Immunocytochemical analysis of intermediate filaments in embryonic heart cells with monoclonal antibodies to desmin

Monoclonal antibodies ( McAbs ) have been generated against a preparation of intermediate filament proteins (IFP) from adult chicken gizzard. Two antibodies, D3 and D76 , have been characterized in detail. They bind specifically to desmin but recognize different epitopes. In the adult chicken, both McAbs produced equivalent immunofluorescent staining patterns, reacting in frozen sections with all forms of muscle tissue, including vascular smooth muscle, but with no other tissue types. In isolated skeletal myofibrils and in longitudinal frozen sections of cardiac and skeletal muscle, desmin was detected with both McAbs at the Z-band and in longitudinally-oriented filament bundles between myofibrils. In contrast to these results in the adult, the intermediate filaments (IF) of embryonic cardiac myocytes in primary cultures were decorated only with McAb D3, whereas McAb D76 was completely unreactive with these cells. Similarly, frozen sections through the heart at early stages of embryonic chick development (Hamburger-Hamilton stages 17-18) revealed regions of myocytes, identified by double immunofluorescence with myosin-specific McAbs , that were unstained with McAb D76 even though similar regions were stained by McAb D3. That McAb D76 reacted with desmin in all adult cardiac myocytes but not with all embryonic heart cells indicates that embryonic and adult cardiac IF are immunologically distinct and implies a conversion in IF immunoreactivity during cardiac development.     

A comparative analysis of intermediate filament proteins in bovine heart Purkinje fibres and gastric smooth muscle

The intermediate filament (IF) composition of muscle cells of various sources is still a controversial issue. In the present study, the IF composition of bovine Purkinje fibres (PFs), atrial and ventricular myocardium, and gastric smooth muscle (SM) has been compared using biochemical and immunocytochemical methods. The Mr of the major IF subunit protein in all four tissues was 55,000. In two-dimensional (2-D) electrophoresis gels of Triton-treated ordinary atrial and ventricular myocardium and the gastric muscular wall, two or three isoelectric isoforms were seen, whereas in PFs up to seven isoforms caused by phosphorylation were observed. In immunofluorescence studies antibodies against the Mr 55,000 subunit of PFs and gastric SM, respectively, both showed identical reactivity with PFs, atrial and ventricular myocytes, gastric SM cells and some SM cells in intramyocardial and gastric muscular wall blood vessels. A small amount of vimentin (Mr 57,000) was also detected in 2-D gel electrophoresis in all four tissues as well as in immunoblotting of PFs with antibodies to vimentin. Immunofluorescence studies using both polyclonal and monoclonal antibodies to vimentin showed that vimentin was present in the endothelium and SM cells of both intramyocardial and gastric muscular wall vessels, sometimes together with desmin in the vascular SM cells, but was never seen in PF, atrial, ventricular or gastric SM cells proper. As expected, vimentin was present in interstitial tissue, i.e., fibroblasts and capillaries. However, interestingly, the monoclonal antibodies, which recognized different antigenic determinants of vimentin, did not give identical staining patterns. Especially the staining of the vascular SM cells differed. Since this staining pattern did not change upon denaturation and unmasking experiments, it seems that the organization of vimentin in different mesenchymal cell types varies. Vimentin was also detected in isolated PFs but here it was located solely in the contaminating interstitial tissue. Thus, desmin is the sole IF protein expressed in PFs, in atrial and ventricular myocytes and in gastric SM cells proper; vimentin alone being present in the interstitial tissue cells, whilst in vascular SM cells desmin and vimentin are coexpressed in various proportions. The variation in number of isoforms of desmin and the heterogeneity in staining of mesenchymal tissues with monoclonal vimentin antibodies probably indicates that the IF cytoskeletons are differently organized in various cell types, even though they contain IFs of the same class.     

