Localization Of Ubiquitin Research Articles

Ubiquitination-coupled liquid phase separation regulates the accumulation of the TRIM family of ubiquitin ligases into cytoplasmic bodies.

Many members of the tripartite motif (TRIM) family of ubiquitin ligases localize in spherical, membrane-free structures collectively referred to as cytoplasmic bodies (CBs) in a concentration-dependent manner. These CBs may function as aggresome precursors or storage compartments that segregate potentially harmful excess TRIM molecules from the cytosolic milieu. However, the manner in which TRIM proteins accumulate into CBs is unclear. In the present study, using TRIM32, TRIM5α and TRIM63 as examples, we demonstrated that CBs are in a liquid droplet state, resulting from liquid-liquid phase separation (LLPS). This finding is based on criteria that defines phase-separated structures, such as recovery after photobleaching, sensitivity to hexanediol, and the ability to undergo fusion. CB droplets, which contain cyan fluorescent protein (CFP)-fused TRIM32, were purified from HEK293 cells using a fluorescence-activated cell sorter and analyzed by LC-MS/MS. We found that in addition to TRIM32, these droplets contain a variety of endogenous proteins and enzymes including ubiquitin. Localization of ubiquitin within CBs was further verified by fluorescence microscopy. We also found that the activation of the intracellular ubiquitination cascade promotes the assembly of TRIM32 molecules into CBs, whereas inhibition causes suppression. Regulation is dependent on the intrinsic E3 ligase activity of TRIM32. Similar regulation by ubiquitination on the TRIM assembly was also observed with TRIM5α and TRIM63. Our findings provide a novel mechanical basis for the organization of CBs that couples compartmentalization through LLPS with ubiquitination.

FBXO2/SCF ubiquitin ligase complex directs xenophagy through recognizing bacterial surface glycan.

Xenophagy, also known as antibacterial selective autophagy, degrades invading bacterial pathogens such as group A Streptococcus (GAS) to defend cells. Although invading bacteria are known to be marked with ubiquitin and selectively targeted by xenophagy, how ubiquitin ligases recognize invading bacteria is poorly understood. Here, we show that FBXO2, a glycoprotein-specific receptor for substrate in the SKP1/CUL1/F-box protein (SCF) ubiquitin ligase complex, mediates recognition of GlcNAc side chains of the GAS surface carbohydrate structure and promotes ubiquitin-mediated xenophagy against GAS. FBXO2 targets cytosolic GAS through its sugar-binding motif and GlcNAc expression on the GAS surface. FBXO2 knockout resulted in decreased ubiquitin accumulation on intracellular GAS and xenophagic degradation of bacteria. Furthermore, SCF components such as SKP1, CUL1, and ROC1 are required for ubiquitin-mediated xenophagy against GAS. Thus, SCFFBXO2 recognizes GlcNAc residues of GAS surface carbohydrates and functions in ubiquitination during xenophagy.

Vps13D functions in a Pink1-dependent and Parkin-independent mitophagy pathway.

Defects in autophagy cause problems in metabolism, development, and disease. The autophagic clearance of mitochondria, mitophagy, is impaired by the loss of Vps13D. Here, we discover that Vps13D regulates mitophagy in a pathway that depends on the core autophagy machinery by regulating Atg8a and ubiquitin localization. This process is Pink1 dependent, with loss of pink1 having similar autophagy and mitochondrial defects as loss of vps13d. The role of Pink1 has largely been studied in tandem with Park/Parkin, an E3 ubiquitin ligase that is widely considered to be crucial in Pink1-dependent mitophagy. Surprisingly, we find that loss of park does not exhibit the same autophagy and mitochondrial deficiencies as vps13d and pink1 mutant cells and contributes to mitochondrial clearance through a pathway that is parallel to vps13d. These findings provide a Park-independent pathway for Pink1-regulated mitophagy and help to explain how Vps13D regulates autophagy and mitochondrial morphology and contributes to neurodegenerative diseases.

Nuclear envelope budding is a response to cellular stress

Nuclear envelope budding (NEB) is a recently discovered alternative pathway for nucleocytoplasmic communication distinct from the movement of material through the nuclear pore complex. Through quantitative electron microscopy and tomography, we demonstrate how NEB is evolutionarily conserved from early protists to human cells. In the yeast Saccharomyces cerevisiae, NEB events occur with higher frequency during heat shock, upon exposure to arsenite or hydrogen peroxide, and when the proteasome is inhibited. Yeast cells treated with azetidine-2-carboxylic acid, a proline analog that induces protein misfolding, display the most dramatic increase in NEB, suggesting a causal link to protein quality control. This link was further supported by both localization of ubiquitin and Hsp104 to protein aggregates and NEB events, and the evolution of these structures during heat shock. We hypothesize that NEB is part of normal cellular physiology in a vast range of species and that in S. cerevisiae NEB comprises a stress response aiding the transport of protein aggregates across the nuclear envelope.

