Cytoplasmic Tubular Structures Research Articles

Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization

Posttranslational modifications (PTMs) such as phosphorylation of RNA-binding proteins (RBPs) regulate several critical steps in RNA metabolism, including spliceosome assembly, alternative splicing, and mRNA export. Notably, serine-/arginine- (SR)-rich RBPs are densely phosphorylated compared with the remainder of the proteome. Previously, we showed that dephosphorylation of the splicing factor SRSF2 regulated increased interactions with similar arginine-rich RBPs U1-70K and LUC7L3. However, the large-scale functional and structural impact of these modifications on RBPs remains unclear. In this work, we dephosphorylated nuclear extracts using phosphatase in vitro and analyzed equal amounts of detergent-soluble and -insoluble fractions by mass-spectrometry-based proteomics. Correlation network analysis resolved 27 distinct modules of differentially soluble nucleoplasm proteins. We found classes of arginine-rich RBPs that decrease in solubility following dephosphorylation and enrich the insoluble pelleted fraction, including the SR protein family and the SR-like LUC7L RBP family. Importantly, increased insolubility was not observed across broad classes of RBPs. We determined that phosphorylation regulated SRSF2 structure, as dephosphorylated SRSF2 formed high-molecular-weight oligomeric species in vitro. Reciprocally, phosphorylation of SRSF2 by serine/arginine protein kinase 2 (SRPK2) in vitro decreased high-molecular-weight SRSF2 species formation. Furthermore, upon pharmacological inhibition of SRPKs in mammalian cells, we observed SRSF2 cytoplasmic mislocalization and increased formation of cytoplasmic granules as well as cytoplasmic tubular structures that associated with microtubules by immunocytochemical staining. Collectively, these findings demonstrate that phosphorylation may be a critical modification that prevents arginine-rich RBP insolubility and oligomerization.

The West Nile virus assembly process evades the conserved antiviral mechanism of the interferon-induced MxA protein

Flaviviruses have evolved means to evade host innate immune responses. Recent evidence suggests this is due to prevention of interferon production and signaling in flavivirus-infected cells. Here we show that the interferon-induced MxA protein can sequester the West Nile virus strain Kunjin virus (WNVKUN) capsid protein in cytoplasmic tubular structures in an expression-replication system. This sequestering resulted in reduced titers of secreted WNVKUN particles. We show by electron microscopy, tomography and 3D modeling that these cytoplasmic tubular structures form organized bundles. Additionally we show that recombinant ER-targeted MxA can restrict production of infectious WNVKUN under conditions of virus infection. Our results indicate a co-ordinated and compartmentalized WNVKUN assembly process may prevent recognition of viral components by MxA, particularly the capsid protein. This recognition can be exploited if MxA is targeted to intracellular sites of WNVKUN assembly. This results in further understanding of the mechanisms of flavivirus evasion from the immune system.

Morphologic Characteristics of Pulmonary Macrophages in Cetaceans: Particular Reference to Pulmonary Intravascular Macrophages as a Newly Identified Type

We examined the morphologic characteristics of pulmonary macrophages in 42 specimens of Odontoceti (Globicephala macrorhynchus, Grampus griseus, Tursiops truncatus, Stenella attenuata, Stenella coeruleoalba, Berardius bairdii), using light and electron microscopes as well as immunohistochemistry with SRA-E5. SRA-E5-positive alveolar macrophages and pulmonary interstitial macrophages contained graphitic soots, indicating the clearance of airborne, aspirated foreign bodies. Pulmonary intravascular macrophages (PIMs), positive with SRA-E5, were present within pulmonary capillaries, attaching to applied endothelial cells by cell junctions. They showed cytoplasmic tubular structures of micropinocytosis vermiformis and erythrophagocytosis, indicating their contributory role in the clearance of blood-borne particles. The uptake of pathogens by PIMs may be associated with the inducement of acute lung injury, especially bacterial infectious pneumonia. This study revealed for the first time the presence of PIMs in cetaceans.