Desmin and titin expression in early postimplantation mouse embryos

The expression of the intermediate filament (IF) constituents desmin, vimentin and keratin, as well as the striated-muscle-specific marker titin, was studied in mouse embryos of 8.0 to 9.5 days post coitum (d.p.c.), using the indirect immunofluorescence technique in combination with polyclonal and monoclonal antibodies. During the development of the embryo, desmin was first detected at 8.25 d.p.c. in the ectoderm, where it was transiently coexpressed with keratin and vimentin. At later stages, the ectoderm contained only keratin and to a certain extent also vimentin IF. At 8.5 d.p.c., desmin was found exclusively in the heart rudiment, and remained present with increasing intensity in the myocardial cells during later cardiogenesis. Striation of desmin in the heart muscle cells was observed in 9.5 d.p.c. embryos. At these stages (8.5-9.5 d.p.c.), triple expression of the IF proteins desmin, vimentin and keratin was evident in these cells. From 9.0 d.p.c. onwards, desmin could be detected in the myotomes as well. Immunoblotting studies of 9.5 d.p.c. mouse embryos confirmed the immunohistochemical data. Titin was found in the early heart anlage at stage 8.25 d.p.c., when no desmin expression was observed in this tissue. At this stage the titin appeared in a punctate pattern, similar to that observed in cardiac myofibrils of early chicken embryos (Tokuyasu and Maher, 1987; J. Cell Biol. 105, 2781-2793). In 8.5 d.p.c. mouse embryos, this punctate titin staining pattern was still observed, while, at this stage, a filamentous staining reaction could be seen with the desmin antibodies. During further development, cross-striation was detected within myocardial cells using the polyclonal titin antibody from 9.0 d.p.c. onwards, i.e. before such striation could be detected with the desmin antibodies. From these data, we conclude that titin synthesis may anticipate desmin expression in the developing mouse myocard, although the level of expression of the former protein remains low until 9.0 d.p.c.     

Site-specific antibodies block kinase A phosphorylation of desmin in vitro and inhibit incorporation of myoblasts into myotubes.

Desmin and vimentin are two type III intermediate filament (IF) proteins, which can be phosphorylated in vitro by cAMP-dependent kinase (kinase A) and protein kinase C, and the in vitro phosphorylation of these proteins appears to favor the disassembled state. The sites of phosphorylation for desmin and vimentin have been mapped to their amino-terminal headpiece domains; in chicken smooth muscle desmin the most kinase A-reactive residues are ser-29 and ser-35. In this study we have examined the phosphorylation of desmin by the catalytic subunit of kinase A by using anti-peptide antibodies directed against residues 26-36. The antibodies, which we call anti-D26, recognize both native and denatured desmin and can discriminate between intact desmin and those derivatives that do not possess residues 26-36. Pre-incubation of desmin with affinity purified anti-D26 blocks total kinase A catalyzed incorporation of 32P into desmin by 75-80%. When antibody-treated IFs are subjected to phosphorylation, no filament break-down is observed after 3 hours. Thus anti-D26 antibodies block phosphorylation of IF in vitro. We have also explored the role of desmin phosphorylation in skeletal muscle cell differentiation using these antibodies. Quail embryo cells, induced to differentiate along the myogenic pathway by infection with avian SKV retroviruses expressing the ski oncogene, were microinjected with affinity purified anti-D26 at the mononucleated, myoblast stage. By 24 h post-injection, the vast majority of uninjected cells had fused into multinucleated myotubes, but all microinjected cells were arrested in the process of incorporating into myotubes and remained mononucleated. This observation suggests that kinase A phosphorylation-induced dynamic behavior of the desmin/vimentin IF cytoskeleton may be one of the many cytoskeletal restructuring events that must take place during myoblast fusion.     

Localization of vimentin and desmin in BHK21/C13 cells and in baby hamster kidney

Specific antibodies against the intermediate filament protein subunits, desmin and vimentin, were used to characterize the fibroblastic tissue culture cell line BHK21/C13 and the cells comprising baby hamster kidney (BHK). The BHK21/C13 cells have previously been shown to contain desmin and vimentin by biochemical techniques. The results from double immunofluorescence analysis show that both immunologically distinct intermediate filament subunit proteins are expressed simultaneously within the same BHK21/C13 cell, and that the filamentous patterns are very similar, if not superimposable even in cells treated with colchicine. There are some cells that may contain vimentin only. Double immunofluorescence on cryostat sections of BHKs and preparations of dissociated kidney cells demonstrate that the cells, most likely smooth muscle, comprising the blood vessel walls contain vimentin and desmin simultaneously. The simultaneous expression of vimentin and desmin is not a phenomenon which is restricted to tissue culture cells. Thus, the simultaneous presence of these two intermediate filament proteins within the BHK21/C13 cell may not be the result of growth in tissue culture.     

