Acquisition of vimentin in astrocytes cultured from postnatal rat brain

Summary Vimentin and glial fibrillary acidic protein (GFAP) represent the principal constituents of intermediate filaments found in astrocytes. In contrast to vimentin—GFAP transition which occurs during glial developmentin situ, vimentin coexists with GFAP in cortical astrocytes allowed to differentiate in culture. To examine whether culture conditions or proliferative activity of the cells is responsible for the expression of vimentin, we generated cultures of GFAP-positive, vimentin-negative astrocytes isolated from 26-day postnatal rat brain cortices. Isolated astrocytes are characterized by a very thin rim of perinuclear cytoplasm and by numerous processes. Antiserum to GFAP labelled major processes and cell somata of some astrocytes, especially those with relatively short and large processes. Within 3 days in culture, all astrocytes accumulated GFAP in hypertrophic cell bodies and many began to express vimentin. Vimentin appeared primarily close to nuclei, and filaments of vimentin extended into proximal segments of the cell processes. In some astrocytes, however, vimentin was always absent. Combined double immunolabelling and histoautoradiography experiments demonstrated that the acquisition of vimentin was independent of the ability of astrocytes to incorporate tritiated thymidine. The results indicate that astrocytes isolated from 26-day postnatal rat brain are heterogeneous with respect to their ability to express vimentin and that vimentin synthesis is not correlated with the growth state of the cells as had been previously suspected.     

Elucidation of GlcNAc‐binding properties of type III intermediate filament proteins,using GlcNAc‐bearing polymers

Vimentin, desmin, glial fibrillary acidic protein (GFAP) and peripherin belong to type III intermediate filament family and are expressed in mesenchymal cells, skeletal muscle cells, astrocytes and peripheral neurons, respectively. Vimentin and desmin possess N‐acetyl‐d ‐glucosamine (GlcNAc)‐binding properties on cell surfaces. The rod II domain of these proteins is a GlcNAc‐binding site, which also exists in GFAP and peripherin. However, the GlcNAc‐binding activities and behaviors of these proteins remain unclear. Here, we characterized the interaction and binding behaviors of these proteins, using various well‐defined GlcNAc‐bearing polymers synthesized by radical polymerization with a reversible addition‐fragmentation chain transfer reagent. The small GlcNAc‐bearing polymers strongly interacted with HeLa cells through vimentin expressed on the cell surface and interacted with vimentin‐, desmin‐, GFAP‐ and peripherin‐transfected vimentin‐deficient HeLa cells. These proteins present high affinity to GlcNAc‐bearing polymers, as shown by surface plasmon resonance. These results show that type III intermediate filament proteins possess GlcNAc‐binding activities on cell surfaces. These findings provide important insights into novel cellular functions and physiological significance of type III intermediate filaments.     

The detection of smooth muscle antibodies reacting with intermediate filaments of desmin type

Some human smooth muscle antibodies (SMA) react with cytoskeletal intermediate filament (IMF) antigens. The smooth muscle tissue contains two types of IMF: vimentin and desmin filaments. In this study, SMA of anti-IMF type in 52 patients' sera have been classified into anti-vimentin filament and anti-desmin filament types according to their immunofluorescence staining patterns on rat testis. This classification is based on the fact that the arterial walls of testis contains both vimentin and desmin whereas the myoid cell layer surrounding the seminiferous tubuli contains only desmin. Four out of the 52 sera gave the anti-desmin staining pattern and 40 sera showed the anti-vimentin type of staining. Thirty-two sera were further classified by using cultured human rhabdomyosarcoma (RD) cells as targets. Nine sera reacted with the intermediate filaments of the RD cells. Among these were 3 out of the 4 sera that gave the anti-desmin filament staining pattern. The anti-desmin specificity of SMA was confirmed in 1 serum by the immunoblotting technique. These results indicate that while human anti-desmin filament antibodies exist, most human SMA of anti-IMF type react with vimentin filaments.     

Subcellular distribution of cytokeratin and vimentin in malignant rhabdoid tumor: three-dimensional imaging with confocal laser scanning microscopy and double immunofluorescence.

