Transcriptome, proteome, and protein synthesis within the intracellular cytomatrix

Summary Despite the knowledge that protein translation and various metabolic reactions that create and sustain cellular life occur in the cytoplasm, the structural organization within the cytoplasm remains unclear. Recent models indicate that cytoplasm contains viscous fluid and elastic solid phases. We separated these viscous fluid and solid elastic compartments, which we call the cytosol and cytomatrix, respectively. The distinctive composition of the cytomatrix included structural proteins, ribosomes, and metabolome enzymes. High-throughput analysis revealed unique biosynthetic pathways within the cytomatrix. Enrichment of biosynthetic pathways in the cytomatrix indicated the presence of immobilized biocatalysis. Enzymatic immobilization and segregation can surmount spatial impediments, and the local pathway segregation may form cytoplasmic organelles. Protein translation was reprogrammed within the cytomatrix under the restriction of protein synthesis by drug treatment. The cytosol and cytomatrix are an elaborately interconnected network that promotes operational flexibility in healthy cells and the survival of malignant cells.


INTRODUCTION
The central problem of cell biology is the organization of biochemical reactions and organelles in the cytoplasm and how organelles need to be assembled in the cytoplasmic space after cell division or depending on cell conditions. Since the 19 th century, scientists have made substantial efforts to understand the physical and chemical properties of the cytoplasm, and debated the nature of the cytoplasm. 1 Still, the structural organization of the cytoplasm remains a contentious topic. For the past hundred years since its inception, the concept that cell cytoplasm is a gel-like substance has been broadly acknowledged. 2 However, the mechanisms of chemical reaction in the cell are routinely explained based on the idea of free diffusion, as if the cells were an aqueous solution. 3 The cytoplasm is a dynamic, crowded, and heterogeneous environment, but this concept gives little help in understanding the cytoplasmic structure and organization. The cytoplasm is involved in protein synthesis, energy generation, nutrient processing, transport, and metabolism, which also contributes to nuclear processes in response to external stimuli or stressors. Capable of managing a myriad of chemical reactions, the structure of the cytoplasm is complex, with enzymes and metabolic pathways organized into clusters, 4 with little space available for soluble species to roam. 5 Current theories suggest that the cytomatrix is an elastic solid within which intermediate filaments anchor organelles-interdependent structures that communicate through contact sites 6 -against fluctuating cytosolic forces. 7 Further, the structure of a large portion of the intracellular water is dictated by interactions with the cell ultrastructure, driving changes in cytoplasmic states from a viscous fluid to an elastic solid. 8 The cytomatrix (CMX) is thought to be a proteinaceous matrix that forms a network with the cytoskeleton to integrate metabolic pathways. 1 The CMX is traversed by channels and delimited by microtubules throughout which it associates with organelles, ER ribosomes, and metabolic protein complexes. 9 Structural proteins are also an integral part of the cytoplasmic matrix, wherein the nucleus and cytoplasm are connected through the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. [10][11][12] The integrity of protein structure and macromolecular complexes within the CMX are supported by the ionic Since it has been established that Li + interacts with peptide groups, 22 lithium salts have been widely investigated as a member of the Hofmeister series. In model polymers such as oligoglycines, which are insoluble in water, lithium salts increase the solubility of polyglycine, a property that likely contributes to the denaturing ability of lithium salts. 23 For alkali cations, the bond dissociation energies for Li-ions are the highest. 24 As well, direct protein-ion interactions are responsible for the Hofmeister effects of ions on protein stability. Further, at low ion concentrations (<100-200 mM), Li-ions specifically interact with biopolymers. 25 Similarly, it has been shown that Li cations exhibit a strong salting-in effect through improved solvation of the amide carbonyl group. 26 The charge-dense Li ions dissolve hydrophobic molecules. Li cations, however, can also interact with a variety of functional groups, suggesting that the apparent Li-induced lowering of hydrophobicity may be a result of specific interactions between the functional groups and Li ions.
Here, we determined compartmentalization of transcriptome, proteome, metabolome, and protein synthesis within the CMX and found that CMX-associated ribosomes are distinct in mRNA transcript assortment. Enrichment of biosynthetic pathways indicated the presence of immobilized biocatalysis within the cytomatrix. Efficient biocatalysis achieved by segregating and immobilizing the metabolome enzymes can overcome spatial impediments to biochemical reactions, which implies the CMX involvement in cytoplasmic organelle formation and assembly. Accordingly, the dynamics of the cytoplasmic organelles may depend on the organization of the CMX. High-throughput analysis and conventional methods revealed different responses from the cytosol and CMX during times of drug-induced protein deficiency. The reprogrammed protein translation within the cytomatrix shows operational flexibility of the cytoplasmic phases that promote cell survival under extreme conditions.

RESULTS
The cytoplasm is a two-phase system Our objective was to isolate the CMX and compare this elastic solid phase of the cytoplasm with the cytosolic viscous fluid, or liquid, phase of the cytoplasm. To this end, our two-step strategy for isolating the CMX (Figure S1A) included the initial separation of the dynamic, viscous fluid fraction of the cytoplasm using a nonionic detergent. To avoid osmotic stress and keep the CMX intact, we used a physiological concentration of potassium ions and a minimal solvent volume. The first dissolved cellular material was the cytoplasmic solution, otherwise known as the cytosol. In the second step, we used a stringent buffered solvent, containing lithium ions, to separate the CMX from the nucleus. Thus, using two buffers we separated the cytosolic, CMX, and nuclear fractions. Two different nonionic detergents (NP-40 and Triton X-100) were used to solubilize the cytoplasmic material. To prevent rupturing the basic structure of organelles, the cytosol and CMX were extracted under physiologically osmotic conditions. During the first step of the separation technique, the mild buffer extracted only the cytosol while the CMX was pelleted with the nucleus as a non-dissolved solid phase ( Figure S1A). The CMX was subsequently isolated from the nucleus using a second, stringent solvent.