Distribution and localization of CMP- N-acetylneuraminic acid hydroxylase and N-glycolylneuraminic acid-containing glycoconjugates in porcine lymph node and peripheral blood lymphocytes*

An immunohistochemical analysis was performed on paraplast-embedded sections of porcine lymph node with antibodies specific for CMP-N-acetylneuraminic acid hydroxylase (h-3 antibody) and glycoconjugate-bound N-glycolylneuraminic acid (Neu5Gc), which appears as a result of the hydroxylase reaction (a-Gc antibody). The observed localization of the enzyme in cells of the perifollicular zone, including lymphocytes, was reflected in a similar distribution of glycoconjugate-bound Neu5Gc. This result confirms previous biochemical investigations on the role of the hydroxylase in regulating Neu5Gc biosynthesis in vitro on a histological level. An analysis of lymphocytes isolated from porcine thymus, spleen, lymph node and peripheral blood revealed differences in the amount of Neu5Gc in the various lymphocytes that correlated well with the activity of the hydroxylase determined in these cells. The largest amount of Neu5Gc and highest activity of the enzyme were detected in the peripheral blood lymphocytes (PBL). Immunohistochemical studies with a-Gc and h-3 antibodies on sections of paraplast-embedded PBL showed that these antigens were located at the cell surface and in the cytosol, respectively. Ultrastructural immunocytochemistry with the h-3 antibody and immunogold labelling was used to investigate the subcellular localization of the hydroxylase. The enzyme was detected in the cytosol in the vicinity of the nuclear membrane and the outer membrane of mitochondria, in particular those close to the nucleus. The antigen was also detected on cytoplasmic tubular structures. In addition, a weak labelling of the Golgi apparatus was also observed occasionally. The possibility that this localization may be related to the availability of the substrate CMP-Neu5Ac and the redox partner cytochrome b5 is discussed.

Hepatitis C virus: detection of intracellular virus particles by electron microscopy.

We previously demonstrated that a human T-cell line, HPBMa10-2 derived from HPBALL, was capable of supporting a productive infection of hepatitis C virus (HCV). We subsequently found Daudi cells, a human B-cell line, to be susceptible to HCV infection. Employing these cell lines infected with HCV as well as liver obtained during the acute phase of hepatitis C from a chimpanzee, we observed intracellular HCV particles by electron microscopy (EM). We detected viruslike particles with a diameter of approximately 50 nm in the cytoplasmic vesicles. In Daudi cells harvested 15 days after virus inoculation, the appearance of cytoplasmic tubular structures which we originally described for chimpanzee hepatocytes in association with HCV infection was noted. Immunoperoxidase EM using antibodies against HCV core and envelope demonstrated that the particles contained the viral antigens.

Hepatitis C virus: Detection of intracellular virus particles by electron microscop

We previously demonstrated that a human T-cell line, HPBMa10-2 derived from HPBALL, was capable of supporting a productive infection of hepatitis C virus (HCV). We subsequently found Daudi cells, a human B-cell line, to be susceptible to HCV infection. Employing these cell lines infected with HCV as well as liver obtained during the acute phase of hepatitis C from a chimpanzee, we observed intracellular HCV particles by electron microscopy (EM). We detected viruslike particles with a diameter of approximately 50 nm in the cytoplasmic vesicles. In Daudi cells harvested 15 days after virus inoculation, the appearance of cytoplasmic tubular structures which we originally described for chimpanzee hepatocytes in association with HCV infection was noted. Immunoperoxidase EM using antibodies against HCV core and envelope demonstrated that the particles contained the viral antigens. (Hepatology 1996 Feb;23(2):205-9)

Identification of cytoplasmic tubular structures in Pneumocystis carinii

Pneumocystis carinii (PC) is an extracellular organism which frequently causes pneumonia in immunocompromised patients. In the United States more than 60% of patients with acquired immunodeficiency syndrome (AIDS) have at least one episode of PC pneumonia. The earliest published ultrastructural study of PC was undertaken by Dickmann et al. in 1954. A few articles which studied the ultrastructure of PC continued to appear in the German medical literature in the late 1950’s and early 1960’s. With the development of immunosuppressive chemotherapy for cancer patients and in later years the onslaught of the AIDS epidemic, the ultrastuctural literature concerned with PC has expanded. The internal morphology of PC has proved to be difficult to preserve and although extensively examined questions remain concerning the intracellular contents of PC. In this study we report the presence of cytoplasmic tubular structures (CTS) in PC. These structures have been identified in hepatocytes of Chimpanzees infected with non-A,non-B hepatitis and in the lymphocytes of humans with adult T-cell leukemia or AIDS.

