Intermediate-sized Filament Proteins Research Articles

The cell\u2013cell junctions of mammalian testes: II. The lamellar smooth muscle monolayer cells of the peritubular wall are laterally connected by vertical adherens junctions\u2014a novel architectonic cell\u2013cell junction system

The testes of sexually mature males of six mammalian species (men, bulls, boars, rats, mice, guinea pigs) have been studied using biochemical as well as light and electron microscopical techniques, in particular immunolocalizations. In these tissues, the peritubular walls represent lamellar encasement structures wrapped around the seminiferous tubules as a bandage system of extracellular matrix layers, alternating with monolayers of very flat polyhedral “lamellar smooth muscle cells” (LSMCs), the number of which varies in different species from 1 to 5 or 6. These LSMCs are complete SMCs containing smooth muscle α-actin (SMA), myosin light and heavy chains, α-actinin, tropomyosin, smoothelin, intermediate-sized filament proteins desmin and/or vimentin, filamin, talin, dystrophin, caldesmon, calponin, and protein SM22α, often also cytokeratins 8 and 18. In the monolayers, the LSMCs are connected by adherens junctions (AJs) based on cadherin-11, in some species also with P-cadherin and/or E-cadherin, which are anchored in cytoplasmic plaques containing β-catenin and other armadillo proteins, in some species also striatin family proteins, protein myozap and/or LUMA. The LSMC cytoplasm is rich in myofilament bundles, which in many regions are packed in paracrystalline arrays, as well as in “dense bodies,” “focal adhesions,” and caveolae. In addition to some AJ-like end-on-end contacts, the LSMCs are laterally connected by numerous vertical AJ-like junctions located in variously sized and variously shaped, overlapping (alter super alterum) lamelliform cell protrusions. Consequently, the LSMCs of the peritubular wall monolayers are SMCs sensu stricto which are laterally connected by a novel architectonic system of arrays of vertical AJs located in overlapping cell protrusions.

On the Formation of Lipid Droplets in Human Adipocytes: The Organization of the Perilipin–Vimentin Cortex

We report on the heterogeneity and diversity of lipid droplets (LDs) in early stages of adipogenesis by elucidating the cell and molecular biology of amphiphilic and cytoskeletal proteins regulating and stabilizing the generation of LDs in human adipose cells. A plethora of distinct and differently sized LDs was detected by a brief application of adipocyte differentiation medium and additional short treatment with oleic acid. Using these cells and highly specific antibodies for LD-binding proteins of the perilipin (PLIN) family, we could distinguish between endogenously derived LDs (endogenous LDs) positive for perilipin from exogenously induced LDs (exogenous LDs) positive for adipophilin, TIP47 and S3-12. Having optimized these stimulation conditions, we used early adipogenic differentiation stages to investigate small-sized LDs and concentrated on LD-protein associations with the intermediate-sized filament (IF) vimentin. This IF protein was described earlier to surround lipid globules, showing spherical, cage-like structures. Consequently - by biochemical methods, by immunofluorescence microscopy and by electron- and immunoelectron microscopy - various stages of emerging lipid globules were revealed with perilipin as linking protein between LDs and vimentin. For this LD-PLIN-Vimentin connection, a model is now proposed, suggesting an interaction of proteins via opposed charged amino acid domains respectively. In addition, multiple sheaths of smooth endoplasmic reticulum cisternae surrounding concentrically nascent LDs are shown. Based on our comprehensive localization studies we present and discuss a novel pathway for the LD formation.

Observations on early development of the murine fetal oral vestibule.

Morphological and immuno/histochemical studies were performed on the vestibular lamina (VL) of gestational day 13 murine fetuses, using light microscopy (LM), confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM), in an effort to elucidate the early development of the oral vestibule. Histochemistry employing LM demonstrated some PAS-positive glycogen particles in embryonic cells of the VL, dental lamina (DL), the primary epithelial band connecting the VL and DL, and the related stomodaeal simple epithelium. On the other hand, Gomori's aldehyde fuchsine method stained certain cystine-containing intracellular granules and intercellular amorphous substances, particularly in the central VL. Intense immunoreactivity for CK-10 intermediate-sized filament proteins was demonstrated in suprabasal and superficial cells of the VL stratified keratinized epithelium. Conversely, reactions for CK-19 filaments were found diffusely in both VL and DL cells retaining the cytokeratin characteristic of the simple epithelium. TEM of the VL revealed an increment in keratinosomes, tonofilaments and desmosomes in the suprabasal layers shifting toward superficial flat parakeratinized cells. The TUNEL method using CLSM detected programmed cell death in the VL, while TEM provided no morphological evidence of necrosis or typical apoptotic features during VL development. The present results indicate that physiological (naturally occurring) cell death and exfoliation of the oral-gingival type multilayered keratinizing epithelium are essential for degeneration and separation of the VL, ultimately leading to formation of the oral vestibule.

