Perinuclear Intermediate Filaments Research Articles

Ultrastructural and Immunohistochemical Characterization of Maternal Myofibroblasts in the Bovine Placenta around Parturition

Myofibroblasts are contractile cells that exhibit features of both fibroblasts and smooth muscle cells. In the synepitheliochorial placenta of the cow myofibroblasts are found in the maternal stroma. However, a deeper understanding of the structure and function of the stromal myofibroblasts in the developed bovine placenta is still missing. Thus, immunohistochemical and ultrastructural analyses in bovine term placentomes, compared to non-pregnant caruncle samples, were conducted. To investigate functional aspects, contractility of placentomal caruncle slices was assessed in an in vitro contraction assay. Additionally, a three-dimensional reconstruction of a bovine placental myofibroblast was created. Immunofluorescent staining revealed a characteristic pattern, including cytoplasmic expression of α-smooth muscle actin, strong perinuclear signal for the intermediate filament vimentin and nuclear progesterone receptor staining. Ultrastructurally, stress fibers, extended cisternae of the rough endoplasmic reticulum and perinuclear intermediate filaments were observed. Moreover, in vitro stimulation with angiotensin-II, but not with prostaglandin F2α, induced contraction of placental caruncle tissue. Altogether, these results indicate that progesterone-responsive myofibroblasts represent a mesenchymal phenotype that is involved in the contractile properties of bovine placental stroma. Therefore, the present findings suggest a potential involvement of myofibroblasts in post-partum events of cattle, i.e., expulsion of fetal membranes and uterine involution.

Malignant Extrarenal Rhabdoid Tumor in a 5-Month-Old Male With Mediastinal and Heart/Great Vessel Involvement

Abstract Introduction/Objective Extrarenal rhabdoid tumor is a high-grade soft-tissue predominately pediatric malignancy with a frequency of 0.15 per million children less than 15 years. It is associated with del(22) (q11.2) with SMARCB1 loss, recognized by loss of INI-1 staining. It has distinct perinuclear hyaline inclusions; however, this feature can be present in other tumors. We describe a malignant extrarenal rhabdoid tumor involving the heart and great vessels to highlight the multiple modalities required for the diagnosis of this tumor in this unusual location. Methods We utilized routine histology, immunohistochemistry, and electron microscopy. Results This 5 -month -old male presented with respiratory distress due to a mass that invaded the heart, surrounding the great vessels. The tumor was composed of sheets of polygonal large cells with light pink to clear cytoplasm, eccentric nuclei with mild anisonucleosis, peripherally marginating chromatin, prominent nucleoli, with 2 mitoses per 10 high power field. Hyaline perinuclear inclusions were inapparent. There were frequent clusters of lymphocytes and eosinophils, with 10 percent necrosis and moderately increased vascularity. The tumor was positive for vimentin, SMA, EMA, MSA, S-100, keratin, and WT1, and negative for INI-1, CD34, ALK, AFP, PLAP, CD30, myogenin, NeuN1, synaptophysin, chromogranin, and NSE. Electron microscopy demonstrated occasional cells with perinuclear collections of intermediate filaments, some with whorls. The strong positivity for vimentin, keratin, SMA, MSA, S-100, negative staining for INA-1 and CD34, and visualization of perinuclear intermediate filament whorls by electron microscopy helped make the diagnosis of extrarenal rhabdoid tumor. Conclusion Malignant extrarenal rhabdomyosarcomas may occur in the middle mediastinum, invade the heart, occur in the first 6 months of life, and not have the typical rhabdoid cells. Diagnoses depend on multiple modalities