Malignant rhabdoid tumor (MRT) is a highly aggressive neoplasm that mostly occurs in childhood, characterized histologically by rhabdoid cells as shown by eosinophilic intracytoplasmic inclusions. Although it is known that rhabdoid cells co-express cytokeratin (CK) and vimentin, the distribution patterns of these two kinds of intermediate filaments and structural relationship between them are still not known. We investigated the subcellular distribution of CKs 8 and 18 and vimentin in MRT cell lines (Tm87-16, STM91-01, TTC549, and TC289) using confocal laser scanning microscopy and double immunofluorescence, in addition to ultrastructural examination. Vimentin was diffusely expressed in the cytoplasm of MRT cells, focally forming a filamentous network. In contrast, CKs 8 and 18 were partially expressed in the cytoplasm of MRT cells, forming globules or a few vague agglomerates. Three-dimensional images in TC289 cells revealed distinct distribution patterns of cytokeratin and vimentin, showing agglomerates of cytokeratins within the vimentin filament network. We conclude that these globules and agglomerates of CKs 8 and 18 correspond with the characteristic ultrastructural finding, showing cytoplasmic bundles of intermediate filaments concentrated in whorled arrays.     

Phosphorylation and reorganization of vimentin by p21-activated kinase (PAK)

BACKGROUND: Intermediate filament (IF) is one of the three major cytoskeletal filaments. Vimentin is the most widely expressed IF protein component. The Rho family of small GTPases, such as Cdc42, Rac and Rho, are thought to control the organization of actin filaments as well as other cytoskeletal filaments. RESULTS: We determined if the vimentin filaments can be regulated by p21-activated kinase (PAK), one of targets downstream of Cdc42 or Rac. In vitro analyses revealed that vimentin served as an excellent substrate for PAK. This phosphorylated vimentin lost the potential to form 10 nm filaments. We identified Ser25, Ser38, Ser50, Ser65 and Ser72 in the amino-terminal head domain as the major phosphorylation sites on vimentin for PAK. The ectopic expression of constitutively active PAK in COS-7 cells induced vimentin phosphorylation. Fibre bundles or granulates of vimentin were frequent in these transfected cells. However, the kinase-inactive mutant induced neither vimentin phosphorylation nor filament reorganization. CONCLUSION: Our observations suggest that PAK may regulate the reorganization of vimentin filaments through direct vimentin phosphorylation.     

Vimentin expression during altered spermatogenesis in rats

The collapse of vimentin caused by some xenobiotics correlates with the loss of structural integrity of the seminiferous epithelium. In this study, we investigated the effect of busulphan (an anticancer drug with toxic effects on dividing germ cells) on vimentin filament distribution in rat seminiferous epithelium and compared it with changes found in testes of unilaterally cryptorchid rats. In the seminiferous epithelium, the vimentin labelling was observed only in the Sertoli cells, showing a stage-specific arrangement of the filaments. Both busulphan treatment and cryptorchism caused altered distribution of vimentin filaments in the Sertoli cells. In both models, the apical vimentin filaments collapsed towards the nuclei and were disorganized in the basal region of the Sertoli cells while the germ cells were diminished in the epithelium. After the busulphan effect subsided (4 weeks after administration), spermatogenesis began to restore and vimentin filaments began to organize in basal and perinuclear regions of Sertoli cells among the spermatogonia and spermatocytes. Vimentin labelling of the sloughed material in the lumen of cryptorchid testes (but not in busulphan treated animals) was observed. We conclude that the Sertoli cell vimentin filaments play an important role in the maintenance of spermatogenesis, their damage is associated with the seminiferous epithelium disintegration and their restoration with a recovery of spermatogenesis after the unfavourable conditions subside.     

Multimeric conformation of type III intermediate filaments but not the filamentous conformation exhibits high affinity to lipid bilayers

Vimentin, desmin, glial fibrillary acidic protein (GFAP) and peripherin, classified as the type III intermediate filament family, maintain the integrity and architecture of various cell types. Recently, we reported their cell surface expression and binding to multivalent N‐acetylglucosamine‐conjugated polymers. Furthermore, the presence of vimentin on the surface of various cell types including malignant tumor cells and fibroblasts has been demonstrated. Type III intermediate filament proteins are traditionally considered intracellular proteins and do not possess signal peptides for cell membrane recruitment. Therefore, the mechanism of their transport to the cell surface is unclear. In the current study, we aimed to elucidate this mechanism by focusing on the relationship between their multimeric structure and lipid bilayer affinity. Blue native polyacrylamide gel electrophoresis demonstrated that cell surface‐expressed type III intermediate filament proteins formed a multimeric mostly including 4–12‐mers but not filamentous structure. Moreover, surface plasmon resonance analysis revealed that the multimeric structure of these recombinant proteins had high affinity to lipid bilayers, whereas their filament‐like large multimeric structure did not. Our results suggest that type III intermediate filaments are incorporated into the cell membrane through alteration from a filamentous to a multimeric structure.     