Regulated docking of nuclear membrane vesicles to vimentin filaments during mitosis.

During mitosis, several types of intermediate-sized filaments (IFs) undergo an extensive remodelling in response to phosphorylation by cdc 2 and other protein kinases. However, unlike the nuclear lamins, the cytoplasmic IFs do not seem to follow a fixed disassembly stereotype and often retain their physical continuity without depolymerizing into soluble subunits. To investigate potential interactions between mitotically modified IFs and other cellular structures, we have examined prometaphase-arrested cells expressing the IF protein vimentin. We demonstrate here that vimentin filaments associate in situ and co-fractionate with a distinct population of mitotic vesicles. These vesicles carry on their surfaces nuclear lamin B, the inner nuclear membrane protein p58, and wheat germ agglutinin (WGA)-binding proteins. Consistent with a tight interaction between the IFs and the mitotic membranes, vimentin, nuclear lamin B, and a 180-kD WGA-binding protein are co-isolated when whole mitotic homogenates are incubated with anti-vimentin or anti-lamin B antibodies immobilized on magnetic beads. The vimentin-associated vesicles are essentially depleted of ER, Golgi and endosomal membrane proteins. The interaction of vimentin with lamin B-carrying membranes depends on phosphorylation and is weakened by dephosphorylation during nuclear reassembly in vitro. These observations reveal a novel interaction between IFs and cellular membranes and further suggest that the vimentin filaments may serve as a transient docking site for inner nuclear membrane vesicles during mitosis.

Development of the rabbit retina

Measures of rabbit eyes and retinal wholemounts were used to evaluate the development of retinal area and shape. The retina is shown to have a horizontal axis about a third longer than the vertical axis just before birth, and to adopt an almost symmetrical shape during postnatal development to adulthood. In general, retinal thickness is shown to decrease after birth, but differently in particular retinal regions: the reduction is marked in the periphery, and less pronounced in the visual streak. As an exception, the myelinated region--after it becomes really myelinated, from 9 days p.p.--even increases in thickness. In all regions of the retina, the absolute and relative thickness of the nuclear layers decreases, whereas the relative thickness of plexiform and fibrous layers increases. Proliferation of cells within the rabbit retina was studied during the first three postnatal weeks. 3H-thymidine incorporation was used to demonstrate DNA synthesis autoradiographically in histological sections as well as in enzymatically isolated retinal cells. A first proliferation phase occurs in the neuroblastic cell layer and ceases shortly after birth in the retinal center, but lasts for about one week in the retinal periphery. We found, however, a few 3H-thymidine-labeled cells as late as in the third postnatal week. These late-labeled cells were found within the nerve fiber layer and in the inner plexiform layer. The latter cells were shown to express antigens detected by antibodies directed to the intermediate-sized filament protein vimentin, which are known to label Müller cells and neuroepithelial stem cells. This was confirmed in our preparation of enzymatically isolated cells; all cells with autoradiographically labeled nuclei revealed a characteristic elongated morphology typical for Müller radial glia (and also for early neuroepithelial stem cells). 3H-thymidine-labeled cells in the nerve fiber layer were most probably astrocytic. In analogy to the brain, we conclude that the mammalian retina undergoes a series of proliferation phases: first an early phase producing both neurons and glial cells, and then a late phase producing glial cells, e.g., in the nerve fiber layer. Most probably, the late phase within the inner nuclear layer is glial as well, i.e., consists of dividing Müller cells; it cannot be excluded, however, that there may remain some mitotically active stem cells.

