共查询到20条相似文献,搜索用时 15 毫秒
1.
We describe a new monoclonal antibody (MAb) directed against glomerular visceral epithelial cells (podocytes), generated by immunization with isolated rat kidney glomeruli. In immunoblotting experiments this MAb (IgG1 subclass) reacted with a 44 KD protein. In cryostat sections of normal rat kidney the MAb stained glomerular podocytes; therefore, we called the antigen pp44 (podocyte protein 44 KD). On 0.5-micron cryostat sections the signal could be more precisely ascribed to the podocyte foot processes, whereas the cell bodies appeared virtually unreactive. On ultra-thin frozen sections pp44 was found within the cytoplasm of podocyte foot processes at their origin from their parent processes. The podocyte cell membrane was not labeled. All other parts of the nephron were unreactive. An additional but weaker immunoreaction was found in the arterial endothelium; the endothelia of other vessels (peritubular capillaries, veins) were negative. In human kidney anti-pp44 revealed the same staining pattern as in rat kidney. The expression of pp44 was also studied in newborn rat kidney. The early stages of glomerular development (renal vesicle, S-shaped body) were negative. pp44 first appeared during the capillary loop stage, i.e., when formation of podocyte foot processes commences. In comparing the present results with published data, pp44 is clearly different from other antigens thus far described in podocytes. From the results of this investigation we conclude that pp44 represents a novel cytoplasmic protein of podocytes. Our data suggest a cytoskeletal role for pp44 in preserving the complex architecture of podocytes. This idea is confirmed by the simultaneous appearance of foot processes and anti-pp44 immunoreactivity during glomerular development. 相似文献
2.
Ceacam6 (carcinoembryonic antigen-related cell adhesion molecule 6 gene) has recently been isolated by differential display followed by RT-PCR and DNA sequence analyses. Ceacam6 is a member of an immunoglobulin superfamily and encodes a protein of 266 amino acid residues possessing one immunoglobulin (Ig)-like domain. RT-PCR analysis showed that Ceacam6 was dominantly expressed in rat testis and its expression level prominently increased after 6 wk of postnatal development in testis. Immunohistochemical analyses using the anti-CEACAM6 antibody revealed that CEACAM6 colocalized with intermediate filaments (vimentin) in Sertoli cells and interstitial cells. The association between CEACAM6 and vimentin was observed throughout postnatal development in rat testis. Transfection experiments performed in COS-7 cells suggested that overexpression of CEACAM6 brought about aggregation of vimentin filament around nuclei with which CEACAM6 colocalized and that the N-terminus region of CEACAM6, including the Ig-like domain, seemed to be required for association with vimentin filaments. Interaction between CEACAM6 and vimentin in rat testis and transfected COS-7 cells was confirmed by immunoprecipitation. Our observations strongly suggested that CEACAM6 might be a novel intermediate filament-associated protein involved in regulation of vimentin architecture in Sertoli cells. 相似文献
3.
V B Zaitsev 《Cellular and molecular biology, including cyto-enzymology》1992,38(8):823-833
Using TEM and immunofluorescence microscope, a study was made on podocytes in vertebrates where an intermediate-sized filament system is replaced by a microtubule system, accompanied by highly developed microfilaments structures. A comparative immunofluorescence study was carried out on cryotome renal sections of plaice (Pleuronectes platessa L.) and rats, using specific antibodies anti-cytokeratins and anti-vimentin. With polyclonal anti-vimentin serum the capillaries of the renal glomeruli showed a bright colour of plaice and only a week one in the rats. Double staining of renal tissue of mongrel rats of different ages (6-7 weeks and 1.8 years old) with antibodies for actin and anti-vimentin polyclonal serum revealed in young rats an intensive fluorescence for actin and a slight fluorescence for the intermediate filaments. Renal glomeruli of old rats demonstrate a strong vimentin-activity and lower actin one. The ultrastructural study of human podocytes showed two different cytoskeleton age-depending types (2, 4, 6, 37 and 65 years old). It is suggested that during individual development and ageing in kidneys of higher animals and human, physiological changes induce morphological cytoskeleton restructuration accompanied by intensive development of intermediate filaments and simultaneous "involution" of microtubules and microfilaments. 相似文献
4.
