Immunocytochemical Detection of Structural and Regulatory Proteins in Rat Adrenal Nuclear Matrix

The nuclear matrix is a specific cell structure consisting of a residual nucleoskeleton that extends from the nucleoli to the nuclear envelope. The nuclear matrix of steroido-genic cells was isolated previously from a purified nuclear fraction. We present here an in situ extraction method, modified Lutz's method, for rat glandular adrenal cell nuclear matrix. This residual organelle was characterized and studied using immunocytochemical methods. The adrenal glands were removed, the cells prepared in suspension and deposited by cytospin onto Poly-L-lysine glass slides. The nuclear matrix was extracted with Nonidet P-40, DNase I and high and low ionic strength buffers. Structural proteins, nuclear lamins, coilin and fibrillarin were detected immunocytochemically. The adrenal fasciculata cells were easily identified by this method because of their large nuclei and abundant lipid droplets in the cytoplasm. After immunocytochemical detection by antibodies against lamins A and C, a marked brown layer at the periphery of the nucleus was observed. The intensity of the staining was lower using the antibody against nuclear lamin B. Immunocytochemical detection of the protein coilin revealed punctuated stained areas, 2-6 per nucleus, that probably correspond to the coiled bodies. The protein fibrillarin was detected at the nucleolus and coiled bodies. Our technique is simple, reveals well preserved adrenal nuclear matrices, and may be a useful method for immunocytochemical analysis and in situ hybridization.     

Process Formation in Astrocytes: Modulation of Cytoskeletal Proteins

Studies on primary astrocytes cultured in vitro have shown that process formation involves changes in cytoskeletal proteins and release of tension on the substratum. Actin filament reorganization has previously been found to be the major cytoskeletal change occurring during process formation. These changes are relatively rapid with breakdown of the actin web and release of contacts occur within 15 min. of cyclic AMP treatment. The former is regulated by myosin light chain (MLC) and actin depolymerizing factor (ADF), with MLC involved in the initial release of contractile tension and ADF in both initial and longer term actin breakdown. Our results show that the dephosphorylation of MLC is due to the phosphorylation and inactivation of myosin light chain kinase (MLCK) in response to cyclic AMP. To further study the mechanisms underlying the process formation in astrocytes we used endothelin-1 (ET-1), a vasopeptide which has been shown to inhibit process formation in astrocytes and sodium fluoride which is a general phosphatase inhibitor. We observe an increase in phosphorylation of MLC on inhibition of process formation. To study the role of adhesion in process formation we used suspension cultures of astrocytes. Our results with the astrocytes in suspension suggest that the process formation in astrocytes is adhesion dependent and the changes in ADF and MLC occur only when there is process formation.     

Androgen Regulates Gene Expression of Cytoskeletal Proteins in Adult Rat Motoneurons

Expression of β-actin and β-tubulin mRNA was examined in androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in adult male rats by in situ hybridization histochemistry using complementary DNAs encoding chick β-actin and mouse β-tubulin, respectively. Both hybridizable β-actin and βtubulin mRNAs were localized in the somata and proximal dendrites of SNB motoneurons. Removal of androgen by castration significantly reduced the expression levels of both β-actin and β-tubulin mRNAs in the SNB motoneurons, whereas the changes were prevented by testosterone treatment. In contrast, castration or testosterone treatment induced little or no change in the expression levels of these mRNAs in the much less androgen-sensitive motoneurons of the retrodorsolateral nucleus (RDLN). These results suggest that androgen regulates the expression of β-actin and β-tubulin genes in the SNB motoneurons and may provide evidence for the molecular mechanisms of hormonally induced neuronal plasticity in the SNB motoneurons.     

