Three-dimensional study of the cytoskeleton in macrophages and multinucleate giant cells by quick-freezing and deep-etching method

The three-dimensional ultrastructure of multinucleate giant cells in subcutaneous granulomas was compared with those of peritoneal macrophages using a quick-freezing and deep-etching method. Subcutaneous granulomas were induced by implanting plastic coverslips in the dorsal subcutaneous tissue of rats. The quick-freezing and deep-etching replicas were prepared from the cells attached to the coverslips. Dense networks of actin filaments were distributed along all peripheral aspects (beneath the plasma membrane, and on free and coverslip-attached surfaces) of the multinucleate giant cells. On the coverslip-attached surface, numerous clathrin-coated pits and vesicles occurred between the actin filaments. In these cells, intermediate filaments, but not actin filaments, were the predominant cytoskeletal components in perinuclear regions and were attached to the cell nucleus, mitochondria and other vesicular cell organelles. A similar distribution of cytoskeletal components was observed in the mononuclear macrophages of the granulomas and the peritoneal macrophages. These results show that the cytoskeletal organization varies in different regions of the cytoplasm of multinucleate giant cells, while the characteristic cytoskeletal arrangement, resembling that of mononuclear macrophages, is maintained.     

Three-dimensional study of the cytoskeleton in macrophages and multinucleate giant cells by quick-freezing and deep-etching method.

The three-dimensional ultrastructure of multinucleate giant cells in subcutaneous granulomas was compared with those of peritoneal macrophages using a quick-freezing and deep-etching method. Subcutaneous granulomas were induced by implanting plastic coverslips in the dorsal subcutaneous tissue of rats. The quick-freezing and deep-etching replicas were prepared from the cells attached to the coverslips. Dense networks of actin filaments were distributed along all peripheral aspects (beneath the plasma membrane, and on free and coverslip-attached surfaces) of the multinucleate giant cells. On the coverslip-attached surface, numerous clathrin-coated pits and vesicles occurred between the actin filaments. In these cells, intermediate filaments, but not actin filaments, were the predominant cytoskeletal components in perinuclear regions and were attached to the cell nucleus, mitochondria and other vesicular cell organelles. A similar distribution of cytoskeletal components was observed in the mononuclear macrophages of the granulomas and the peritoneal macrophages. These results show that the cytoskeletal organization varies in different regions of the cytoplasm of multinucleate giant cells, while the characteristic cytoskeletal arrangement, resembling that of mononuclear macrophages, is maintained.     

In vitro fusion of newt macrophages

Spontaneous formation of multinucleate giant cells is often observed in in vitro cultures of peritoneal adherent macrophages from the newts, Notophthalmus viridescens and Taricha granulosa (urodele amphibians). The frequency of such giant cells in these cultures is increased by the addition of phorbol myristic acetate at the initiation of the cultures. This high frequency of multinucleate cells permitted us to evaluate whether multinucleate giant cells arise by cell fusion and/or by repeated nuclear division without cytokinesis. Cell fusion is readily detectable by scanning electron microscopy. To determine whether nuclear division without cytokinesis also occurs, some cultures were treated with colchicine to arrest mitotic figures; others were pulsed with tritiated thymidine to detect DNA synthesis. Mitotic figures were not seen in acridine orange-stained samples. In monolayers that were processed for autoradiography, only a few nuclei were marked with tritium. These observations suggest that nuclear division does not contribute significantly, if at all, to the formation of multinucleate giant cells from cultured newt peritoneal macrophages.     

Inflammatory giant cells: immunophagocytosis and rosette formation

Mouse inflammatory giant cells formed after subcutaneous implantation of coverslips were exposed to sheep red blood cells opsonized with isologous antibodies. The maximal number of engulfed erythrocytes in numerous multinuclear cells exceeded that encountered in subcutaneous macrophages, but, on a per nucleus basis, the giant cells appeared less phagocytic.     