Cytomorphologic and immunocytochemical characteristics of reactive renal tubular cells in renal glomerular disease 总被引:1,自引:0,他引:1
OBJECTIVE: To investigate the morphologic characteristics and immunocytochemical reaction to vimentin of the reactive renal tubular cells (RRTCs) in renal glomerular disease. STUDY DESIGN: We prospectively evaluated the urine cytology of renal glomerular disease in 40 patients who underwent renal biopsy. The cytology and renal biopsy specimens were analyzed for vimentin immunostaining. RESULTS: A total of 40 urine samples from the 40 patients were cytologically analyzed, and RRTCs were found in 25 samples (25 of 40, 62.5%). These RRTCs showed clear or vacuolated cytoplasm, intracytoplasmic pigmented granules (hemosiderin or lipofuscin) and large nuclei with round to irregular nuclear contours and prominent nucleoli. These cells were seen singly and in acinar clusters. Occasionally RRTCs were embedded in a cast (RRTC cast). Immunocytochemicalstudy revealed RRTCs to be positive for vimentin (25 of 25, 100%). CONCLUSION: Frequently observed characteristic cytomorphologic features of RRTCs included RRTC cast, acinar cluster, vacuolated cytoplasm and intracytoplasmic pigmented granules. A diagnosis of RRTCs can be suggested based on these cytomorphologic features. However, a definitive diagnosis will require immunocytochemical confirmation for vimentin. 相似文献
5.
V A Krutovskikh S M Troianovski? 《Biulleten' eksperimental'no? biologii i meditsiny》1985,100(8):240-241
Cryostatic sections of rat large bowel tumors induced by 1,2-dimethylhydrazine were stained with monoclonal antibodies against different proteins of intermediate filaments: (a) against prekeratin (mol. mass 49 000, PK49) found in many epithelial cells and (b) against vimentin, a constituent of intermediate filaments of mesenchymal cells. Immunofluorescence study showed that large bowel tumor cells as well as normal cells of this organ contain PK49 but not vimentin. High sensitivity of the method allowed one to clearly identify small invasive nodules and groups of tumor cells not visible in usual histologic preparations. Moreover, in some cases single atypical tumor cells were identified in tumor stroma and in the submucosal layer underlying the tumor, that were indistinguishable from normal mesenchymal cells at the light microscopy level. 相似文献
6.
S Liu W Shi H Xiao X Liang C Deng Z Ye P Mei S Wang X Liu Z Shan Y Liang B Zhang W Wang Y Liu L Xu Y Xia J Ma Z Li 《PloS one》2012,7(7):e41331
Background
Glomerulosclerosis correlates with reduction in podocyte number that occurs through mechanisms which include apoptosis. Podocyte injury or podocyte loss in the renal glomerulus has been proposed as the crucial mechanism in the development of glomerulosclerosis. However, the mechanism by which podocytes respond to injury is poorly understood. TNF and TNF receptor superfamilies are important in the pathogenesis of podocyte injury and apoptosis. The ligand of receptor activator of NF-kappaB (RANKL) and receptor activator of NF-kappaB (RANK) are members of the TNF and receptor superfamilies. We investigated whether RANK - RANKL is a receptor - ligand complex for podocytes responding to injury.Methodology/Principal Findings
In this study, RANKL and RANK were examined in human podocyte diseases and a rat model of puromycin aminonucleoside nephrosis (PAN). Compared with controls, RANK and RANKL were increased in both human podocyte diseases and the rat PAN model; double immunofluorescence staining revealed that RANK protein expression was mainly attributed to podocytes. Immunoelectron microscopy showed that RANK was localized predominantly at the top of the foot process membrane and the cytoplasm of rat podocyte. In addition, RANK was upregulated in mouse podocytes in vitro after injury induced by puromycin aminonucleoside (PA). Knockdown of RANK expression by small interference RNA (siRNA) exacerbated podocyte apoptosis induced by PA. However, RANKL inhibited significantly the apoptosis of podocytes induced by PA.Conclusions/Significance
These findings suggest the increase in RANK–RANKL expression is a response to podocyte injury, and RANK–RANKL may be a novel receptor–ligand complex for the survival response during podocyte injury. 相似文献7.
The importance of protein phosphatases in maintaining the integrity of intermediate filaments is supported by the fact that intermediate filaments would undergo a massive reorganization in cells treated with inhibitors of protein phosphatases 1 and 2A. Herein we used okadaic acid to investigate the differential roles of protein phosphatases 1 and 2A in the maintenance of intermediate filament integrity in 9L rat brain tumor cells. Protein phosphatase 2A activity was substantially inhibited after treatment with 400 nM okadaic acid for 2 h, whereas the activity of protein phosphatase 1 was only slightly affected. Furthermore, protein phosphatase 2A shows selective specificity toward phosphovimentin, which was immunologically precipitated from isotopically labeled and okadaic acid-treated cells. Further biochemical fractionation and microscopic studies revealed that vimentin intermediate filaments were colocalized with protein phosphatase 2A, but not protein phosphatase 1, in control cells. On okadaic acid treatment, vimentin filament disassembled and protein phosphatase 2A redistributed throughout the cytoplasm, suggesting that these two proteins separate from each other, whereas protein phosphatase 2A was inhibited. This working hypothesis was further supported by treatment with a low concentration (40 nM) of okadaic acid, which causes the same phenomenon. Taken together, our results showed that protein phosphatase 2A could be assigned to the intermediate filaments to serve the physiological role in maintaining the proper phosphorylation level of intermediate filaments in normal cells. This finding should pave the way for the elucidation of the regulatory mechanism of intermediate filament organization governed by protein phosphorylation. 相似文献
8.