Age-dependent changes in the proteome following complete spinal cord transection in a postnatal South American opossum (Monodelphis domestica).

Recovery from severe spinal injury in adults is limited, compared to immature animals who demonstrate some capacity for repair. Using laboratory opossums (Monodelphis domestica), the aim was to compare proteomic responses to injury at two ages: one when there is axonal growth across the lesion and substantial behavioural recovery and one when no axonal growth occurs. Anaesthetized pups at postnatal day (P) 7 or P28 were subjected to complete transection of the spinal cord at thoracic level T10. Cords were collected 1 or 7 days after injury and from age-matched controls. Proteins were separated based on isoelectric point and subunit molecular weight; those whose expression levels changed following injury were identified by densitometry and analysed by mass spectrometry. Fifty-six unique proteins were identified as differentially regulated in response to spinal transection at both ages combined. More than 50% were cytoplasmic and 70% belonged to families of proteins with characteristic binding properties. Proteins were assigned to groups by biological function including regulation (40%), metabolism (26%), inflammation (19%) and structure (15%). More changes were detected at one than seven days after injury at both ages. Seven identified proteins: 14-3-3 epsilon, 14-3-3 gamma, cofilin, alpha enolase, heart fatty acid binding protein (FABP3), brain fatty acid binding protein (FABP7) and ubiquitin demonstrated age-related differential expression and were analysed by qRT-PCR. Changes in mRNA levels for FABP3 at P7+1day and ubiquitin at P28+1day were statistically significant. Immunocytochemical staining showed differences in ubiquitin localization in younger compared to older cords and an increase in oligodendrocyte and neuroglia immunostaining following injury at P28. Western blot analysis supported proteomic results for ubiquitin and 14-3-3 proteins. Data obtained at the two ages demonstrated changes in response to injury, compared to controls, that were different for different functional protein classes. Some may provide targets for novel drug or gene therapies.

HSP70 and p62/SQSTM 1 regulate differently autophagy clearance in ARPE‐19 cells

Abstract Purpose Prior to proteolysis, heat shock proteins (HSPs) attempt to refold misfolded proteins. If this process is not successful proteins are degradated in proteasomes or in lysosomes. In the present study, the roles of the HSP70 and the p62/SQSTM 1 in autophagy clearance were evaluated in ARPE‐19 cells after proteasome inhibitor treatment. Methods The HSP70, p62/SQSTM 1 and ubiquitin localization were analyzed by immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to analyze the morphological changes. HSP70 and p62/SQSTM 1 levels were modulated using RNA interference techniques. Cell viability was measured by colorimetric assay. Results The proteasome inhibitor MG‐132 evoked the accumulation of perinuclear aggregates positive for HSP70, p62/SQSTM 1 and ubiquitin‐protein conjugates. We observed that the aggregation was reversible: a cessation of proteasome inhibition led to clearance of the deposits via autophagy. Interestingly, p62/SQSTM 1 mRNA depletion delayed autophagy clearance and significantly increased cell death in conjunction with proteasome inhibition. The hsp70 RNA interference did not change autophagy clearance, but increased cell death in response to proteasome inhibition. Conclusion The HSP70 and p62/SQSTM 1 regulate differently autophagy clearance in RPE cells. Autophagy seems to be an important mechanism to clean proteasome inhibitor –induced protein aggregates.

Ontogeny in terminal buds of Abies nordmanniana (Pinaceae) characterized by ubiquitin

Meristematic activity in the bud meristem of Abies nordmanniana was visualized by ubiquitin immunohistochemical localization from before bud break and throughout shoot expansion. Ubiquitin was detected in meristematic cells either in the cytosol or nucleus, or both, depending on tissue type and developmental stage. During winter dormancy, ubiquitin was only observed in the protodermal/hypodermal layers, but at bud break in mid May, the signal expanded to the entire shoot tip. At the end of May, a clear zonation in ubiquitin localization appeared that lasted about one month. Throughout this period, ubiquitin was barely detectable in a central group of cells that might indicate an organizing center with stem cells. At the end of June, coinciding with the transition from scale leaf to needle primordia production, ubiquitin again was more prevalent in the peripheral cell layers. During shoot expansion, a strong ubiquitin signal developed in the axil of all needles. Most of these signals later disappeared, except for those few axils where buds actually developed. A strong ubiquitin signal was also observed in cells lining the young resin ducts. Our data showed that ubiquitin may be used as a marker for metabolic activity associated with seasonal development in the apical meristem.

