Specific types of prosomes are associated to subnetworks of the intermediate filaments in PtK1 cells.

Prosomes are small ribonucleoprotein (RNP) particles of unique morphology in the electron microscope but of variable protein and RNA composition, depending on the differentiation state of the cells studied. They were initially observed as subcomplexes of untranslated mRNP. In previous studies, we found that prosomes are associated to the intermediate filaments (IF) of cytokeratin type in HeLa and PtK1 cells. Here we have studied in detail the association of prosomal antigens with the IF networks in PtK1 cells. Contrary to our earlier conclusions, in these cells the vimentin fibers also carry prosomes which, thus, distribute in between the two types of networks. During the selective collapse of the IF induced by acrylamide, and upon recovery after the withdrawal of the drug, no dissociation of the prosome and IF networks of cytokeratin- and vimentin-type could be observed. These data show that even in a dynamic situation, prosome and IF antigens do not dissociate, indicating strongly that they are located on one and the same structure. Furthermore, the differential distribution of specific prosomal antigens between both types of intermediate filament networks indicates that prosomes do not ubiquitously populate the intermediate filaments but occupy subnetworks of either vimentin or cytokeratin type.     

2,5-hexanedione aggregates vimentin-, but not keratin-, intermediate filaments of PtK1 cells

2,5-hexanedione (2,5HD) induces focal accumulation of neurofilaments in nerve axons and juxtanuclear aggregation of vimentin-intermediate filaments (vimentin-IF) in cultured human skin fibroblasts. It has been postulated that 2,5HD prevents the cross-filament associations of intermediate filaments (IF) with microtubules which are required for their transport. If this is true, only subclasses of IF which depend on microtubules for their cellular distribution should be affected by 2,5HD-treatment and the aggregates formed should resemble the juxtanuclear coils which form following dissolution of microtubules by colchicine. We have tested this hypothesis in PtK1 cells which contain two separate networks of IF: vimentin-IF which aggregate in the presence of colchicine, and keratin-filaments (keratin-IF) whose distribution is not altered by depolymerization of microtubules. Treatment of confluent monolayers of PtK1 cells with 2,5HD (4 to 6 mM for 14 to 21 days) induced aggregates of vimentin-IF which resembled those induced by colchicine (5 X 10(-6)M for 48 hours), but had no effect on the distribution of keratin-IF.     

The intermediate-sized filaments in rat kangaroo PtK2 cells. II. Structure and composition of isolated filaments.

When cultured cells of the rat kangaroo cell line PtK2 grown on plastic or glass surfaces are lysed and extracted with combinations of low and high salt buffers and the non-ionic detergent Triton X-100 cytoskeletal preparations are obtained that show an enrichment of 6 to 11 nm thick filaments. The arrays of these filaments have been examined by various light and electron microscopic techniques, including ultrathin sectioning, whole mount transmission electron microscopy, negative staining, and indirect immunofluorescence microscopy. In addition, 6 to 11 nm filaments isolated from these cells with similar extraction procedures and with centrifugation techniques have been examined by electron microscopy. The arrays of these isolated intermediate-sized filaments, their ultrastructure and their specific decoration by certain antibodies present in normal rabbit sera as well as by guinea pig antibodies against purified bovine prekeratin is demonstrated. When preparations enriched in these intermediate-sized filaments are examined by SDS-polyacrylamide gel electrophoresis a corresponding enrichment of three polypeptide bands with apparent molecular weights of about 45 000, 52 000 and 58 000 (the latter component sometimes appears split into two bands) is observed, besides some residual actin and a few high molecular weight bands. The morphology of the isolated filaments, their immunological reaction with antibodies decorating prekeratin-containing structures, and the sizes of their constitutive polypeptides suggest that these filaments are closely related to prekeratin-containing filaments observed in a variety of epithelial cells.     