Association of glycosphingolipids with intermediate filaments of mesenchymal, epithelial, glial, and muscle cells.

We reported recently that two glycosphingolipids (GSLs), globoside (Gb4) and ganglioside GM3, colocalized with vimentin intermediate filaments of human umbilical vein endothelial cells. To determine whether this association is unique to endothelial cells or to vimentin, we analyzed a variety of cell types. Double-label immunofluorescent staining of fixed, permeabilized cells, with and without colcemid treatment, was performed with antibodies against glycolipids and intermediate filaments. Globoside colocalized with vimentin in human and mouse fibroblasts, with desmin in smooth muscle cells, with keratin in keratinocytes and hepatoma cells, and with glial fibrillary acidic protein (GFAP) in glial cells. Globoside colocalization was detected only with vimentin in MDCK and HeLa cells, which contain separate vimentin and keratin networks. GM3 ganglioside also colocalized with vimentin in human fibroblasts. Association of other GSLs with intermediate filaments was not detected by immunofluorescence, but all cell GSLs were detected in cytoskeletal fractions of metabolically labelled endothelial cells. These observations indicate that globoside colocalizes with vimentin, desmin, kertain and GFAP, with a preference for vimentin in cells that contain both vimentin and keratin networks. The nature of the association is not yet known. Globoside and GM3 may be present in vesicles associated with intermediate filaments (IF), or bound directly to IF or IF associated proteins. The prevalence of this association suggests that colocalization of globoside with the intermediate filament network has functional significance. We are investigating the possibility that intermediate filaments participate in the intracellular transport and sorting of glycosphingolipids.     

Assembly of amino-terminally deleted desmin in vimentin-free cells

To study the role of the amino-terminal domain of the desmin subunit in intermediate filament (IF) formation, several deletions in the sequence encoding this domain were made. The deleted hamster desmin genes were fused to the RSV promoter. Expression of such constructs in vimentin- free MCF-7 cells as well as in vimentin-containing HeLa cells, resulted in the synthesis of mutant proteins of the expected size. Single- and double-label immunofluorescence assays of transfected cells showed that in the absence of vimentin, desmin subunits missing amino acids 4-13 are still capable of filament formation, although in addition to filaments large numbers of desmin dots are present. Mutant desmin subunits missing larger portions of their amino terminus cannot form filaments on their own. It may be concluded that the amino-terminal region comprising amino acids 7-17 contains residues indispensable for desmin filament formation in vivo. Furthermore it was shown that the endogenous vimentin IF network in HeLa cells masks the effects of mutant desmin on IF assembly. Intact and mutant desmin colocalized completely with endogenous vimentin in HeLa cells. Surprisingly, in these cells endogenous keratin also seemed to colocalize with endogenous vimentin, even if the endogenous vimentin filaments were disturbed after expression of some of the mutant desmin proteins. In MCF-7 cells some overlap between endogenous keratin and intact exogenous desmin filaments was also observed, but mutant desmin proteins did not affect the keratin IF structures. In the absence of vimentin networks (MCF-7 cells), the initiation of desmin filament formation seems to start on the preexisting keratin filaments. However, in the presence of vimentin (HeLa cells) a gradual integration of desmin in the preexisting vimentin filaments apparently takes place.     

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.     

Changes in the distribution of intermediate-filament types in Japanese quail embryos during morphogenesis