We found that the viscous fluid dynamic phase of the cytoplasm was salt and detergent sensitive and could be extracted using the nonionic detergent NP-40 or its analog. To prevent damage to the elastic solid cytomatrix, we used potassium salt, which replicates the natural ionic environment of the cell. Contrary to the cytosol, the CMX was salt and detergent resistant but sensitive to lithium salt. Thus, we used LiCl to dissociate the CMX from the nucleus and to break up the macromolecular complexes and Triton X-100/DOX for solubilization. Due to the strong salting-in or dissolving effect of Li cations, our method required a precise calculation of the lithium salt concentration to ensure that dissociating the CMX did not rupture the nucleus. The KCl concentration was also calculated to avoid the osmotic stress to keep the CMX intact during the cyto-solution (cytosol) extraction. A saline solution containing 0.9% sodium chloride (154 mM NaCl) was used as the standard to calculate the LiCl and KCl salt concentrations in our solvents.
In aqueous solutions, salts are dissociated to ions and ions are surrounded by a hydration or solvation shell. The relative ionic radius 28 is a solvation shell of ions in solution that surround proteins or protein complexes in the cell. Because the ionic ratio of a protein solution depends on the relative ionic radius, we developed an equation to calculate the concentration of ions to replace sodium ions in the standard isotonic solution based on the relative ionic radius ( Figure S1B). We found that solutions containing 120 mM KCl and 200 mM LiCl do not cause osmotic pressure. Further, our calculated concentration was similar to the concentration values obtained experimentally for erythrocyte hemolysis, 29 in which the isotonic molarity of the LiCl solution was 0.189 M. Experimentally, it was confirmed that 0.2 M LiCl salt does not rupture the nucleus, but if this concentration were to be exceeded, the nuclear contents would spill out.
The solubilization of lipid bilayers using detergents is a slow process at 4 C, 30 and can involve several steps. The sequence of events that must occur include: 1) relatively rapid penetration of detergent monomers into the outer lipid monolayer; 2) transmembrane equilibration of detergent monomers between the two monolayers; 3) saturation of the lipid bilayer by detergents and consequent permeabilization of the membrane; and 4) transition of the entire lipid bilayer to thread-like mixed micelles. 31,32 Mixing the collected cells via rotation with Buffer A for 30 min provided time for the micelles, which envelop lipids and proteins immersed in lipid bilayers, to form ( Figure S1C). Therefore, water-soluble extracts of the cytosolic cellular contents, and lipids of the ER, plasma membrane (PM), and outer nuclear membrane (ONM), would be contained within the suspended micelles and dispersed in a liquid colloid. Hence, the formation and stabilization of the micelles prevent post-lysis reassortment. 33 To validate the fractionation method, we compared our protein extraction procedure with a widely used cell lysis buffer from Cell Signaling Technology (cat# 9803). The percent of proteins in each fraction was determined by dividing the concentration of proteins found in each fraction by the total protein concentration. Approximately 70% of the total colorectal cancer cell (HCT-15) proteins were detected in the cytosol. Further, the remaining proteins were roughly equally distributed between the nuclear and CMX fractions ( Figure S1D). Our results were consistent with previous findings in four other cancer and normal cell lines. 27 Notably, the concentration of proteins in the cytosol fraction was approximately 10% greater using the conventional cell lysis buffer containing 150 mM NaCl, indicating that CMX protein recovery was divided between the cytosolic and the nuclear fractions ( Figure S1E).
To distinguish the cytomatrix from the cytosol we studied marker proteins of the corresponding fractions. Western blot analysis of proteins in the cytosolic, CMX, and nuclear fractions from HCT-15 colon cancer cells shows the differential distribution of well-known cellular markers ( Figure 1A). The nuclear envelope proteins SUN2 and emerin were found in the CMX and nuclear fractions. With a lower molecular weight, iScience Article emerin was spotted only in the CMX. Conversely, the nucleolar protein B23 was found predominantly in the nuclear fraction. The nuclear pore complex protein Nup 98 was detected exclusively in the CMX, suggesting a potential new marker for this compartment. The NPC attached to the INM 17 cannot be extracted to the cytosol by the mild isotonic first buffer, even if the ONM dissolves.
The GM130 protein was previously identified in the Golgi matrix fraction as an inter-cisternal structural component. 34 Because it is known that the Golgi matrix is detergent and salt resistant, 18 we did not expect GM130 35 to be detected in the cytosol fraction. To this end, we found that GM130 was the most abundant protein detected within the CMX fraction, indicating that the Golgi matrix could be an integral part of the CMX. Cellular markers also were visualized by immunofluorescence. After cytosol extraction, keratin filaments and the LINC complex proteins (i.e., SUN2 and Nesprin-1) were localized to the nuclear surface ( Figures 1B-1D). The nucleus is well protected by the LINC complex proteins and keratin, which allowed us to separate the core nucleus from the CMX. A previous study demonstrated a keratin cage formation in cultured monolayers of living cells by time-lapse imaging 36 that supports our finding of the keratin encapsulation of the nucleus.
