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PubMed2005

Molecular dynamics simulations of complexes between wild-type and mutant anthrax protective antigen variants and a model anthrax toxin receptor.

Bacillus anthracis, a spore-forming infectious bacterium, produces a toxin consisting of three proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA). LF and EF possess intracellular enzymatic functions, the net effect of which is to severely compromise host innate immunity. During an anthrax infection PA plays the critical role of facilitating entry of both EF and LF toxins into host cell cytoplasm. Crystal structures of all three of the anthrax toxins have been determined, as well as the crystal structure of the (human) von Willebrand factor A (integrin VWA/I domain) -- an anthrax toxin receptor. A theoretical structure of the complex between VWA/I and PA has also been reported. Here we report on the results of 1,000 psec molecular dynamics (MD) simulations carried out on complexes between the Anthrax Protective Antigen Domain 4 (PA-D4) and the von Willebrand Factor A (VWA/I). MD simulations (using Insight II software) were carried out for complexes containing wild-type (WT) PA-D4, as well as for complexes containing three different mutants of PA-D4, one containing three substitutions in the PA-D4 "small loop" (residues 679-693) (D683A/L685E/Y688C), one containing a single substitution at a key site at the PA-D4 -- receptor interface (K679A) and another containing a deletion of eleven residues at the C-terminus of PA (Delta724-735). All three sets of PA mutations have been shown experimentally to result in serious deficiencies in PA function. Our MD results are consistent with these findings. Major disruptions in interactions were observed between the mutant PA-D4 domains and the anthrax receptor during the MD simulations. Many secondary structural features in PA-D4 are also severely compromised when VWA complexes with mutant variants of PA-D4 are subjected to MD simulations. These MD simulation results clearly indicate the importance of the mutated PA-D4 residues in both the "small loop" and at the carboxyl terminus in maintaining a PA conformation that is capable of effective interaction with the anthrax toxin receptor.

Antigens, Bacterial↗

PubMed1987

Characterization of the bioluminescent organelles in Gonyaulax polyedra (dinoflagellates) after fast-freeze fixation and antiluciferase immunogold staining.

To characterize the microsources of bioluminescent activity in the dinoflagellate Gonyaulax polyedra, an immunogold labeling method using a polyclonal antiluciferase was combined with fast-freeze fixation and freeze substitution. The quality of the preservation and the specificity of the labeling were greatly improved compared to earlier results with chemical fixation. Two organelles were specifically labeled: cytoplasmic dense bodies with a finely vermiculate texture, and mature trichocysts, labeled in the space between the shaft and the membrane. The available evidence indicates that the dense bodies are the light-emitting microsources observed in vivo. The dense bodies appear to originate in the Golgi area as cytoplasmic densifications and, while migrating peripherally, come into contact with the vacuolar membrane. Mature organelles protrude and hang like drops in the vacuolar space, linked by narrow necks to the cytoplasm. These structural relationships, not previously apparent with glutaraldehyde fixation, suggest how bioluminescent flashes can be elicited by a proton influx from a triggering action potential propagated along the vacuolar membrane. Similar dense bodies were labeled in the active particulate biochemical fraction (the scintillons), where they were completely membrane bound, as expected if their necks were broken and resealed during extraction. The significance of the trichocyst reactivity remains enigmatic. Both organelles were labeled with affinity-purified antibody, which makes it unlikely that the trichocyst labeling is due to a second antibody of different specificity. But trichocysts are not bioluminescent; the cross-reacting material could be luciferase present in this compartment for some other reason, or a different protein carrying similar antigenic epitopes.

Animals↗

PubMed1995

c-Src regulates the simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP following epidermal growth factor stimulation.

