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Showing papers on "Cellular compartment published in 1996"


Journal ArticleDOI
TL;DR: The data indicated that the ability of L. pneumophila to establish the intracellular infection of amoebae is dependent on its capacity to reside and multiply within a phagosome surrounded by the rough endoplasmic reticulum.
Abstract: Legionella pneumophila is an intracellular parasite of protozoa and human phagocytes. To examine adaptation of this bacterium to parasitize protozoa, the sequence of events of the intracellular infection of the amoeba Hartmannella vermiformis was examined. The previously described uptake phenomenon of coiling phagocytosis by human monocytes was not detected. A 1 h postinfection with wild-type strain AA100, mitochondria were observed within the vicinity of the phagosome. At 2.5 h postinfection, numerous vesicles surrounded the phagosomes and mitochondria were in close proximity to the phagosome. At 5 h postinfection, the bacterium was surrounded by a ribosome-studded multilayer membrane. Bacterial multiplication was evident by 8 h postinfection, and the phagosome was surrounded by a ribosome-studded multilayer membrane until 15 h postinfection. The recruitment of organelles and formation of the ribosome-studded phagosome was defective in an isogenic attenuated mutant of L. pneumophila (strain AA101A) that failed to replicate within amoebae. At 20 h postinfection with wild-type strain AA100, numerous bacteria were present in the phagosome and ribosome were not detected around the phagosome. These data showed that, at the ultrastructural level, the intracellular infection of protozoa by L. pneumophila is highly similar to that of infection of macrophages. Immunocytochemical studies provided evidence that at 5 h postinfection the phagosome containing L. pneumophila acquired an abundant amount of the endoplasmic reticulum-specific protein (BiP). Similar to phagosomes containing heat-killed wild-type L. pneumophila, the BiP protein was not detectable in phagosomes containing the mutant strain AA101A. In addition to the absence of ribosomes and mitochondria, the BiP protein was not detected in the phagosomes at 20 h postinfection with wild-type L. pneumophila. The data indicated that the ability of L. pneumophila to establish the intracellular infection of amoebae is dependent on its capacity to reside and multiply within a phagosome surrounded by the rough endoplasmic reticulum. This compartment may constitute a rich source of nutrients for the bacteria and is probably recognized as cellular compartment. The remarkable similarity of the intracellular infections of macrophages and protozoa by L. pneumophila strongly supports the hypothesis that adaptation of the bacterium to the intracellular environment of protozoa may be the mechanism for its ability to adapt to the intracellular environment of human alveolar macrophages and causes pneumonia.

198 citations


Journal ArticleDOI
TL;DR: The results strongly support the role of mitotic nuclei in determining septal placement, and suggest that cell size control is post-mitotic in A. nidulans.
Abstract: The mycelium of Aspergillus nidulans is composed of multinucleate cellular compartments delimited by crosswalls called septa. Septum formation is dependent on mitosis and requires the recruitment of actin to the site of septum formation. Employing a collection of temperature sensitive nuclear distribution (nudA2, nudC3 and nudF7), nuclear division (nimA5, hfaB3), and septation (sepD5, sepG1) mutants, we have investigated the interdependency among nuclear positioning, mitosis, and cell growth in structuring the cellular compartments of A. nidulans. The cellular compartments of nud+ strains were highly uniform with regard to nuclear distribution and averaged 38 microns in length. Incubation of nud mutants at semi-restrictive temperature resulted in aberrant nuclear distribution that appeared to direct the formation of variable-sized cellular compartments, ranging from 5 microns to greater than 81 microns. In germinating spores, the first septum forms at the basal end of the germ tube following the third round of nuclear division. Germlings must undergo mitosis in order to form a septum. Temperature-sensitive mitotic mutants were used to show that a single nuclear division is sufficient to activate septum formation, provided a critical cell size has been attained. In mitotic mutants and wild-type cells, delays in nuclear division resulted in the misplacement of the first septum. These results strongly support the role of mitotic nuclei in determining septal placement, and suggest that cell size control is post-mitotic in A. nidulans.