The endothelial vesicle system in cryofixed frog mesenteric capillaries analysed by ultrathin serial sectioning

Conventional EM sections of chemically fixed capillary endothelial cells reveal numerous apparently free smooth plasmalemmal vesicles. However, the method of ultrathin (less than 150 A) serial sectioning has shown that the smooth vesicle profiles arise merely as a result of the EM thin sectioning of two sets of complex vesicular invaginations from the luminal and abluminal cell surfaces, which end blindly in the cytoplasm. While 50-70% of the total population of vesicular profiles appear to lack connections to the cell surface in conventional (500-700 A thick) EM thin sections less than 1% truly free vesicles can be found by the ultrathin serial section analyses. In the present study it is examined whether similar conclusions apply to endothelial cells which were directly frozen by slam-freezing and subsequently freeze-substituted. The three-dimensional organization of the plasmalemmal vesicular system was analyzed in four series of 19, 18, 13, and 10 ultrathin sections (approximately 110 A thick) of capillaries from frog mesenteries quickly excised from decapitated frogs (Rana pipiens). None of 920 vesicular profiles (diameter 500-1,200 A) which appeared free in individual thin sections of the series represented free vesicles; all profiles either communicated with other vesicles, the cell surface, or in rare cases turned out to be part of cytoplasmic tubular membrane structures. It is concluded that free smooth plasmalemmal vesicles are very rare in rapidly frozen as well as in directly fixed frog capillary endothelium. The volume density of profiles (13-15%), the proportion of apparently free vesicle profiles (70%), and interconnected profiles (20%) were similar to the picture previously found in single EM sections of frog mesenteric capillaries. No transendothelial channels were found in the four series of ultrathin sections of capillaries. However, continuities between the luminal and abluminal cell surfaces were seen in the endothelium of venules. Furthermore, in the ultrathin series of the capillaries, vesicular units belonging to the two sets of invaginations and cytoplasmic tubular membrane structures were in more cases found in very close contact-as fused to share one unit membrane. If this finding is representative for the in vivo situation, it may reflect that the vesicular system represents a highly dynamic system with possibilities for mixing of membranes, cellular traffic of lipid, membrane proteins, and receptors between internal compartments and the cell surfaces, as well as occasional exchange of macromolecules between blood and tissue through rare temporary connections between the two sets of surface invaginations, without actually moving vesicles.

Glucagon receptors in endothelial and Kupffer cells of mouse liver.

To determine whether hepatic sinusoidal cells contain glucagon receptors and, if so, to study the significance of the receptors in the cells, binding of [125I]-glucagon to nonparenchymal cells (mainly endothelial cells and Kupffer cells) isolated from mouse liver was examined by quantitative autoradiography and biochemical methods. Furthermore, the pathway of intracellular transport of colloidal gold-labeled glucagon (AuG) was examined in vivo. Autoradiographic and biochemical results demonstrated many glucagon receptors in both endothelial cells and Kupffer cells, and more receptors being present in endothelial cells than in Kupffer cells. In vivo, endothelial cells internalized AuG particles into coated vesicles via coated pits and transported the particles to endosomes, lysosomes, and abluminal plasma membrane. Therefore, receptor-mediated transcytosis of AuG occurs in endothelial cells. The number of particles present on the abluminal plasma membrane was constant if the amount of injected AuG increased. Therefore, the magnitude of receptor-mediated transcytosis of AuG appears to be regulated by endothelial cells. Kupffer cells internalized the ligand into cytoplasmic tubular structures via plasma membrane invaginations and transported the ligand exclusively to endosomes and lysosomes, suggesting that the ligand is degraded by Kupffer cells.

Mutations in the nuclear lamin proteins resulting in their aberrant assembly in the cytoplasm.

We have constructed a series of mutations in the human A lamin cDNA to identify and alter the nuclear localization signal using an in vivo functional assay system. The nuclear localization signal in the lamin proteins has both structural and functional similarities with that of the SV40 large T-antigen. Mutations within this functional domain result in the assembly of cytoplasmic tubular structures, and the behavior of these mutants suggests a post-translational dimerization of the lamin proteins prior to their transport into the nucleus. In the course of this work other regions of the carboxy terminus of the A/C lamin proteins have been implicated in the proper assembly and structure of the nuclear envelope.

Ultrastructural and immunohistochemical findings in oral hairy leukoplakia.

Three cases of HL from the lateral border of the tongue of male homosexual AIDS patients were investigated by thin section electron microscopy. Keratinocytes contained condensed chromatin in their pyknotic nuclei and a few organelles in the oedematous cytoplasm. Chromatin was in close association to the nuclear membrane and showed a punched-out appearance. Particles typical of the herpes virus group were abundant in the upper two thirds of the epithelium in all three cases. Virus particles were seen frequently in the nuclei of the ballooned keratinocytes, but rarely in cells containing Candida albicans. Viral nucleocapsids were observed budding at the inner nuclear membrane, thereby acquiring the prospective viral envelope. Complete, enveloped virions were found in the endoplasmic reticulum and in the extracellular space. These virions were identified immunohistochemically as Epstein-Barr virus (EBV) using two monoclonal antibodies directed against EBV capsid and membrane antigen, respectively. Candida albicans was observed in the stratum corneum and in the upper layer of the stratum spinosum. Special cytoplasmic tubular structures arranged in parallel bundles were found in koilocytotic cells in addition to characteristic membrane structures composed of undulating convoluted membranes. Epithelial basement membranes were always intact.