Analysis of C3H10T1/2 murine fibroblasts overexpressing wild type and dominant negative variants of c-Src has demonstrated a requirement for c-Src in EGF-induced mitogenesis. Correlating with the ability of c-Src variants to potentiate or inhibit EGF-dependent DNA synthesis is the phosphotyrosine content of multiple cellular proteins, including p190-RhoGAP, a protein thought to regulate growth factor-induced actin cytoskeleton remodeling by modulating the activity of the small GTP binding protein, Rho. Because the in vivo phosphotyrosine content of p190 varies with the level of active c-Src and not with EGF treatment, p190 is considered to be a preferred substrate of c-Src. To determine whether tyrosyl phosphorylation of p190 (by c-Src) could influence EGF-dependent actin remodeling, we used conventional and confocal immunofluorescence microscopy to examine the intracellular distribution of p190, actin, and p120RasGAP in EGF-stimulated or unstimulated 10T1/2 Neo control cells and cells that stably overexpress wild-type (K+) or kinase-defective (K-) c-Src. We found that in all cell lines, EGF induced a rapid and transient condensation of p190 and RasGAP into cytoplasmic, arclike structures. However, in K+ cells the rate of appearance and number of cells exhibiting arcs increased when compared with control cells. Conversely, K- cells exhibited delayed arc formation and a reduction in number of cells forming arcs. EGF-induced actin stress fiber disassembly and reassembly occurred with the same kinetics and frequency as did p190 and RasGAP rearrangements in all three cell lines. These results, together with the documented Rho-GAP activity intrinsic to p190 and the ability of Rho to modulate actin stress fiber formation, suggest that c-Src regulates EGF-dependent actin cytoskeleton reorganization through phosphorylation of p190.

Actins↗

PubMed2006

Gawky is a component of cytoplasmic mRNA processing bodies required for early Drosophila development.

In mammalian cells, the GW182 protein localizes to cytoplasmic bodies implicated in the regulation of messenger RNA (mRNA) stability, translation, and the RNA interference pathway. Many of these functions have also been assigned to analogous yeast cytoplasmic mRNA processing bodies. We have characterized the single Drosophila melanogaster homologue of the human GW182 protein family, which we have named Gawky (GW). Drosophila GW localizes to punctate, cytoplasmic foci in an RNA-dependent manner. Drosophila GW bodies (GWBs) appear to function analogously to human GWBs, as human GW182 colocalizes with GW when expressed in Drosophila cells. The RNA-induced silencing complex component Argonaute2 and orthologues of LSm4 and Xrn1 (Pacman) associated with 5'-3' mRNA degradation localize to some GWBs. Reducing GW activity by mutation or antibody injection during syncytial embryo development leads to abnormal nuclear divisions, demonstrating an early requirement for GWB-mediated cytoplasmic mRNA regulation. This suggests that gw represents a previously unknown member of a small group of genes that need to be expressed zygotically during early embryo development.

Animals↗

PubMed1969

The fine structure of Acanthamoeba castellanii (Neff strain). II. Encystment.

Encysting cells of Acanthamoeba castellanii, Neff strain, have been examined with the electron microscope. The wall structure and cytoplasmic changes during encystment are described. The cyst wall is composed of two major layers: a laminar, fibrous exocyst with a variable amount of matrix material, and an endocyst of fine fibrils in a granular matrix. The two layers are normally separated by a space except where they form opercula in the center of ostioles (exits for excysting amebae). An additional amorphous layer is probably present between the wall and the protoplast in the mature cyst. Early in encystment the Golgi complex is enlarged and contains a densely staining material that appears to contribute to wall formation. Vacuoles containing cytoplasmic debris (autolysosomes) are present in encysting cells and the contents of some of the vacuoles are deposited in the developing cyst wall. Lamellate bodies develop in the mitochondria and appear in the cytoplasm. Several changes are associated with the mitochondrial intracristate granule. The nucleus releases small buds into the cytoplasm, and the nucleolus decreases to less than half its original volume. The cytoplasm increases in electron density and its volume is reduced by about 80%. The water expulsion vesicle is the only cellular compartment without dense content in the mature cyst. The volume fractions of lipid droplets, Golgi complex, mitochondria, digestive vacuoles, and autolysosomes have been determined at different stages of encystment by stereological analysis of electron micrographs. By chemical analyses, dry weight, protein, phospholipid, and glycogen are lower and neutral lipid is higher in the mature cyst than in the trophozoite.

Amoeba↗

PubMed1969

Choline metabolism and membrane formation in rat hepatoma cells grown in suspension culture. II. Phosphatidylcholine synthesis during growth cycle and fluctuation of mitochondrial density.

