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Integrin and spectrin homologues, and cytoplasm-wall adhesion in tip growth.

Saprolegnia ferax contains an integrin homologue, identified by crossreactivity with antiserum to the consensus sequence of human/chick/Xenopus cytoplasmic domain beta 1-integrin, which is highly conserved. In non-reduced samples, this integrin was larger than the reported size range for beta 1-integrins, at 178 kDa. In reduced samples, there was a reducing agent-concentration-dependent conversion from 178 kDa to 120 kDa, well within the reported size range for beta 1-integrins in other organisms. The integrin antiserum stained plasma membrane-associated patches, which had a shallow tip-high gradient. This population was reduced and its distribution perturbed in hyphae whose growth rate was reduced by half with tetrapentyl ammonium chloride. The expected integrin function in cytoplasm-cell wall attachment was shown by differential resistance to plasmolysis-induced separation, which positively correlated with integrin abundance. However, when there was separation, remnants of cytoplasm stayed attached to the wall. These were enriched in actin and integrin. Saprolegnia also has a spectrin homologue identified by crossreactivity with an erythrocyte spectin antibody, which has a size (246 kDa) similar to other organisms. This spectrin had a superficially similar distribution to that of integrin, but it did not participate in cytoplasm-wall anchoring. These data suggest that Saprolegnia hyphae have a plasma membrane which is strengthened by spectrin, and cytoplasm which is attached to the cell wall by integrin.

Animals↗

A localisation signal in the 3' untranslated region of c-myc mRNA targets c-myc mRNA and beta-globin reporter sequences to the perinuclear cytoplasm and cytoskeletal-bound polysomes.

There is increasing evidence that in mammalian cells some mRNAs are localised to specific parts of the cytoplasm and a proportion of mRNAs and polyribosomes are associated with the cytoskeleton. It has been shown previously that c-myc mRNA is present in the perinuclear cytoplasm and associated with the cytoskeleton, and that this localisation is dependent upon the 3' untranslated region of the mRNA. The present studies show that in transfected fibroblasts the c-myc 3' untranslated region is able to localise beta-globin reporter sequences to the perinuclear cytoplasm. Studies with constructs containing deletions within the 3' untranslated region identify the region between bases 194 and 280 as critical for localisation. Transfection of cells with constructs in which this region is linked to beta-globin sequences showed that it was sufficient to localise the chimaeric transcripts to the perinuclear cytoplasm and to cytoskeletal-bound polyribosomes. Transfection with constructs containing a mutated AUUUA sequence within the 194-280 base region showed that this conserved AUUUA is required for targeting of both c-myc mRNA and a chimaeric transcript of beta-globin transcripts linked to the c-myc 3' untranslated region. The region between bases 194 and 280 did not induce instability of beta-globin transcripts and the AUUUA mutation had little effect upon mRNA stability. We propose that this 86 nt region of the 3' untranslated region contains a localisation signal to target c-myc mRNA so that it is retained on cytoskeletal-bound polysomes in the perinuclear cytoplasm; a conserved AUUUA sequence appears to be a critical part of this signal.

Animals↗

Dynamic association of cytoplasmic dynein heavy chain 1a with the Golgi apparatus and intermediate compartment.

Microtubule motors, such as the minus end-directed motor, cytoplasmic dynein, play an important role in maintaining the integrity, intracellular location, and function of the Golgi apparatus, as well as in the translocation of membrane between the endoplasmic reticulum and Golgi apparatus. We have immunolocalised conventional cytoplasmic dynein heavy chain to the Golgi apparatus in cultured vertebrate cells. In addition, we present evidence that cytoplasmic dynein heavy chain cycles constitutively between the endoplasmic reticulum and Golgi apparatus: it colocalises partially with the intermediate compartment, it is found on nocodazole-induced peripheral Golgi elements and, most strikingly, on Brefeldin A-induced tubules that are moving towards microtubule plus ends. The direction of movement of membrane between the endoplasmic reticulum and Golgi apparatus is therefore unlikely to be regulated by controlling motor-membrane interactions: rather, the motors probably remain bound throughout the whole cycle, with their activity being modulated instead. We also report that the overexpression of p50/dynamitin results in the loss of cytoplasmic dynein heavy chain from the membrane of peripheral Golgi elements. These results explain how dynamitin overexpression causes the inhibition of endoplasmic reticulum-to-Golgi transport complex movement towards the centrosomal region, and support the general model that an intact dynactin complex is required for cytoplasmic dynein binding to all cargoes.