The nuclear membrane is resistant to high salts and detergents due to nuclear encapsulation with filamentous proteins. 15 Finally, in addition to the LINC complex, approximately 200 proteins protect the INM, 37 which prevented a rupture of the nucleus and allowed us to separate the CMX from the nucleus with the second Li + containing stringent buffer.
Transmission electron microscopy (TEM) was used to visualize the outcome of fractionation at the ultrastructural level. The resulting micrographs revealed that intact HCT-15 cells had a well-defined perinuclear space, nuclear envelope, Golgi, mitochondria, and ribosomes ( Figures 1E and 1F). After removing the cytosol, the nucleus had a higher electron density, likely due to cytoplasmic lipid deprivation that contrasted the nucleus on the cytoplasmic background ( Figures 1G-1I). The INM, the carcass of the PM and ribosomes were clearly visible. The outer nuclear membrane was dissolved. Notably, the buffer used to extract the CMX from the nucleus caused chromatin condensation ( Figure 1J). Our results indicate that the CMX buffer partially dissolved the LINC complex bridge, 38 separating the CMX from the nuclear matrix. Thus, the CMX appeared to stretch from the INM to the lipid-depleted carcass of the PM.
The isolation of the CMX was validated in two additional digestive system cell lines, AGS and HepG2 cells ( Figure 1K). Calnexin (CNX) and calreticulin (CalR) are chaperons that control proper protein folding in the ER in cooperation with the BiP, ERp57, PDI, and Ero-1-La proteins. 39,40 Except for CNX ( Figure 1K, middle panel), which is equally distributed between the cytosol and CMX, all other members of the protein folding process were detected predominantly in the cytosol ( Figures 1A and 1K).
CalR is a soluble protein that is retained in the ER lumen, whereas CNX is localized to the cytoplasm via a singlepass transmembrane domain that couples it to the outer face of the ER membrane. 39 However, CNX can translocate to the lumen during the protein folding process, as evidenced by an equal distribution of CNX within the cytosol and CMX. The ER is composed of distinct structures that include tubules, matrices, and sheets. 41 Further, the ER membrane proteins tubulin, reticulon, p180, and kinectin, as well as the DP1, REEP, and Climp63 proteins and the ER luminal protein calumenin-1 maintain ER sheet morphology and shape. 42-44 Besides well-visualized lipid membranes, the ER is an organized proteinaceous net, the hidden morphology of which can be revealed by the cryo-ET. 45 We did not expect our mild isotonic buffer to rupture the proteinaceous carcass of the ER, but rather to extract the transporting luminal proteins and the lipid membrane. To this end, we detected ER luminal proteins ( Figure 1K) and the dynamic lipid layers in the cytosol following extraction, as evidenced by TEM, the depletion of lipids after cytosol extraction in the CMX ( Figures 1G and 1H).

Assessment of protein synthesis in the cytosol and the cytomatrix
To address differential mRNA translation in the cytosol and CMX, we needed a method that statistically detects ribosome biogenesis and protein biosynthesis. For ribosome biogenesis, ribosomal proteins iScience Article were an obvious choice; moreover, ribosomal protein L7a (rpL7a) was a large subunit protein. The rpL7a belongs to the ''druggable genome'' protein family, the genome-encoding proteins that small molecules can regulate. Because the mRNA translation is controlled by 80S formation and 60S availability 54 and the nucleolar hypertrophy is an indicator of ribosome biogenesis, 55 the rpL7a was an appropriate choice. In addition, rpL7a is a highly conserved ribosome protein localized on the surface of the 60S 56,57 and contains two distinct RNA binding domains 58 and an FKBP25 binding domain, 59 suggesting that rpL7a may play an essential role in 60S subunit biogenesis. Notably, the localization of rpL7a on the surface of the 60S ribosomal subunit enables the use of rpL7a as a readout for the ribosomal large subunit biogenesis and protein expression.
We preliminarily tested the effects of several compounds that suppress diverse signaling pathways: pp242, Torin 1 (mTOR inhibitors), PF-4708671 (RSK), Bio (GSK-3), Rotenone (mitochondrial electron transport), MG132 (26S proteasome), and Geldanamycin (Hsp90) on the ribosomal protein translation (Figures S2A-S2J). We expressed GFP-tagged rpL7a in HCT-15 cells and monitored the resulting fluorescence in living cells. Although the compounds inhibit different signaling pathways, all compounds upregulated rpL7a translation. Additionally, rpL7a displayed the perinuclear localization and accumulation in the nucleus. rpL7a contains a domain II (residues 52-100) that directs nuclear and nucleolar localization. 60 The increase of rpL7a biosynthesis in the cytoplasm, in response to chemical agents, has likely partially delayed the nuclear entry of an excessive number of proteins at the nuclear envelope, presenting itself as a bright rim around the nucleus ( Figures S2C-S2J). Consequently, the intensification of the 60S biogenesis occurring in the nucleus, was revealed as nucleolar hypertrophy, due to an excessive accumulation of rpL7a. Highthroughput tests confirmed the number of nucleoli and average nucleoli size was significantly increased in treated cells ( Figures S2K-S2M). Since all compounds elicited similar results, we selected the mTOR kinase inhibitor (mTOR-KI), pp242, 61 for further analysis.