Cytochalasin B-induced redistribution of cytokeratin filaments in PtK1 cells

Indirect immunofluorescence demonstrated a dramatic reorganization of cytokeratin filaments produced by cytochalasin B (CB) treatment of PtK1 cells. Much of the normal cytokeratin network became arranged into a latticework consisting of bundles of cytokeratin filaments that radiated from, and interconnected, distinct foci. Electron microscopy showed foci to be dense granular regions through which bundles of cytokeratin filaments looped. Composition of the foci included actin, myosin, and alpha-actinin, as shown by labeling with rhodamine phalloidin or specific antisera. Simultaneous treatment with CB and colchicine was not required for lattice formation, but did produce more extensive development than did CB alone. In cells treated only with CB, the microtubule network remained intact, even in regions of extensive lattice formation. These results contrast sharply with those of Knapp et al (J. Cell Biol. 97:1788 [1983b]), who found lattice formation dependent upon simultaneous CB and colchicine treatment. Time-course and dose-response studies of CB treatment showed lattice formation to follow disruption of stress fibers and the concentration of actin into distinct patches that marked the location of lattice foci. Overall results suggest a structural association between microfilaments and cytokeratin filaments that produces the lattice pattern upon CB-induced disruption of stress fibers. Lattice formation was not limited to a specific cell-cycle stage, since G1, G2, and M cells displayed the lattice. Treatment of cells with dihydro-CB and experiments with enucleated cells showed that lattice formation was dependent upon neither the inhibition of sugar transport nor the nuclear extrusion effects of CB.     

Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments

We report here on the in vivo assembly of alpha-internexin, a type IV neuronal intermediate filament protein, in transfected cultured cells, comparing its assembly properties with those of the neurofilament triplet proteins (NF-L, NF-M, and NF-H). Like the neurofilament triplet proteins, alpha-internexin coassembles with vimentin into filaments. To study the assembly characteristics of these proteins in the absence of a preexisting filament network, transient transfection experiments were performed with a non-neuronal cell line lacking cytoplasmic intermediate filaments. The results showed that only alpha-internexin was able to self-assemble into extensive filamentous networks. In contrast, the neurofilament triplet proteins were incapable of homopolymeric assembly into filamentous arrays in vivo. NF-L coassembled with either NF-M or NF-H into filamentous structures in the transfected cells, but NF-M could not form filaments with NF-H. alpha- internexin could coassemble with each of the neurofilament triplet proteins in the transfected cells to form filaments. When all but 2 and 10 amino acid residues were removed from the tail domains of NF-L and NF-M, respectively, the resulting NF-L and NF-M deletion mutants retained the ability to coassemble with alpha-internexin into filamentous networks. These mutants were also capable of forming filaments with other wild-type neurofilament triplet protein subunits. These results suggest that the tail domains of NF-L and NF-M are dispensable for normal coassembly of each of these proteins with other type IV intermediate filament proteins to form filaments.     

Decidual cells in the human ovary at term: II. Morphometric analysis of cytoplasmic processes and organelles

Morphometric analysis of human ovarian decidual cells was performed with a Videoplan computer, and mean values were established for the area and perimeter of cellular processes and organelles. Two-hundred forty electron micrographs representing 160 cells were analyzed. The mean decidual cell area was 218.7 microns2, of which 34.5 microns2 was occupied by the nucleus (15.8% of the cytoplasmic area); the nucleus contained 1.74 micron2 of nucleolar material (0.8%). The endoplasmic reticulum occupied 13.63 microns2 (6.2%). Mitochondria occupied 7.3 microns2 (3.3%) and the Golgi network 5.49 microns2 (2.5%). Decidual secretory bodies occupied 0.91 micron2 (0.42%) and cytoplasmic processes 1.89 micron2 (0.94%). The remainder of the cytoplasm, containing inclusions and cytoskeleton, represented 71% of the cell area. Perimeter measurements indicated an average decidual cell was surrounded by 87.8 microns of plasma membrane. The mean nuclear membrane measured 28.3 microns (representing 32.3% of the plasma membrane, pm, or 4.1% of total cellular membranes, cm). Outer mitochondrial membranes measured 156.6 microns (178% pm, 23.5% cm); endoplasmic reticulum membranes measured 350.3 microns (400% pm, 52.6% cm); Golgi membrane measured 30.77 microns (35% pm; 4.5% cm) and membrane surrounding secretory bodies measured 9.8 microns (11.2% pm; 1.4% cm). A mean of 280 secretory bodies per ovarian decidual cell was calculated. The plasma membranes of evaginated cytoplasmic processes represented 22.3% of the total pm (19.6 microns or 2.9% cm). A mean of seven such processes was observed per 87.8 microns of plasma membrane (160/cell). These morphometric data provide a baseline for comparisons of human ovarian decidual cells with uterine decidua, in vivo and in vitro, as well as with decidual cells of other species.     