Vimentin-Ser82 as a memory phosphorylation site in astrocytes

In astrocytes, the PGF(2alpha) or ionomycin treatment induces the phosphorylation at Ser38 and Ser82 of vimentin, a type III intermediate filament, by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). We found here that vimentin phospho-Ser82 was dephosphorylated much slower than phospho-Ser38. Vimentin phospho-Ser38 was dephosphorylated quickly by purified PP1 catalytic subunit (PP1c) in vitro, whereas phospho-Ser82 was insensitive to PP1c. Because PP1c directly bound to vimentin through a VxF motif (Val83-Asp84-Phe85), the PP1c active site appeared to be unable to approach phospho-Ser82, leading to the prolongation of the phosphorylation at Ser-82. In astrocytes, PP1calpha was in vivo associated with vimentin filaments. The repetitive treatment by ionomycin at a short interval resulted in the sustained elevation of Ser82 phosphorylation, leading to the marked disassembly of vimentin filaments. Taken together, these results suggest that vimentin is a novel member of binding partner of PP1c in astrocytes, and vimentin-Ser82 may act as a memory phosphorylation site.     

Vimentin Intermediate Filaments: the Central Base in Sinus Endothelial Cells of the Rat Spleen

The ultrastructural distribution of vimentin intermediate filaments (IFs) and localizations of the related proteins in sinus endothelial cells of the rat spleen was examined by confocal laser scanning and electron microscopy with detergent extraction, myosin‐fragment 1 decoration, and immunogold labeling to elucidate their functions in endothelial cells. Vimentin IFs were extremely abundant over stress fibers in the basal part of the endothelial cells. Some of them were intermingled with actin filaments in stress fibers, and were associated with coated vesicles. Plectin was predominantly localized in the layers of vimentin and stress fibers of the endothelial cells, but rarely in the vicinity of adherens junctions in the lateral part and focal adhesions in the basal part of the cells. Neither plakoglobin nor desmoplakin, which is coupled VE‐cadherin to vimentin IFs, was detected in sinus endothelial cells. Vinculin was localized in the basal membranes of the endothelial cells. These data suggest that abundant vimentin IFs are associated with stress fibers by plectin in the basal part of the cells and form cytoskeletal cores of sinus endothelial cells only partially supported by the ring‐shaped basal lamina to have roles in scaffolding and the mechanical stabilization of the endothelial cells. Furthermore, taken in connection with recently revealed functions of vimentin and plectin, vimentin might play a cytoskeletal core of sinus endothelial cells. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Presence and distribution of vimentin in cynomolgus monkey trabecular cells

The role of the trabecular meshwork in the ocular outflow tract has made it the object of considerable study. Recent work has examined the presence and function of microfilaments and microtubules in the cytoskeleton of cultured cynomolgus monkey trabecular cells. In this study, we used an indirect immunofluorescence technique to investigate the presence and distribution of the intermediate filament vimentin in cultured cynomolgus monkey trabecular cells. The cytoskeletal active agents cytochalasin B, colchicine, nocodozole, and taxol were also employed to investigate the role of vimentin in these cells. Vimentin formed a network of filaments that radiated throughout the cytoplasm from the nucleus to the cellular projections and cell membrane. The extensiveness of the vimentin network, and the cell shape, were observed to vary according to the degree of cell confluence, the degree of cell spread, and the degree of cell/cell contact. Cells in the less-confluent periphery had extensive vimentin networks and greater cell spreading and were polygonal in shape. Cells in the more confluent areas had a less-extensive vimentin network, underwent less cell spreading, and were primarily fusiform in shape. The change in cellular morphology induced by colchicine, nocodozole and taxol was proportional to the extensiveness and the degree of change of the vimentin network. Our observations have identified a proportional association between the extensiveness of the vimentin network, changes in the vimentin organization, and alterations in cellular morphology that is suggestive of a role for vimentin in determining cellular structure and shape.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intermediate filaments and desmosomal plaque proteins in testicular seminomas and non-seminomatous germ cell tumours as revealed by immunohistochemistry