Mark C. Willingham Nancy D. Richert Angelina V. Rutherford 《Experimental cell research》1987,171(2):284-295
Using a monoclonal antibody, we have detected an antigen present in a unique fibrillar structure in the cytoplasm of cultured cells by immunofluorescence. These structures have been identified by transmission electron microscopy and ultrastructural immunocytochemistry as large single paracrystalline arrays of individual filaments morphologically similar to intermediate filaments. The antibody detects these structures in fibroblastic and epithelioid cultured cell lines of mouse, rat, bovine, and human origin but not of avian origin. Only a small percentage of the cells in a culture contains these structures; each cell usually contains only one, although two or more have been observed in a single cell. The structures are elongated vermiform arrays of filaments in the cytoplasm (approximately 0.5 X 3 microns) which have a thread-like or toroidal appearance. Because of this shape, we have named the putative antigen recognized by this antibody "nematin." Double-label experiments showed that these structures had no relationship to tubulin or vimentin. Immunocytochemical localization in human tissues revealed a high concentration of a reactive antigen in the stratum granulosum of skin and in what probably are neuroglial cells in the central nervous system. This monoclonal antibody may detect a novel intermediate filament protein and/or a shared determinant of different intermediate filament proteins. 相似文献
9.
The subcellular localization of protein kinase C (PKC)-δ was determined in HL60 cells differentiated toward monocytes/macrophages by treatment with TPA. PKC-δ was detected in the nucleus and cytoplasm of differentiated HL60 cells and, more specifically, associated with structures resembling intermediate filaments. Indirect immunostaining revealed that PKC-δ colocalized with vimentin in the cytosol and perinuclear region of these cells. Immunoprecipitation studies showed that PKC-δ was in an active (autophosphorylated) state in differentiated HL60 cells and that vimentin immunoprecipitated from these cells was also phosphorylated. Treatment of HL60 cells with the PKC-specific inhibitor chelerythrine decreased the phosphorylation of vimentin. These data suggest that vimentin is a substrate for PKC-δ and that this PKC isoenzyme may play a specific role in the regulation of shape change and cell adhesion during HL60 differentiation. 相似文献
10.
The subcellular localization of protein kinase C (PKC)-δ was determined in HL60 cells differentiated toward monocytes/macrophages by treatment with TPA. PKC-δ was detected in the nucleus and cytoplasm of differentiated HL60 cells and, more specifically, associated with structures resembling intermediate filaments. Indirect immunostaining revealed that PKC-δ colocalized with vimentin in the cytosol and perinuclear region of these cells. Immunoprecipitation studies showed that PKC-δ was in an active (autophosphorylated) state in differentiated HL60 cells and that vimentin immunoprecipitated from these cells was also phosphorylated. Treatment of HL60 cells with the PKC-specific inhibitor chelerythrine decreased the phosphorylation of vimentin. These data suggest that vimentin is a substrate for PKC-δ and that this PKC isoenzyme may play a specific role in the regulation of shape change and cell adhesion during HL60 differentiation. 相似文献
11.
Process formation of podocytes: morphogenetic activity of microtubules and regulation by protein serine/threonine phosphatase PP2A 总被引:6,自引:0,他引:6
Kobayashi N Reiser J Schwarz K Sakai T Kriz W Mundel P 《Histochemistry and cell biology》2001,115(3):255-266
Podocytes possess major processes containing microtubules (MTs) and intermediate filaments and foot processes containing actin filaments (AFs) as core cytoskeletal elements. Although the importance of these cytoskeletal elements for maintaining podocyte processes was previously shown, so far no data are available concerning the developmental regulation of podocyte process formation. A conditionally immortalized mouse podocyte cell line, which can be induced to develop processes similar to those found in vivo, was treated with various reagents to disrupt cytoskeletal elements or to inhibit protein phosphatases. MTs colocalized with vimentin intermediate filaments but not with AFs. After AF disassembly, major processes were maintained, whereas after depolymerization of MTs, podocytes lost their processes, rounded up, and maintained only actin-based peripheral projections. Suppression of MT elongation by nanomolar vinblastine or inhibition of serine/threonine phosphatase PP2A with okadaic acid abolished process formation. PP2A was expressed in undifferentiated but not in differentiated podocytes. One- and two-dimensional western blot analyses revealed a dose-dependent increase in serine/threonine phosphorylation after okadaic acid treatment. Hence, morphogenetic activity of MTs induces podocyte process formation via serine/threonine protein dephosphorylation by PP2A. These results may open new avenues for understanding the signaling mechanism underlying podocyte cytoskeleton alterations during development and in glomerular diseases. 相似文献
12.