126 citations


Journal ArticleDOI
TL;DR: Members of all classes of proteins--cytoskeletal components, secreted growth factors, glycolytic enzymes, kinases, transcription factors, chaperones, transmembrane proteins, and extracellular matrix proteins--have been identified in cellular compartments other than their conventional sites of action.
Abstract: Members of all classes of proteins--cytoskeletal components, secreted growth factors, glycolytic enzymes, kinases, transcription factors, chaperones, transmembrane proteins, and extracellular matrix proteins--have been identified in cellular compartments other than their conventional sites of action. Some of these proteins are expressed as distinct compartment-specific isoforms, have novel mechanisms for intercompartmental translocation, have distinct endogenous biological actions within each compartment, and are regulated in a compartment-specific manner as a function of physiologic state. The possibility that many, if not most, proteins have distinct roles in more than one cellular compartment has implications for the evolution of cell organization and may be important for understanding pathological conditions such as Alzheimer's disease and cancer.

81 citations


Journal ArticleDOI
TL;DR: A classification is proposed that distinguishes between stable, preexisting membrane compartments and vesicles that are, by definition, transient organelles that are able to “mature”, a process defined as an irreversible set of biochemical events which lead to a physiologically distinct end-state of the vesicle prior to its vectorial fusion with a target compartment.
Abstract: Two different mechanisms have been proposed to explain transport along the endocytic and biosynthetic transport routes in cells. The first involves stable compartments connected by vesicular traffic while the second argues that the key organelles (early endosomes or the cis Golgi) form de novo by fusion of vesicles and subsequently mature into later forms. In the first part of this article, I propose a classification that distinguishes between stable, preexisting membrane compartments and vesicles that are, by definition, transient organelles. In this scheme, compartments, but not vesicles, are capable of homotypic fusion while vesicles, but not compartments, are able to “mature”, a process defined as an irreversible set of biochemical events which lead to a physiologically distinct end-state of the vesicle prior to its vectorial fusion with a target compartment. In the second part, I summarize my current ideas about the ultrastructural organization of the ER-Golgi region. Finally, I review the cell biology of selected examples of different vesicle types in order to exemplify the fascinating diversity of functions that this class of membrane organelles has evolved.

77 citations


Journal ArticleDOI
TL;DR: The other subject reviewed in this paper is cell surface glycoconjugates, as they are expressed in relation to specific cell types present in various organs and during cellular differentiation processes.
Abstract: High resolution immunolabeling applying the protein A-gold technique and carbohydrate cytochemistry using lectin-gold labeling on Lowicryl K4M and thawed-frozen thin sections are most useful approaches for the detection of protein antigens and lectin binding sites in intracellular organelles and the plasma membrane. They provided the basis for modern electron microscopic studies on protein glycosylation reactions and the identification of their subcellular localization as reviewed here. These studies have demonstrated organelle subcompartments and the cell type-specific compartmentation of endoplasmic reticulum and Golgi apparatus-associated glycosylation reactions. The other subject reviewed in this paper is cell surface glycoconjugates, as they are expressed in relation to specific cell types present in various organs and during cellular differentiation processes.

72 citations


Journal ArticleDOI
TL;DR: It is concluded that if VDAC molecules are present at nonmitochondrial locations in mammalian cells, these are unlikely to be the known products of the HVDAC1 or HVDac2 genes.
Abstract: Higher eukaryotes, including mammals and plants, express a family of VDAC proteins each encoded by a distinct gene. Two human genes encoding VDAC isoforms (HVDAC1 and HVDAC2) have been characterized in greatest detail. These genes generate three proteins that differ primarily by the addition of distinct N terminal extensions in HVDAC2 and HVDAC2′, a splice variant of HVDAC2, relative to HVDAC1. Since N terminal sequences have been demonstrated to target many proteins to appropriate subcellular compartments, this observation raises the possibility that the N terminal differences found in HVDAC isoforms may lead to targeting of each protein to different cellular locations. Consistent with this hypothesis, a large number of reports have provided evidence consistent with the notion that HVDAC1 and its homolog in related mammalian species may specifically be present in the plasma membrane or other nonmitochondrial cellular compartments. Here, we review this information and conclude that if VDAC molecules are present at nonmitochondrial locations in mammalian cells, these are unlikely to be the known products of the HVDAC1 or HVDAC2 genes.