Summary Seminomas and non-seminomatous testicular germ cell tumours were studied for the presence of cytokeratin and vimentin filaments and desmosomes using immunohistochemical methods. In the majority of the classical seminomas and in seminomatous areas of mixed tumours most tumour cells appeared to lack cytokeratin filaments. Some seminomas contained a focally variable proportion of cells exhibiting cytokeratin-positive structures while other cases contained only few seminoma cells with a well developed fibrillar cytokeratin network. Gel electrophoresis of cytoskeletal proteins from microdissected regions revealed cytokeratin polypeptides nos. 8 and 18 typical of simple epithelia. In one seminoma, however, all, or almost all, tumour cells contained cytokeratin filaments. This finding is in line with the assumption of transitional forms between seminoma and embryonal carcinoma. Despite the lack - or variable expression - of cytokeratin filaments most seminoma cells contained desmosomes, although often few in number and irregularly distributed at the circumference of the cells. Loosely arranged and often very sparse vimentin fibrils were found in many, but not all seminoma cells. Double label immunofluorescence microscopy suggested that the majority of desmosomes was associated with intermediate filaments of the vimentin type. In contrast, in carcinoma cells of malignant teratomas, in well differentiated epithelial cells of intermediate-type malignant teratomas and in trophoblastic cells present in trophoblastic-type malignant teratomas cytokeratin filament bundles as well as desmosomes were decorated. The arrangement and density of the cytokeratin filament skeleton and of desmosomes varied with degree of maturation of the tissue. The most regular distribution and intensive staining of cytokeratin filaments and desmoplakin was found in mature tissues. Vimentin was demonstrated in mesenchymal areas and stroma cells. The results show that seminomas are distinguished from most other germ cell and non-germ cell tumours by the presence of true desmosomes together with scanty vimentin filaments in most tumour cells. In addition, they indicate that seminoma cells can be heterogenous in their cytoskeletal complement and may include cells with cytokeratin expression, indicative of a multi-potential character of the initially transformed cell(s).Supported in part by Fonds Zur Förderung der wissenschaftlichen Forschung (grant No. 4708 to H.D) and by the Deutsche Forschungsgemeinschaft (grants Mo 345/3 and Fr 308-17)     

Coexpression of keratin and vimentin in damaged and regenerating tubular epithelia of the kidney.

Most renal cell carcinomas coexpress vimentin and keratin, while renal tubular epithelia express only keratin. Investigation of the intermediate filament composition of tubular epithelia in diseased rat and human kidneys now shows that altered tubular epithelia unequivocally coexpress keratin and vimentin. In rats, pronounced coexpression of vimentin and keratin was observed in chronic nephrosis induced by daunomycin, and the extent of coexpression seemed to increase with the incidence of altered collapsed and cystically dilated tubules and with the degree of tubular epithelial proliferation. It was also seen during tubular regeneration after acute tubulotoxic injury induced by mercury chloride poisoning, with vimentin expression being lost in fully regenerated tubular epithelium. In man, expression was seen in chronically and irreversibly damaged kidneys. Thus, vimentin can be expressed temporarily in acutely and reversibly damaged kidneys and chronically in irreversibly damaged kidneys. Vimentin could perhaps be regarded as an indicator of the regenerating and proliferating activity of tubular lesions.     

Normal tubular regeneration and differentiation of the post-ischemic kidney in mice lacking vimentin.

Proliferation and dedifferentiation of tubular cells are the hallmark of early regeneration after renal ischemic injury. Vimentin, a class III intermediate filament expressed only in mesenchymal cells of mature mammals, was shown to be transiently expressed in post-ischemic renal tubular epithelial cells. Vimentin re-expression was interpreted as a marker of cellular dedifferentiation, but its role in tubular regeneration after renal ischemia has also been hypothesized. This role was evaluated in mice bearing a null mutation of the vimentin gene. Expression of vimentin, proliferating cell nuclear antigen (a marker of cellular proliferation), and villin (a marker of differentiated brush-border membranes) was studied in wild-type (Vim+/+), heterozygous (Vim+/-), and homozygous (Vim-/-) mice subjected to transient ischemia of the left kidney. As expected, vimentin was detected by immunohistochemistry at the basal pole of proximal tubular cells from post-ischemic kidney in Vim+/+ and Vim+/- mice from day 2 to day 28. The expression of the reporter gene beta-galactosidase in Vim+/- and Vim-/- mice confirmed the tubular origin of vimentin. No compensatory expression of keratin could be demonstrated in Vim-/- mice. The intensity of proliferating cell nuclear antigen labeling and the pattern of villin expression were comparable in Vim-/-, Vim+/- and Vim+/+ mice at any time of the study. After 60 days, the structure of post-ischemic kidneys in Vim-/- mice was indistinguishable from that of normal non-operated kidneys in Vim+/+ mice. In conclusion, 1) the pattern of post-ischemic proximal tubular cell proliferation, differentiation, and tubular organization was not impaired in mice lacking vimentin and 2) these results suggest that the transient tubular expression of vimentin is not instrumental in tubular regeneration after renal ischemic injury.     