Cytokeratin filaments and desmonsomes in the epithelioid cells of the perineurial and arachnoidal sheaths of some vertebrate species

Abstract Using electron microscopy and immunohistochemistry with a large panel of antibodies to various cytoskeletal proteins we have noted that the singel- or multilayered sheaths of epithelioid cells (“neurothelia”) surrounding periopheral nerves (perineurial cells) or structres of the central nerous system, including the optic nerve (arachnoid cells), show remarkable interspecies differences in their cytoskeletal complements. In two anuran amphibia examined ( Xenopus laevis, Rana ridibunda), the cells of both froms of neuraothelia, i.e., perineurial and arachnoid, are interconnnected hy true desmosomes and are rich in intermediate-sized filaments (IFs) of the cytokeratin type. Among higher vertebrates, a similar situation is found in the bovine and chicken nervous systems, in which the arachnoid cells of the meninges contain desmosomes and IFs of both the cytokeratin (apparently with restricted epitope accessibilities in the chicken) and the vimentin tupe, whereas the perineurial cells of many nerves contain cytokeratin IFs, often together with vimentin, but no desmosomes. In contrast, in rat arachnoidal and perineurial cells significant reactions have been observed neither for cytokeratins nor for desmosomes. In the human nervous system, cytokeratins and desmosomes have also not been seen in thevarious perineuria studied whereas desmosomes are frequent in arachnoidal cell layers which are dominated by vimentin IFs and only in certain small regions of the brain contain some additional cytokeratins. The occurrence of cytokeratins in the tissues found positive by immunohistochemistry has been confirmed by gel electrophoresis of cytoskeletal proteins, followed by immunoblottting. Our results emphasize both similarities and differences between the neurothelia on the one hand and epithelia or endothelia on the other, justifying classificaiton as a separate kind of tissue, i. e., neurothelium. The observatons of interspecies differences lead tp the challenging conclusion thaht neither desmosomes nor cytokeratins are essential for the basic functions of neurothelial sheaths nor does the specific type of IF protein expressed in these cells appear to matter in this respect. The results are also discussed in relation to the cytoskeletal characteristicis of other epithelioid tissues and of human neurothelium-derived tumors.

Intermediate filament expression in small cell lung cancer; poor correlation to in vitro data

A panel of monoclonal antibodies, detecting different intermediate sized filament proteins, was prospectively applied on all specimens derived from S.C.L.C. patients attending our clinic in 1985. Reactivity with the antibodies was subsequently correlated to clinical data. The results indicate a heterogeneous pattern of reactivity of the assessed antibodies. However this heterogeneity is not a straightforward extension of the intermediate sized filament expression in SCLC cell lines.

Cytoskeletal components of lymphoid organs

Abstract Using light and electron microscopic immunolocalization with antibodies to cytoskeletal proteins, we have characterized the nonlymphoid cells of various human lymphoid organs (lymph nodes, tonsils, spleen). In all these tissues, the lymphoid follicles contain a three-dimensional meshwork of “dendritic reticulum cells” which are characterized by the presence of desmosomal junctions, as demonstrated by positive punctate staining with antibodies to the desmosome-specific proteins desmoplakin I and desmoglein, and by intermediate-sized filaments (IFs) of the vimentin type only. In contrast, the extrafollicular regions are characterized by an extended meshwork of other types of reticulum cells, which also contain vimentin IFs but lack desmosomal proteins. In addition, a considerable, although variable proportion of these extrafollicular reticulum cells forms IFs containing cytokeratins 8 and 18 and/or desmincontaining IFs. The occurrence of cytokeratins 8 and 18 in lymph nodes has also been shown by gel electrophoresis and immunoblotting. Results of double-label immunolocalization indicate that some of the extrafollicular reticulum cells coexpress all three kinds of IF protein. A large proportion of these cells also synthesizes another marker of myogenic differentiation, i.e., the isoform of α-actin specific for smooth muscle. This proportion includes some cells that are negative for desmin. Comparison of the distribution of cells expressing cytokeratins and/or desmin with that of reticulum cells showing strong alkaline phosphatase activity (as a marker for the so-called “fiber-associated (fibroblastic) reticulum cells”) suggests that the former represent a subset of the latter. The biological meaning of these different patterns of expression in reticulum cells and of the resulting cell-type heterogeneity as well as possible implications of these observations for tumor diagnosis, notably of lymph-node metastases and lymphomas, are discussed.

Synaptophysin: a reliable marker for medulloblastomas.