Cytoskeletal dynamics in rabbit synovial fibroblasts: I. Effects of acrylamide on intermediate filaments and microfilaments

Rabbit synovial fibroblasts respond to changes in cell shape and cytoskeletal architecture by altering specific gene expression. We have tested the ability of acrylamide, a neurotoxin that alters the distribution of intermediate filaments in cultured PtK1 cells, to induce metalloprotease expression in synovial fibroblasts. Cells treated with 2-20 mM acrylamide for 5 to 24 h underwent shape changes similar to cells treated with the tumor promoter phorbol myristate acetate. Intermediate filaments visualized with anti-vimentin antibodies did not collapse into a perinuclear cap in these rounded cells, but were still present in the extended cell processes. Unexpectedly, when actin was visualized in acrylamide-treated cells, extensive dissociation and clumping of microfilaments was observed. Concentrations of acrylamide greater than 10 mM were cytotoxic, but cells recovered completely after 24 h incubation with 5 mM acrylamide. Like other agents that alter cell shape and actin distribution in synovial fibroblasts, acrylamide also induced expression of the secreted metalloprotease collagenase. Although some recent evidence suggests that acrylamide may be able to exert its collagenase-inducing effects extracellularly, perhaps through transmembrane matrix receptors, our observation that this neurotoxin dramatically alters protein synthesis in synovial fibroblasts suggests that direct effects on cell metabolism may also play a role in acute acrylamide intoxication.     

Changes in the organization of non-epithelial intermediate filaments induced by triethyl lead chloride

The in vivo effect of triethyl lead chloride (TriEL) (10(-6)-10(-8) M) on the organization of non-epithelial intermediate filaments (vimentin and desmin filaments) was studied by indirect immunofluorescence microscopy employing different mammalian cell lines. The in vitro effect of TriEL on filament formation as well as on the structure of preformed filaments was investigated by electron microscopy. TriEL induces perinuclear coil formation of intermediate filaments in SV40-transformed human fibroblasts and baby hamster kidney (BHK21) cells. The rearrangements observed are not correlated with significant changes in the microtubular system as tested by double labelling of both filament systems. The effect of TriEL is reversible. Assembly of intermediate filaments in vitro is disturbed in the presence of TriEL such that only short filaments and various kinds of fragments are formed. When preformed filaments are incubated in the presence of TriEL, unravelling of fibres into protofilamentous strands is observed. Possible mechanisms of TriEL-filament interaction are discussed.     

Role of adenovirus E1B proteins in transformation: altered organization of intermediate filaments in transformed cells that express the 19-kilodalton protein.

Cooperation of the nuclear oncogene E1A with the E1B oncogene is required for transformation of primary cells. Expression vectors were constructed to produce the 19-kilodalton (19K) and 55K E1B proteins under the direction of heterologous promoters in order to investigate the role of individual E1B proteins in transformation. Coexpression of E1A and either the 19K or 55K E1B gene products was sufficient for the formation of transformed foci in primary rat cells at half the frequency of an intact E1B gene, suggesting that the 19K and 55K proteins function via independent pathways in transformation. Furthermore, the effects of Ha-ras and the E1B 19K gene product were additive when cotransfected with E1A, suggesting that the 19K protein functions in transformation by a mechanism independent from that of ras as well. Although expression of E1A and either E1B protein was sufficient for the subsequent growth of cells in long-term culture, the 19K protein was required to support growth in semisolid media. As the 19K protein has been shown to associate with and disrupt intermediate filaments (IFs) when transiently expressed with plasmid vectors (E. White and R. Cipriani, Proc. Natl. Acad. Sci. USA, 86:9886-9890, 1989), the organization of IFs in transformed cells was investigated. Primary rat cells transformed by plasmids encoding E1A plus the E1B 19K protein showed gross perturbations of IFs, whereas cell lines transformed by plasmids encoding E1A plus the E1B 55K protein or E1A plus Ha-ras did not. These results suggest that an intact IF cytoskeleton may inhibit anchorage-independent growth and that the E1B 19K protein can overcome this inhibition by disrupting the IF cytoskeleton.     