The incorporation of methyl-labeled choline into phosphorylcholine and phosphatidylcholine of cellular membranes by Novikoff rat hepatoma cells (line N1S1-67) during growth in suspension culture was investigated. Upon initiation of a fresh culture at 10(5) cells/ml, the rate of synthesis of phosphorylcholine by the cells was four to five times greater than that of the synthesis of phosphatidylcholine. While the rate of synthesis of the latter remained relatively constant, the rate of phosphorylation of choline decreased progressively during the course of the growth cycle of the culture to 10-20% of the initial rate when the culture reached stationary phase at 3 x 10(6) cells/ml. The decrease in phosphorylcholine synthesis during the growth cycle was not due to depletion of choline in the medium or a decrease in its concentration, but was correlated with a decrease in choline kinase activity of the cells as measured in cell-free extracts. Newly synthesized phosphatidylcholine was detectable in cells only as an integral part of cellular membranes. Its distribution among various cytoplasmic membrane structures separated by isopycnic centrifugation in sucrose density gradients remained relatively constant during the growth cycle. About 50% was associated with the mitochondria, and the remainder with plasma membrane fragments and other membranous structures with mean densities of about 1.15 and 1.13 g/cm(3), respectively. However, the density of the mitochondria increased from about 1.167 g/cm(3) in early exponential phase cells to about 1.190 g/cm(3) in stationary phase cells. The finding that the density of the entire propulation of mitochondria changed simultaneously and progressively is in agreement with the view that mitochondria grow by addition of phospholipids and structural proteins and increase in number by division.

Animals↗

PubMed1984

Synthesis and assembly of the cytoskeleton of Naegleria gruberi flagellates.

When Naegleria gruberi flagellates were extracted with nonionic detergent and stained by the indirect immunofluorescence method with AA-4.3 (a monoclonal antibody against Naegleria beta-tubulin), flagella and a network of cytoskeletal microtubules (CSMT) were seen. When Naegleria amebae were examined in the same way, no cytoplasmic tubulin-containing structures were seen. Formation of the flagellate cytoskeleton was followed during the differentiation of amebae into flagellates by staining cells with AA-4.3. The first tubulin containing structures were a few cytoplasmic microtubules that formed at the time amebae rounded up into spherical cells. The formation of these microtubules was followed by the appearance of basal bodies and flagella and then by the formation of the CSMT. The CSMT formed before the cells assumed the flagellate shape. In flagellate shaped cells the CSMT radiate from the base of the flagella and follow a curving path the full length of the cell. Protein synthetic requirements for the formation of CSMT were examined by transferring cells to cycloheximide at various times after initiation. One-half the population completed the protein synthesis essential for formation of CSMT 61 min after initiation of the differentiation. This is 10 min after the time when protein synthesis for formation of flagella is completed and 10-15 min before the time when the protein synthesis necessary for formation of the flagellate shape is completed.

Amoeba↗

PubMed1959

The mammalian cell-virus relationship. II. Adsorption, reception, and eclipse of poliovirus by HeLa cells.

Phases of attachment, reception or penetration, and eclipse of Type 1 poliovirus infecting HeLa cells in monolayer were studied. Firm attachment was not completely dependent on salt concentration, and was sensitive to temperature change. Like attachment, progressive resistance of adsorbed virus to inactivation by externally applied antibody was temperature-sensitive. Penetration was shown to be independent of physiologic integrity of cells. Virus in process of penetration was not affected by ribonuclease. Eclipse of adsorbed virus was not dependent on metabolic activity or physical integrity of HeLa cells. Debris from poliovirus-susceptible cells inactivated the virus in a manner similar to the kinetic course of virus adsorption by intact cells, and released cell-associated infective virus in similar amounts. All cells insusceptible to poliovirus infection failed to yield active debris. Virus inactivation by debris, like virus reception by intact cells, was temperature-sensitive. Debris could not inactivate virus adsorbed to cells, or alter the progressive incapacity of antibody to neutralize penetrating virus. The active debris factor was insoluble, was not associated with cell nuclei, was inactivated by fat solvents and trypsin treatment, and was destroyed by beat inactivation or sonic disruption. Anti-HeLa serum applied to cells before exposure to virus reduced the rate of virus adsorption, while antiserum treatment immediately following virus adsorption was ineffective. These findings suggested that the capacity of HeLa and other susceptible cells to adsorb, receive, and eclipse poliovirus was associated with organized cytoplasmic lipoprotein structures not possessed by insusceptible cells. The reaction of virus with receptor substance contained in debris was not readily reversed by treatment shown not to affect virus and to destroy activity of uncombined debris. Sensitivity of poliovirus adsorption by HeLa cells to change in environmental salt concentration or temperature was dependent on the method of measurement.