VIMENTIN EXPRESSION IN CERVICAL CARCINOMAS: ASSOCIATION WITH INVASIVE AND MIGRATORY POTENTIAL

Vimentin is an intermediate filament protein normally expressed in mesenchymal cells, but evidence is accumulating in the literature which suggests that the aberrant expression of vimentin in epithelial cancer cells might be related to local invasiveness and metastatic potential. Vimentin expression has previously been associated with invasive properties in an in vitro model consisting of a set of HPV-33-transformed cervical keratinocyte cell lines.^1,2 In the present study, in order to emphasize those in vitro findings, the expression of vimentin has been investigated in cervical neoplasms of different grades, using immunohistochemistry. A clear association is reported between vimentin expression and metastatic progression, since vimentin was detected in all invasive carcinomas and lymph node metastases, but not in CIN III lesions. These in vivo results are compared with present and previous data obtained in vitro on cervical keratinocyte cell lines, where vimentin expression also correlated with in vitro invasiveness.     

Cytokeratin-Containing Globular Filamentous Bodies in Renal Oncocytoma

Eighty-four cortical neoplasms were studied for cytokeratin and vimentin expression by immunohis-tochemistry and for intermediate filament aggregates by electron microscopy. Twenty oncocytomas expressed cytokeratin, 16 in a distinctive punctate pattern. These same 16 tumors also contained small globular filamentous bodies (GFB) by electron microscopy. The GFB were characterized by a matrix of intermediate-sized filaments with incorporation of diverse cell organelles such as endoplasmic reticulum, lysosomes, mitochondria, and lipid. The GFB were not within a unit membrane. Although 11 of 64 carcinomas also contained intermediate filament aggregates, only 2 of these solely expressed cytokeratin, and this was restricted to a few cells in small foci. Small GFB were also present in 5 carcinomas by electron microscopy. Three mixed clear and granular cell carcinomas contained only rare cells, whereas 2 sarcomatoid carcinomas, both of rhabdoid cell phenotype, contained numerous GFB that coexpressed vimentin and cytokeratin. Cytokeratin-containing GFB are common in oncocytomas but are uncommon in carcinoma, and, when numerous, may provide a diagnostically useful immunohistochemical feature with which to distinguish oncocytoma from its carcinoma congeners.     

Intermediate filaments cytokeratin and vimentin in ovarian sex cord-stromal tumours with correlative studies in adult and fetal ovaries

The expression of the intermediate filaments cytokeratin and vimentin were studied immunohistochemically in a series of ovarian sex cord-stromal tumours (26 adult and juvenile granulosa cell tumours, 11 thecomas, six fibromas, three Sertoli-Leydig cell tumours and 1 sex cord tumour with annular tubules). Contrary to previous reports, granulosa cell tumours expressed cytokeratins as well as vimentin. Thecomas and fibromas expressed vimentin only. In Sertoli-Leydig cell tumours and the sex cord tumour with annular tubules, both cytokeratins and vimentin were detected. Correlative studies in adult ovaries showed that patterns of expression in non-neoplastic granulosa, thecal and stromal cells correspond to their neoplastic counterparts. Investigation of fetal ovaries demonstrated that these patterns of intermediate filament expression exist from relatively early stages of development. Ovarian surface epithelium and rete ovarii, like granulosa cells, co-expressed cytokeratin and vimentin. The demonstration of cytokeratins in granulosa cells and the reported presence of desmosomes and tonofilaments, suggests the epithelial nature of these cells although not clarifying their histogenesis. The presence of both these intermediate filaments in granulosa and Sertoli-Leydig cell tumours as well as in some ovarian carcinomas which may mimic them, limits their value in differential diagnosis between these tumour groups.     

Cytokeratin-containing globular filamentous bodies in renal oncocytoma.