We examined the distribution of intermediate filaments in early quail embryos in order to determine whether these cytoskeletal proteins play a role in the epithelial-mesenchymal transitions that commonly occur during embryogenesis, e.g., the separation of neural-crest cells from the neural epithelium. The distribution of cytokeratins, vimentin, and desmin was examined in frozen sections of quail embryos at stages during which dramatic reorganizations of tissues take place. All embryonic tissues were found to contain either vimentin or cytokeratins, but the distribution of these cytoskeletal proteins was characteristic neither of the cellular organization (e.g., epithelium vs. mesenchyme) nor of the germ-layer derivation of the tissues. Cytokeratin monoclonal antibodies stained most embryonic epithelia (defined here as being sheet-like tissue with an underlying basement membrane), including epidermis and extraembryonic membranes derived in part from the ectoderm, splanchnopleure and kidney tubules derived from mesoderm, and endoderm. Cytokeratin antibodies did not stain some epithelia, including the neural tube, neural plate, and dermatome/myotome. Whereas the cytokeratin antibodies exclusively stained epithelia, the vimentin antibodies labeled both epithelial (the neural tube, dermatome/myotome, and somatic and splanchnic mesoderm) and mesenchymal tissues (the sclerotome and neural-crest cells), regardless of their germ-layer derivation. In early embryos, antibodies against desmin only stained the myotome and, in 4-day embryos, the heart and mesenchyme around the pharynx. As the distribution of intermediate-filament types did not reflect tissue organization or germ-layer derivation, we propose that the distribution of intermediate filaments in early avian embryos reflects the motile capacity of an embryonic cell and/or the presence of specialized cell junctions, i.e., desmosomes.     

The distribution of desmin (100 A) filaments in primary cultures of embryonic chick cardiac cells.

Using antibodies to desmin, the major component of the 100Å-filaments from smooth muscle cells, we studied by indirect immunofluorescence the distribution of this protein in primary cultures of embryonic chick cardiac cells. We show that desmin is a component of cytoplasmic filamentous structures which comprise a network distinct from actin filament bundles and microtubules. Exposure of these cells to colcemid results in a rapid disaggregation of microtubules, and a slow aggregation of the desmin-containing filaments towards the nuclear area with the ultimate formation of a perinuclear ring. In differentiated skeletal or cardiac muscle cells, in addition to its cytoplasmic filamentous distribution, desmin is found intimately associated with the Z lines of sarcomeres. We further show that approx. 50% of the cells in these primary cardiac cultures are unreactive with desmin antibodies. Similarly the majority of the cells in a number of established cell lines from various species grown in tissue culture, are unreactive to desmin antibodies in indirect immunofluorescence, despite the fact that these cells are known to contain cytoplasmic 100Å-filaments. These results indicate that desmin occurs in at least two distinct cytoplasmic distribution in cardiac cells. They also demonstrate the existence of immunological and biochemical differences in the major component of 100Å-filaments between muscle and non-muscle cells as evidenced by the failure of non-muscle cells to react with antibodies to chick smooth muscle desmin.     

The distribution of desmin (100 Å) filaments in primary cultures of embryonic chick cardiac cells

Using antibodies to desmin, the major component of the 100Å-filaments from smooth muscle cells, we studied by indirect immunofluorescence the distribution of this protein in primary cultures of embryonic chick cardiac cells. We show that desmin is a component of cytoplasmic filamentous structures which comprise a network distinct from actin filament bundles and microtubules. Exposure of these cells to colcemid results in a rapid disaggregation of microtubules, and a slow aggregation of the desmin-containing filaments towards the nuclear area with the ultimate formation of a perinuclear ring. In differentiated skeletal or cardiac muscle cells, in addition to its cytoplasmic filamentous distribution, desmin is found intimately associated with the Z lines of sarcomeres. We further show that approx. 50% of the cells in these primary cardiac cultures are unreactive with desmin antibodies. Similarly the majority of the cells in a number of established cell lines from various species grown in tissue culture, are unreactive to desmin antibodies in indirect immunofluorescence, despite the fact that these cells are known to contain cytoplasmic 100Å-filaments. These results indicate that desmin occurs in at least two distinct cytoplasmic distribution in cardiac cells. They also demonstrate the existence of immunological and biochemical differences in the major component of 100Å-filaments between muscle and non-muscle cells as evidenced by the failure of non-muscle cells to react with antibodies to chick smooth muscle desmin.     

