Vimentin-containing Intermediate Filaments Research Articles

Treatment of cultured human astrocytes and vascular endothelial cells with protein kinase CK2 inhibitors induces early changes in cell shape and cytoskeleton

Ubiquitous protein kinase CK2 is a key regulator of cell migration, proliferation and tumor growth. CK2 is abundant in retinal astrocytes, and its inhibition suppresses retinal neovascularization in a mouse retinopathy model. In human astrocytes, CK2 co-distributes with GFAP-containing intermediate filaments, which implies its association with cytoskeleton. Contrary to astrocytes, CK2 is co-localized in microvascular endothelial cells (HBMVEC) with microtubules and actin stress fibers, but not with vimentin-containing intermediate filaments. Specific CK2 inhibitors (TBB, TBI, TBCA and DMAT) and nine novel CK2 inhibiting compounds (TID43, TID46, Quinolone-7, Quinolone-39, FNH28, FNH62, FNH64, FNH68 and FNH74) were tested at 10-200 μM for their ability to induce morphological alterations in cultured human astrocytes (HAST-40), and HBMVEC (For explanation of the inhibitor names, see "Methods" section). CK2 inhibitors caused dramatic changes in shape of cultured cells with effective inhibitor concentrations between 50 and 100 μM. Attached cells retracted, acquired shortened processes, and eventually rounded up and detached. CK2 inhibitor-induced morphological alterations were completely reversible and were not blocked by caspase inhibition. However, longer treatment or higher inhibitor concentration did cause apoptosis. The speed and potency of the CK2 inhibitors effects on cell shape and adhesion were inversely correlated with serum concentration. Western analyses showed that TBB and TBCA elicited a significant (about twofold) increase in the activation of p38 and ERK1/2 MAP kinases that may be involved in cytoskeleton regulation. This novel early biological cell response to CK2 inhibition may underlie the anti-angiogenic effect of CK2 suppression in the retina.

A Nuclear SH3 Domain-binding Protein That Colocalizes with mRNA Splicing Factors and Intermediate Filament-containing Perinuclear Networks

A protein (SNP70) has been isolated that binds to the Src homology domain 3 of p47(phox), p85alpha, and c-src. Cloning and sequencing of the polypeptide revealed it to be a 70-kDa protein that has a number of potential domains, including Src homology 3 binding motifs and several nuclear localization signals. Immunofluorescence using anti-peptide antibodies revealed SNP70 to be primarily concentrated in the nucleus but excluded from nucleoli, in interphase cells. However, it was distributed throughout the cytoplasm in dividing cells. Extraction and subfractionation experiments indicated that SNP70 did not bind directly to DNA but did bind to poly(G)-rich oligonucleotides and was resistant to extraction with non-ionic detergents but was solubilized by treatment with RNase, high salt, or ammonium sulfate. Double-immunofluorescence experiments showed that SNP70 co-localized with two pre-mRNA splicing factors SC35 and U2B" within the nucleus. A population of SNP70 was found outside the nucleus, and double-immunofluorescence and immunoelectron microscopy demonstrated that it associated with vimentin-containing intermediate filaments, particularly those surrounding the nucleus. The data suggest that SNP70 associates with nuclear or perinuclear filaments and may play a role in the regulation of pre-mRNA processing.

Hsp70 and antifibrillogenic peptides promote degradation and inhibit intracellular aggregation of amyloidogenic light chains.

In light chain (LC) amyloidosis an immunoglobulin LC assembles into fibrils that are deposited in various tissues. Little is known about how these fibrils form in vivo. We previously showed that a known amyloidogenic LC, SMA, can give rise to amyloid fibrils in vitro when a segment of one of its beta sheets undergoes a conformational change, exposing an Hsp70 binding site. To examine SMA aggregation in vivo, we expressed it and its wild-type counterpart, LEN, in COS cells. While LEN is rapidly oxidized and subsequently secreted, newly synthesized SMA remains in the reduced state. Most SMA molecules are dislocated out of the ER into the cytosol, where they are ubiquitinylated and degraded by proteasomes. A parallel pathway for molecules that are not degraded is condensation into perinuclear aggresomes that are surrounded by vimentin-containing intermediate filaments and are dependent upon intact microtubules. Inhibition of proteasome activity shifts the balance toward aggresome formation. Intracellular aggregation is decreased and targeting to proteasomes improved by overexpression of the cytosolic chaperone Hsp70. Importantly, transduction into the cell of an Hsp70 target peptide, derived from the LC sequence, also reduces aggresome formation and increases SMA degradation. These results demonstrate that an amyloidogenic LC can aggregate intracellularly despite the common presentation of extracellular aggregates, and that a similar molecular surface mediates both in vitro fibril formation and in vivo aggregation. Furthermore, rationally designed peptides can be used to suppress this aggregation and may provide a feasible therapeutic approach.