167 The pathogenic implications of prelamin a accumulation in cardiomyocytes: a model of premature cardiac senescence?

Introduction Mutations in the LMNA gene, which encodes the nuclear intermediate filament proteins lamins A and C, lead to a number of premature ageing syndromes, including Hutchinson Gilford Progeria syndrome (HGPS) and Emery Dreifuss muscular dystrophy (EDMD) as well as dilated cardiomyopathy (DCM). Some causal mutations disrupt lamin A processing, resulting in the accumulation of the lamin A precursor, prelamin A, however it is not clear to what extent accumulated prelamin A contributes to DCM. Here we identify DCM patients with cardiomyocyte accumulation of prelamin A and explore the impact of prelamin A accumulation in vivo . Methods We generated a novel line of targeted transgenic mice that accumulate prelamin A specifically in cardiomyocytes (PLA Tg mice), by expressing an uncleavable form of prelamin A, driven by Cre expression from the myosin light chain 2 ventricular (MLC2v) promoter. Results Immunofluorescence staining of human DCM biopsies showed the presence of nuclear prelamin A in cardiomyocytes. PLA Tg mice were born without any obvious phenotype but manifested retarded growth by 3 weeks of age and succumbed to heart failure at ˜5 weeks. At 4 weeks, MRI showed a marked dilatation of the cardiac chambers and a decline in cardiac function in vivo . Ejection fraction (EF) was substantially depressed in PLA Tg micecompared with wildtype indicating DCM and heart failure. Cardiac histology showed marked cardiomyocyte disarray and profound fibrosis. Biochemical and microscopic analyses indicated disruption of the linkers of nucleoskeleton to cytoskeleton complex and perinuclear intermediate filament network and was supported by electron micrographs showing nuclear morphology defects. Myocardial infiltration of CD45 positive cells coincided with the expression of γ-H2AX, phosphorylated ATM and increased NF-κB signalling, which suggested an inflammatory response initiated by DNA damage. This may be related to the senescence associated secretory phenotype (SASP) as senescence associated β-galactosidase was also expressed in PLA Tg myocardium. Conclusion We have demonstrated prelamin A accumulation in the nuclei of human DCM biopsies and show that overexpression of prelamin A in a transgenic mouse model leads to an early decline in cardiac function and premature myocardial senescence.

218 A TRANSGENIC MODEL OF PRELAMIN A ACCUMULATION LEADS TO CARDIAC DYSFUNCTION IN MICE

Ageing is a potent risk factor for cardiovascular (CV) disease. Mutations in the LMNA gene, which encodes the nuclear intermediate filament proteins lamins A & C, lead to a number of premature ageing syndromes, e.g. Emery Dreifuss muscular dystrophy (EDMD), which can lead to dilated cardiomyopathy (DCM) and heart failure. Some of these mutations affect lamin A processing, resulting in the accumulation of the lamin A precursor, prelamin A. Importantly, recent studies have shown that the mechanisms of premature ageing observed in patients with lamin A mutations may also occur during normal ageing processes. This led us to postulate that accumulation of prelamin A in the heart may mimic ‘age-related’ cardiac dysfunction. To test this, we generated a novel line of targeted transgenic mice that accumulate prelamin A specifically in cardiomyocytes, by expressing a modified prelamin A gene driven by the myosin light chain 2 ventricular (MLC2v) promoter. These mice were born without any obvious phenotype but manifested retarded growth by 3 weeks of age and had a significantly attenuated lifespan (∼5 weeks) with the cause of death appearing to be heart failure. Their failure to thrive was also underscored by a muscular dystrophy like appearance. At 4 weeks age, echocardiography showed a marked dilatation of the cardiac chambers and a decline in cardiac function in vivo, e.g. ejection fraction (EF) was substantially depressed in transgenic mice (20.9±3.6%) compared with wildtype (56.7±11.2%, P=0.01), indicating DCM and heart failure. Cardiac histology showed marked cardiomyocyte disarray, profound fibrosis (Picro-sirius Red staining), and expression of senescence markers (senescence-associated beta-galactosidase). Analyses of molecular events indicated severe disruption of the LINC complex and perinuclear intermediate filament network as a number of components (SUN 2, Nesprin 2, Desmin) underwent significant expression changes and/or were mislocalised. Additionally, immunohistochemistry and western blot for CD45 and CD68 confirmed increased numbers of inflammatory cells in hearts overexpressing prelamin A. In conclusion, prelamin A accumulation in cardiomyocytes causes a pathogenic response leading to decline in cardiac function and, ultimately, death. Further investigation aims to establish the precise mechanisms underpinning the response of the heart to accumulation of prelamin A.