mTOR-KI inhibits mTOR-dependent protein translation and directly affects autophagy and PI3K-Akt signaling. 62,63 Treating cell lines with pp242 shifted the localization of several proteins toward the CMX and induced posttranslational modifications ( Figure 2A, right panel). Determining whether proteins are translated in the CMX or translocated is central to characterizing CMX function. To test whether motor proteins facilitate cytosol/CMX protein translocation, HCT-15 cells were treated with dynein and kinesin inhibitors, and proteins in the cytosol and CMX were compared ( Figures S3A and S3B). No significant protein translocation from the cytosol to the CMX or vice versa following treatment with Dynarrestin and K858 were detected. Proteins involved in the mRNA and protein recycling pathways, including LAMP1, 64 DDX6/RCK, 65 DCP1B, 66 IRE1a, 47 and BiP, 67 were exclusively localized within the cytosol ( Figure 2B). mTOR inhibitors remove ribosomes from polysomes and prevent polysome aggregation. 68 We used polysome profiling to compare differences of the cytosol and CMX under pp242 treated conditions and normal growth conditions in HCT-15 cell lines. After inhibitor treatment, the polysome profiles in the cytosol and CMX were reversed ( Figures 2C and 2D). In the CMX, polysomes and large ribosomal subunit levels increased in response to inhibitor treatment. In the cytosol, the 60S and polysome levels decreased and monosome (80S) levels increased in response to pp242 treatment. The ratio of the small subunit 18S rRNA in the cytosol and CMX, determined by RT-PCR, was 1.8 to 1 ( Figure S3C). To address the 40S subunit dynamics at the normal growth condition and pp242 treatment, the mRNA of small ribosomal protein rpS6 level was monitored using single-molecule fluorescence in situ hybridization ( Figure S3D). Even after pp242 treatment for 2 h, the cells had higher levels of rpS6 mRNA signal. A steady state abundance of rpL7a and the rpS6 mRNA expression evidenced the rise of ribosome biogenesis under the pp242 treatment. However, these results do not clarify the translated spectrum of the proteins.
To further address observed protein synthesis dynamics, we quantified protein synthesis using a radiolabeled amino acid to detect newly synthesized proteins. Incorporating L-[ 3 H] phenylalanine into proteins revealed that the mTOR inhibitor pp242 decreased de novo protein synthesis in the cytosol by 20%, which aligns with prior reports of global protein translation suppression. 61,69 However, the incorporation of labeled amino acids increased in CMX proteins by 50% ( Figure 2E). The latter result indicated that de novo protein synthesis increased in the CMX when global protein translation was suppressed in the cytosol, the latter of which also was detected by polysome profiling (Figures 2C and 2D) iScience Article radiolabeled amino acids during protein synthesis-we showed that protein biosynthesis could differ in the CMX and the cytosol following pp242 treatment. These data may indicate an unknown cell survival mechanism based on the translational capabilities found in the CMX.
Next, we sought to determine whether the increased CMX protein translation following the pp242 treatment could be explained by an mTOR-independent pathway. The switch from eIF4E1-dependent to eIF4E3-dependent protein translation has been demonstrated in a few cell lines. 70 After validating the specificity of an anti-eIF4E3 antibody ( Figure 2F), we treated HCT-15 cells with increasing concentrations of pp242 to determine the effects of mTOR-KI on eIF4E3 expression. A dose-dependent increase in eIF4E3 protein levels in the CMX was observed ( Figure 2G). Inhibiting 4E-BP1 phosphorylation using the same compound in the cytosol and CMX appears to be in the inverse relationship with the eIF4E3 level in the CMX ( Figure 2H). Results indicate the possibility of activating other protein synthesis regulatory pathways in CMX ( Figure 2I).

Mass-spectrometry-based proteome profiling of the cytomatrix
Large-scale protein identification with mass spectrometry-based proteomics is an appropriate way of categorizing the proteome content of the CMX. Proteins that could form the structural foundation of the CMX, the cytoskeleton-associated proteins, actins, keratins, tubulins, and other cellular microfibrils were identified ( Table 1). The Golgi, ER, nuclear pore complex proteins, and nuclear and plasma membrane proteins also were enriched in CMX. Thus, mass spectrometry data confirmed immunoblotting results and supported the notion that the CMX is enriched with the Golgi and ER matrices proteome. Enzymes and iScience Article substrates of protein glycosylation, prenylation, and protein-lipid modification are the types of posttranslational modification (PTM) that were identified within CMX, confirming the presence of the ER-Golgi matrix constituency. These also happen to be the most abundant types of PTM of proteins that can promote the immobilization and segregation of protein and enzyme complexes. 71 Extracellular matrix proteins, desmosomes, cell junction proteins, integrins, and receptors also were enriched. Interestingly, along with the normal cell signaling pathway proteins, the CMX contained a broad spectrum of proto-oncoproteins, which suggests the essential role of CMX in cancer development. A full spectrum of the CMX's functional proteome is represented as a pie chart ( Figure S4A). The CMX proteome was grouped based on the biological function of proteins (Table S1).
Cytosolic enzyme spectrum is comprised of protein and RNA degradation pathways, lipid, nucleotide, and amino acid metabolism, and glycolysis, which can be categorized as the nutrient and energy processing cycle of the cell ( Figure S4B).