Reorganization of Structural Proteins in Vascular Smooth Muscle Cells Grown in Collagen Gel and Basement Membrane Matrices (Matrigel): A Comparison with Their in Situ Counterparts

When smooth muscle cells are enzyme-dispersed from tissues they lose their original filament architecture and extracellular matrix surrounds. They then reorganize their structural proteins to accommodate a 2-D growth environment when seeded onto culture dishes. The aim of the present study was to determine the expression and reorganization of the structural proteins in rabbit aortic smooth muscle cells seeded into 3-D collagen gel and Matrigel (a basement membrane matrix). It was shown that smooth muscle cells seeded in both gels gradually reorganize their structural proteins into an architecture similar to that of their in vivo counterparts. At the same time, a gradual decrease in levels of smooth muscle-specific contractile proteins (mainly smooth muscle myosin heavy chain-2) and an increase in β-nonmuscle actin occur, independent of both cell growth and extracellular matrix components. Thus, smooth muscle cells in 3-D extracellular matrix culture and in vivo have a similar filament architecture in which the contractile proteins such as actin, myosin, and α-actinin are organized into longitudinally arranged “myofibrils” and the vimentin-containing intermediate filaments form a meshed cytoskeletal network. However, the myofibrils reorganized in vitro contain less smooth muscle-specific and more nonmuscle contractile proteins.

Heterogeneous distribution of isoactins in cultured vascular smooth muscle cells does not reflect segregation of contractile and cytoskeletal domains.

We have previously demonstrated that alpha-smooth muscle (alpha-SM) actin is predominantly distributed in the central region and beta-non-muscle (beta-NM) actin in the periphery of cultured rabbit aortic smooth muscle cells (SMCs). To determine whether this reflects a special form of segregation of contractile and cytoskeletal components in SMCs, this study systematically investigated the distribution relationship of structural proteins using high-resolution confocal laser scanning fluorescent microscopy. Not only isoactins but also smooth muscle myosin heavy chain, alpha-actinin, vinculin, and vimentin were heterogeneously distributed in the cultured SMCs. The predominant distribution of beta-NM actin in the cell periphery was associated with densely distributed vinculin plaques and disrupted or striated myosin and alpha-actinin aggregates, which may reflect a process of stress fiber assembly during cell spreading and focal adhesion formation. The high-level labeling of alpha-SM actin in the central portion of stress fibers was related to continuous myosin and punctate alpha-actinin distribution, which may represent the maturation of the fibrillar structures. The findings also suggest that the stress fibers, in which actin and myosin filaments organize into sarcomere-like units with alpha-actinin-rich dense bodies analogous to Z-lines, are the contractile structures of cultured SMCs that link to the network of vimentin-containing intermediate filaments through the dense bodies and dense plaques.

Ubiquitin-Related Proteins Regulate Interaction of Vimentin Intermediate Filaments with the Plasma Membrane

Integrin-associated protein (IAP, CD47) is a plasma membrane receptor for thrombospondins and signal regulatory proteins (SIRPs) that has an essential role in host defense through its association with integrins. The IAP gene encodes alternatively spliced carboxy-terminal cytoplasmic tails that have no previously described function. IAP cytoplasmic tails can bind two related proteins that mediate interaction between IAP and vimentin-containing intermediate filaments, named p roteins l inking I AP with c ytoskeleton (PLICs). Integrins interact with PLICs indirectly, through IAP. Transfection of PLICs induces redistribution of vimentin and cell spreading in IAP-expressing cells. This novel connection between plasma membrane and cytoskeleton is likely to be significant in many adhesion-dependent cell functions.