Novel Interaction Partners of the CD2BP2-GYF Domain

The GYF domain of CD2BP2 serves as an adapter that recognizes proline-rich sequences in intracellular proteins. Although the T cell adhesion molecule CD2 and the core splicing protein SmB/B' were previously shown to interact with CD2BP2-GYF, we are now using a general approach to identify putative GYF domain target sites within the human proteome. The phage display-derived recognition motif for CD2BP2-GYF is PPG(W/F/Y/M/L). SPOT analysis confirmed that the GYF domain interacts with peptides from human proteins containing the consensus site. Epitope mapping by NMR spectroscopy performed for several peptides revealed a conserved binding surface. A direct interaction of the CD2BP2-GYF domain with the novel protein interaction partners PI31 and NPWBP was verified by yeast two-hybrid analysis.

Papillary tumor of the pineal region.

Primary papillary tumors of the central nervous system are rare. We have encountered a series of six papillary tumors of the pineal region with distinctive features that appear to represent a clinicopathologic entity. The tumors occurred in four women and two men, ranging in age from 19 to 53 years. Imaging studies showed a large well-circumscribed mass in the pineal region. The tumors were characterized by an epithelial-like growth pattern, in which the vessels were covered by a layer of tumoral cells. In papillary areas, the neoplastic cells were large, columnar or cuboidal, with a clear cytoplasm. Nuclei, round or infolded, were found generally at the basal pole of tumoral cells. Immunohistochemically, the tumor cells showed strong staining for cytokeratin, S-100 protein, neuron-specific enolase, and vimentin but only weak or no staining for epithelial membrane antigen and glial fibrillary acid protein. Ultrastructural examination of two cases revealed abundant rough endoplasmic reticulum with distended cisternae filled with secretory product, microvilli, and perinuclear intermediate filaments. The morphofunctional features of these papillary tumors of the pineal region, remarkably uniform within this series, are similar to those described for ependymal cells of the subcommissural organ, and the papillary tumors of the pineal region may be derived from these specialized ependymocytes.

The architecture of microtubular network and Golgi orientation in osteoclasts—major differences between avian and mammalian species

In the present study, we analyze multinuclear osteoclasts obtained from several avian and mammalian species and describe the reorganization of their microtubular architecture and Golgi complex orientation during osteoclast differentiation and activation for bone resorption. In nonresorbing quail and chicken multinuclear osteoclasts, microtubules radiate from multiple centrosomal microtubule-organizing centers (MTOCs), whose number is equal to the number of nuclei. However, centrosomal MTOCs disappear at the time of cell activation for bone resorption and the Golgi membranes redistribute to circumscribe nuclei. In contrast to avian osteoclasts, both resorbing and nonresorbing rat, rabbit, and human osteoclasts have no or few centrosomal MTOCs. Instead, after cold-induced depolymerization, regrowing microtubules nucleate from the perinuclear area where immunofluoresce and immunoelectron scanning microscopy reveal pericentriolar matrix protein pericentrin associated with vimentin filaments. Furthermore, the circumnuclear reorganization of MTOCs and the Golgi is a result of mammalian osteoclast maturation and occur before any resorptive activity of the mononuclear osteoclasts and their fusion into multinucleated cells. Our results show that unlike previously suggested, the nuclear surfaces of mammalian osteoclasts act as the microtubule anchoring sites similarly to nuclear surfaces in multinucleated myotubes and suggest the role of perinuclear intermediate filament network in orchestrating the microtubular cytoskeleton.

Aspects of the functional morphology of the ductus epididymidis in domestic anseriform and galliform birds.