Role of microtubule organization in centrosome migration and mitotic spindle formation in PtK1 cells

Summary Quinacrine, an acridine derivative, has previously been shown to disrupt lateral associations between non-kinetochore microtubules (nkMTs) of opposite polarity in PtK₁ metaphase spindles such that the balance of spindle forces is significantly altered. We extended the analysis of the spatial relationship of spindle microtubules (MTs) in this study by using quinacrine to compare ATP-dependent requirements for early prometaphase centrosome separation and spindle formation. The route used for centrosome migration can take a variety of pathways in PtK₁ cells, depending on the location of the centrosomes at the time of nuclear envelope breakdown. Following quinacrine treatment centrosome separation decresased by 1.9 to 14.0 m depending on the pathway utilized. However, birefringence of the centrosomal region increased approximately 50% after quinacrine treatment. Quinacrine-treated mid-prometaphase cells, where chromosome attachment to MTs had occurred, showed a decrease in spindle length of approximately 6.0 m with only a slight increase in astral birefringence. Computer-generated reconstructions of quinacrine-treated prometaphase cells were used to confirm changes in MT reorganization. Early-prometaphase cells showed more astral MTs (aMTs) of varied length while mid-prometaphase cells showed only a few short aMTs. Late prometaphase cells again showed a large number of aMTs. Our results suggest that: (1) quinacrine treatment affects centrosome separation, (2) recruitment of nkMTs by kinetochores is quinacrine-sensitive, and (3) development of the prometaphase spindle is dependent on quinacrine-sensitive lateral interactions between nkMTs of opposite polarity. These data also suggest that lateral interactions between MTs formed during prometaphase are necessary for centrosome separation and normal spindle formation but not necessarily chromosome motion.Abbreviations aMT(s) astral microtubule(s) - DIC differential interference contrast - MT(s) microtubule(s) - kMT(s) kinetochore microtubule(s) - NEB nuclear envelope breakdown - nkMT(s) non-kinetochore microtubule(s)     

Use of cell enucleation in studying the stability of cytoplasmic organelles and cytoplasm organization

A study was made of the viability and ultrastructure of cytoplasts produced by enucleation of cytochalasin-induced A9 cells in suspension. These cytoplasts are in general as viable as cells enucleated in the monolayer. The organization of the cytoplasm, i.e. a specific distribution of cytoplasmic organelles, is conserved for at least 24 hours in the absence of the nucleus. This fact may reflect a high degree of autonomy of the cytoskeleton.     

Nuclear and cytoplasmic organization in Xenopus oocytes after disruption of actin filaments by latrunculin

Actin-containing filaments have been visualized inside Xenopus oocyte nuclei by a combination of fluorescence and transmission electron microscopy. It was shown that these filaments contact nucleoli, spherical bodies, and nuclear pore complexes. The incubation of oocytes with actin-depolymerizing agent, latrunculin, caused membrane vesiculation in cytoplasm and the disruption of nucleoplasm and the integrity of the nuclear envelope. We suggest that actin-containing filaments are important cell components involved in the regulation of nucleus-cytoplasm interactions, as well as of cellular transport of components during the growth of Xenopus oocytes.     