Effect of bacterial wall lipopolysaccharide (LPS) on morphology, motility, and cytoskeletal organization of microglia in cultures

We examined the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the morphology, cell motility, cytoskeletal organization, and phagocytic activity of microglia in tissue cultures initiated from neopallia of newborn C3H/OuJ mice. Normally, the microglia in our cultures are non-migratory and Mac-1 positive, have ameboid cell morphology, no polarity, many short processes that extend into lamellipodia in opposing directions, and undulating cell membrane projections. When 1-5 micrograms/ml LPS is added to such cultures, some cells acquire polarity by forming a large lamellipodium and begin to migrate. Two hours later migration ceases; the membrane undulations stop; and the cells become non-polar, assume a large, round, flat shape, and gradually develop many microspikes all over the cell body. Those cells that do not transform into large, round, flat cells enlarge and extend numerous lamellipodia in opposing directions. We found that the cytoskeleton of microglia is composed of actin, vimentin-containing intermediate filaments (IF) and microtubules (MT). Vimentin-containing IF and MT form dense networks that radiate into the cell periphery, whereas F-actin is diffusely arranged throughout the cytoplasm. The LPS-treated cells show changes in the organization of the main components of the cytoskeleton. F-actin is reorganized by the formation of bundles underneath and parallel to the cell membrane and other bundles projecting into the cores of the microspikes. The vimentin-containing IF dense network reorganizes into two condensed rings, with fine strands of IF extended between the two rings and the MT networks become less dense and extend throughout the cytoplasm. The LPS treatment potentiates the phagocytic activity of the microglia. However, approximately 30% of microglia lose the expression of MHC class II antigens.

The influence of vinyl-4-cyclohexene dioxide on the vimentin-containing intermediate filaments and the microtubular cytoskeleton of cultured mouse fibroblasts: Immunofluorescence investigations

The exposure of 3T3 mouse fibroblasts to vinyl-4-cyclohexene dioxide (VCD), a compound used as a chemical intermediate and as a reactive diluent for diepoxides and epoxy resins, induces changes in cellular shape and cytoplasmic organization. Toxicological effects on the cytoskeletal elements were investigated by culturing 3T3 cells in medium containing VCD for increasing times (15, 30, 60 and 180 min). The immunofluorescence investigations demonstrated that both vimentin-containing intermediate filaments and microtubules were compromised by the treatment in a time-dependent manner. Increasing the duration of exposure to VCD increased cellular damage.

The thermoresistant state: Protection from initial damage or better repair?

The induction of and recovery from heat-induced perturbations in several cellular parameters were examined in normal, transiently thermotolerant, and permanently heat-resistant HA-1 Chinese hamster fibroblasts. The initial heat-induced perturbations in total cellular protein synthesis, RNA synthesis, vimentin-containing intermediate filaments, and nuclear protein mass were similar in the three different cell types which display various levels of thermal resistance as determined by clonogenic survival. The posthyperthermia recovery from the heat-induced perturbations in all of the cellular parameters was more rapid in both the permanently heat-resistant cells and in the transiently thermotolerant cells. This response was observed in cells in which transient thermotolerance was induced by either a mild heat shock or exposure to sodium arsenite. The development and decay of the capacity for more rapid recovery from the initial heat-induced perturbations in total cellular protein and RNA synthesis paralleled the development and decay of clonogenic thermotolerance. Overall, these results support the notion that more rapid recovery from similar levels of heat-induced perturbations in various cellular parameters are a salient feature of both the transiently and permanently heat-resistant state.

Heat-resistant variants of the Chinese hamster ovary cell: alteration of cellular structure and expression of vimentin.

Three heat-resistant mutant cell lines (78-1, 78-2, 78-3) were previously selected from Chinese hamster ovary cells. In this study, we investigated whether the differences in intrinsic thermal sensitivity result from alteration of stress protein levels or cellular structural changes. Although there was no significant difference in the levels of stress proteins, i.e., constitutive HSP70 in wild type and three heat-resistant mutant strains, there were marked differences in the amounts of vimentin among the cell lines. Two-dimensional gel electrophoresis and Western blot showed a 2.3-2.9-fold increase in the level of vimentin in the mutant cells under normal growth conditions. Northern blot also revealed higher amounts of vimentin mRNA in the mutant cells. Electron microscopy and immunofluorescence suggest that increased amounts of the vimentin-containing intermediate filaments are correlated with the heat-resistant phenotypes.