Specific attachment of desmin filaments to desmosomal plaques in cardiac myocytes

Intercellular junctions which are similar in ultrastructure and protein composition to typical desmosomes have so far only been found in epithelial cells and in heart tissue, specifically in the intercalated disks of cardiac myocytes and at cell boundaries between Purkinje fiber cells. In epithelial cells the cytoplasmic side of desmosomes, the 'desmosomal plaque', represents a specific attachment structure for the anchorage of intermediate filaments (IF) of the cytokeratin type. Cardiac myocytes do not contain cytokeratin filaments. In primary cultures of rat cardiac myocytes, we have examined by immunofluorescence and electron microscopy, using single and double label techniques, whether other types of IF are attached to the desmosomal plaques of the heart. Antibodies to desmoplakin, the major protein of the desmosomal plaque, have been used to label specifically the desmosomal plaques. It is shown that the desmoplakin-containing structures are often associated with IF stained by antibodies to desmin, i.e., the characteristic type of IF present in these cells. Like cytokeratin filaments in epithelial cells, desmin filaments attach laterally to the desmosomal plaque. They also remain attached to these plaques after endocytotic internalization of desmosomal domains by treatment of the cells with EGTA. These desmin filaments do not appear to attach to junctions of the fascia adherens type and to nexuses (gap junctions). These observations show that anchorage at desmosomal plaques is not restricted to IF of the cytokeratin type and that IF composed of either cytokeratin or desmin, specifically attach, in a lateral fashion, to desmoplakin-containing regions of the plasma membrane. We conclude that special domains exist in these two IF proteins that are involved in binding to the desmosomal plaque.     

A high molecular weight intermediate filament-associated protein in BHK-21 cells is nestin, a type VI intermediate filament protein. Limited co-assembly in vitro to form heteropolymers with type III vimentin and type IV alpha-internexin

BHK-21 fibroblasts contain type III vimentin/desmin intermediate filament (IF) proteins that typically co-isolate and co-cycle in in vitro experiments with certain high molecular weight proteins. Here, we report purification of one of these and demonstrate that it is in fact the type VI IF protein nestin. Nestin is expressed in several fibroblastic but not epithelioid cell lines. We show that nestin forms homodimers and homotetramers but does not form IF by itself in vitro. In mixtures, nestin preferentially co-assembles with purified vimentin or the type IV IF protein alpha-internexin to form heterodimer coiled-coil molecules. These molecules may co-assemble into 10 nm IF provided that the total amount of nestin does not exceed about 25%. However, nestin does not dimerize with types I/II keratin IF chains. The bulk of the nestin protein consists of a long carboxyl-terminal tail composed of various highly charged peptide repeats. By analogy with the larger neurofilament chains, we postulate that these sequences serve as cross-bridgers or spacers between IF and/or other cytoskeletal constituents. In this way, we propose that direct incorporation of modest amounts of nestin into the backbone of cytoplasmic types III and IV IFs affords a simple yet flexible method for the regulation of their dynamic supramolecular organization and function in cells.     

Differential organization of desmin and vimentin in muscle is due to differences in their head domains

In most myogenic systems, synthesis of the intermediate filament (IF) protein vimentin precedes the synthesis of the muscle-specific IF protein desmin. In the dorsal myotome of the Xenopus embryo, however, there is no preexisting vimentin filament system and desmin's initial organization is quite different from that seen in vimentin-containing myocytes (Cary and Klymkowsky, 1994. Differentiation. In press.). To determine whether the organization of IFs in the Xenopus myotome reflects features unique to Xenopus or is due to specific properties of desmin, we used the injection of plasmid DNA to drive the synthesis of vimentin or desmin in myotomal cells. At low levels of accumulation, exogenous vimentin and desmin both enter into the endogenous desmin system of the myotomal cell. At higher levels exogenous vimentin forms longitudinal IF systems similar to those seen in vimentin-expressing myogenic systems and massive IF bundles. Exogenous desmin, on the other hand, formed a reticular IF meshwork and non-filamentous aggregates. In embryonic epithelial cells, both vimentin and desmin formed extended IF networks. Vimentin and desmin differ most dramatically in their NH2- terminal "head" regions. To determine whether the head region was responsible for the differences in the behavior of these two proteins, we constructed plasmids encoding chimeric proteins in which the head of one was attached to the body of the other. In muscle, the vimentin head- desmin body (VDD) polypeptide formed longitudinal IFs and massive IF bundles like vimentin. The desmin head-vimentin body (DVV) polypeptide, on the other hand, formed IF meshworks and non-filamentous structures like desmin. In embryonic epithelial cells DVV formed a discrete filament network while VDD did not. Based on the behavior of these chimeric proteins, we conclude that the head domains of vimentin and desmin are structurally distinct and not interchangeable, and that the head domain of desmin is largely responsible for desmin's muscle- specific behaviors.     