The structure of the ductus epididymidis, and its capacity to take up luminal particulate material (India ink) were studied, using scanning and transmission electron microscopy, in the domestic fowl, turkey and Japanese quail (Order Galliformes) and drake (Order Anseriformes). The epithelium appeared contractile and stable structurally, especially in the drake in which, among other cytoskeletal features, abundant perinuclear intermediate filaments radiated peripherally in the principal, nonciliated (Type III) cell. The Golgi complex was well developed, except in the turkey in which it was extremely inconspicuous. Sparsely granulated profiles were the main component of the elaborate endoplasmic reticulum, which was best developed in the drake and least in the turkey. The cell in the turkey was uniquely laden with large aggregates of lipid droplets. Generally, characteristics of a cell active in the synthesis and merocrine secretion of proteinaceous material were evident. There was no evidence of apocrine-like secretion in well-fixed tissues. The cell lacked the capacity to take up luminal India ink particles, and displayed very poorly developed endocytic structural features, which are also probably only selectively and minimally absorptive. Ultrastructural features varied between and within the orders studied, necessitating further studies.

Changes in the distribution of intermediate filaments in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development.

Intermediate filaments (IFs) are components of the cytoskeleton. In mammalian Sertoli cell, IFs are formed by vimentin. Previous studies have shown some characteristics of its distribution in Sertoli cells, however, very little is known of its distributional changes during the seminiferous epithelium cycle and during postnatal development. Immunohistochemical and electron microscopic methods were used to determine the distribution of vimentin-type IFs in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development. The distribution of IFs in adult rat Sertoli cell showed distinct cyclic changes during the seminiferous epithelium cycle. At stages I-VI, bundles of IFs extend from the perinuclear region to the supranuclear and apical regions of the Sertoli cell. These apical extensions became shorter at stage VII, and at stages VIII-X IFs were observed only in the perinuclear region. Short apical extensions reappeared at stages XI-XII; and at stages XIII-XIV, they extended again into the apical region. During this cycle, IFs were always closely associated with the heads of elongate spermatids. IFs were also shown to be in close apposition to some specialized structures on the cell membrane, such as the ectoplasmic specialization between adjacent Sertoli cells. During postnatal (p.n.) development, IFs were mainly observed at the basal nuclear region on p.n. day 7. The IFs in the supranuclear or apical regions first appeared at p.n. day 14 and gradually increased during the development. The perinuclear IFs network was fully established by p.n. day 28 and the adult distribution pattern of the IFs was established by p.n. day 42. Vimentin-type IFs in rat Sertoli cells are a delicate endocellular network, which is centered in the perinuclear region and extends to the apical region of the cell. During the seminiferous epithelium cycle, the distribution of IFs changes in a stage-dependent manner and is closely related to the location of the heads of elongate spermatids. During postnatal development, IFs gradually increase in numbers and the main distribution area is transferred from the basal nuclear to the perinuclear and supranuclear regions.

Reversible Large-Body Formation from Nuclear Bodies upon Amino Acid(s) Starvation in T24 Cells

AP435 dot, a nuclear dot-like structure that is recognized by a monoclonal antibody AP435 MAb and that seems to correlate with perinuclear intermediate filaments, was identified as a nuclear body by double immunofluorescent staining with AP435 MAb and the nuclear-body-specific antibody αSp100 or mAb 5E10. In T24 cells, nuclear bodies usually appear as small entities with an apparent diameter ranging from 0.2 to 0.7 μm, and several to 20 or more of them are present per nucleus. After long culture without a change in the medium, however, nuclear bodies disappeared while one or more large doughnut-shaped bodies appeared, which had apparent outer diameters of 0.7–1.8 μm. When the medium was changed or medium components were added, large bodies disappeared and many nuclear bodies of normal size reappeared within several hours. Large-body formation was not related to the arrest of DNA synthesis, as revealed by double labeling with AP435 MAb and anti-cyclin antibody. Among the medium components, only an amino acid mixture induced the change from large bodies to nuclear bodies. Large-body formation was also observed in long-cultured HeLa cells. These results suggest that nuclear bodies reversibly aggregate or reorganized to form large bodies upon amino acid(s) starvation.