A peptide area-based quantification (iBAQ) showed a relative protein level of the CMX and the cytosol proteome (Table S2). Differences in the corresponding proteomes were calculated as fold change in the CMX compared with the cytosol and vice versa, the results of which indicated an inverse relationship between the cytosolic and CMX proteome. Hierarchical clustering of the proteins detected with the respective proteomes revealed differences between the cytosol and CMX proteomes, as well as between the control and TOR-KI treated cells (Figures 3A and 3B). KEGG and Reactome pathway analyses likewise revealed the effect of mTOR-KI on the proteomes (Figures 3C and 3D). iScience Article Differences of the cytosol and cytomatrix and correspondence of RNA-seq and Ribo-seq RNA-seq and Ribo-seq are powerful tools that identify differentially expressed genes (DEGs). DEGs permit over-representation (or enrichment) analysis (ORA), a statistical method that determines whether genes from pre-defined sets are present more than expected. ORA of the pre-defined gene sets Hallmark (Table S3), KEGG (Table S4), Reactome (Table S5), and GOBP (Table S6) permitted analysis of specific pathway distribution within the cytosol or CMX.
To distinguish pathways of the cytosol and CMX, we prepared ribosomal footprint and mRNA libraries for sequencing from untreated and pp242-treated HCT-15 cells. Using ribosome profiling (Ribo-Seq), which is based on the deep sequencing of ribosome-protected footprints of translated mRNA, 72 the translation differences were compared between the cytosol and the CMX. RNA-seq and Ribo-seq datasets were used to establish a transcriptome and translatome map of the cytosol and CMX, respectively. The expression of approximately 13,000 genes was analyzed (Table S7).
Using ORA, the pathway distribution was analyzed within the cytosol and cytomatrix, and the correspondence of pathways between the RNA-seq and Ribo-seq, at normal and drug-treated conditions. The Ribo-seq and RNA-seq data obtained by CMX over cytosol (CMX) revealed that transcriptome and translatome profiles differ in the CMX (Table S8). Data acquired of pp242 treated cells also showed transcriptome and translatome differences for the CMX (Table S9) and Cytosol (Table S10). The CMX translatome pathway differences are also present when comparing cells under pp242 treated and normal growth conditions (Table S11). Results indicate the flexibility of transcription and translation, which in turn may reflect the state of cells.
Additionally, the GOBP pathway differences were analyzed to determine corresponding pathways of transcription and translation between compartments (Tables S12-S14). The highest p-values for pathways identified by Ribo-seq for CMX corresponded to the lowest or undetected p-values of the cytosol (Table S12). The opposite result was obtained by comparing cytosolic pathways with the CMX (Table S13). The parallel Ribo-seq and RNAseq analysis of the p values of the CMX and cytosolic pathways revealed that the lowest correlation occurs with iScience Article the RNA-seq dataset (Table S14). The data also showed the overlapped pathways between the cytosol and the CMX. Overall results indicated that Ribo-seq analysis could be more reliable in showing real translation than RNA-seq, which prompted us to use ribosome footprints for the pathway analysis.
Heatmaps for RNA-seq and Ribo-seq datasets, where the red color represents enriched by upregulated and the blue color is downregulated genes set, showed the distribution of transcripts in the cytosolic and CMX fractions that differed according to their respective Z score ( Figures 4A and 4B). These hierarchical heatmaps also revealed differences between the pp242-treated and untreated cells for both transcriptome and translatome.
Venn diagrams represent the ratio of differentially transcribed (RNA-seq), translated (Ribo-seq), and overlapped transcript sets ( Figure 4C). A low overlap rate was observed in the cytosol and CMX when the dataset was acquired for pp242 treated cells. The overlapped transcripts were higher in the CMX (CMX over Cyt) under normal growth conditions. These differences may reflect translational alteration between the cytosol and CMX compartments in normal and drug-treated cell states. The dynamics of the ribosome footprint and RNA-seq analyses indicated that some of the transcribed mRNAs (15%-16%) might not have been translated, which supports the notion that Ribo-seq analysis is more reliable in the identification of the enriched (ORA) pathways.
ORA of the select pathways of the Reactome and Hallmark collections are shown in Figures 4D and S5. RNA-seq, common, and Ribo-seq pathway dynamics were compared across the three groups of experimental conditions (I-III), which revealed the distribution of upregulated (red) and downregulated (blue) pathways. Experimental condition I shows the dynamics of cytosolic pathway distribution (Figures 4D-I and S5-I), which differed from the CMX dynamics (condition II). Experimental condition III, which identified iScience Article CMX pathways under normal growth conditions, revealed upregulated and downregulated pathways that were specific to the CMX. The results revealed differences in upregulated and downregulated pathways, as well as transcription and translation, between the CMX and cytosol. Thus, ribosome footprint analysis exposed the highest upregulated pathway enrichment for the CMX (condition III, Ribo-seq).
Using ribosome footprint analysis, we assessed only the upregulated pathways to reveal translation differences between the cytosol and CMX (Figures 4E, 4F, S6A, and S6B). The CMX KEGG pathway analysis showed that some pathways enriched at the normal growth conditions differ from the CMX pathways of cells treated with the mTOR inhibitor ( Figure 4E). Comparing the cytosolic and CMX pathways shows that pp242 treatment prompted pathway overlaps between the cytosol and CMX ( Figure 4F). Differences were observed in the spectrum of pathways between the cytosol and CMX as well as pp242 treated and the normal growth conditions. Thus, the spectrum of pathways between the cytosol and CMX is altered depending on cell conditions.