The Changes of Cytoskeletal Proteins in Plasma of Acrylamide-Induced Rats

Acrylamide (ACR) is a known industrial neurotoxic chemical that can induce neurodegeneration. Cytoskeletal protein aggregation is a pathological hallmark of neurodegenerative disorders. This study was an initial exploration on cytoskeletal proteins in plasma as potential biomarkers of ACR neurotoxicity. Low and high ACR groups received 20 mg/kg and 40 mg/kg ACR by intraperitoneal injection in adult Wistar rats and control group received physiological saline. Rats were all killed after 8 weeks to evaluate the levels of neurofilament(NF)-L, NF-M, NF-H, β-actin, α-tubulin, β-tubulin, tau, MAP₂ proteins in plasma using both SDS-PAGE and western blotting. Compared with the control, the levels of NF-L, NF-M, NF-H, β-actin, tau, MAP₂ proteins decreased and the level of α-tubulin increased in high ACR group, the levels of α-tubulin, β-tubulin and MAP₂ increased in low ACR group. The results suggested that the changes of these proteins might be relevant to the neurotoxicity of ACR. Some of the cytoskeletal proteins in plasma might be used as marker of biological effect in ACR induced neuropathy.     

Changes in Organization and Axonal Transport of Cytoskeletal Proteins During Regeneration

Changes in solubility and transport rate of cytoskeletal proteins during regeneration were studied in the motor fibers of the rat sciatic nerve. Nerves were injured by freezing at the midthigh level either 1-2 weeks before (experiment I) or 1 week after radioactive labeling of the spinal cord with L-[35S]methionine (experiment II). Labeled proteins in 6-mm consecutive segments of the nerve 2 weeks after labeling were analyzed following fractionation into soluble and insoluble populations with 1% Triton at 4 degrees C. When axonal transport of newly synthesized cytoskeleton was examined in the regenerating nerve in experiment I, a new faster component enriched in soluble tubulin and actin was observed that was not present in the control nerve. The rate of the slower main component containing most of the insoluble tubulin and actin together with neurofilament proteins was not affected. A smaller but significant peak of radioactivity enriched in soluble tubulin and actin was also detected ahead of the main peak when the response of the preexisting cytoskeleton was examined in experiment II. It is thus concluded that during regeneration changes in the organization take place in both the newly synthesized and the preexisting axonal cytoskeleton, resulting in a selective acceleration in rate of transport of soluble tubulin and actin.     

Time-dependent Alteration of Cytoskeletal Proteins in Cerebral Cortex of Rat During 2,5-Hexanedione-induced Neuropathy

To investigate the mechanisms of the axonopathy induced by 2,5-hexanedione (2,5-HD), male Wistar rats were administered at a dosage of 400 mg/kg/day 2,5-HD (five times per week). The rats produced a slightly, moderately, or severely abnormal neurological changes, respectively, after 2, 4, or 8 weeks of treatment. The cerebrums were Triton-extracted and ultracentrifuged to yield a pellet fraction and a corresponding supernatant fraction. The relative levels of six cytoskeletal proteins (NF-L, NF-M, NF-H, α-tubulin, β-tubulin, and β-actin) in both fractions were determined by immunoblotting. The results showed that NFs content in HD-treated rats demonstrated a progressive decline as the intoxication of HD continued. As for microtubule proteins, the levels of α-tubulin and β-tubulin demonstrated some inconsistent changes. The content of α-tubulin kept unchangeable, while the content of β-tubulin increased significantly at the late stage of HD exposure. Furthermore, the content of β-actin in both fractions remained unaffected throughout the study. These findings suggest that HD intoxication resulted in a progressive decline of NFs, which was highly correlated with the development of HD-induced neuropathy.     

Localization of Human Cytomegalovirus Structural Proteins to the Nuclear Matrix of Infected Human Fibroblasts

The intranuclear assembly of herpesvirus subviral particles remains an incompletely understood process. Previous studies have described the nuclear localization of capsid and tegument proteins as well as intranuclear tegumentation of capsid-like particles. The temporally and spatially regulated replication of viral DNA suggests that assembly may also be regulated by compartmentalization of structural proteins. We have investigated the intranuclear location of several structural and nonstructural proteins of human cytomegalovirus (HCMV). Tegument components including pp65 (ppUL83) and ppUL69 and capsid components including the major capsid protein (pUL86) and the small capsid protein (pUL48/49) were retained within the nuclear matrix (NM), whereas the immediate-early regulatory proteins IE-1 and IE-2 were present in the soluble nuclear fraction. The association of pp65 with the NM resisted washes with 1 M guanidine hydrochloride, and direct binding to the NM could be demonstrated by far-Western blotting. Furthermore, pp65 exhibited accumulation along the nuclear periphery and in far-Western analysis bound to proteins which comigrated with proteins of the size of nuclear lamins. A direct interaction between pp65 and lamins was demonstrated by coprecipitation of lamins in immune complexes containing pp65. Together, our findings provide evidence that major virion structural proteins localized to a nuclear compartment, the NM, during permissive infection of human fibroblasts.     