Bettina S. Buchmaier Asima Bibi Gerhard A. Müller Gry H. Dihazi Marwa Eltoweissy Jenny Kruegel Hassan Dihazi 《PloS one》2013,8(7)
Osmotic stress has been shown to regulate cytoskeletal protein expression. It is generally known that vimentin is rapidly degraded during apoptosis by multiple caspases, resulting in diverse vimentin fragments. Despite the existence of the known apoptotic vimentin fragments, we demonstrated in our study the existence of different forms of vimentin VIM I, II, III, and IV with different molecular weights in various renal cell lines. Using a proteomics approach followed by western blot analyses and immunofluorescence staining, we proved the apoptosis-independent existence and differential regulation of different vimentin forms under varying conditions of osmolarity in renal cells. Similar impacts of osmotic stress were also observed on the expression of other cytoskeleton intermediate filament proteins; e.g., cytokeratin. Interestingly, 2D western blot analysis revealed that the forms of vimentin are regulated independently of each other under glucose and NaCl osmotic stress. Renal cells, adapted to high NaCl osmotic stress, express a high level of VIM IV (the form with the highest molecular weight), besides the three other forms, and exhibit higher resistance to apoptotic induction with TNF-α or staurosporin compared to the control. In contrast, renal cells that are adapted to high glucose concentration and express only the lower-molecular-weight forms VIM I and II, were more susceptible to apoptosis. Our data proved the existence of different vimentin forms, which play an important role in cell resistance to osmotic stress and are involved in cell protection against apoptosis. 相似文献
13.
Modulation of vimentin containing intermediate filament distribution and phosphorylation in living fibroblasts by the cAMP-dependent protein kinase 总被引:16,自引:11,他引:5
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Microinjection of the purified catalytic subunit of the cAMP-dependent protein kinase (A-kinase) into living rat embryo fibroblasts leads to dramatic changes in vimentin intermediate filament (IF) organization, involving the collapse of the filaments into tight bundles. In some cell types, this rearrangement of the IF proceeds further, leading to an apparent loss of filament integrity, resulting in a punctate staining pattern throughout the cytoplasm. Both these types of IF rearrangement are fully reversible, and similar to structural changes previously described for IF during mitosis. As shown by electron microscopy, in rat embryo fibroblasts these changes in IF structure do not involve the loss of the 10-nM filament structure but instead correspond to the bundling together of 25 or more individual filaments. Metabolic pulse labeling of injected cells reveals that accompanying these changes in IF organization is a dramatic increase in vimentin phosphorylation which appears maximal when the IF are fully rearranged. However, this increase in IF phosphorylation is not accompanied by any significant increase in soluble vimentin. Analysis of the sites of phosphorylation on vimentin from injected cells by either V8 protease cleavage, or two-dimensional tryptic peptide mapping, revealed increased de novo phosphorylation of two vimentin phosphopeptides after microinjection of A-kinase. These data strongly suggest that the site-specific phosphorylation of vimentin by A-kinase is responsible for the dynamic changes in IF organization observed after injection of the kinase into living cells, and may be involved in similar rearrangement of the IF previously described during mitosis or after heat shock. 相似文献
14.
Andrew J Dawson Tim J King Ian Wilmut Linda M Harkness Brian G Kelly Susan M Rhind 《The journal of histochemistry and cytochemistry》2004,52(12):1657-1664
Kidneys from lambs derived by nuclear transfer are frequently abnormal and are characterized by an enlarged pelvis and narrow medulla, consistent with lower urinary tract obstruction and development of variable hydronephrosis. The precise pathogenesis of this entity is unknown. Immunohistochemical staining for intermediate filaments was used to further characterize the lesions seen in this condition and was compared with age-matched control tissue. Major findings were upregulation of cytokeratin on damaged tubules, desmin and vimentin in undifferentiated mesenchyme, and smooth muscle actin in mesenchyme and on smooth muscle "collars" around dilated tubules. In addition, some cases showed reexpression of vimentin and desmin on proximal tubular epithelial cells. Taken together, these findings provide a valuable database for tracking the expression of intermediate filaments throughout renal development in sheep and have further characterized the nature of the response to injury by the developing kidney, a response that is characterized by proliferation of mesenchyme and both reexpression and upregulation of intermediate filaments within renal cells. In addition, the study has confirmed that the changes in cloned lamb nephropathy are established by day 85 of development. 相似文献
15.