Using the upregulated Reactome pathways, we analyzed differences between the cytosol and CMX ( Figures S6A and S6B). The cytosolic pathways included translation, cell cycle, and respiratory pathways, but the overall PTM pathways overlapped ( Figure S6A). The CMX pathways involved the ER-Golgi function that include transport, CNX/CalR cycle, lipid and steroid metabolism, and N-linked glycosylation, but the vesicle and membrane transport pathways overlapped ( Figure S6B). The compartmental differences exposed functional dynamics, which supports previous selective western blotting data ( Figures 1K and  2A). Transcribed and translated mRNAs, which have been used for RNA-seq and Ribo-seq analyses, could be associated with the CMX through mRNA binding complexes such as FUBP1/2, hnRNPK, and PTBP1/2 and other RNA binding proteins ( Figure 1K and Table S1).

Implications of the cytosolic and cytomatrix pathway differences
Using ribosome footprints, we compared the enriched GOBP pathways in the CMX and cytosol (Figures 5A-5D). The enrichment of RNA catabolism and the ubiquitin-proteasome pathways identified by the Ribo-seq analysis in the cytosol (Figures 5A and 5B) was supported by the mRNA and protein degradation markers detected by the western blot analysis within this compartment ( Figures 1K, 2A, and 2B). Cytosolic enrichment of the upregulated pathways also included a small molecule, amide, organic acid, organophosphate, and nucleotide metabolism that can be categorized as a form of cellular nutrition. Specific transport, pathways involved in protein localization to organelles, translation, and organization of large protein complexes were enriched in the cytosol as well. The DNA repair, cell cycle transition, and apoptosis pathways were also enriched in the cytosol ( Figures 5A and 5B), which corresponds with the KEGG and Reactome pathway Ribo-seq data analysis ( Figures 4F and S6A).
Interestingly, the enrichment of the upregulated pathways within the cytomatrix differed from the cytosol ( Figures 5C and 5D). Distribution of the carbohydrate, phospholipid, glycerolipid, and glycoprotein biosynthetic pathways within the CMX indicated immobilized biocatalysis. In living cells, bio-macromolecules are exposed to a densely crowded environment where a myriad of metabolic reactions that could exclude each other coincide. The immobilization of biosynthetic pathways provides the separation of chemical reactions and efficient biocatalysis. The upregulated pathways also included lipid, glycerolipid, carbohydrate, and glycoprotein metabolism. Consequently, the upregulated pathway enrichment also involved glycosylation and O-linked glycosylation, localization of proteins to membrane, and transmembrane transport (Figure 5C). Golgi organization, endomembrane system organization, actin filament-based processes, and cell junction organization pathways were also enriched in the CMX ( Figure 5D). Response to hormones and Growth Factors, Receptor signaling pathways, intracellular and extracellular cell-to-cell signaling pathways were enriched as well. Apparently, these chains of the enriched pathways within the CMX form a logical system responsible for the cell phenotype. Accordingly, cell differentiation, cell projection organization and cell morphogenesis pathways are enriched in the cytomatrix ( Figure 5D).
Although upregulated pathways in the CMX and cytosol appeared to be distinct, they nonetheless complement each other, which is essential for proper cell function. The examples of complementing pathways are the transporting systems and protein localization of the cytosol and CMX (Figures 5B and 5C).
The highest number of enriched pathways in the CMX were structural, metabolic, and signaling ( Figure S7). Pathways related to protein glycosylation, transport, and translocation were also abundant in the CMX. The CMX pathways derived from the GOBP ribosome footprint are presented in iScience Article and downregulated pathways that were enriched in the CMX and the cytosol are shown in Table S16. We observed that the relationship between the CMX and cytosol was inverse-downregulated pathways enriched in the CMX corresponded to upregulated pathways enriched in the cytosol.
A good correspondence was found between the ribosome footprints ( Figure 5C) and protein expression, referring to the PTM, carbohydrate, and lipid metabolism pathways for example (Table 1). Ribo-seq pathway analysis, mass spectrometry proteome profiling, and western blotting experiments confirmed that the ER and Golgi matrices are integral parts of the CMX. Along with the cytoskeleton fibers, the ER and Golgi matrices are detergent and salt-resistant and form an elastic solid phase in contrast to mobile cytosol, which is detergent sensitive. Thus, the cytomatrix proteins and enzymes catalyze glycosylation, prenylation, or lipid transfer to proteins while remaining within the cytomatrix and forming protein cargo or secretory complexes. Cells would not be efficient if these enzymes were secreted with their cargo complexes to the extracellular space. Therefore, immobilized CMX enzymes are involved in lipid, carbohydrate, and glycolipid synthesis. These immobilized enzymes could also lead to glycosylation or lipidation posttranslational modifications, followed by the endomembrane system (ER) and Golgi organizations as well as transmembrane transport and ECM organization ( Figure 5D). Thus, the cytomatrix integrates ER-Golgi-specific pathways with the cell surface receptors and signaling pathways.

DISCUSSION
In the mid-1970s, microscopy, immunocytochemistry, and cell fractionation methods revealed the cytoskeleton-associated ribosomes; however, the biological implication of this finding remained unclear due to the heterogeneous nature of the cytoskeletal fraction. 73 Nevertheless, the considerable physiological significance of the mRNAs/polysomes association with the cytoskeleton has been stated. Still, it was also noted that the full implications of the molecular assembly must be awaited until detailed knowledge of iScience Article the types of synthesized proteins is revealed. Keith R. Porter proposed the concept of a cytoplasmic structure termed the ''microtrabecular lattice'' (MTL), which was described as a three-dimensional meshwork. [74][75][76] Since being proposed, however, the use of histological techniques has raised questions as to whether the MTL is a valid construct or artifact. 77,78 The central principle underlying MTL was not just whether structure and order exist within the cytoplasmic matrix, but also whether the existing structure and order extend beyond the well-defined cytoskeletal fibers. 79 Undoubtedly, the nano-compartments observed by HVEM 74 could be the reason for the mRNA/polysome presence and distinct protein synthesis capacity of the CMX. Nano-compartmentalization concentrates the required components in a confined space, allowing an efficient formation of the end products.