Transient change of organization of vimentin filaments during mitosis as demonstrated by a monoclonal antibody

A monoclonal antibody specific for vimentin is described which, by immunofluorescence and immunoelectron microscopy, decorates fibrillar and/or granular structures in mitotic and early postmitotic cells but does not react with vimentin filaments of interphase stages of various cultured cells (rat vascular smooth muscle-derived cell line RVF-SM; SV40-transformed human fibroblasts; bovine kidney epithelial cells of line MDBK). These observations indicate that the organization of vimentin filaments varies during the cell cycle, undergoing a perimitotic change of filament organization. These changes of vimentin filaments are described in relation to those reported for cytokeratin filaments of various epithelial and carcinoma cells. The possible functional implications of filament protein rearrangements both during the cell cycle and in cell differentiation processes are discussed.     

Hemmung der Cytokinese und Bildung von Riesenzellen beiPoterioochromonas malhamensis durch organische Bleiverbindungen und andere Agenzien

Summary Organic lead compounds inhibit cytokinesis of the chrysophycean flagellatePoterioochromonas malhamensis leading to giant, multinucleate cells. This action on cytokinesis is compared with the long-time effects of various compounds with better known subcellular activities.Calcium (10 mM), and cytochalasin B (up to 100 g/ml) do not visibly influence cytokinesis. Caffeine (1 mM) totally inhibits multiplication of the algae whereas calcium has only a slight and cytochalasin has no effect on this parameter.The other reference-compounds (colchicine, sodium cacodylate, deuterium oxide, local anesthetics, and sodium dodecylsulfate) all inhibit cell multiplication, simultaneously leading to giant multinucleate cells, obviously by inhibition of cytokinesis.The most potent inhibitor of cytokinesis is triethyl lead which was shown to be 250× more effective than colchicine in respect to the molar concentrations.The comparison of the effects of tetraethyl lead and triethyl lead with the reference agents leads to the conclusion that organic lead compounds might inhibit cytokinesis ofPoterioochromonas malhamensis by disintegrating peripheral microtubules and/or by interfering with structures and functions of membranes.

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Verwendete Abkürzungen im Text CB Cytochalasin B - KE Karminessigsäure - KV kontraktile Vakuole - LV Leukosinvakuole - MT Mikrotubuli - SDS Na-Dodecyl-sulfat - TEL Tetraäthylblei - TriEL Triäthylblei     

The Association of Tau-Like Proteins with Vimentin Filaments in Cultured Cells

There is increasing evidence that the different polymers that constitute the cytoskeleton are interconnected to form a three-dimensional network. The macromolecular interaction patterns that stabilize this network and its intrinsic dynamics are the basis for numerous cellular processes. Within this context,in vitrostudies have pointed to the existence of specific associations between microtubules, microfilaments, and intermediate filaments. It has also been postulated that microtubule-associated proteins (MAPs) are directly involved in mediating these interactions. The interactions of tau with vimentin filaments, and its relationships with other filaments of the cytoskeletal network, were analyzed in SW-13 adenocarcinoma cells, through an integrated approach that included biochemical and immunological studies. This cell line has the advantage of presenting a wild-type clone (vim+) and a mutant clone (vim−) which is deficient in vimentin expression. We analyzed the cellular roles of tau, focusing on its interactions with vimentin filaments, within the context of its functional aspects in the organization of the cytoskeletal network. Cosedimentation experiments of microtubular protein with vimentin in cell extracts enriched in intermediate filaments, combined with studies on the direct interaction of tau with nitrocellulose-bound vimentin and analysis of tau binding to vimentin immobilized in single-strand DNA affinity columns, indicate that tau interacts with the vimentin network. These studies were confirmed by a quantitative analysis of the immunofluorescence patterns of cytoskeleton-associated tubulin, tau, and vimentin using flow cytometry. In this regard, a decrease in the levels of tau associated to the cytoskeletal network in the vim− cell mutant compared with the wild-type clones was observed. However, immunofluorescence data on SW-13 cells suggest that the absence of a structured network of vimentin in the mutant vim− cells does not affect the cytoplasmic organization formed by microtubules and actin filaments, when compared with the wild-type vim+ cells. These studies suggest that tau associates with vimentin filaments and that these interactions may play a structural role in cells containing these filaments.     