Human malignant mesothelial cells: variability of ultrastructural features in established and nude mice transplanted cell lines.

The aim of this study was to determine, by transmission electron microscopy, the differentiation features of 21 human malignant mesothelioma cell lines (HMCLs) established from 13 specimens of 12 confirmed human malignant mesotheliomas, and of tumours induced in nude mice injected with 16 HMCLs. Fifty per cent of HMCLs showed typical mesothelial differentiation (long and slender microvilli, desmosomes, perinuclear intermediate filaments); 29 per cent did not show differentiation; and the remainder were poorly differentiated. Three human tumour specimens gave several different HMCLs; the cell lines obtained from a given tumour exhibited variable mesothelial differentiation. Eleven HMCLs were compared with the native tumour. Four were similar to the tumour and seven were less well differentiated, in most cases in relation to their microvilli. With six HMCLs, tumours induced in nude mice were less well differentiated than the corresponding cell lines, whereas with four HMCLs, tumours were equally or better differentiated. However, in most nude mice tumours, typical mesothelial microvilli were present. These results show that cell lines established from malignant mesothelioma may exhibit dedifferentiated features. However, while the variability in ultrastructural differentiation may result from the culture microenvironment, it could also be related to the state of differentiation, of the native tumour sample and to tumour cell heterogeneity.

A Nuclear Dot-like Structure That Has a Relationship with Perinuclear Intermediate Filaments

A new type of nuclear dot-like structure was found that seems to associate with perinuclear intermediate filaments (IFs). A monoclonal antibody AP435 MAb (IgM), reactive with desmin and vimentin, recognized from several to dozens of small nuclear dots (AP435 dots) as well as cytoplasmic IFs in various mammalian cells under immunofluorescence microscopy. AP435 dots were sensitive to treatment with 0.5% Triton X-100 while IFs were resistant. The dots disappeared during mitosis in some cells. They colocalized with neither centromeres nor structures involved in RNA processing. Many AP435 dots seem to associate with perinuclear IFs, though they were observed even in cells with no IFs. In cells possessing IFs AP435 dots separated from the bulk of the IFs and remained in the nuclear region both after treatment with colchicin and after treatment with cytochalasin B followed by centrifugation. Confocal laser-scanning fluorescence microscopy revealed the dots to be inside the nucleus. AP435 dots are small intranuclear structures that seem to interact directly or indirectly with perinuclear IFs.

Actin filaments associated with the basal Sertoli cell surface in the alligator and turtle

We have studied, using electron microscopy, the basal cytoplasm of Sertoli cells in the alligator ( Alligator mississippiensis) and the turtle ( Pseudemys scripta). Filament bundles occur immediately adjacent to the basal plasma membrane. The diameter of these filaments is smaller than that of perinuclear intermediate filaments, but is similar to that of filaments present in peritubular cells. In the alligator, the filament bundles appear to form a branching network over the base of the cell and are related to cisternae of the endoplasmic reticulum. In the turtle. filaments occur either in distinct bundles within folds of the plasma membrane or as a more uniform layer over the base of the cell. In fixed frozen sections, labelled with rhodamine phalloidin, linear bands of fluorescence occur in regions which correspond to those containing the filament bundles observed by electron microscopy. We conclude that the basal filaments in alligator and turtle Sertoli cells are actin and speculate that these filaments may be involved in cell/substratum adhesion.

Combined ultrastructural and immunohistochemical studies on a mouse rhabdomyosarcoma.