Non-A, non-B hepatitis associated with the administration of intravenous immunoglobulin—transmission studies in chimpanzees

Recent reports have described serum aminotransferase elevations in hypogammaglobulinaemic patients following prophylactic infusions of intravenous immunoglobulins. Whether these serum enzyme elevations represent viral non-A, non-B hepatitis (NANBH) or are related to hepatocyte damage secondary to non-infectious mechanisms has not always been clear. We inoculated two chimpanzees with a serum pool obtained from five hypogammaglobulinaemic patients with persistent alanine aminotransferase (ALT) elevations following prophylactic treatment with an intravenous immunoglobulin (Gammonativ ®, KabiVitrum). Both chimpanzees developed ALT elevations post-inoculation but with quite different patterns. One (1278) had elevated ALT levels at weeks 5–13 after serum inoculation whereas the other (1279) showed a biphasic pattern of ALT elevations during weeks 11–13 and 21–27. The earliest serum ALT elevations were accompanied by marked lymphocytic infiltration of the liver in the absence of characteristic cytoplasmic tubular structures in hepatocytes examined by electron microscopy. Chimpanzee 1279, however, developed tubular structures of the C-III type concomitantly with its later ALT elevations, suggesting the possibility that the serum inoculum contained two NANBH agents and thus that the intravenous immunoglobulin itself may have been contaminated with two NANBH agents.

Liver disease in aids

Abnormal liver chemistries, unexplained fevers, or hepatomegaly prompted 36 liver biopsies on 34 patients with the acquired immunodeficiency syn-drome. The most common finding was the presence of hepatic granulomas, seen in 13 of the biopsy specimens. Eight of these granulomas were illdefined, and 5 were more clearly associated with mycobacterial disesse. Portal fibrosis and fatty infiltration were comon, but a paucity of significant inflammatory activity was seen despite elevated aspartate aminotransferase levels, perhaps related to the underlying imunoincompetent status. Other noteworthy histopathologic findings included 1 patient each with peliosis hepatitis and cryptococcal hepatitis. Electron-microscopic evidence of cytoplasmic tubular structures or viral particles were seen within the hepatocytes of 2 patients. It is concluded that a broad spectrum of hepatic histopathology may be seen in the acquired immunodeficiency syndrome, and that liver biopsy may be diagnostically valuable in the clincial investigation of such patients.

The spectrum of liver disease in the acquired immunodeficiency syndrome

Abnormal liver chemistries, unexplained fevers, or hepatomegaly prompted 36 liver biopsies on 34 patients with the acquired immunodeficiency syndrome. The most common finding was the presence of hepatic granulomas, seen in 13 of the biopsy specimens. Eight of these granulomas were ill-defined, and 5 were more clearly associated with mycobacterial disease. Portal fibrosis and fatty infiltration were common, but a paucity of significant inflammatory activity was seen despite elevated aspartate aminotransferase levels, perhaps related to the underlying immunoincompetent status. Other noteworthy histopathologic findings included 1 patient each with peliosis hepatis and cryptococcal hepatitis. Electron-microscopic evidence of cytoplasmic tubular structures or viral particles were seen within the hepatocytes of 2 patients. It is concluded that a broad spectrum of hepatic histopathology may be seen in the acquired immunodeficiency syndrome, and that liver biopsy may be diagnostically valuable in the clinical investigation of such patients.

Transmission of non-A, non-B hepatitis agent to chimpanzees from patients of epidemic hepatitis.

Two chimpanzees were inoculated intravenously with acute-phase sera obtained from two patients with epidemic hepatitis. They developed histopathologically confirmed hepatitis. Electron microscopic examination of the liver showed peculiar cytoplasmic tubular structures in the hepatocytes. These ultrastructural findings were similar to those described for the livers of chimpanzees inoculated with the F strain of non-A, non-B hepatitis agent derived from a posttransfusion hepatitis case. The chimpanzee that had recovered from hepatitis caused by the F strain of non-A, non-B hepatitis agent was re-challenged with the serum from one of the patients. The chimpanzee developed neither clinical signs nor histological changes of hepatitis. These results suggested that non-A, non-B hepatitis agent was involved not only in post-transfusion hepatitis but also in epidemic hepatitis.