Location and Distribution of Non-collagenous Matrix Proteins in Musculoskeletal Tissues of Rat

The study assessed immunohistochemically the location and distribution of various non-collagenous matrix proteins (fibronectin, laminin, tenascin-C, osteocalcin, thrombospondin-1, vitronectin and undulin) in musculoskeletal tissues of rat. Fibronectin and thrombospondin-1 were found to be ubiquitous in the studied tissues. High immunoreactivity of these proteins was found in the extracellular matrix of the anatomical sites where firm bindings are needed, i.e. between muscle fibres and fibre bundles, between the collagen fibres of a tendon and at myotendinous junctions, osteotendinous junctions and articular cartilage. Tenascin-C was found in the extracellular matrix of regions where especially high forces are transmitted from one tissue component to the other, such as myotendinous junctions and osteotendinous junctions. Laminin was demonstrated in the basement membranes of the muscle cells and capillaries of the muscle–tendon units. Osteocalc in immunoreactivity concentrated in the extracellular matrix of areas of newly formed bone tissue, i.e. in the subperiosteal and subchondral regions, osteoid tissue and mineralized fibrocartilage zone of the osteotendinous junction. Mild vitronectin activity could be seen in the extracellular matrix of the osteotendinous and myotendinous junctions, and high activity around the bone marrow cells. Undulin could be demonstrated in the extracellular matrix (i.e. on the collagen fibres) of the tendon and epimysium only. However, it was co-distributed with fibronectin and tenascin-C. Together, these findings on the normal location and distribution of these non-collagenous proteins in the musculoskeletal tissues help to form the basis of knowledge against which the location and distribution of the these proteins in various pathological processes could be compared.     

Purmorphamine Promotes Matrix Mineralization and Cytoskeletal Changes in Human Umbilical Cord Mesenchymal Stem Cells

Human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs) were subjected to in vitro osteogenic differentiation using a novel combination of signaling molecules including BMP-2 and purmorphamine. Differentiation outcomes were assessed by calcein staining and by microscopic examination of the cytoskeleton. Calcein staining showed appreciable degree of calcium mineralization in cell culture, and changes in the morphological attributes of differentiating cells were observed vis-a-vis the actin cytoskeleton. Finally, positive calcein staining, altered cytoskeletal profile, and stress fiber formation in treated cells demonstrated, for the first time, a potentially synergistic interplay between BMP-2 and the hedgehog agonist, purmorphamine. This study lends support to the notion of combining small doses of potent molecules that can act as safe, less toxic inducers of osteogenic differentiation of human umbilical cord mesenchymal stem cells with respect to bone regeneration.     

Association of Nuclear Matrix Proteins with Granular and Threaded Nuclear Bodies in Cell Lines Undergoing Apoptosis

The granules which appear in the nucleolar area in apoptotic HL-60 cells after camptothecin administration (Zweyeret al., Exp. Cell Res.221, 27–40, 1995) were detected also in several other cell lines induced to undergo apoptosis by different stimuli, such as MOLT-4 treated with staurosporine, K-562 incubated with actinomycin D, P-815 exposed to temperature causing heat shock, Jurkat cells treated with EGTA, U-937 growing in the presence of cycloheximide and tumor necrosis factor-α, and HeLa cells treated with etoposide. Using immunoelectron microscopy techniques, we demonstrate that, besides the already described nuclear matrix proteins p125 and p160, these granules contain other nucleoskeletal polypeptides such as proliferating cell nuclear antigen, a component of ribonucleoprotein particles, a 105-kDa constituent of nuclear spliceosomes, and the 240-kDa nuclear mitotic apparatus-associated protein referred to as NuMA. Moreover, we also found in the granules SAF-A/hn-RNP-U and SATB1 proteins, two polypeptides that have been reported to bind scaffold-associated regions DNA sequencesin vitro,thus mediating the formation of looped DNA structuresin vivo.Fibrillarin and coilin are not present in these granules or the PML protein. Thus, the granules seen during the apoptotic process apparently are different from coiled bodies or other types of nuclear bodies. Furthermore, these granules do not contain chromatin components such as histones and DNA. Last, Western blotting analysis revealed that nuclear matrix proteins present in the granules are not proteolytically degraded except for the NuMA polypeptide. We propose that these granules might represent aggregates of nuclear matrix proteins forming during the apoptotic process. Moreover, since the granules are present in several cell lines undergoing apoptosis, they could be considered a previously unrecognized morphological hallmark of the apoptotic process.     