GCP6 binds to intermediate filaments: a novel function of keratins in the organization of microtubules in epithelial cells

In simple epithelial cells, attachment of microtubule-organizing centers (MTOCs) to intermediate filaments (IFs) enables their localization to the apical domain. It is released by cyclin-dependent kinase (Cdk)1 phosphorylation. Here, we identified a component of the gamma-tubulin ring complex, gamma-tubulin complex protein (GCP)6, as a keratin partner in yeast two-hybrid assays. This was validated by binding in vitro of both purified full-length HIS-tagged GCP6 and a GCP6(1397-1819) fragment to keratins, and pull-down with native IFs. Keratin binding was blocked by Cdk1-mediated phosphorylation of GCP6. GCP6 was apical in normal enterocytes but diffuse in K8-null cells. GCP6 knockdown with short hairpin RNAs (shRNAs) in CACO-2 cells resulted in gamma-tubulin signal scattered throughout the cytoplasm, microtubules (MTs) in the perinuclear and basal regions, and microtubule-nucleating activity localized deep in the cytoplasm. Expression of a small fragment GCP6(1397-1513) that competes binding to keratins in vitro displaced gamma-tubulin from the cytoskeleton and resulted in depolarization of gamma-tubulin and changes in the distribution of microtubules and microtubule nucleation sites. Expression of a full-length S1397D mutant in the Cdk1 phosphorylation site delocalized centrosomes. We conclude that GCP6 participates in the attachment of MTOCs to IFs in epithelial cells and is among the factors that determine the peculiar architecture of microtubules in polarized epithelia.     

Alteration in the arrangement of the keratin-type intermediate filaments during mitosis in cultured human keratinocytes

The behavior of the keratin-type intermediate filaments (KIFs) during mitosis was characterized in cultured human keratinocytes by immunofluorescence microscopy using polyclonal antibodies to keratin. The structural relationship of KIFs with microtubules (MTs) was also studied at the same time using a monoclonal antibody to alpha-tubulin. The KIFs and MTs showed similar but different cytoskeletal networks and underwent structural rearrangements independently during the cell cycle. KIFs in keratinocytes formed two different arrangements during meta- and anaphase: a global aggregation of filaments around the spindle and a fibrous array radiating from the central, global aggregation of filaments to the cell periphery where they were connected with those of the adjacent cells at desmosomal sites. These radiating fibrous portions of KIFs appeared to play a role in retaining the cell in its correct relationship to the surrounding cells during mitosis. This behavior of KIFs in normal keratinocytes was different from the KIF-alterations which had been previously described in SV40-transformed keratinocytes and other cells which expressed two different IFs (keratin and vimentin).     

Roles of actin filaments in cytoplasmic streaming and organization of transvacuolar strands in root hair cells ofHydrocharis

Summary Effects of cytochalasin B and mycalolide-B on cytoplasmic streaming, organizations of actin filaments and the transvacuolar strand were studied in root hair cells ofHydrocharis, which shows reverse fountain streaming. Both toxins inhibited cytoplasmic streaming and destroyed the organizations of actin filaments and transvacuolar strands. However, we found a great difference between these toxins with respect to reversibility. The effects of cytochalasin B were reversible but not those of mycalolide B. The present results suggest that actin filaments work as a track of cytoplasmic streaming and as a cytoskeleton to maintain the transvacuolar strand. The usefulness of root hair cells ofHydrocharis in studying the dynamic organization of actin filaments of plant is discussed.Abbreviations CB cytochalasin B - DMSO dimethylsulfoxide - ML-B mycalolide B     

Effects of griseofulvin on mitosis in PtK1 cells

The concentration dependent effects of griseofulvin (GF) on mitosis in PtK₁ cells were studied using a combination of time lapse cinematography and polarization and electron microscopy. Low concentrations of GF (4×10^–5 M) allowed a substantial number of cells to enter and complete an apparently normal mitosis. At higher concentrations of GF (1×10^–4 M and 2.5×10^–4 M) all cells entering mitosis were arrested. Typical c-mitotic chromosome arrays were observed at 1×10^–4 M GF with microtubules present but no spindle formed. At 2.5×10^–4 M GF chromosomes did not orient toward a common center to form a c-mitotic figure, but instead remained in a loosely clustered grouping at the center of the cell. Electron microscopy showed microtubules to be absent but revealed an irregularly shaped electron dense cloud around the centrioles. Quantitative polarization microscopy of metaphase cells perfused with GF showed rapid loss of spindle birefringence after exposure to the drug. Coinciding with loss of birefringence the spindle shrank rapidly with a pronounced shortening of pole to pole distance.