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.     

Distribution of vimentin and desmin filaments in smooth muscle tissue of mammalian and avian aorta

The presence of intermediate filament proteins in vascular tissue cells has been examined by immunofluorescence microscopy on frozen sections of the aortic wall of diverse vertebrates (rat, cow, human and chicken) and by gel electrophoresis of cytoskeletal proteins from whole aortic tissue or from stripped tunica media of cow and man. Most cells of the aortic wall in these species contain vimentin filaments, including smoooth muscle cells of the tunica media. In addition, we have observed aortic cells that are positively stained by antibodies to desmin. The presence of desmin in aortic tissue has also been demonstrated by gel electrophoresis for rat, cow and chicken. In aortic tissue some smooth muscle cells contain both types of intermediate filament proteins, vimentin and desmin. Bovine aorta contains, besides cells in which vimentin and desmin seem to co-exist, distinct bundles of smooth muscle cells, located in outer regions of the tunica media, which contain only desmin. The results suggest that (i) intermediate-sized filaments of both kinds, desmin and vimentin, can occur in vascular smooth muscle in situ and (ii) smooth muscle cells of the vascular system are heterogeneous and can be distinguished by their intermediate filament proteins. The finding of different vascular smooth muscle cells is discussed in relation to development and differentiation of the vascular system.     

Microinjection of monoclonal antibodies to vimentin, desmin, and GFA in cells which contain more than one IF type

Microinjection of antibodies to vimentin into fibroblast cell lines causes intermediate filaments (IFs) to build perinuclear caps. We have extended these findings by microinjection of monoclonal antibodies specific for different IF types to non-epithelial cell lines of human origin, which co-express two different IF proteins. Thus GFA and vimentin IgGs have been microinjected in separate experiments into a glioma cell line, desmin and vimentin IgGs into RD cells, and vimentin IgGs into a cell line which co-expresses neurofilaments and vimentin. In all instances, microinjection of a single antibody causes the formation of perinuclear caps in which the two different IF proteins co-localize, suggesting that vimentin and the second IF type present in each cell line localize to the same 10-nm filaments. Immunoelectron microscopy using desmin and vimentin antibodies made in different species and appropriate second antibodies labelled with 5 and 20 nm gold particles confirm this result for RD cells. When Fab' fragments of the vimentin IgGs are microinjected into different cell types, formation of perinuclear caps is observed in immunofluorescence microscopy. In RD cells immunoelectron microscopy shows that the Fab' fragments induce caps which appear less dense than the caps seen after microinjection of IgGs.     

Cytoskeletal components of lymphoid organs

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 desmin-containing 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 alpha-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.     

Diagnosis of human childhood rhabdomyosarcoma of antibodies to desmin, the structural protein of muscle specific intermediate filaments

Four embryonal rhabdomyosarcomas, one tumor diagnosed as an undifferentiated sarcoma, probably a rhabdomyosarcoma, and six different non-muscular sarcomas were investigated with antibodies specific for different intermediate filament types. The tumor cells in the rhabdomyosarcomas and the undifferentiated tumor were stained clearly by antibodies to desmin, the intermediate filament type characteristic of muscle. The staining of tumor cell by antibodies to vimentin, the intermediate filament type characteristic of certain cell types of mesenchymal origin including myoblasts, was different in these 5 cases. In one case of embryonal rhabdomyosarcoma nearly all tumor cells were stained, but in the remaining cases few or no tumor cells were positive with the vimentin antibody. In these rhabdomyosarcomas not only the large rhabdomyoblasts, but also the small undifferentiated cells were labeled by antibodies to desmin. In the latter cell type the desmin filaments were arranged typically in coils. In contrast, tumor cells in the non-muscular mesenchymal sarcomas were stained only by antibodies to vimentin but not by antibodies to desmin or prekeratin. The retention of the desmin marker characteristic of normal muscle in cases of rhabdomyosarcoma not only allowed the undifferentiated desmin-positive sarcoma to be classified as rhabdomyosarcoma but also suggests that the use of antibodies to desmin could be very helpful in the future for the diagnosis of undifferentiated rhabdomyosarcomas.