51 citations


Journal ArticleDOI
TL;DR: An intracellular binding protein (FGFBP) for FGF-2 that undergoes a juxtanuclear localization coincident with the nuclear translocation of the growth factor during G1 of the cell cycle is characterized.
Abstract: Fibroblast growth factor-2 (FGF-2) is a potent autocrine mitogen for fetal epiphyseal growth plate chondrocytes and exhibits a transient nuclear translocation during G1 of the cell cycle. We have characterized an intracellular binding protein (FGFBP) for FGF-2 that undergoes a juxtanuclear localization coincident with the nuclear translocation of the growth factor. Chondrocytes were isolated from the proliferative zone of the ovine fetal proximal tibial growth plate at 50-130 days gestation by collagenase digestion and were maintained in monolayer at early passage number. Cells were growth restricted by serum starvation for 48 h, and the synchronized culture was restarted into the cell cycle in the presence of 2% FBS. Cells were removed between 4-26 h of incubation, and fractions representing the plasma membrane, cytoplasm, nuclear membrane, and nuclear contents were separated by differential centrifugation. FGFBPs were separated using FGF-2 affinity chromatography. Ligand blot analysis using 125I-labeled FGF-2 showed that a FGFBP of 46-48 kDa (represented by a double band) was present on the nuclear membrane at mid to late G1, and Western blot showed this to be immunologically related to a part of the extracellular domain of the high affinity FGF receptor 1 (FGFR1). Immunocytochemistry with intact cell cultures showed that this protein underwent a juxtanuclear distribution through mid to late G1. Immunoprecipitation was performed to monitor newly synthesized FGFR1 migration throughout the cell cycle. Synchronized cells were cultured in medium containing 35S-labeled methionine/cysteine, and the cellular compartments were separated before immunoprecipitation using an antibody raised against the extracellular domain of FGFR1. Newly synthesized FGFR1-related proteins appeared throughout G1 and migrated multidirectionally within the cell; intact receptor of 125-145 kDa accumulated at the plasma membrane, while both intact receptor and truncated FGFR1 of 46-48 kDa were detected on the nuclear membrane, but not within the nucleus. Cells were incubated with protamine sulfate to prevent the binding of endogenous, cell membrane-associated FGF-2 to high affinity FGFRs and their subsequent internalization. This did not alter the juxtanuclear accumulation of truncated FGFR1 in late G1, suggesting that this was not derived from the plasma membrane. The truncated FGFR1 may mediate the nuclear translocation of FGF-2 during late G1.

49 citations


Journal ArticleDOI
TL;DR: A very early expression of lipid body lipoxygenase was found by studying the rate of de novo synthesis of lip oxygengenase forms during germination and good agreement was observed between the amino acid sequence deduced from the cDNA sequence and the peptide structures analyzed biochemically.
Abstract: Lipid bodies are cellular compartments containing triacylglycerols. They are encompassed by a phospholipid monolayer and decorated with characteristic proteins. In plants, lipid bodies are synthesized during seed formation but acquire new proteins during seed germination. In germinating cucumber (Cucumis sativus) seeds, the set of newly synthesized proteins appearing in the lipid bodies at the early stage of triacylglycerol mobilization comprises a special form of lipoxygenase. We isolated the lipid body lipoxygenase and characterized fragments prepared by limited proteolysis and cleavage with cyanogen bromide. A very early expression of lipid body lipoxygenase was found by studying the rate of de novo synthesis of lipoxygenase forins during germination. This allowed a clear distinction of this enzyme from other lipoxygenase isoforms. Hence, for determining the molecular structure of lipid body lipoxygenase we analyzed a cDNA prepared from mRNA of cotyledons at day 1 of germination. From the cDNA sequence, oligonucleotides were derived that specifically detected lipid body lipoxygenase mRNA on nothern blots. The very early expression of lipid body lipoxygenase was corroborated by this approach. Good agreement was observed between the amino acid sequence deduced from the cDNA sequence and the peptide structures analyzed biochemically. In particular, the cleavage products of cyanogen bromide treatment indicated that we had isolated the lipid body lipoxygenase cDNA. The sequence data show a lipoxygenase form characterized by a molecular mass of 99655 Da, which is significantly higher than the molecular masses of the cytosolic forms. Compared to the cytosolic forms that exhibit a molecular mass of 95 kDa, the lipid body form has an N-terminal extension of 34 amino acid residues. No evidence for a cotranslational or post-translational proteolytic processing was obtained by the size comparison of the in vitro translated lipoxygenase and the lipid body form.