Cytoskeletal reorganizations responsible for the phorbol ester-induced formation of cytoplasmic processes: possible involvement of intermediate filaments.

The tumor promoter phorbol 12-myristate 13-acetate (PMA) induces characteristic reversible changes of cell shape in certain fibroblastic lines: motile lamellas are transformed into noncontractile narrow processes; simultaneously, the actin microfilament network of lamellas is locally disorganized. This reaction to PMA may be regarded as a prototype of reorganizations involving formation of stable cytoplasmic processes. Specific drugs, Taxol and Colcemid, were used to study the role of microtubules and vimentin-containing intermediate filaments (IF) in the development of PMA-induced reorganizations. PMA readily induced formation of noncontractile processes in Taxol-treated fibroblasts; these cells had a profoundly altered microtubular system but noncollapsed IF. A short (1 hr) exposure to PMA induced formation of processes in control cells but not in the Colcemid-treated cells, which had depolymerized microtubules and IF that collapsed around the nucleus. Longer (3-4 hr) exposure of the Colcemid-treated cells to PMA induced partial reversal of the IF collapse; those parts of the peripheral lamellas that contained IF were transformed into narrow noncontractile processes. It is suggested that the local interaction of IF with the actin system is an essential step in the formation of processes from lamellas. The microtubular system controls distribution of IF in the cytoplasm and thus plays an indirect role in the reorganization of the actin cortex.

Cytoskeleton, adhesion, and extracellular matrix of fetal human retinal pigmented epithelial cells in culture.

Small sheets of human retinal pigmented epithelial (RPE) cells isolated from 6- to 9-week-old fetal human eyes were used to establish RPE cells in culture. After 4-5 days in culture the RPE cells in various parts of a colony were examined by immunofluorescence and fluorescence microscopy to determine the distribution of the cytoskeletal proteins tubulin, myosin, F-actin, vimentin, and cytokeratin and the extracellular matrix proteins laminin and type IV collagen. The extracellular matrix components laminin and type IV collagen, which are abundant underneath the RPE cells in the centre of the colony, progressively decrease in amount towards the edge of the colony. Microtubules labeled by antisera to tubulin were present in the cytoplasm of RPE cells throughout the colony. F-actin and myosin were found in stress fibres in the RPE cells at the edge of the colony and in compact circumferential rings circumscribing the apical ends in RPE cells in the centre of the colony. Examination of cell-substratum adhesion patterns by interference reflection microscopy showed that focal contacts were present only in RPE cells at the edge of the colony where they were located at the ends of the stress fibres. The large focal adhesions previously observed in colonies of chick RPE cells, however, were not seen in human RPE cells. Human RPE cells throughout the colony had cytokeratin-containing intermediate filaments as well as vimentin-containing intermediate filaments, whereas only the latter were observed in chick RPE cells. These results indicate that in the general organization of the cytoskeleton and in the distribution of the extracellular matrix components, laminin and type IV collagen, colonies of human RPE cells are similar but not identical to colonies of chick RPE cells.

Early appearance of type II astrocytes in developing human fetal brain

To compare rat and human glial development, we examined the cellular composition of human fetal brain in short-term cultures and fresh cell suspensions from fetal ages ranging from 7 to 16 weeks, utilizing the cell type-specific markers which define glial subsets in rats. Here we report that unlike the early rat central nervous system (CNS), 7–10 week human fetal brain contains mostly astrocytes that can be characterized as type II rather than type I. Type I astrocytes become more prevalent in 16-week gestational age human brain. Although cells morphologically and immunocytochemically similar to the rat 02-A cell are found in human fetal brain and spinal cord, these cells were not induced to express galactocerebroside in serum-free media and did not have vimentin-containing intermediate filaments as do rat 02-A cells. These results suggest that functional differences may exist between rat type I and type II astrocytes and phenotypically similar cells found in humans.

Effects of elevated intracellular magnesium on cytoskeletal integrity.