Preparation and some properties of multinucleate cells of Saccharomyces cervisiae after colchicine treatment

For the purpose of forming cells possessing more than three nuclei and of determining the factors inducing multinucleation, cells of Saccharomyces cerevisiae were treated with 0, 0.3, 0.5, and 1.0% [w/v] colchicine solution, with and without shaking. When the cells were treated with 1.0% [w/v] colchicine solution, the number of cells containing two to eight nuclei was the largest. The multinucleate cells could grow on potato dextrose agar medium and their multinucleate nature did not disappear for at least three generations. This means that such cells are genetically stable. The proliferation rate of the multinucleate cells was not superior to that of the original strain. However, by monitoring the weight loss of the flask, it was possible to indirectly estimate the increase in the alcohol production of the multinucleate cell. It was concluded that the shaking treatment and higher colchicine concentrations contributed to multinucleation.     

A quantitative cytochemical investigation of osteoclasts and multinucleate giant cells

Summary Quantitative cytochemical, immunocytochemical, autoradiographic and electron cytochemical investigations have been used to compare osteoclasts with multinucleate giant cells that had been freshly obtained from the same animal. The levels of -acid galactosidase activity, the DNA in individual nuclei and the cellular protein content were similar in both cell types. However, osteoclasts generally possessed greater acid phosphatase and NADH dehydrogenase activity but lower levels of fluoride-inhibited non-specific esterase activity than multinucleate giant cells. The acid phosphatase activity in multinucleate giant cells was completely inhibited by 100 mM tartrate, but in osteoclasts only a 20% reduction in activity was observed. Formation of multinucleate giant cells in a bone microenvironment (thin bone slices) did not increase their content of tartrate-resistant acid phosphatase activity. Moreover, in osteoclasts, endogenous peroxidase activity was undetectable but present in several granules within the cytoplasm of multinucleate giant cells. Osteoclasts and multinucleate giant cells displayed a similar microtubular distribution, but calcitonin, which induced rearrangement of microtubules and cellular contraction in osteoclasts, had no effect on multinucleate giant cells. Thus, these investigations reveal both similarities and differences between these two syncytia and support the hypothesis that osteoclasts and multinucleate giant cells are related. Possibly osteoclasts arise from monocyte progenitors before commitment to a macrophage lineage has occurred.     

Phagocytosis and giant cell formation at 0° C by macrophage (MØ) of Notothenia coriiceps

Macrophages adhered to round glass coverslips that had been implanted into the abdominal cavity of Notothenia coriiceps , collected from Admiralty Bay, King George Island, and removed and incubated in vitro for 4 h at 0° C at various intervals. The macrophages phagocytosed yeast Saccharomyces cerevisiae cells suspended in saline. Bi-nuclear macrophages were first observed after 6 h. The formation of giant cells and their phagocytic activity was observed only in fish which had been injected with 1 ml of Bacillus of Calmette and Guerin (BCG)-suspension 24 h before coverslip implantation and the coverslips removed 15 days after the implantation. Phagocytosis and the formation of giant cells in an Antarctic fish at 0° C is described for the first time.     

Effect of acrylamide on the cytoskeleton and apoptosis of bovine lens epithelial cells

Acrylamide, a known disrupter of intermediate filaments, has been used to produce the collapse of vimentin filaments in bovine lens epithelial (BEL) cells, and its potential modulation of staurosporine-induced apoptosis has been investigated. In BEL cells, short treatments with acrylamide caused the collapse of vimentin filaments and microtubules and the almost complete disappearance of stress fibers, with thick f-actin bundles remaining in the cell periphery. Actin organization was less affected in cells pretreated with colchicine and in spreading cells, suggesting that extended microtubules and vimentin filaments are required for acrylamide to produce its maximal effects. Acrylamide alone slightly increased apoptosis compared to controls. However, simultaneous exposure to acrylamide and staurosporine for 8h produced significantly less apoptosis than staurosporine alone, and preincubation with acrylamide followed by staurosporine markedly reduced apoptosis at 8 and 24h of treatment. Acrylamide seems therefore to have a dual effect on BEL cell survival.     