36 citations


Journal ArticleDOI
TL;DR: P pH within lysosomes and possibly other acidic cellular compartments of astrocytes is increased by cell swelling, which may have important consequences for astroCyte function.

34 citations


Journal ArticleDOI
TL;DR: A variety of in-vivo and in-Vitro approaches are beginning to reveal how this motility may be regulated, and identify which motors are involved in each function.

32 citations


Journal ArticleDOI
01 Aug 1996-Glia
TL;DR: The results suggest that M and L astrocytes are heterogeneous concerning the ability to synthesize GAGs and distribute them among the different cellular compartments and might be at least partially responsible for the differential effects of L and M glial cultures on the growth of midbrain neurons.
Abstract: Sulfated glycosaminoglycans (S-GAGs) were isolated from the pericellular (P), intracellular (I), and extracellular (E) compartments of astrocytes cultures from lateral (L) and medial (M) sectors of embryonic mouse midbrain; these sectors differ in their ability to support neurite growth (L, permissive, M, non-permissive for growth) and laminin deposition patterns (L, fibrillar; M, punctate pattern). The total amount of S-GAGs in M cultures was twice that in L cultures and was particularly high in the P compartment of M glia. Both glial cultures showed heparan sulfate (HS) in the three cellular compartments but chondroitin sulfate (CS) GAGs were vestigial in I and P compartments of L glia. Our results suggest that M and L astrocytes are heterogeneous concerning the ability to synthesize GAGs and distribute them among the different cellular compartments. Together with other data (Garcia-Abreu et al: J Neurosci Res 40:471, 1995; Garcia-Abreu et al: Neuroreport 6:761, 1995), the present results suggest that this heterogeneous features might be at least partially responsible for the differential effects of L and M glial cultures on the growth of midbrain neurons and may also be involved in complex ways in the guidance of axons at the brain midline.

Journal ArticleDOI
TL;DR: Immunoelectronmicroscopy of epimastigote, spheromastigotes, and metacyclic cells obtained in vitro showed anti-HSP60 reactive proteins in the mitochondria and near the kinetoplast and after heat shock all the three cell forms analyzed presented gold particles associated with the cellular membrane.

Journal ArticleDOI
TL;DR: The engineered Ca2+ sensitive photoprotein aequorin is engineered to monitor selectively [Ca2+] within defined subcellular compartments, namely the cytosol, nucleus and endoplasmic reticulum, and represents a novel method to monitor changes in free [Ca1+] in cellular organelles.

Book ChapterDOI
Y. Anraku1
01 Jan 1996
TL;DR: The importance of being acid in these cellular compartments by the function of vacuolar type H + -ATPases is presented where not only normal vegetative growth of cells, but also cell transformation and differentiation, metamorphosis, and apoptosis are regulated by thefunction and components of this unique family of Vacuolar-type H - ATPases.
Abstract: Publisher Summary This chapter describes the structure and function of the yeast vacuolar membrane H + -ATPase. The yeast vacuolar membrane H + -ATPase is the first member of a well defined “vacuolar type” ATPase family that has been successively identified in various endocytic and exocytic membrane compartments of eukaryotic cells. The vacuolar type H + -ATPase characterized thus far are multisubunit complexes composed of an integral membrane V 0 sector and a peripherally associated V 1 sector with similar subunit compositions and conserved functional motifs. The chapter discusses the reason vacuolar-type H + -ATPase are destined to be delivered to diverse organelles in different organisms. The eukaryotic vacuolar type H + -ATPase perform a diversity of functions in establishing and maintaining pH and cation homeostasis in the lumens of organelles, in the cytosol, and even in the extracellular milieu. The importance of being acid in these cellular compartments by the function of vacuolar type H + -ATPases is presented where not only normal vegetative growth of cells, but also cell transformation and differentiation, metamorphosis, and apoptosis are regulated by the function and components of this unique family of vacuolar type H + -ATPases.