Synaptophysin is an acidic, integral membrane glycoprotein (Mr 38,000) of presynaptic vesicles in various neurons and neuroendocrine cells, and in tumours derived from such cells. By indirect immunofluorescence microscopy of cryostat sections, using the monoclonal antibody SY 38 to synaptophysin, a consistent positive immunoreactivity was observed in all medulloblastomas (n = 6) and neuroblastomas (n = 3) as well as a ganglioneuroma and a glioneuronal hamartoma. The presence of synaptophysin in medulloblastomas was confirmed biochemically by immunoblotting experiments. For purpose of comparison, the expression of intermediate-sized filament (IF) proteins was also examined. While neurofilament proteins were consistently expressed in the neuroblastomas (3/3), the ganglioneuroma and the glioneuronal hamartoma, IF distribution in medulloblastomas was variable. A neurofilament-positive type of tumour (1/6) could be distinguished from vimentin-expressing neoplasms (4/6) by immunocytochemistry. These data indicate that synaptophysin is a reliable marker for medulloblastomas as well as other differentiated and undifferentiated neuronal tumours and in this respect is superior to the more heterogeneous expression patterns of IF proteins in these tumours.

Characterization of dimer subunits of intermediate filament proteins

The fundamental subunit of the various types of intermediate-sized filaments (IF) has been shown to be a tetramer that is thought to represent a double dimer, i.e. an array of two laterally packed coiled-coils of α-helices. The two-chain state of intact IF proteins had up to this point not been isolated and characterized as has been done for other fibrous α-helical coiled-coil proteins. Using buffers containing 3 m-guanidinium hydrochloride we prepared dimers by depolymerization of IF or by reconstitution from fully denatured molecules. Dimers of desmin (from chicken gizzard), vimentin (from bovine lens tissue and cultured human fibroblasts) and the neurofilament protein NF-L (from bovine brain) as well as in vitro formed homodimers of human and rat cytokeratins numbers 8 (A), 18 (D) and 19 (“40K”), are characterized by ultracentrifugation techniques (sedimentation velocity and equilibrium), electron microscopy and chemical cross-linking. The results show that IF proteins form discrete complexes of two polypeptide chains in parallel orientation and probably in coiled-coil configuration, which apparently have a high tendency to further associate into double dimers. Implications of these results for concepts of IF organization and IF protein assembly are discussed.

Cytokeratin expression in simple epithelia: I. Identification of mRNA coding for human cytokeratin no. 18 by a cDNA clone

To study the regulation of the expression of cyto-keratins characteristic of simple epithelia, i.e., human cytokeratins nos. 7, 8, 18, and 19, we prepared several cDNA clones coding for these proteins and their bovine counterparts. In the present study, we describe a cDNA clone of the mRNA coding for human cytokeratin no. 18, which was isolated from an expression library using the monoclonal antibody, K G 8.13. This clone (756 nucleotides, excluding the poly A portion), encodes approximately one-half of the mRNA (approximately 1.4 kb), identifies one mRNA band in Northern-hybridization blots, and specifically selects one mRNA species coding for cytokeratin no. 18, as demonstrated by translation in vitro. Comparison of the deduced amino acid sequence - confirmed by direct amino-acid-sequence analyses of some polypeptide fragments produced by cleavage with cyanogen bromide - indicated that cytokeratin no. 18 is a member of the acidic (type I) subfamily of cytokeratins. It has only limited sequence homologies in common with other intermediate-sized filament proteins, and these are essentially restricted to certain domains of the a-helical rod portion. The carboxyterminal tail sequence does not contain glycine-rich elements, thus distinguishing this cytokeratin from those acidic (type I) cytokeratins that are characterized by this feature. The similarities and differences between cytokeratin no. 18 and previously described epidermal cytokeratins are discussed in relation to the differences in the stability of the complexes which this cytokeratin forms with basic (type 11) cytokeratins, as well as in relation to possible functional differences of cytokeratins in simple and stratified epithelia.

Identification of a distinct soluble subunit of an intermediate filament protein: tetrameric vimentin from living cells.

Intermediate-sized filaments (IF) are among the most insoluble intracellular protein polymer structures. We have analyzed the small amounts of soluble vimentin, an IF protein, present in cytosol fractions obtained from lysis of cultured cells [rat RVF-SM cells, simian virus 40-transformed human fibroblasts, and human rhabdomyosarcoma (RD line) cells]. The molecular form of this soluble vimentin was determined by sucrose density gradient centrifugation, using vimentin-specific antibodies for subsequent ELISA and immunoblotting analyses. The majority of the soluble vimentin appeared in a distinct form indistinguishable in its sedimentation behavior from reconstituted tetrameric subunits of purified vimentin arrested at low ionic strength. The tetrameric coiled-coil nature of the soluble form of vimentin was indicated by the digestion pattern with chymotrypsin and by chemical crosslinking with copper-1,10-phenanthroline and dimethylsuberimidate. The competence of this soluble vimentin to assemble into IF at higher salt concentrations was demonstrated by electron microscopy. Pulse-chase experiments showed that the soluble form was not an exclusively posttranslational intermediate. We propose that in the living cell a small pool of a distinct soluble tetrameric form of vimentin exists which may exchange with polymeric IF vimentin.