Adsorption↗

PubMed2004

The structure of resting bacterial populations in soil and subsoil permafrost.

The structure of individual cells in microbial populations in situ of the Arctic and Antarctic permafrost was studied by scanning and transmission electron microscopy methods and compared with that of cyst-like resting forms generated under special conditions by the non-spore-forming bacteria Arthrobacter and Micrococcus isolated from the permafrost. Electron microscopy examination of microorganisms in situ revealed several types of bacterial cells having no signs of damage, including "dwarf" curved forms similar to nanoforms. Intact bacterial cells in situ and frozen cultures of the permafrost isolates differed from vegetative cells by thickened cell walls, the altered structure of cytoplasm, and the compact nucleoid, and were similar in these features to cyst-like resting forms of non-spore-forming "permafrost" bacterial strains of Arthrobacter and Micrococcus spp. Cyst-like cells, being resistant to adverse external factors, are regarded as being responsible for survival of the non-spore-formers under prolonged exposure to subzero temperatures and can be a target to search for living microorganisms in natural environments both on the Earth and on extraterrestrial bodies.

Arthrobacter↗

PubMed2000

Death of the autoimmune thyrocyte: is it pushed or does it jump?

Programmed cell death or apoptosis is central both in physiology during development and in disease. The mechanism of apoptosis is under the control of antiapoptotic survival genes of the Bcl-2 family and proapoptotic death receptors of the TNF superfamily (Fas, TNFR, TRAILR). Following death signal, the death receptor binds to its own receptor and initiates, through binding of adaptors, a cascade of events mediated by the autoproteolytic activation of specific enzymes called caspases. This enzyme activation is ultimately responsible for the dissembly of basic nuclear and cytoplasmic cell structures leading to cell death. In certain cell systems, antiapoptotic genes of the Bcl-2 family prevent the proapoptotic pathway. One of their roles is to maintain mitochondrial function integrity. In autoimmune destructive thyroiditis high levels of apoptosis have been demonstrated particularly within the destructed follicles near the infiltrated areas in comparison to Graves' disease and non autoimmune glands. In Hashimoto's thyroiditis Fas expression has been found increased on thyrocytes and in vitro can be modulated by proinflammatory cytokines. FasL expression on thyrocytes remains controversial. Thyroid cells from Graves' disease and multinodular glands are known to kill Fas expressing target cells although Hashimoto's thyrocytes are not efficient effector cells. Intrathyroidal lymphocytes from Hashimoto's thyroids maintain functional killer activity. These findings would suggest that intrathyroidal lymphocytes could be responsible for thyrocyte death in vivo. Whether this mechanism is Fas/FasL, TRAIL/TRAILR dependent can not be confirmed as specific blocking reagents were not able to inhibit cell induced death. In Hashimoto's thyroiditis an impairment of Bcl-2 and Bcl-X anitapoptotic genes on thyrocytes has also been detected. Bcl-X expression can be down-regulated in vitro by incubation with cytokines. These findings suggest that thyrocyte death may not exclusively be the result of specific interactions between death receptor and their ligands but it may involve simultaneous impairment of protective genes of the Bcl-2 family. Whether the impairment of the Bcl-2 family is a direct consequence of environmental stimuli or is the result of an intrinsic thyrocyte (mitochondrial?) alteration is as yet not known.

Animals↗

PubMed2001

Yeast rab GTPase-activating protein Gyp1p localizes to the Golgi apparatus and is a negative regulator of Ypt1p.