Ubiquitin Signals in the Developing Acrosome during Spermatogenesis of Rat Testis: An Immunoelectron Microscopic Study

The localization of ubiquitin (UB) signals in the acrosomes of rat spermiogenic cells was investigated by immunoelectron microscopy using two anti-UB antibodies: UB1, reacting with ubiquitinated proteins and free UB; and FK1, recognizing polyubiquitinated proteins but not monoubiquitinated proteins or free UB. Labeling of UB by UB1 (UB1 signal) was detected in the acrosomes at any stage of differentiation. In step 1 spermatids, UB1 signals were detected on the cytoplasmic surface and in the matrix of transport vesicles located between the trans-Golgi network and the acrosome. Weak signals were detected in acrosomal granules within acrosome vesicles that had not yet attached to the nucleus. In step 4-5 spermatids, the acrosome vesicles had enlarged and attached to the nucleus. Strong gold labeling was noted in a narrow space between the outer acrosomal membrane and the developing acrosomal granule, where a dense fibrous material was observed on routine electron microscopy, whereas the acrosomal granule was weakly stained by UB1 antibody. In step 6-8 spermatids, UB1 signals were detected in the fibrous material that expanded laterally to form a narrow electronless dense zone between the acrosomal granule and the outer acrosomal membrane. Labeling in the acrosomal granule increased. In step 9-11 spermatids, UB1 signals were confined to the narrow zone from the tip of the head to the periphery of the ventral fin. The matrix of the acrosome was weakly stained. In epididymal sperm, UB1 labeling in the acrosome decreased without any pretreatment, whereas staining was noted in a spot in the neck region and in the dorsal fin after trypsin digestion. On the other hand, the staining pattern with FK1 was quite different from that with UB1. The trans-Golgi network was weakly stained but the cis-Golgi network was strongly stained. The dense fibrous material just beneath the outer membrane was never stained with FK1. The results suggest that UB on the surface of transport vesicles is involved in anterograde transport from the Golgi apparatus to the acrosome. The physiological role of UB in acrosomes is not clear. Two candidates for monoubiquitinated proteins in the acrosome, which have a UB-interacting motif, were found by cyber screening.

Immunocytochemical localization of ubiquitin and proteasomes in spermatids during spermiogenesis of Chara vulgaris (Charophyceae)

Immunocytochemistry with anti-ubiquitin and anti-proteasome antibodies revealed antigenic signals in spermatids of Chara vulgaris in all ten spermiogenesis phases defined on the basis of morphological features. The strongest antigenic signals, indicating the presence of both ubiquitin and proteasome, were observed mainly in the cytoplasm in early and mid-spermiogenesis (phases II – V). During the transition of histone into protamine-type proteins, stronger anti-ubiquitin and anti-proteasome antigenic signals were present in the nucleus than in the cytoplasm (phases VII – VIII). In late spermiogenesis both types of signals were very scarce. We hypothesiZe that the roles of the proteasome/ubiquitin system may be: (1) participation in the degradation of short-lived proteins that appear temporarily after gene expression is triggered and initiates the programme of major ultrastructural changes in spermatids in early spermiogenesis; and (2) participation in the transition of histones into protamine-type proteins during late spermiogenesis, and in the remodelling of chromatin into extremely compact forms characteristic of generative cells.