Light microscopic and ultrastructural observations, using immunoperoxidase staining for myoglobin and actin were performed on pleomorphic rhabdomyosarcoma in an ICR-male mouse aged 98 weeks, which arose in the striated muscle of the lumbar region. The tumor consisted of three types of cells showing varying degrees of myofibrinogenesis. The small round or elongated mononucleated cells (type I), which predominated in number, were immature mesenchymal cells. Some of these cells contained small amount of perinuclear intermediate filaments (9-11 nm in diameter). The eosinophilic plump cells (type II) exhibited one or two irregular nuclei and broad cytoplasm containing both types of myofilaments, actin and myosin filaments, not arranged to form myofibrils. The multinucleated giant cells (type III) alone possessed a Well developed element of tumoral myofibrils, Z-band. C-type virus like particles were rarely found in cytoplasmic vesicles or intercellular spaces of all types of cells. Myoglobin-positive staining was detected in most of the type III cells, and a few of the type II cells, while actin-positive staining was seen in most of both types cells. However, the type I cells were consistently negative for these two markers.

Primary neuroendocrine (Merkel cell?) carcinoma of the skin. I. A clinicopathologic and ultrastructural study of 43 cases.

The clinical and pathologic features of 43 cases of primary neuroendocrine carcinoma of the skin are reported. These tumors arise in the dermis and subcutaneous tissues of elderly individuals. The head and neck are the most common primary sites followed by the lower and upper extremities and trunk. Characteristic histologic features include round cells with scanty amphophilic cytoplasm and vesicular nuclei with multiple small nucleoli. The cells are arranged in sheets, solid nests, or anastomosing trabeculae. Collections of perinuclear intermediate filaments, cytoplasmic dense-core membrane-bound secretory granules, complex intercellular junctions, and cytoplasmic spinous processes are the principal fine structural features. These ultrastructural findings are similar to those of the normal cutaneous Merkel cell. The natural history of this neoplasm is characterized by local recurrence in 30% of cases, regional lymph node metastases in 65% of cases, and distant metastases in 40%. One-third of the patients were dead because of their tumors. Treatment of extensive local or distant metastatic disease with chemotherapy or radiotherapy resulted in only short-term palliative response.

Association of mitochondria with intermediate filaments and of polyribosomes with cytoplasmic actin.

Anti-mitochondrial autoantibody and fluorescent derivatives of insulin stain phase-dense mitochondria in acetone fixed monolayers of fibroblasts. Double fluorochrome studies show mitochondria in close topographic association with intermediate filaments. In cells treated with vinblastine or colchicine, mitochondria are relocated in sites closely associated with coils of perinuclear intermediate filaments. In contrast, autoantibody to polyribosomes stains granules aligned in the long axis of well spread embryonic cells, in the direction of actin-containing fibrils, an arrangement that is lost in cells pretreated with the actin filament disrupting drug cytochalasin B. In more mature fibroblasts, antiribosomal antibody reacts with phase-dense rough endoplasmic reticulum and this staining pattern is not affected by cytochalasin B. The observations suggest that mitochondria are associated with intermediate filaments and that free polyribosomes, but not polyribosomes attached to rough endoplasmic reticulum, are associated with cytoplasmic actin.

Malignant soft tissue tumors (malignant fibrous histiocytoma, pleomorphic liposarcoma, and pleomorphic rhabdomyosarcoma): An electron microscopic study

A comparative ultrastructural analysis of malignant soft tissue tumors (malignant fibrous histiocytoma, pleomorphic liposarcoma, and pleomorphic rhabdomyosarcoma) revealed similar ultrastructural features in this group of tumors. However, by electron microscopy these tumors can be differentiated on the basis of cytoplasmic and extracytoplasmic features (myosin filaments, lipid droplests and perinuclear intermediate filaments, for example). This is even true of less well differentiated tumors and tumor cells. These findings support and amplify the concept of a common histogenesis for tumors of mesenchymal origin. Paradoxical features observed by light microscopy wasrant further study by electron microscopy if the correct diagnosis is to be made in atypical cases, such as apparent malignant fibrous histiocytoma with cross striations.