Age-Related Changes of Myelin Proteins in the Rat Peripheral Nervous System

Age-related changes in amounts of myelin proteins from rat sciatic nerve or spinal root were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). In the aged peripheral nerve myelin, the relative amounts of band 105K and proteins X and Y increased, whereas those of proteins P0 and P1 and band 190K decreased. Band 105K purified by preparative SDS-PAGE exhibited three bands of 105K, 28K, and 21K at the second electrophoresis. A repeated SDS-PAGE did not improve the purity of bank 105K, but increased the ratio of 21K to 28K. Compared with P0 protein, band 105K has a very similar peptide map pattern and amino acid composition, as well as the identical NH2 terminal residue, isoleucine. These findings suggest that band 105K is an aggregate form of P0 protein and its fragment, 21K. The 21K protein is a distinct entity from X protein. The quantitative and qualitative alterations in myelin proteins, as we report here, may reflect continuing demyelination and remyelination in aged peripheral nerves.     

Wistar Furth Rat Megakaryocytes Lack Dense Compartments and Intercellular Plaques,Membranous Structures Rich in Cytoskeletal Proteins

Wistar Furth (WF) rats have an abnormal thrombopoietic phenotype with morphologically aberrant megakaryocytes, larger than normal mean platelet volume, and platelet alpha-granule protein deficiency. Here, ultrastructural comparisons of WF rat megakaryocytes to those of rats (Wistar) with normal platelet formation during stimulated megakaryocytopoiesis following 5-fluorouracil administration, have revealed a previously unrecognized membrane structure in normal rat megakaryocytes, and two additional abnormalities in WF megakaryocytes. The novel structures were zones of electron density on the cytoplasmic face of apposed plasma membranes of adjoining normal megakaryocytes. These modified focal adhesion-type contacts were distributed at intervals between adjacent megakaryocytes, and were spaced by deposits of extracellular material. These structures also were present between apposed plasma membranes of Wistar rat megakaryocytes in unperturbed marrows, but were absent between megakaryocytes of WF rats. The second WF rat megakaryocyte abnormality is the absence of cytoplasmic dense compartments, another specialized membranous structure that is continuous with the megakaryocyte demarcation membrane system. Both the intercellular plaques and dense compartments of Wistar rat megakaryocytes were found to be rich in cytoskeletal proteins including actin, α-actinin, talin, and vinculin as indicated by ultrastructural immunogold labeling. We hypothesize that an abnormality in cytoskeletal protein function may be responsible for the lack of these structures in the WF rat.     

Expression of Cytoskeletal Proteins in Neurons of the Rat Sensorimotor Cortex upon Hypoperfusion of the Brain and Sensitization by Cerebral Antigen

Neurophysiology - Relatively mild disturbances in the blood supply of the rat brain provided by ligation of one common carotid artery leading to relatively mild disturbances of the cerebral blood...     

