic responses to certain cellular stresses (e,g. heat shock) [40]. Separately, it would appear to be detrimental for cells to exhibit sustained elevations in their nucleolar volume, which is inversely correlated with longevity [49]. Indeed, nucleolar size trends are smaller in humans if they adopt a healthier lifestyle and calorific reduction [49]. Pathological interest in the nucleolus is also concerned with neurodegeneration-related cellular events and cancerrelated pathways [28]. Significantly, aging is associated with both an elevated nucleolar volume [47,49] and higher steady-state levels of 5-InsP 7 [50]; these two phenomena are directly linked in the current study.

Data Availability Statement:
The data that support the findings of this study are all provided within the body of the manuscript.