A family of related proteins in yeast Saccharomyces cerevisiae is known to have in vitro GTPase-activating protein activity on the Rab GTPases. However, their in vivo function remains obscure. One of them, Gyp1p, acts on Sec4p, Ypt1p, Ypt7p, and Ypt51p in vitro. Here, we present data to reveal its in vivo substrate and the role that it plays in the function of the Rab GTPase. Red fluorescent protein-tagged Gyp1p is concentrated on cytoplasmic punctate structures that largely colocalize with a cis-Golgi marker. Subcellular fractionation of a yeast lysate confirmed that Gyp1p is peripherally associated with membranes and that it cofractionates with Golgi markers. This localization suggests that Gyp1p may only act on Rab GTPases on the Golgi. A gyp1Delta strain displays a growth defect on synthetic medium at 37 degrees C. Overexpression of Ypt1p, but not other Rab GTPases, strongly inhibits the growth of gyp1Delta cells. Conversely, a partial loss-of-function allele of YPT1, ypt1-2, can suppress the growth defect of gyp1Delta cells. Furthermore, deletion of GYP1 can partially suppress growth defects associated with mutants in subunits of transport protein particle complex, a complex that catalyzes nucleotide exchange on Ypt1p. These results establish that Gyp1p functions on the Golgi as a negative regulator of Ypt1p.

Carboxypeptidases↗

PubMed2005

Tracking retrograde flow in keratocytes: news from the front.

Actin assembly at the leading edge of the cell is believed to drive protrusion, whereas membrane resistance and contractile forces result in retrograde flow of the assembled actin network away from the edge. Thus, cell motion and shape changes are expected to depend on the balance of actin assembly and retrograde flow. This idea, however, has been undermined by the reported absence of flow in one of the most spectacular models of cell locomotion, fish epidermal keratocytes. Here, we use enhanced phase contrast and fluorescent speckle microscopy and particle tracking to analyze the motion of the actin network in keratocyte lamellipodia. We have detected retrograde flow throughout the lamellipodium at velocities of 1-3 microm/min and analyzed its organization and relation to the cell motion during both unobstructed, persistent migration and events of cell collision. Freely moving cells exhibited a graded flow velocity increasing toward the sides of the lamellipodium. In colliding cells, the velocity decreased markedly at the site of collision, with striking alteration of flow in other lamellipodium regions. Our findings support the universality of the flow phenomenon and indicate that the maintenance of keratocyte shape during locomotion depends on the regulation of both retrograde flow and actin polymerization.

Actins↗

PubMed2006

The Ca2+-binding protein ALG-2 is recruited to endoplasmic reticulum exit sites by Sec31A and stabilizes the localization of Sec31A.

The formation of transport vesicles that bud from endoplasmic reticulum (ER) exit sites is dependent on the COPII coat made up of three components: the small GTPase Sar1, the Sec23/24 complex, and the Sec13/31 complex. Here, we provide evidence that apoptosis-linked gene 2 (ALG-2), a Ca(2+)-binding protein of unknown function, regulates the COPII function at ER exit sites in mammalian cells. ALG-2 bound to the Pro-rich region of Sec31A, a ubiquitously expressed mammalian orthologue of yeast Sec31, in a Ca(2+)-dependent manner and colocalized with Sec31A at ER exit sites. A Ca(2+) binding-deficient ALG-2 mutant, which did not bind Sec31A, lost the ability to localize to ER exit sites. Overexpression of the Pro-rich region of Sec31A or RNA interference-mediated Sec31A depletion also abolished the ALG-2 localization at these sites. In contrast, depletion of ALG-2 substantially reduced the level of Sec31A associated with the membrane at ER exit sites. Finally, treatment with a cell-permeable Ca(2+) chelator caused the mislocalization of ALG-2, which was accompanied by a reduced level of Sec31A at ER exit sites. We conclude that ALG-2 is recruited to ER exit sites via Ca(2+)-dependent interaction with Sec31A and in turn stabilizes the localization of Sec31A at these sites.

Apoptosis Regulatory Proteins↗

PubMed1977

Lamella-particle complexes in nuclei of owl monkey kidney cells infected with herpesvirus saimiri.

A complex consisting of ribosome-like particles and a striated lamella occurring in stacked, tubular, and twisted ribbon-like configurations was observed in the nuclei of confluent monolayer cultures of primary owl monkey kidney cells infected with Herpesvirus saimiri. They were similar to the cytoplasmic cylindroid structures seen in some human leukemias and in the lymphoma of the northern pike.