Increasing the intracellular magnesium concentration of PtK2 cells by 1 mM or more resulted in the disassembly of the interphase microtubule array over a period of 5 min after microinjection. This effect was found to be both transient and fully reversible, with the microtubule arrays reforming after further incubation. These effects were studied using immunofluorescence microscopy of fixed cells, and also in living cells using rhodamine-tubulin or rhodamine-conjugated anti-tubulin antibodies and image intensification and enhancement techniques. Simultaneously and accompanying the disassembly of the microtubule arrays the F-actin stress fibres also disappeared, usually leaving the peripheral and perinuclear F-actin microfilaments intact. In contrast, increasing intracellular magnesium appeared to have no effect on the vimentin-containing intermediate filaments of PtK2 cells. These effects on the cytoskeleton were specific to magnesium and could not be mimicked by either microinjection of injection buffer of equivalent ionic strength or sham injection. Raising the intracellular free calcium to the same extent resulted in the disassembly of the microtubule network, but appeared to have no effect on the F-actin stress fibres.

Actin cortex and microtubular system in morphogenesis: cooperation and competition.

Actin cortex and microtubules determine two different types of morphological organization of the cytoplasm. Cooperation and competition between these two organizations may produce a diversity of final forms. Actin cortex alone, without the other cytoskeletal systems, is sufficient for the organization of vectorized pseudopod-forming cytoplasmatic units termed 'actinoplasts'. Reversible segregation of cytoplasm into actinoplasts and microtubule-rich stable domains ('tubuloplasts') is suggested to play an important role in many types of morphogenesis. Segregation of this type can be induced in fibroblasts of certain lines by the tumour promotor, tetradecanoyl phorbol-acetate (TPA). Self-organization of each actinoplast leads to the development of microfilament bundles associated with focal contacts. Analysis of the evolution of these bundle-contact structures during spreading of mouse fibroblasts suggests that their patterns are determined by the degree of centripetal tension within the actin cortex; the microtubular system stabilizes these patterns. Pseudopodial activity of the edges of the actinoplasts can be controlled by environmental factors. One particular type of control, i.e. the effect of cell-cell contact, is discussed. Actin cortex and microtubules seem to have alternative effects on the distribution of vimentin-containing intermediate filaments.

A putative protein-sequestration site involving intermediate filaments for protein degradation by autophagy. Studies with microinjected purified glycolytic enzymes in 3T3-L1 cells.

Several glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase) were radiolabelled by [125I]iodination, conjugation with 125I-labelled Bolton & Hunter reagent and reductive [3H]methylation, and their degradative rates after microinjection into 3T3-L1 cells compared with that of the extracellular protein bovine serum albumin. Although the albumin remains largely cytosolic in recipient cells, the glycolytic enzymes rapidly (less than 30 min) become insoluble, as measured by detergent and salt extractions. The microinjected glycolytic enzymes appear to form disulphide-linked aggregates, are found in a cell fraction rich in vimentin-containing intermediate filaments and histones (nuclear-intermediate-filament fraction), and are degraded slowly by a lysosomal mechanism, as judged by the effects of inhibitors (NH4Cl, leupeptin, 3-methyladenine). 125I-labelled bovine serum albumin appears to be degraded rapidly and non-lysosomally. Prolonged treatment (96 h) of cultured cells with leupeptin results in the accumulation of pulse-labelled ([35S]methionine for 24 h) endogenous cell proteins in the detergent-and salt-non-extractable residue, but NH4Cl and 3-methyladenine do not have this effect. The findings are in terms of the interpretation of experiments involving microinjection of proteins to study intracellular protein protein degradation by autophagy.

Cytoskeleton Organization Is Aberrantly Rearranged in the Cells of B Chronic Lymphocytic Leukemia and Hairy Cell Leukemia

The organization of actin-containing microfilaments and vimentin-containing intermediate filaments has been investigated in B chronic lymphocytic leukemia (B-CLL), hairy cell leukemia (HCL), and normal B cells cultured in vitro under basal conditions and after induction with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In uninduced B-CLL cells, F-actin was predominantly associated with dot-shaped structures scattered over the ventral membrane representing spotty close contact adhesion sites analogous to "podosomes" described in other cell types. On TPA induction, podosomes became clustered in sharply defined areas sitting in the cell center beneath the nucleus. In some cells, long actin-containing protrusions appeared. In HCL cells, F-actin was associated with thin microvilli responsible for the "hairy" appearance; occasional cells showed scattered podosomes. On TPA induction, HCL cells sprouted long dendritic processes rich in submembraneous F-actin, which made intertwined networks. Therefore, in both B-CLL and HCL cells, adhesion structures were present and the capacity for adhesion in vitro was marked, which might explain some peculiar clinical features of the diseases. Adhesion structures and adhesive properties never appeared in normal B cells. These data further support the notion that B-CLL and HCL, although clinically different, may share common biological features and suggest that in these disorders, cytoskeleton modifications may represent a hallmark of transformation.