beta-Internexin, a ubiquitous intermediate filament-associated protein

In this article we show a Triton-insoluble, intermediate filament-associated protein of approximately 70 kD to be expressed ubiquitously in diverse mammalian cell types. This protein, assigned the name beta-internexin, exhibits extreme homology in each of the various cell lines as demonstrated by identical limited peptide maps, similar mobilities on two-dimensional gels, and detection in Triton-soluble and -insoluble extracts. beta-Internexin also shares some degree of homology with alpha-internexin, an intermediate filament-associated protein isolated and purified from rat spinal cord, which accounts for the immunologic cross-reactivity displayed by these polypeptides. Light microscopic immunolocalization of beta-internexin with a monoclonal antibody (mAb-IN30) reveals it to be closely associated with the vimentin network in fibroblasts. The antigen is also observed to collapse with the vimentin reticulum during the formation of a juxtanuclear cap induced by colchicine treatment. Ultrastructural localization, using colloidal gold, substantiates the affinity of beta-internexin for cytoplasmic filaments and, in addition, demonstrates its apparent exclusion from the intranuclear filament network. We examine also the resemblance of beta-internexin to a microtubule-associated polypeptide and the constitutively synthesized mammalian heat shock protein (HSP 68/70).     

Immunofluorescence microscopic demonstration of vimentin filaments in asteroid bodies of sarcoidosis. A comparison with electron microscopic findings

Asteroid bodies in multinucleate giant cells from sarcoid granulomas were investigated by immunofluorescence and electron microscopy. The following points have been established: 1. Asteroid bodies are made up of individual components of the so-called cytoskeleton, predominantly vimentin filaments. Microtubules are involved in smaller amounts in the formation of the asteroid bodies. 2. They arise within the area of the cytosphere. The body of the asteroid includes the centrioles while the arms of the asteroid usually extend into the Golgi area and occasionally up to the cell nuclei. 3. Asteroid bodies result from aggregation of the flexible filamentous and microtubular systems of the centrosphere. The processes of aggregation probably result from local fluid shifts and sol-gel transformations. 4. The stellate form of the aggregations is determined by the preexistent radial arrangement of the elements of the cytosphere. 5. The prevailing specific environment of the underlying granulomatous disease, together with the internal characteristics of the structure and function of the giant cells, in particular in states of exhaustion may play a part in their development.     

De novo synthesis and specific assembly of keratin filaments in nonepithelial cells after microinjection of mRNA for epidermal keratin

Poly(A)+ RNA isolated from bovine muzzle epidermis was microinjected into nonepithelial cells containing only intermediate-sized filaments of the vimentin type. In recipient cells keratin polypeptides are synthesized and assemble into intermediate-sized filaments at multiple dispersed sites. We describe the time course and the pattern of de novo assembly of keratin filaments within living cells. These filaments were indistinguishable, by immunofluorescence and immunoelectron microscopic criteria, from keratin filament arrays present in true epithelial cells. The presence of extended keratin fibril meshworks in these injected cells is compatible with cell growth and mitosis. Double immunolabeling revealed that newly assembled keratin was not codistributed with microfilament bundles, microtubules or vimentin filaments. We suggest that assembly mechanisms exist which in vivo sort out newly synthesized cytokeratin polypeptides from vimentin.     

去核过程中CHO细胞波形纤维的分布

本文用豚鼠抗小牛晶状体波形纤维蛋白的血清抗体,对经细胞松驰素B(CB)和秋水仙素等药物处理后再离心去核的CHO细胞及其核体、胞质体进行了间接免疫荧光染色,并对核体做了电镜观察。CB处理后离心的细胞的免疫荧光染色显示,去核过程中核的后方始终伴有强烈的荧光,核体上也有强荧光斑。在核体的电镜材料中同样观察到了中等纤维。经CB和秋水仙素合并处理后离心的细胞,去核效果比仅用CB处理有明显的增强,免疫荧光染色表明,核后的荧光并不因秋水仙素处理而消失。实验结果表明:1.微丝对维持细胞表面的完整性有重要作用,CB能破坏微丝故有利于离心去核。2.中等纤维与核之间存在密切联系,这种联系在核膜的某些区域比较集中、牢固,不易为离心力所破坏。3.微管对核固着作用有重要意义,细胞核可能通过中等纤维与微管相连而抛锚在胞质中,故秋水仙素可增强去核作用。微管对维持细胞表面强度可能也有一定作用。     