Animals↗

PubMed1980

Intermediate-sized filaments in cultured rat liver tumor cells with Mallory body-like cytoplasm abnormalities.

The endoskeletal structure of tumor cells with a characteristic cytoplasmic abnormality initiated in rat liver by the in vivo adminisration of the carcinogen diethylnitrosamine was studied in clonal cell lines established and propagated in vitro. The bulk of the cytoplasmic and plasma membrane protein was removed by extraction with Triton X-100, and subsequently the juxtanuclear detergent-insoluble fraction containing filaments of 100-150 A was released into citrate buffer at pH 2.8. Analysis of this fraction by sodium dodecyl sulfate acrylamide gel electrophoresis revealed the pressence of five major proteins that banded with apparent molecular weights of about 66, 57, 52, 48 , and 43 x 10(3), the last of which comigrated with actin. The proteins thus resembled those from intermediate-sized filaments of both the vimentin (57 x 10(3)) and the prekeratin types obtained from various vertebrate cells. They also appeared to be related to the polypeptides of intermediate-sized filaments from Mallory bodies induced by griseofulvin in the livers of mice and to some of the polypeptides seen in isolates of Mallory bodies from human alcoholics. These results indicated that a major component of the carcinogen-induced lesion consisted of intermediate-sized filaments. The possible significance in cell transformation of this stably maintained aggregate of filaments that binds concanavalin A and displaces the nucleus is discussed. The close resemblance of this lesion to that seen in the cells of cirrhotic livers of alcoholics (Mallory's alcoholic hyalin) raises a question regarding the possible oncogenic status of such cells in humans.

Animals↗

PubMed1991

An RNA molecule copurifies with RNase P activity from Xenopus laevis oocytes.

Utilizing a procedure for the purification of RNase P from Xenopus laevis germinal vesicle (GV) extracts, according to which the contamination by a large, cytoplasmic, cylindrical structure (1) is avoided, we demonstrate that the X.laevis enzyme, like the HeLa RNase P, is precipitated by anti-Th antibodies and an RNA molecule (XL RNA), 320 nucleotides long, copurifies with the activity. The sequence of XL RNA is 60% homologous to HeLa H1 RNA, therefore the two molecules seem related.

Animals↗

PubMed2000

Cytopathology of pleural mesotheliomas.

Pleural mesothelioma is a rare and highly malignant tumor that has shown increasing incidence. In approximately 90% of patients the tumor manifests initially as recurrent, unilateral, bloody pleural effusions; in the remaining 10% of patients the tumor is detected by chest radiography. As a result, pleural mesotheliomas can be diagnosed by cytologic examination of pleural effusions or transthoracic fine-needle aspirates; the cytologic manifestations of such an examination are described in detail. Additionally, the handling of cytologic specimens, diagnostic problems, and the value and limitations of immunocytochemical and electron microscopic studies and other ancillary techniques in the cytologic identification of pleural mesotheliomas are discussed.

Cytodiagnosis↗

PubMed2001

Central liponeurocytoma.

The 2000 World Health Organization has included cerebellar liponeurocytoma in the category of glioneuronal tumors of the central nervous system. Once termed medullocytoma and considered an embryonal tumor, a variant of medulloblastoma, its indolent behavior and morphologic features prompted this nosologic change. Biphasic in appearance, the tumor consists of well-differentiated neurons with the cytology of neurocytes in addition to a population of lipidized cells resembling mature adipose tissue. Such tumors occur in older adults and have a relatively good prognosis. Linking the concept of liponeurocytoma to its occurrence in the cerebellum unnecessarily obscures the existence of similar neoplasms at other sites, such as among classic central neurocytomas of the lateral and third ventricles. Indeed, two such cases have briefly been reported. To these, we add a third example, the first to be ultrastructurally examined. Our case provides evidence that the lipid vacuoles progressively accumulate and coalesce within cells retaining neurocytic features. Thus, these distinctive lesions are the result of tumoral lipidization, rather than true adipose metaplasia.

Biomarkers, Tumor↗