Journal ArticleDOI
TL;DR: The results indicate that p35nck5a is a physiological activator of Cdk5 in immature neurons and further suggest that Cdk 5 has another function in mature neurons.

Journal ArticleDOI
TL;DR: The localization of heat shock messages in the processes of these neural cell types could provide a mechanism for local control of synthesis of heatshock proteins in cellular compartments that are remote from the cell body.
Abstract: Heat shock proteins are essential cellular proteins that may play important roles in cellular repair and/or protection. This report focuses on the expression of two members of the hsp70 multigene family, namely, constitutive hsc70 mRNA and stress-inducible hsp70 mRNA in the control and hyperthermic rabbit brain. The intracellular localization of these heat shock mRNAs was examined using high-resolution nonradioactive in situ hybridization. The distribution of hsc70 mRNA and hsp70 mRNA was examined in (1) neuronal cell bodies and their dendritic processes and (2) oligodendrocytes and their cellular processes. In control animals, hsc70 mRNA was detected in the apical dendritic processes and cell bodies of cortical layer II and V neurons, CA3 and CA4 neurons, deep cerebellar neurons, and brainstem neurons. A time course analysis of hsc70 mRNA, after a physiologically relevant increase in body temperature of 2.6 degrees C, revealed more distal transport of this constitutive message into dendrites of these neuronal populations. In the same neuronal populations, basal levels of hsp70 mRNA were observed in the cell body; however, this mRNA was not detected in dendritic processes in control or hyperthermic animals. After hyperthermia, hsp70 mRNA was strongly induced in oligodendrocytes and transported to the processes of these glial cells. The localization of heat shock messages in the processes of these neural cell types could provide a mechanism for local control of synthesis of heat shock proteins in cellular compartments that are remote from the cell body.

Journal ArticleDOI
TL;DR: Analysis of the distribution of cytoplasmic dynein and kinesin under these conditions indicates that immunolocalization data alone are not reliable indicators of sites of likely function for these microtubule-based motors.
Abstract: While immunolocalization methods have been used as a reasonable means to judge where a given molecule may be active in the cellular milieu, the correlation between distribution and function for proteins involved in intracellular transport may not be clear cut. To address the question of specificity and reproducibility of immunolocalization of microtubule-based motor proteins, we have co-localized cytoplasmic dynein and kinesin by immunofluorescence microscopy using two specific antibodies for each motor molecule. The results indicate that cytoplasmic dynein and kinesin appear to co-localize on a small subset of vesicles, but largely reside or accumulate on morphologically distinct organelles. In addition, anti-kinesin antibodies differing in their epitope specificity label different cellular compartments. To address the question of whether the distribution of motor molecules is representative of organelles that are undergoing active transport, we have altered the activity of vesicle trafficking pathways in fibroblasts using several different methods, including cytoplasmic acidification and disruption of cellular compartments with brefeldin A, nocodazole and okadaic acid. Analysis of the distribution of cytoplasmic dynein and kinesin under these conditions indicates that immunolocalization data alone are not reliable indicators of sites of likely function for these microtubule-based motors.

Journal ArticleDOI
TL;DR: Results show that GH is specifically taken up in the anterior pituitary by the somatotropes, lactotropes and gonadotropic cells, where, after binding to the plasma membrane, it is internalized into several cellular compartments, including the nucleus.
Abstract: In order to determine the processing of growth hormone (GH) by its pituitary target cells, male rats were injected intracardially with 125I-bGH and their pituitaries removed at specific time intervals (2–120 min). Autoradiographic analysis performed at the electron-microscopic level showed that only three cell populations specifically took up 125I-bGH: somatotropes, lactotropes and gonadotropes. Specificity was demonstrated by concomitant injection with an excess of unlabeled bGH. A time course study indicated that eight compartments had distinct labeling patterns. The plasma membrane was highly labeled after as little as 2 min, and showed biphasic labeling 2 and 60 min after injection. The secretory granules of the somatotropes were more intensely labeled than those of the other cell populations. The rough endoplasmic reticulum was more intensely labeled in the gonadotropes. The Golgi apparatus was specifically labeled only in the gonadotropes. The mitochondria showed the highest degree of labeling at 15 and 120 min after injection. The lysosome compartment showed triphasic labeling, with maxima at 2, 30 and 120 min after injection. The labeling of the nuclear membrane showed a biphasic pattern, firstly at 15 min, then at 120 min after injection, except in the gonadotropes, and the labeling in the nuclear matrix showed similar biphasic pattern and maxima. These results show that GH is specifically taken up in the anterior pituitary by the somatotropes, lactotropes and gonadotropes, where, after binding to the plasma membrane, it is internalized into several cellular compartments, including the nucleus. The differences in cellular localization and processing between these cell types may reflect different paracrine and autocrine roles for GH.