Ubiquitin Activity Following Forebrain Ischemia/Reperfusion in the Rat

Domorakova I., E. Mechirova, J. Burda, M. Ferikova: Ubiquitin Activity Following Forebrain Ischemia/Reperfusion in the Rat. Acta Vet. Brno 2004, 73: 45-49. Cerebral ischemia/reperfusion leads to selective neuronal death in specific brain areas. The effects of forebrain ischemia produced by four-vessel occlussion were investigated in rats. Immunohistochemical localization of ubiquitin in the brain cortex and hippocampus was studied. Using antibodies against ubiquitin we have found that ubiquitin immunoreactivity (UIR) was normally present in all neurons of hippocampus and in the cerebral cortex. After 20 min of ischemia and 24 h of reperfusion the number of ubiquitin positive cells was decreased in CA2 and CA3 regions of hippocampus and ubiquitin-negative neurons were found in the most vulnerable region of CA1 subfield. Cerebral cortex showed significant changes of ubiquitin immunoreactivity in the nuclei and perikarya of pyramidal neurons. Seven days of reperfusion after 20 min of ischemia showed recovery of UIR in the hippocampal CA2, CA3 regions and in granule cells layer of the dentate gyrus. The most vulnerable CA1 pyramidal cells showed morphological changes of the cell bodies and irregularities of the cell membrane. Interestingly, number of those cells display intensive UIR in their perikarya and in the nuclei as well . Comparison of UIR in CA1 pyramidal cells after ischemia/reperfusion with those in the control sections showed increase in ubiquitin reaction. Remarkable UIR was also found in all layers in the cerebral cortex. These data suggest that recovery of ubiquitin immunoreactivity in some regions of hippocampus and cerebral cortex may be a prerequisite to neuronal survival after ischemia /reperfusion. In CA1 region the accumulation of ubiquitinated proteins in pyramidal cells was visualized without their degradation. The stress protein ubiquitin is one of the regulatory proteins playing a role in ischemic tolerance. Ischemia, hippocampus, cerebral cortex, ubiquitin, rat Specific neuronal populations in the brain are selectively vulnerable to ischemic insults. Ischemic cell death is characterized by a delay between the insult and manifestation of major cell damage. This delay varies greatly, depending on the nature of the insult and of the brain region being affected (Lipton 1999). Two different types of cell death can be discerned. One type progresses rapidly over a period of hours whereas the second type requires several days to mature (Kirino 1982). The hippocampus is a brain structure displaying both these features. Following transient periods of cerebral ischemia in the rat a rapid degeneration of the neurons in the dentate gyrus takes place. The CA1 neurons, on the other hand, show light microscopical signs of degeneration during the first days following the insult, demonstrating delayed neuronal death (Kirino 1982; Pulsinelli and Brierley 1982). Although total protein synthesis of the brain during ischemia/reperfusion is severely inhibited ( Araki et al. 1990), synthesis of specific proteins, such as heat shock proteins (HSP70) is induced. Protective role of HSP in ischemic tolerance has been reported by Kirino et al. (1991) and Liu et al. (1992). Ubiquitin, a low molecular weight heat shock protein found in all eukaryotic cells, is bound to short-lived and denatured proteins produced by various forms of injury. Ubiquitin

Increased ubiquitin immunoreactivity in unstable atherosclerotic plaques associated with acute coronary syndromes

ObjectivesThe current study was designed to examine whether ubiquitin expression is higher in unstable than in stable lesions of patients with acute coronary syndrome (ACS). BackgroundThe ubiquitin system has been identified as the nonlysosomal pathway of protein degradation; it is involved in a number of biologic processes crucial to cell and tissue integrity and therefore, might be potentially involved in the rupture of unstable coronary plaques. MethodsWe conducted an autopsy-based study of 25 consecutive patients with fatal ACS. Lesions of both infarct-related and noninfarct-related segments from the same patients were examined for the expression and localization of ubiquitin by use of immunohistochemistry and a semiquantitative grading scale. ResultsUbiquitin immunoreactivity was higher in infarct-related than in noninfarct-related lesions (1.4 ± 0.5 vs. 1.1 ± 0.6, p = 0.03). Compared with areas adjacent to the plaque (0.6 ± 0.7), ubiquitin immunoreactivity was higher in areas around the lipid core (2.5 ± 0.8, p < 0.001), plaque shoulders (1.6 ± 1.1, p < 0.001), and fibrous cap regions (1.6 ± 1.1, p < 0.001). Within the plaque area, co-localization of ubiquitin immunoreactivity with T cells and macrophages was found. In areas adjacent to the plaque, ubiquitin immunoreactivity co-localized with neointima cells and media smooth muscle cells. ConclusionsIn patients with ACS, ubiquitin immunoreactivity is enhanced in unstable, infarct-related lesions, predominantly in plaque regions of tissue degradation. Based on these findings, this study suggests a role for the ubiquitin system in the destabilization and rupture of coronary atherosclerotic plaques in humans.

Immunohistochemical localisation of ubiquitin and the proteasome in sunflower (Helianthus annuus cv. Giganteus).