16.
Summary Some intermediate filament (IF) proteins expressed in the development of glia include nestin, vimentin, and glial fibrillary acidic protein (GFAP). However, GFAP is the major intermediate filament protein of mature astrocytes. To determine the organization of GFAP in glial cells, rat GFAP cDNA tagged with enhanced green fluorescent protein (EGFP) was transfected into the rat C6 glioma cell line. After selection, two stable C6-EGFP-GFAP cell lines were established. Stable C6-EGFP-GFAP cell lines with or without heat shock treatment were analyzed by immunocytochemistry, electron microscopy, and Western blot analysis. In the transient transfection study, EGFP-GFAP transiently expressed in C6 cells formed punctate aggregations in the cytoplasm right after transfection, but gradually a filamentous structure of EGFP-GFAP was observed. The protein level of nestin in the C6-EGFP-GFAP stable clone was similar to that in the pEGFP-C1 transfected C6 stable clones and non-transfected C6 cells, whereas the level of vimentin was reduced in Western blotting. Interestingly, the expression level of small heat shock protein αB-crystallin in C6-EGFP-GFAP cells was also enhanced after transfection. Immunostaining patterns of C6-EGFP-GFAP cells showed that GFAP was dispersed as a fine filamentous structure. However, after heat shock treatment, GFAP formed IF bundles in C6-EGFP-GFAP cells. In the meantime, αB-crystallin also colocalized with IF bundles of GFAP in C6-EGFP-GFAP cells. The heat-induced GFAP reorganization we found suggested that small heat shock protein αB-crystallin may play a functional role regulating the cytoarchitecture of GFAP. 相似文献
17.
The presence and distribution of vimentin, a subunit of intermediate filaments, in the neural lobe and in the pars intermedia of the cat, rabbit and rat pituitary glands were investigated immunocytochemically. In the pars intermedia, our study revealed the presence of vimentin in glial-like cells located between glandular secretory cells of the three species and in the cells of the marginal layer of the cat and rat hypophyseal cleft. In the neural lobe of the cat and rabbit pituitary glands, there was a large amount of cell processes and immunoreactive pituicytes. In contrast, in the rat neural lobe, few pituicytes exhibited immunoreactivity, and these were located principally in the posterior region near the pituitary stalk. The significance of immunoreactive vimentin in these cells is discussed. 相似文献
18.
19.
Vimentin Dephosphorylation by Protein Phosphatase 2A Is Modulated by the Targeting Subunit B55 总被引:6,自引:0,他引:6
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Patric Turowski Timothy Myles Brian A. Hemmings Anne Fernandez Ned J. C. Lamb 《Molecular biology of the cell》1999,10(6):1997-2015
The intermediate filament protein vimentin is a major phosphoprotein in mammalian fibroblasts, and reversible phosphorylation plays a key role in its dynamic rearrangement. Selective inhibition of type 2A but not type 1 protein phosphatases led to hyperphosphorylation and concomitant disassembly of vimentin, characterized by a collapse into bundles around the nucleus. We have analyzed the potential role of one of the major protein phosphatase 2A (PP2A) regulatory subunits, B55, in vimentin dephosphorylation. In mammalian fibroblasts, B55 protein was distributed ubiquitously throughout the cytoplasm with a fraction associated to vimentin. Specific depletion of B55 in living cells by antisense B55 RNA was accompanied by disassembly and increased phosphorylation of vimentin, as when type 2A phosphatases were inhibited using okadaic acid. The presence of B55 was a prerequisite for PP2A to efficiently dephosphorylate vimentin in vitro or to induce filament reassembly in situ. Both biochemical fractionation and immunofluorescence analysis of detergent-extracted cells revealed that fractions of PP2Ac, PR65, and B55 were tightly associated with vimentin. Furthermore, vimentin-associated PP2A catalytic subunit was displaced in B55-depleted cells. Taken together these data show that, in mammalian fibroblasts, the intermediate filament protein vimentin is dephosphorylated by PP2A, an event targeted by B55. 相似文献
20.
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. 相似文献