Evidence for cycling of aggregate-containing tubules in toad urinary bladder.

Antidiuretic hormone (ADH) promotes the fusion of cytoplasmic tubular structures with the luminal membrane of receptor tissues such as toad urinary bladder. To determine whether fusion is a continuous cyclic process, bladders were stimulated with ADH with colloidal gold in the luminal bathing medium. After as little as 15 min of stimulation, gold-filled tubules were seen in the cytoplasm, evidence that cycling was indeed taking place. Serial sections confirmed that these tubules had no connection with the luminal membrane, and had returned to the cytoplasm. Cessation of ADH stimulation, followed by a second stimulation, greatly reduced the number of gold-filled cytoplasmic tubules, suggesting that many tubules were capable of refusion. Mean fusion event diameter underwent significant changes, enlarging at 15 min, and contracting at 60 min. Thus, ADH initiates a process of continuous cycling of cytoplasmic tubules between cytoplasm and luminal membrane.

Alteration of luminal membrane structure by antidiuretic hormone.

The final step in the action of antidiuretic hormone (ADH) is the insertion of aggregates of water-conducting particles into the luminal membrane of the receptor cell. The aggregates arise from cytoplasmic tubular structures that fuse with the luminal membrane. This review presents a number of questions about this process, along with current attempts to answer them. The following topics are addressed: 1) the exact role of the cytoskeleton in promoting tubular fusion, 2) the nature of the translocation process leading to fusion, 3) the point in the sequence at which ADH enters, 4) the composition and structure of the water-conducting particles. Some of the answers require a more complete understanding of the structure of the apical portion of ADH-sensitive cells; recent morphological studies are therefore included.

Cytoplasmic tubular structures in the diagnosis of oral discoid lupus erythematosus

Biopsies from oral lesions of 5 patients with discoid lupus erythematosus (DLE), 5 with lichen planus (LP), 5 with leukoplakia (LEUK) and 3 patients with uncertain diagnoses termed DLE?, LP? were examined in the electron microscope for presence of cytoplasmic tubular structures (CTS) in vascular endothelium. Five vessels were examined in each of two randomly selected sections from each biopsy, without knowledge of the clinical diagnosis. All five DLE biopsies showed presence of CTS in 4-9 out of 10 vessels, as compared with none of 10 vessels in biopsies from LP or LEUK, the difference being significant. Further, CTS occurred in 4 out of 10 vessels in one DLE?, LP? biopsy from a patient with serologic signs of systemic lupus erythematosus. As CTS seem to be absent in oral lesions of LP and LEUK, which are the most important differential diagnoses for oral DLE, the identification of CTS in vascular endothelium sing electron microscopy may be an auxiliary diagnostic procedure in oral DLE.

Stabilization of vasopressin-induced membrane events by bifunctional imidoesters.

Vasopressin increases the water permeability of the luminal membrane of the toad bladder epithelial cell. This change in permeability correlates with the occurrence in luminal membranes of intramembrane particle aggregates, which may be the sites for transmembrane water flow. Withdrawal of vasopressin is ordinarily associated with a rapid reduction of water flow to baseline values and a simultaneous disappearance of the particle aggregates. The bifunctional imidoesters dithiobispropionimidate (DTBP) and dimethylsuberimidate (DMS), which cross-link amino groups in membrane proteins and lipids, slow the return of water flow to baseline after vasopressin withdrawal. Cross-linking is maximal at pH 10, and is reduced as pH is lowered. Freeze-fracture studies show persistence of luminal membrane particle aggregates in cross-linked bladders and a reduction in their frequency as water flow diminishes. Fusion of aggregate-containing cytoplasmic tubular membrane structures with the luminal membrane is also maintained by the imidoesters. Reductive cleavage of the central S-S bond of DTBP by beta-mercaptoethanol reverses cross-linking, permitting resumption of the rapid disappearance of the vasopressin effect. Bladders that have undergone DTBP cross-linking and beta-mercaptoethanol reduction respond to a second stimulation by vasopressin. Thus, the imidoesters provide a physiologic and reversible means of stabilizing normally rapid membrane events.

Unusual structures in the epidermal Langerhans´ cells of normal human skin

Two unusual structures were found in the epidermal Langerhans´ cells of normal human adult skin: (1) an intramitochondrial crystalline lattice structure composed of numerous electron-dense, fine particles approximately 90 A in diameter, with a lucent spacing of nearly 90 A; (2) bundles of unusual cytoplasmic tubular structures, approximately 120 A wide.