Until now, there have been no significant attempts to chemically isolate the cytomatrix, which was predicted almost five decades ago. 76 We used a two-step process to isolate the cytomatrix. First, the mild solvent separated the liquid phase (cytosol), and then the stringent solvent was applied to isolate the solid phase (cytomatrix) from the nucleus. The cytosol is a viscous fluid phase, consisting of water-soluble molecules and lipid-associated cargo, that is easily extracted with a nonionic detergent from the solid cytomatrix. Since the elastic solid phase is Li + sensitive, a stringent buffer containing LiCl salt isolated the CMX. In choosing the name for the new elastic solid phase, we followed Dr. Porter's CMX terminology. 74 Coordination of the cytosol and CMX functions appear to play an essential role in fine-tuning cellular processes in healthy cells. High-throughput analysis and conventional methods revealed different responses from the cytosol and CMX during times of drug-induced protein deficiency. Of note, inhibiting mTOR evidently stimulated protein translation within CMX, suggesting the tenacity for protein synthesis. The inhibition of mTORC1 is required to initiate the autophagy process and proteolysis. 80 The proteolysis that occurs in the cytosol, by providing amino acids, may stimulate protein synthesis in the CMX. The reprogrammed protein translation within the cytomatrix is likely a cell survival mechanism. Our data indicate that the CMX may potentially be essential in numerous metabolic reactions including energy generation, cell cycle, DNA replication and repair, and RNA processing depending on cell status and conditions. These functions could be a backup mechanism of the CMX in extreme conditions. Moreover, the compartmentalization of metabolic processes within the cytosol and CMX could support cancer cell survival and drug resistance as well as other pathological events. We propose that under normal growth conditions, the CMX is responsible for the cellular phenotype, whereas routine functions that support cell maintenance, such as nutrient processing and sustaining housekeeping reactions, occur in the cytosol.
To this end, the CMX appeared to be critical for performing specific tasks such as PTM of proteins, particularly glycosylation, which is the most abundant and diverse protein modification and is considered a special function of the ER and Golgi. The oligo/polysaccharide moiety could anchor (glue) the post-translationally modified proteins and protein complexes to form a matrix similar to the cell surface proteins embedded in a matrix of glycans. 81 This concept of immobilized biocatalysis in the CMX is explained in Figure360 Video. We suggest that the cytomatrix filaments and motor proteins, such as actin, myosin, tubulin, and others, engages the cytosolic motion (a ripple) in reaction to extracellular receptor-ligand binding (Table 1, Figure 5D). Recent data have shown that the cytoplasmic Rho GTPases and actomyosin dynamics with the extracellular matrix can orchestrate the signaling output. 82 The viscosity of the cytosol requires fluctuating cytoplasmic motion 7 to transduce a cascade mechanism of immobilized biocatalysis and nuclear processes. Thus, the enzyme-substrate binding and immobilized biocatalysis could be regulated by cytosolic movement and dynamics of the specific receptor-ligand action.
The Golgi matrix is a detergent and salt-resistant complex. 18 Our mild KCl-containing buffer would not dissociate Golgi, which has membrane clusters that are formed mainly by thick anastomosing tubular structures. 83 The ER-to-Golgi interwoven tubular network is a stabilizing factor 84 for the endoplasmic matrix organization. The ER proteinaceous carcass revealed by the cryo-ET 45  iScience Article the CMX in the biosynthetic-secretory pathway, that affirms the ER-Golgi complex as a segment of the elastic solid phase. In addition, the role of ER-Golgi as a segment of CMX in transport and cellsurface events (cell adhesion, ECM formation) implies a connection of the intracellular matrix with the extracellular matrix. Thus, a matrix system segregating biocatalysis performs efficient reactions and signaling.
The coinciding metabolic reactions that could exclude each other may interrupt biochemical reactions in a confined space. However, the immobilization of biosynthetic pathways, via the CMX, provides separated chemical reactions and efficient biocatalysis, that overcomes spatial impediments. Efficient biocatalysis, achieved by segregating and immobilizing the metabolome enzymes through the lipid and carbohydrate PTM, may form corresponding organelles that perform specific reactions and processes within the cytoplasm. Thus, the presence or absence of organelles in tissue-specific cells and reorganization of organelles after cell division depends on biosynthetic pathway immobilization. The dynamics of the cytoplasmic organelles, apart from DNA containing mitochondria, may depend on the organization of the CMX. Consequently, the cytomatrix organizes Golgi-ER pathways into a singular integrated system along with the receptors and signaling pathways. As such, we define the CMX as the flexible solid state of the cytoplasm, and a separate but interdependent entity from the viscous fluid (liquid) state of the cytosol. Immobilized biocatalysis integrates the intracellular and extracellular matrices and receptors with the nuclear processes, thus removing the spatial hindrances for biochemical processes, which elucidates cellular mechanics and cytoplasm organization.