Isolation of the intermediate filament protein vimentin by chromatofocusing

A novel, simple and relatively rapid method is described for the isolation of the intermediate-sized filament protein vimentin from eye lens tissue. Chromatofocusing is applied as the sole purification step. The apparent isoelectric point of the protein in 6 M urea and at 22°C is 4.9. Electrophoretic mobility on one- and two-dimensional polyacrylamide gels, solubility in 6 M urea and amino acid composition were used for identification

Attachment of vimentin filaments to desmosomal plaques in human meningiomal cells and arachnoidal tissue.

Desmosomal proteins are co-expressed with intermediate-sized filaments (IF) of the cytokeratin type in epithelial cells, and these IF are firmly attached to the desmosomal plaque. In meningiomal and certain arachnoidal cells, however, vimentin IF are attached to desmosomal plaques. Meningiomas obtained after surgery, arachnoid "membranes", and arachnoid granulations at autopsy, as well as meningiomal cells grown in short-term culture have been examined by single and double immunofluorescence and immunoelectron microscopy using antibodies to desmoplakins, vimentin, cytokeratins, glial filament protein, neurofilament protein, and procollagen. In addition, two-dimensional gel electrophoresis of the cytoskeletal proteins has been performed. Using all of these techniques, vimentin was the only IF protein that was detected in significant amounts. The junctions morphologically resembling desmosomes of epithelial cells have been identified as true desmosomes by antibodies specific for desmoplakins and they provided the membrane attachment sites for the vimentin IF. These findings show that anchorage of IF to the cell surface at desmosomal plaques is not restricted to cytokeratin IF as in epithelial cells and desmin IF as in cardiac myocytes, suggesting that binding to desmosomes and hemidesmosomes is a more common feature of IF organization. The co-expression of desmosomal proteins and IF of the vimentin type only defines a new class of cell ("desmofibrocyte") and may also provide an important histodiagnostic criterion.

Protein complexes of intermediate-sized filaments: melting of cytokeratin complexes in urea reveals different polypeptide separation characteristics.

Subunit complexes of cytokeratin polypeptides from intermediate-sized filaments (IF) of various tissues and cultured cells from rat, cow, and man were solubilized in low-salt buffer containing 4 M urea and exposed to increasing concentrations of urea, followed by urea gradient electrophoresis or two-dimensional gel electrophoresis at different urea concentrations. Correspondingly, cytokeratin polypeptides dissociated in 9.5 or 10 M urea were dialyzed into lower concentrations of urea and allowed to reassociate into specific complexes. It was found that the polypeptide constituents of a given cytokeratin complex dissociate in the form of a rather sharp "melting curve" and that dissociated polypeptides reassociate in the same mode of dependence on urea concentration. The midpoint of melting in urea (Um) is a characteristic property of a given complex of cytokeratin polypeptides. Um values differ markedly between different cytokeratin complexes, ranging from 5.9 to 9.0 M urea. The results also show that cytokeratins do not form complexes with vimentin, another type of IF protein. The data suggest that certain cytokeratin polypeptides are complementary and contain sequences that direct their association into specific complexes forming IF subunits.

Phosphorylation of keratin and vimentin polypeptides in normal and transformed mitotic human epithelial amnion cells: behavior of keratin and vimentin filaments during mitosis.

Analysis by means of two-dimensional gel electrophoresis (IEF) of [32P]orthophosphate-labeled proteins from mitotic and interphase transformed amnion cells (AMA) has shown that keratins IEF 31 (Mr = 50,000; Hela protein catalogue number), 36 (Mr = 48,500), 44 (Mr = 44,000), 46 (Mr = 43,500), as well as vimentin (IEF 26; Mr = 54,000) are phosphorylated above their interphase level during mitosis. Similar studies of normal human amnion epithelial cells (AF type) confirmed the above observations except in the case of keratin IEF 44 whose relative proportion was too low to be analyzed. Immunofluorescent staining of methanol/acetone-treated mitotic transformed amnion cells with a mouse polyclonal antibody elicited against human keratin IEF 31 showed a dotted staining (with a fibrillar background) in all of the cells in late anaphase/early telophase (characteristic "domino" pattern) and in a sizeable proportion of the cells in other stages of mitosis. Normal mitotic amnion cells on the other hand showed a fine fibrillar staining of keratins at all stages of mitosis. Similar immunofluorescent staining of normal and transformed mitotic cells with vimentin antibodies revealed a fibrillar distribution of vimentin in both cell types. Taken together the results indicate that the transformed amnion cells may contain a factor(s) that modulates the organization of keratin filaments during mitosis. This putative factor(s), however, is most likely not a protein kinase as transformed amnion cells and amnion keratins are modified to similar extents. It is suggested that in general the preferential phosphorylation of intermediate-sized filament proteins during mitosis may play a role in modulating the various proposed associations of these filaments with organelles and other cellular structures.