Immunocytochemical studies of desmin and vimentin in pericapillary cells of chicken

The composition of intermediate filaments in pericytes was examined by immunofluorescent and immunoelectron microscopic labeling of frozen sections of various chicken microvascular beds in situ. Pericytes in capillaries of cardiac muscle, exocrine pancreas, and kidney (peritubular capillary) were found to contain both desmin and vimentin. In some capillaries where pericytes do not exist, cells apposed to endothelial cells--the Ito cell in the hepatic sinusoid and the reticular cell in the splenic sinusoid--were shown to contain both of the intermediate filament proteins. In contrast, podocytes and mesangial cells around renal glomerular capillaries contained only vimentin. The presence of desmin supports the hypothesis that pericytes may have a contractile apparatus similar to that of vascular smooth muscle cells. Our results also revealed that even in microvascular beds where pericytes are not found, cells having both desmin and vimentin exist next to endothelial cells and may assume similar functions to pericytes.     

TRANSFORMATION OF MONOCYTES IN TISSUE CULTURE INTO MACROPHAGES, EPITHELIOID CELLS, AND MULTINUCLEATED GIANT CELLS : An Electron Microscope Study

The sequential transformation of chicken monocytes into macrophages, epithelioid cells, and multinucleated giant cells in vitro was studied by electron microscopy after fixation and embedment in situ. The following changes occur. In the nucleus, margination of chromatin, evident in monocytes, decreases in later forms. Nucleoli become more complex and nuclear pores increase in number. In cytoplasm, a progressive increase in volume of the ectoplasm and endoplasm occurs in culture. Lysosomes increase in number and size prior to phagocytosis. During phagocytosis (most active from 1 to 3 days of culture) lysosome depletion occurs. Lysosomes are present in greatest number and show maximal structural variation in the epithelioid and young giant cells. Aging giant cells lose lysosomes. All stages possess variably large quantities of rough-surfaced endoplasmic reticulum and free ribosomes. The Golgi apparatus, small in monocytes, increases in size and complexity. Massive accumulations of lysosomes within the Golgi apparatus of macrophages and epithelioid cells suggest that lysosomes originate there. In giant cells, multiple Golgi regions occur, often ringing the nuclei. Monocytes and macrophages have few mitochondria. Mitochondria of epithelioid cells are larger, more numerous, and may have discontinuous outer membranes. Mitochondria are most numerous in giant cells where they increase with age and become polymorphous. Cytoplasmic filaments are approximately 50 to 60 A in diameter and of indeterminate length. They occur both singly and in bundles which touch cytoplasmic vesicles and mitochondria. Few filaments occur in monocytes and macrophages. A large increase in the number of filaments occurs in epithelioid cells, where filaments (90 to 100 A) surround the cytocentrum as a distinctive annular bundle often branching into the cytoplasm. The greatest concentration of filaments occurs in aged giant cells. Pseudopodia are always present. They are short and filiform in monocytes and giant cells, and broad, with abundant micropinocytotic vesicles, in macrophages and epithelioid cells. At every stage, the cell membrane contains dense cuplike structures. These may represent the membranous residue of lysosomes which have discharged to the outside, analogous to merocrine secretion. Contiguous epithelioid cells display elaborate cytoplasmic interdigitation. In places, the plasma membranes break down and epithelioid cells fuse to form giant cells.     