Journal ArticleDOI
TL;DR: This review will focus mainly on the observations that suggest that changes in the cytosolic demand for inorganic phosphate may participate in the selfregulation of phosphate transport and on some of the novel mechanisms by which this could occur.

Journal ArticleDOI
TL;DR: The results imply that phosphodiester double-stranded oligonucleotides can enter cellular compartments of interest for their potential biologic function and, thus, provide a potential tool to control gene transcription.
Abstract: The cellular binding, uptake, and intracellular distribution of structured double-stranded phosphodiester oligonucleotides (decoys) have been examined in T lymphocytes using fluorescein-labeled molecules. Intracellular localization of hairpin and dumbbell decoys was similar to that of single-stranded oligonucleotides. At short incubation times, oligonucleotides were localized only in cytoplasmic vesicles, whereas at longer times, they were also found in the nucleus. Cellular uptake was dependent on temperature, time, and extracellular concentration. Oligonucleotide efflux was similar for all types of molecules and was very rapid (t1/2 = 10-15 minutes). These results imply that phosphodiester double-stranded oligonucleotides can enter cellular compartments of interest for their potential biologic function and, thus, provide a potential tool to control gene transcription.

Journal ArticleDOI
TL;DR: The suggestion that the fragmentation of incoming SFV nucleocapsids in Aedes albopictus cells might be the part of the mechanism leading to the release of viral RNA into the cytosol during early stages of productive infection is suggested.
Abstract: The fate of Semliki Forest virus (SFV) nucleocapsid, especially the capsid protein (C-protein), was investigated during the early stages of a productive infection in mosquitoAedes albopictus cells. Infection of the cells resulted in a time dependent accumulation of a C-protein derived fragment. This fragmentation of incoming viral nucleocapsid was prevented by NH4Cl, an agent generally used to elevate the pH in acidic intracellular compartments, suggesting that a low intravesicular pH is required for this process. Density gradient analysis of the postnuclear cell lysate demonstrated that the fragmentation was associated with a cellular compartment showing a density of 1.14±0.02 g/ml. This cellular compartment was devoid from a lysosomal marker enzyme and represented the timely preceding cellular fraction through which SFV passed before encountering a lysosomal fraction. Furthermore, the intracellular distribution of the viral,3H-uridine-labeled RNA suggested that the same fraction might represent a key cellular compartment in which the separation of the viral RNA from the viral structural proteins is primed. In conclusion, these data lead to the suggestion that the fragmentation of incoming SFV nucleocapsids inAedes albopictus cells might be the part of the mechanism leading to the release of viral RNA into the cytosol during early stages of productive infection.

Book ChapterDOI
TL;DR: This chapter describes the recent advances in the understanding of some of the major mechanisms responsible for precise regulation of intracellular pH, and the functional significance of cytosolic and organellar pH.
Abstract: Publisher Summary The regulation of intracellular pH is of paramount importance in the maintenance of many cellular functions. Intracellular (cytosolic) pH is regulated at a level that is different from the extracellular pH. In addition, different compartments within the cell display pH values that differ by up to 3 units from the cytosolic pH. The prevalence of specific pH levels within the particular cellular compartments is not fortuitous. Precise control over intracellular pH, both cytosolic and intraorganellar, is critical to the proper functioning of the cell. Intracellular membrane traffic, endocytosis and receptor recycling, and cytosolic enzyme function are but a few of the myriad processes that are pH dependent. This chapter describes the recent advances in the understanding of some of the major mechanisms responsible for this precise regulation. In addition, a brief methodological section is also provided. Finally, the functional significance of cytosolic and organellar pH is discussed in some detail.