Morphological study of the mammalian stress response: characterization of changes in cytoplasmic organelles, cytoskeleton, and nucleoli, and appearance of intranuclear actin filaments in rat fibroblasts after heat-shock treatment.

Using both electron microscopy and immunological methods, we have characterized a number of changes occurring in rat fibroblasts after heat-shock treatment. Incubation of the cells for 3 h at 42 degrees-43 degrees C resulted in a number of changes within the cytoplasm including: a disruption and fragmentation of the Golgi complex; a modest swelling of the mitochondria and subtle alterations in the packing of the cristae; and alterations in cytoskeletal elements, specifically a collapse and aggregation of the vimentin-containing intermediate filaments around the nucleus. A number of striking changes were also found within the nuclei of the heat-treated cells: (a) We observed the appearance of rod-shaped bodies consisting of densely packed filaments. Using biochemical and immunological methods, these nuclear inclusion bodies were shown to be comprised of actin filaments. (b) Considerable alterations in the integrity of the nucleoli were observed after the heat-shock treatment. Specifically, there appeared to be a general relaxation in the condensation state of the nucleoli, changes in both the number and size of the granular ribonucleoprotein components, and finally a reorganization of the nucleolar fibrillar reticulum. These morphological changes in the integrity of the nucleoli are of significant interest since previous work as well as studies presented here show that two of the mammalian stress proteins, the major stress-induced 72-kD protein and the 110-kD protein, localize within the nucleoli of the cells after heat-shock treatment. We discuss these morphological changes with regards to the known biological and biochemical events that occur in cells after induction of the stress response.

Disruption of the three cytoskeletal networks in mammalian cells does not affect transcription, translation, or protein translocation changes induced by heat shock.

Mammalian cells show a complex series of transcriptional and translational switching events in response to heat shock treatment which ultimately lead to the production and accumulation of a small number of proteins, the so-called heat shock (or stress) proteins. We investigated the heat shock response in both qualitative and quantitative ways in cells that were pretreated with drugs that specifically disrupt one or more of the three major cytoskeletal networks. (These drugs alone, cytochalasin E and colcemid, do not result in induction of the heat shock response.) Our results indicated that disruption of the actin microfilaments, the vimentin-containing intermediate filaments, or the microtubules in living cells does not hinder the ability of the cell to undergo an apparently normal heat shock response. Even when all three networks were simultaneously disrupted (resulting in a loose, baglike appearance of the cells), the cells still underwent a complete heat shock response as assayed by the appearance of the heat shock proteins. In addition, the major induced 72-kilodalton heat shock protein was efficiently translocated from the cytoplasm into its proper location in the nucleus and nucleolus irrespective of the condition of the three cytoskeletal elements.

Disruption of the three cytoskeletal networks in mammalian cells does not affect transcription, translation, or protein translocation changes induced by heat shock.

Mammalian cells show a complex series of transcriptional and translational switching events in response to heat shock treatment which ultimately lead to the production and accumulation of a small number of proteins, the so-called heat shock (or stress) proteins. We investigated the heat shock response in both qualitative and quantitative ways in cells that were pretreated with drugs that specifically disrupt one or more of the three major cytoskeletal networks. (These drugs alone, cytochalasin E and colcemid, do not result in induction of the heat shock response.) Our results indicated that disruption of the actin microfilaments, the vimentin-containing intermediate filaments, or the microtubules in living cells does not hinder the ability of the cell to undergo an apparently normal heat shock response. Even when all three networks were simultaneously disrupted (resulting in a loose, baglike appearance of the cells), the cells still underwent a complete heat shock response as assayed by the appearance of the heat shock proteins. In addition, the major induced 72-kilodalton heat shock protein was efficiently translocated from the cytoplasm into its proper location in the nucleus and nucleolus irrespective of the condition of the three cytoskeletal elements.