Identification of Nuclear Proteins that Are Developmentally Regulated in Embryonic Rat Brain

Abstract: To identify nuclear proteins that might play a role in the acquisition of neuronal phenotype, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to analyze nuclear proteins expressed over the course of embryonic rat brain development. Metabolically labeled rat brain nuclear proteins from embryonic day 14 (E14) were compared with proteins from embryonic day 20 (E20). Over this period, the rat brain develops from a collection of relatively homogeneous precursor cells into a complex structure containing many different classes of neurons. Computer-assisted analysis of 2D-PAGE fluorograms identified 11 proteins that show increases in their rate of synthesis between E14 and E20. Twenty proteins that consistently appear at E20 are not detectable on fluorograms of E14 nuclear proteins, even after long exposures, and thus may be considered to appear de novo. Fifty-eight proteins show consistent down-regulation between E14 and E20, and of these, 19 are not detectable on fluorograms of E20 nuclear proteins. The electrophoretic properties of many of these proteins suggest that they are previously unreported, developmentally regulated nuclear proteins. Some of the developmentally regulated, brain-enriched nuclear proteins identified here may play a role in regulating the expression of neural genes important for cellular differentiation in the mammalian CNS.     

Synthesis and Turnover of Cytoskeletal Proteins in Cultured Astrocytes

Abstract: We previously reported that the cytoskeleton of rat astrocytes in primary culture contains vimentin, glial fibrillary acidic protein (GFAP), and actin. These proteins were found in a fraction insoluble in Triton X-100 and thought to be assembled in filamentous structures. We now used primary astrocyte cultures to study the kinetics of synthesis and turnover of these cytoskeletal proteins. The intermediate filament proteins were among the most actively synthesized by astrocytes. High levels of synthesis were detectable by the third day of culture in the early log phase of growth, and the pattern of labeling at day 3 was similar to that at 14 days when the cultures had reached confluency. In short-term incorporation experiments vimentin, GFAP, and actin in the Triton-insoluble fraction were labeled within 5 min after exposure of the cultures to radioactive leucine. We did not detect any saturation of labeling for up to 6 h of incubation. The turnover of filament proteins studied by following the decay of radioactivity from prelabeled vimentin, GFAP, and cytoskeletal actin displayed biphasic decay kinetics for all three proteins. In the initial phase a fast-decaying pool with a half-life of 12–18 h contributed about 40% of the total activity in each protein. A major portion, about 60%, of each protein, however, decayed much more slowly, exhibiting a half-life of about 8 days.     

Highly Purified pp60src Induces the Actin Transformation in Microinjected Cells and Phosphorylates Selected Cytoskeletal Proteins In Vitro

The src gene product of Rous sarcoma virus (pp60(src)) was highly purified from a rat tumor cell line and shown to have physiological actin transformation activity in a cellular microinjection assay that measures the dissolution of actin microfilament bundles in vivo. The purified pp60(src) fraction consisted of two major proteins, seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels: a 60,000-dalton (60K) protein, identified as pp60(src) by immunoprecipitation with tumor-bearing rabbit immunoglobulin G (IgG) and peptide mapping, and an unrelated 65K protein. There was no evidence for proteolytic cleavage of pp60(src). A 7,000-fold purification of the tyrosine-specific protein kinase activity of pp60(src) was achieved by this procedure. Purified pp60(src) phosphorylated tumor-bearing rabbit IgG heavy chains, casein, histones H1 and H2B, tubulin, and microtubule-associated proteins when assayed in vitro. When incubated with [gamma-(32)P]ATP in the absence of exogenous phosphoacceptor substrates, purified pp60(src) became labeled with (32)P at the tyrosine residues exclusively. Phosphatase and cyclic AMP-dependent protein kinase activities were undetectable in the purified fraction. Microinjection of highly purified pp60(src) into the cytoplasm of normal Swiss 3T3 mouse fibroblasts caused rapid and reversible dissolution of actin stress fibers, as visualized by indirect immunofluorescence with actin antibodies. The actin-disrupting activity was thermolabile and sensitive to inhibition by coinjection of tumor-bearing rabbit IgG, and purified to about the same extent (8,000-fold) as did the IgG kinase activity of pp60(src), thus implicating pp60(src) as the active agent. Examination of actin-associated proteins as substrates for the pp60(src) kinase in vitro showed that vinculin was phosphorylated directly by pp60(src), although to a small extent. Actin, myosin, and tropomyosin were not phosphorylated. Thus, pp60(src) purified by this procedure retains native functional properties and provides a useful probe for analyzing transformation-dependent changes in actin cytoarchitecture.