Eighty-four cortical neoplasms were studied for cytokeratin and vimentin expression by immunohistochemistry and for intermediate filament aggregates by electron microscopy. Twenty oncocytomas expressed cytokeratin, 16 in a distinctive punctate pattern. These same 16 tumors also contained small globular filamentous bodies (GFB) by electron microscopy. The GFB were characterized by a matrix of intermediate-sized filaments with incorporation of diverse cell organelles such as endoplasmic reticulum, lysosomes, mitochondria, and lipid. The GFB were not within a unit membrane. Although 11 of 64 carcinomas also contained intermediate filament aggregates, only 2 of these solely expressed cytokeratin, and this was restricted to a few cells in small foci. Small GFB were also present in 5 carcinomas by electron microscopy. Three mixed clear and granular cell carcinomas contained only rare cells, whereas 2 sarcomatoid carcinomas, both of rhabdoid cell phenotype, contained numerous GFB that coexpressed vimentin and cytokeratin. Cytokeratin-containing GFB are common in oncocytomas but are uncommon in carcinoma, and, when numerous, may provide a diagnostically useful immunohistochemical feature with which to distinguish oncocytoma from its carcinoma congeners.     

Expression of intermediate filament proteins in fetal and adult human kidney: modulations of intermediate filament patterns during development and in damaged tissue.

The expression of intermediate filament proteins, particularly individual cytokeratins (CKs), vimentin, and glial filament protein, was immunohistochemically investigated using frozen sections and Carnoy-fixed, paraffin-embedded tissue from normal fetal and adult human kidneys as well as from pathologically altered kidneys. In fetal kidneys, the co-expression of CKs and vimentin was detected in the visceral and parietal epithelium of the glomerulus, the proximal tubules, the thin loops of Henle, and the collecting ducts. In contrast, in the tubules of normal adult kidneys, the presence of vimentin and CKs was nearly always mutually exclusive. While CKs 8 and 18 were present in all tubular epithelia, CKs 19 and 7 each exhibited a distinctive distribution pattern, there being a striking alteration between positive and negative segments and, not infrequently, intratubular heterogeneities. In certain segments, particular cell types (e.g., "plica cells," intercalated cells) could thus be recognized. In tubular epithelia altered by various injurious conditions, novel or enhanced expression of vimentin, CK 19 and CK 7, and, less frequently, CK 17 and glial filament protein was noted in certain segments. The increase in intermediate filament protein expression in altered (particularly proximal) tubules appeared to parallel the reduction in the degree of differentiation. Vimentin was never detected in distal tubules. The present results reveal a considerable similarity between the intermediate filament patterns in non-neoplastic proximal tubules of fetal and damaged kidney tissue and those in clear-cell and chromophilic renal cell carcinomas. They also serve to illustrate that the analysis of both fetal development and reactive cell changes may significantly contribute to our understanding of differentiation phenomena in malignant tumors.     

Expression and organization of muscle specific proteins during the early developmental stages of the rabbit heart

Summary The expression and intracellular distribution patterns of muscle-specific proteins were studied during rabbit embryo development (7–13 dpc) using monoclonal antibodies against titin, myosin, tropomyosin and actin, as well as the intermediate filament proteins desmin, keratin and vimentin. From our panel, titin appeared to be the first muscle-specific protein to be exclusively expressed in the embryonic rabbit heart. Upon differentiation (myocyte and myotube formation), titin reorganizes from dot-like aggregates into a cross-striated pattern (in 9- to 30-somite embryos) via a transiently filamentous distribution. When the expression and organization of the other muscle proteins was studied in relation to titin, it became apparent that tropomyosin followed upon titin with respect to its exclusive expression in the heart anlagen and its organization into a striated pattern. Myosin and desmin were organized into cross-striated patterns after titin and tropomyosin, but this arrangement had not reached its final form in 13-dpc embryos. Actin, keratin and vimentin were distributed in cytoplasmic filaments in the embryonic stages we investigated. Since the first pulsations are already detected in 3-somite embryos, we conclude that the organization of titin, tropomyosin, myosin and desmin into a striated pattern does not seem to be essential for the initiation of muscle cell contraction in the heart anlagen. Furthermore, this study shows that, in comparison with studies on mouse, chick and rat, the sequence of expression of muscle-specific and intermediate filament proteins during cardiomyogenesis is species-dependent, and that their expression and organization varies in time in different regions of the developing heart.Abbreviations IFP intermediate filament proteins - PBS phosphate-buffered saline - FITC fluorescein isothiocyanate - TRITC tetramethylrhodamine isothiocyanate - TxRd texas red - dpc days post conception