Antibodies to intermediate filament proteins in the immunohistochemical identification of human tumours: an overview

Intermediate-sized filament proteins (IFP) are tissue specific in that antibodies to keratin, vimentin, desmin, glial fibrillary acidic protein (GFAP) and the neurofilament proteins can distinguish between cells of epithelial and mesenchymal origin as well as of myogenic and neural origin respectively. Malignant cells retain their tissue-specific IFP, which makes it possible to use these antibodies in tumour diagnosis. Carcinomas are exclusively detected by antibodies to keratin. Monoclonal antibodies to keratin have allowed the differentiation between subgroups of epithelial tumours until now between adenocarcinomas and squamous cell carcinomas. Lymphomas, melanomas and several soft tissue tumours are distinctly recognized by antibodies to vimentin. On the other hand, rhabdomyosarcomas and leiomyosarcomas are positive for desmin, while astrocytomas give a strong reaction with GFAP antibodies. Thus, antibodies to IFP are useful tools for differential diagnosis in surgical pathology.

Evidence for coordinated phosphorylation of keratins and vimentin during mitosis in transformed human amnion cells: Phosphate turnover of modified proteins

Two-dimensional gel electrophoresis (IEF) analysis of short-term [ 32P]orthophosphate-labelled intermediate-sized filament proteins (keratins and vimentin) from transformed mitotic amnion cells (AMA), have shown that these proteins are modified coordinately and that the half life of the phosphate is about 13 min for the keratins and 11 min for vimentin. These results support the notion that the preferential modification of intermediate-sized filament proteins during mitosis may play a role in modulating filament associations with organelles and other cellular structures.

The cytoskeleton in tumor cells

During the past few years several laboratories investigated the occurrence of cytoskeletal components in epithelial and mesenchymal cells by electron microscopy and/or immunocytochemical methods in a number of tumor types growing in vitro or in the body. Since it is well established that antibodies to different intermediate-sized filament proteins can distinguish cells and tissues of epithelial, mesenchymal, muscle, astrocytic and neural origin special attention has been paid to the behaviour of these filaments in neoplastic cells recently. While the organisation of the cytoskeleton in tumor cells growing in vitro is very variable, regularities relevant for the diagnosis and the determination of the histogenetic origin of tumors have been observed in tumor cells growing in the body. In general, ultrastructural and immunological features of intermediate filaments are maintained during neoplastic transformation in the body. Thus immunofluorescence microscopy with antibodies to cytoskeletal proteins is a powerful tool for the classification and differential diagnosis of tumors, especially for the distinction between epithelial and mesenchymal tumors, including metastases. The concept that presence of an excess of contractile proteins such as actin is an important prerequisite for the metastatic spread of malignant cells has not been unequivocally supported by more recent results. However, an accumulation of various types of intermediate filaments (e.g. prekeratin, vimentin, acidic glial fibrillar protein) has been shown in different tumor types. The further elucidation of this alteration could contribute to a better understanding of the molecular mechanisms of neoplastic cell transformation.

Effect of the ionic environment on the incorporation of the intermediate-sized filament protein vimentin into residual cell structures upon treatment of Ehrlich ascites tumour cells with triton X-100. II. Ultrastructural analysis.

Ehrlich ascites tumour cells were extracted in buffers containing Triton X-100 and mono-di- and polyvalent cations and then analysed by phase-contrast and electron microscopy. The results of this ultrastructural analysis confirm those of the biochemical analysis in the accompanying paper that the stability of intermediate-sized filaments is dependent on the ionic environment. Furthermore, the organization of filaments in long parallel arrays is dependent on the presence of divalent cations and can be inhibited, to some extent, by the presence of monovalent cations. The stability of other detergent-resistant structures, the boundary lamina, microfilaments, microtubules, centrioles, polyribosomes and the nuclear cortex, is also affected by the ionic environment but to a lesser extent.