Distribution of Cytoskeletal Proteins in Neomycin-Induced Protrusions of Human Fibroblasts

The organization of actin, tubulin, and vimentin was studied in protruding lamellae of human fibroblasts induced by the aminoglycoside antibiotic neomycin, an inhibitor of the phosphatidylinositol cycle. Neomycin stimulates the simultaneous protrusion of lamellae in all treated cells, and the lamellae remain extended for about 15–20 min, before gradually withdrawing. The pattern and distribution of actin, tubulin, and vimentin during neomycin stimulation were analyzed by fluorescence and electron microscopy. F-actin in the newly formed lamellae is localized in a marginal band at the leading edge. Tubulin is colocalized with F-actin in the marginal band, but the newly formed lamellae are initially devoid of microtubules. Over a period of 10 to 20 min after the addition of neomycin, microtubules grow into the lamellae from the adjacent cytoplasm, while the intensity of tubulin staining of the marginal band decreases. Distribution of vimentin remains unchanged in neomycin-treated cells and vimentin filaments do not enter the new protrusions. Treatment of cells with colchicine and Taxol do not inhibit neomycin-induced protrusion but protrusions are no longer localized at the ends of cell processes and occur all around the cell periphery. We conclude that actin filaments are the major component of the cytoskeleton involved in generating protrusions. Microtubules and, possibly, intermediate filaments control the pattern of protrusions by their interaction with actin filaments.     

Antibodies as probes of cellular differentiation and cytoskeletal organization in the mouse blastocyst

Indirect immunofluorescence microscopy has been used to detect cytoskeletal proteins, which allow a distinction between the two cell types present in the mouse blastocyst: i.e. the cells of the inner cell mass (ICM) and the outer trophoblastic cells. Antibodies against three classes of intermediate-sized filaments (cytokeratins, desmin and vimentin), as well as antibodies against actin and tubulin were studied. Antibodies against prekeratin stain the outer trophoblastic cells but not the ICM in agreement with the findings on adult tissues that cytokeratins are a marker for various epithelial cells. Interestingly, vimentin filaments typical of mesenchymal cells as well as of cells growing in culture seem to be absent in both cell types of the blastocyst. Thus, the cytokeratins of the trophoblastic cells seem to be the first intermediate-sized filaments expressed in embryogenesis. Antibodies to tubulin and actin show that microtubules and microfilaments are ubiquitous structures, although microfilaments have a noticeably different organization in the two cell types. In addition, since early embryogenic multipotential cells show close similarities to teratocarcinomic cells, a comparison is made between the cells of the blastocyst, embryonal carcinoma cells (EC cells) and an epithelial endodermal cell line (PYS2 cells) derived from EC cells. EC cells display vimentin filaments whereas PYS2 cells show both vimentin and cytokeratin filaments. The results emphasize the usefulness of antibodies specific for different classes of intermediate filaments in further embryological studies, and suggest that cells of the blastocyst and EC cells differ with respect to vimentin filaments.     

Integrity of intermediate filaments is associated with the development of acquired thermotolerance in 9L rat brain tumor cells

Withangulatin A (WA), a newly discovered withanolide isolated from an antitumor Chinese herb, has been shown to be a vimentin intermediate filament-targeting drug by using immunofluorescence microscopy. Together with cytochalasin D and colchicine, these drugs were employed to investigate the importance of vimentin intermediate filaments, actin filaments, and microtubules in the development of acquired thermotolerance in 9L rat brain tumor cells treated at 45°C for 15 min (priming heat-shock). Acquired thermotolerance was abrogated in cells incubated with WA before the priming heat-shock but it could be detected in cells treated with WA after the priming heat-shock. In contrast, cytochalasin D and colchicine do not interfere with the development of thermotolerance at all. The intracellular localizations of vimentin and the constitutive heat-shock protein70 (HSC70) in treated cells were examined by using immunofluorescence microscopy and detergent-extractability studies. In cells treated with WA before the priming heat-shock, vimentin IFs were tightly aggregated around the nucleus and unable to return to their normal organization after a recovery under normal growing conditions. In contrast, the IF network in cells treated with WA after the priming heat-shock was able to reorganize into filamentous form after a recovery period, a behavior similar to that of the cells treated with heat-shock only. HSC70 was found to be co-localized with vimentin during these changes. It is suggested that the integrity of intermediate filaments is important for the development of thermotolerance and that HSC70 may be involved in this process by stabilizing the intermediate filaments through direct or indirect binding.