This study provides an immunohistochemical demonstration of the involvement of the ubiquitin- and proteasome-dependent pathway during differentiation and organogenesis in plants. The localisation of ubiquitin and the proteasome was studied in meristems, leaves, stems and roots of sunflower (Helianthus annuus L. cv. Giganteus). By using a new technique that enhances very low antigen signals, we obtained information on the structural distribution of the ubiquitin- and proteasome-dependent pathway, and of the importance of this pathway during organogenesis and plant development. Ubiquitin and the proteasome showed overall similarities in their cellular localisation. The highest antigenic signal was observed in the root and shoot apical meristems, in leaf primordia and vascular tissue. The cambium showed less expression than the apical meristems. During adventitious root formation in cuttings, no sign of increased expression was observed within dedifferentiating tissue, but as organogenesis progressed, the antigenic signal of ubiquitin and the proteasome gradually increased in the developing roots. Comparison of immunochemical results and Western blots demonstrated that important changes in the cellular antigen signal could only be detected by immunochemistry.

Dual enhancement of double immunofluorescent signals by CARD: participation of ubiquitin during formation of neurofibrillary tangles.

Amplification with catalyzed reporter deposition (CARD) greatly enhances peroxidase signals, which has been utilized to amplify immunohistochemical labelings including fluorochromes. Here we describe a strategy to amplify each of two immunofluorescent signals without crosstalk on double-stained histological sections from human autopsied brains with Alzheimer's disease (AD). One of the two primary antibodies (anti-Abeta or anti-PHF-tau) was probed by a species-specific secondary antibody conjugated with horseradish peroxidase (HRP), which was visualized by FITC-labeled tyramide. After inactivation of HRP, the other primary antibody was probed by another species-specific secondary antibody conjugated with HRP. Amplification with biotinylated tyramide was followed by streptavidin-conjugated Cy-5, which specifically labeled the latter epitope. It was found that Abeta and PHF-tau were localized to senile plaques and neurofibrillary tangles (NFTs), respectively, which verified lack of crosstalk on the double-stained section. Localization of ubiquitin and PHF-tau was looked for at higher magnification in NFT-bearing neurons. Although these two epitopes were colocalized in some neurons, ubiquitin was not always present in PHF-tau positive NFTs. Discrepancy between PFH-tau and ubiquitin, verified inter- and intracellularly, may represent different stages of NFT formation. This is the first report of successful CARD amplification of two different fluorescent signals on double-labeling immunohistochemistry, which is now proved to be powerful in detecting epitopes in relation to AD-related lesions. Improved intensity over tenfold of the two fluorescent signals without crosstalk will expand the application of the multilabeling method with fluorochromes.

Localization of ubiquitin and ubiquitin cross-reactive protein in human and baboon endometrium and decidua during the menstrual cycle and early pregnancy.

We have examined the distribution of ubiquitin and the related ubiquitin cross-reactive protein (UCRP) in paraffin-embedded sections of human and baboon endometrium and decidua by immunoperoxidase or immunofluorescence cytochemistry with antibodies raised against ubiquitin, UCRP, CD45, and insulin-like growth factor-binding protein-1. Anti-ubiquitin immunoreactivity was present in the nonpregnant endometrium, particularly in the glandular epithelial cells, and up-regulated in endometrial stromal cells as they decidualized at the beginning of pregnancy. Anti-UCRP immunoreactivity was absent from nonpregnant tissue but accumulated to high levels in decidual cells during pregnancy. Western blotting indicated that immunoreactivity was primarily due to the presence of ubiquitin and UCRP conjugated to other proteins, and that although levels of ubiquitin-protein conjugates do not change substantially during pregnancy, decidualization is accompanied by the appearance of conjugates of UCRP. Baboon uterine tissues demonstrated a similar distribution of the two proteins, which indicates that the baboon may be a useful model for study of the role of the ubiquitin system and UCRP in the establishment of pregnancy in humans.