Limitations of the study
The high-throughput experiments (Ribo-seq, RNA-seq, and proteomics) were only performed in HCT-15 cells. Comparative studies such as between normal and cancer cells are needed to expand the results obtained using this new approach.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Buffer ionic composition calculation
Standard approaches to separating the aqueous cytoplasm 94 have not been sufficient for isolating the intracellular CMX. The sequential fractionation of the cytoplasm to obtain cytosol and membrane-bound proteins 95 has likewise proven insufficient for CMX separation. A hypotonic solution is often used to isolate the nucleus. The PM of mammalian cells easily ruptures with a low salt hypotonic solution. 96 The osmotic shock caused by a change in the solute concentration (hypotonic or hypertonic) can rupture PM and organelles. Rupturing the PM and other organelles allows the cytoplasmic contents of the cell to leak out while leaving the nucleus intact. 97 In hypertonic conditions up to 0.6M NaCl, solubilization of nucleosomes was achieved. 98 High salt disrupts charged-based protein-DNA, and protein-protein interactions and chromatin-associated proteins become more soluble. 99 To isolate the cytosol and CMX at physiological osmotic conditions, we developed an equation to calculate the concentration of ions needed to replace sodium ions in a buffer based on the relative ionic radii for the Hofmeister series of monovalent cations (Figure S1B). Physiological or isotonic saline is a solution of 154 mM (0.90% w/v) NaCl, 308 mOsm/L. The relative radii used for the Hofmeister series monovalent cations were as follows: Li = 90 pm., Na = 116 pm., and K = 152 pm. 28 The relative ionic radius is a solvation shell of ions in solution that surround proteins or protein complexes in the cell. The ionic ratio of protein solutions depends on the relative radius of ions. The concentration of replacing cations was calculated using the equation Cr = 18/Ri, where Cr is the replacing concentration and Ri is the ionic radius. Because the dominant positive cation within the cell is potassium, 100 we used potassium chloride to mimic the natural ionic environment of the cell. Using this equation, we found that 120 mM KCl and 200 mM LiCl corresponded to 154 mM NaCl. These concentrations of ions were used sequentially in the isolation of the cytosol and the CMX. We used a minimal solvent volume for cell lysis to induce less damage to the CMX and to maintain the ionic balance.

Cell fractionation
We treated HCT-15 cells with 500 nM of pp242 every 6 h for 24 h unless otherwise stated. Control cells were grown under normal growth conditions. Experiments were performed in duplicate using 10 cm dishes. At least three 10 cm dishes were used for the CMX isolation experiment.
1. The growth media from control untreated HCT-15 cells at an exponential growth phase and pp242treated cells at cell growth arrest were aspirated and 10 cm dishes with cells were placed on ice in tilted position. All the experiments were performed on ice or in cold room that slows down metabolic processes ( Figure S1C).
2. Cells were washed with ice-cold PBS twice allowing the PBS to flow down from tilted dishes and be aspirated. HCT-15 cells were fractionated as described above. Cellular and subcellular fractions were fixed in 3% glutaraldehyde (PBS, pH 7.3) for two days, washed with sodium phosphate buffer (pH 7.3), post-fixed with 1% osmium tetroxide for 1 h, and dehydrated through a series of graded alcohol baths. Standard TEM preparation techniques were used to obtain sample blocks that were cut in ultra-thin sections (80 nm) for mounting onto 100 mesh copper grids. The grids were stained with 2% alcoholic uranyl acetate and Reynold's lead citrate. The grids were examined with a Zeiss CEM 902 transmission electron microscope, and images were captured using an AMT602 digital camera.

High throughput nucleolar hypertrophy assessment
To analyze nucleolar morphology of HCT-15 cells, we performed high speed imaging with optimum focusing capabilities. We seeded 20,000 cells in a 384 well-plate (Greiner) and the next day, we treated cells with 500 nM of pp242, 1 mM of MG132, and 1 mM of Rotenone for 24 h. Cells were fixed with 4% PFA, permeabilized by 1% Triton X-100 for 1 h, blocked with goat serum, incubated with anti-L7a antibody overnight, and detected using Alexa 488-conjugated secondary antibody. Nuclei were stained with DAPI. At least 200 cell nuclei were analyzed with four repeats. Cells were imaged on an IC200 high-throughput image cytometer (VALA Sciences) using a plan APO 403 objective. A single plane was acquired for the DAPI channel, and a z stack series (10 mm, 1 mm increment) was acquired for the GFP channel. Image analysis was performed using a custom-made algorithm developed in MATLAB. We analyzed two fields-of-view (FOV) per well (average of 46 cells per FOV). For each FOV, DAPI-labelled nuclei were segmented using a watershed transform strategy. For nucleolus segmentation, the best focused GFP-labelled nucleoli plane was selected, and objects were identified by first performing morphological top-hat filtering, followed by computing the regional maxima of the H-maxima transform. The mean number of nucleoli per nucleus, average size of nucleoli, and mean fluorescence intensity of nucleoli were then calculated.

RT-PCR
For the RT-PCR, total RNA was isolated from cytosol and CMX of HCT-15 cells using RNeasy Mini Kit (QIAGEN). qScript cDNA Super-Mix (QuantaBio) was used for cDNA synthesis. Beta-actin and 18S rRNA primers (Invitrogen) were used for PCR reaction.

QUANTIFICATION AND STATISTICAL ANALYSIS
Data are presented as the mean G SD. One-way ANOVA was used to detect significant differences between the means of more than two independent groups using Prism 6 (GraphPad). We independently performed t-tests to confirm statistically significant differences between two groups. A p value of <0.05 was considered as significant.