Subcellular localization of ubiquitin in plant protoplasts and the function of ubiquitin in selective degradation of outer-wall plasmodesmata in regenerating protoplasts

Immunocytochemical localizations in Vicia faba L. protoplasts and cultures of regenerating Solanum nigrum L. protoplasts support former observations that in plant cells ubiquitin occurs within the cytoplasm, the nucleus, the chloroplasts and at the plasmalemma, but not within the vacuole or the cell wall. Immunoresponses were also observed within mitochondria and associated with the endoplasmic reticulum, which is in accordance with previous findings on animal cells. Moreover, the tonoplast membrane system was found to be labelled. For regenerating S. nigrum protoplasts, evidence is given that ubiquitin plays a role in selective degradation even of whole subcellular structures. Most of the discontinuous plasmodesmata formed in the newly deposited outer cell walls during the early stages of culture disappear later on, except for those near the periphery of division walls or of non-division walls, which are probably used for the formation of continuous cell connections during further culture. Outer-wall plasmodesmata which are destined to disappear show high immunoreactivity to ubiquitin antibody, but no conspicuous immunolabelling was observed with the remaining plasmodesmata. Thus, the selective disintegration of whole plasmodesmatal structures is obviously regulated by ubiquitination of plasmodesmatal proteins. A model for the mechanism of degradation of outer-wall plasmodesmata during extension growth of the cell wall is presented.

Localization of ubiquitin to differentiating vascular tissues

The selective degradation of proteins, an essential process of any developmental program, may entail conjugation of the protein to be destroyed to the polypeptide ubiquitin. Experiments were designed to localize ubiquitin as a first step in determining whether this molecule is crucial for certain developmental processes in plant tissues and cells. Antibodies to ubiquitinated protein were detected on tissue prints of cross sections of bean petioles (Phaseolus vulgaris, Fabaceae), cotton hypocotyls (Gossypium hirsutum, Malvaceae), and Coleus stems (Coleus x hybridus, Lamiaceae). For most of the material investigated, there appears to be an accumulation of ubiquitin antibodies in vascular tissues, but not preferentially in the abscission zone of bean petioles. Vascular localization was confirmed using immunohistochemical methods on fixed and sectioned internodal tissues of Coleus. Antibodies to ubiquitin are detected in parenchyma cells of the cortex and pith, but are most concentrated in the xylem, especially secondary xylem, and in the cambial region, and in the phloem. Thus, ubiquitin accumulates in certain vascular tissues, some of which may be undergoing programmed cell death. Ubiquitin can also be detected in nondifferentiating cells, and its level is elevated in rapidly dividing cambial cells.

Localization of ubiquitin in anthers and pistils of Nicotiana

Ubiquitin-conjugated compounds were localized in anthers and pistils of Nicotiana alata by immuno-cytochemistry. In young anthers, antibodies to ubiquitin bound to callose cell walls surrounding pollen mother cells and to organelles in the endothecium. At the freespore stage, antibodies bound to circular-cell cluster cells subtending the stomium and to organelles and cell walls of endothecial cells. Near anther dehiscence, locular material was labeled. In pistils, cell walls of stylar transmitting tissue were labeled in a beaded pattern. Antibodies bound to a thin layer surrounding ovules, to the lining of embryo sacs, to cytoplasm of eggs and synergids, and to starch grains in central cells. Sites of localization were tissue- and time-specific, suggesting a regulatory role for ubiquitin in development of reproductive structures in flowering plants.

P-338 - The relationship between catalase activity and ubiquitin localization in gingiva of acatalasemic canine

P-338 - The relationship between catalase activity and ubiquitin localization in gingiva of acatalasemic canine

Immunoelectron microscopy of Rosenthal fibers.

Seventeen intracerebral gliomas containing Rosenthal fibers (RF) were studied by an immunoperoxidase method for localization of ubiquitin (UB), glial fibrillary acidic protein (GFAP), desmin and vimentin (VIM). The majority of RF showed an immunohistochemically negative core surrounded by a ring of overlapping reactions for UB, GFAP and VIM. Many RF were entirely negative for UB and intermediate filaments (IF). Immunoelectron microscopic localization of UB and GFAP was performed on seven selected tumors. UB was found in all RF and on IF in the proximity of RF. GFAP reaction was localized on astrocytic IF, including those trapped within RF, and within the granular component of some RF. In contrast to the light microscopic studies, neither GFAP- nor UB-negative RF were found on immunoelectron microscopy. VIM reaction on IF and a few RF was demonstrated in one tumor processed at low temperature into Lowicryl; it was much weaker than that for GFAP. Many cells with RF contained lysosome-like inclusions with material displaying electron density similar to adjacent RF; few of these inclusions were reactive for UB. It is concluded that RF formation is associated with ubiquitination of astrocytic IF. GFAP- and VIM-immunoreactive IF and products of their disintegration contribute to RF material. It is also suggested that the lysosomal system of astrocytes partially degrades RF.