Amino Acid Sequence↗

Roles of the cytoplasmic and transmembrane domains of syntaxins in intracellular localization and trafficking.

Syntaxins are target-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (t-SNAREs) involved in docking and fusion of vesicles in exocytosis and endocytosis. Many syntaxin isoforms have been isolated, and each one displays a distinct intracellular localization pattern. However, the signals that drive the specific intracellular localization of syntaxins are poorly understood. In this study, we used indirect immunofluorescence analysis to examine the localization of syntaxin chimeras, each containing a syntaxin transmembrane domain fused to a cytoplasmic domain derived from a different syntaxin. We show that the cytoplasmic domains of syntaxins 5, 6, 7 and 8 have important effects on intracellular localization. We also demonstrate that the transmembrane domain of syntaxin 5 is sufficient to localize the chimera to the compartment expected for wild-type syntaxin 5. Additionally, we find that syntaxins 6, 7 and 8, but not syntaxin 5, are present at the plasma membrane, and that these syntaxins cycle through the plasma membrane by virtue of their cytoplasmic domains. Finally, we find that di-leucine-based motifs in the cytoplasmic domains of syntaxins 7 and 8 are necessary for their intracellular localization and trafficking via distinct transport pathways. Combined, these results suggest that both the cytoplasmic and the transmembrane domains play important roles in intracellular localization and trafficking of syntaxins.

Amino Acid Sequence↗

The NITY motif of the beta-chain cytoplasmic domain is involved in stimulated internalization of the beta3 integrin A isoform.

Beta3 integrin adhesion molecules play important roles in wound repair and the regulation of vascular development and three beta3 integrin isoforms (beta3-A, -B, -C) have been described so far. Surface expression of beta3 integrins is dynamically regulated through internalization of beta3 integrins, however, the molecular mechanisms are understood incompletely. To evaluate the role of the cytoplasmic domain of beta3 integrins for internalization, we have generated single chain chimeras with variant and mutated forms of beta3 cytoplasmic domains. Upon transient transfection into chinese hamster ovary cells, it was found that the beta3-A chimera had strongly reduced cell surface expression compared with the corresponding beta3-B, or beta3-C fusion proteins, or the tail-less constructs, whereas steady state levels of all chimeras were near identical. Studies employing cytoplasmic domain mutants showed that the NITY motif at beta3-A 756-759 is critical for plasma membrane expression of beta3-A. Furthermore, delivery of beta3-A to the cell surface was specifically modulated by the cytoplasmic protein beta3-endonexin, a previously described intracellular protein. Coexpression of the native, long form of beta3-endonexin, which does not interact with the beta3 tail, acted as a dominant negative inhibitor of beta3-A-internalization and enhanced steady-state surface expression of the beta3-A-chimera. Furthermore, anti-beta3 antibody-induced internalization of the native beta3 integrin (alpha(IIb)beta3 was dramatically reduced for the Tyr(759)-Ala substitution mutant (alpha(IIb)beta3) (Y759A) and expression of the long isoform of beta3-endonexin substantially decreased the internalization of wild-type alpha(IIb)beta3. Thus, the NITY motif of the beta-chain cytoplasmic domain is involved in stimulated internalization of the beta3 integrin A isoform and beta3-endonexin appears to couple the beta3-A isoform to a specific receptor-recycling pathway.

Amino Acid Sequence↗

Relation of cytoplasmic calcium to contractility in Physarum polycephalum.

In a dumbbell-shaped plasmodium of Physarum polycephalum showing active shuttle streaming Ca was precipitated with potassium pyroantimonate (K[Sb(OH)6]), and the distribution of Ca between the cytoplasm and cellular organelles, especially vacuoles, was examined by electron microscopy. The contracting half-mass, where many empty vacuoles were present, was rich in the small Ca precipitates located in the cytoplasm. The relaxing half-mass, where many Ca-containing vacuoles were present, was poor in the cytoplasmic Ca precipitates. One half-mass of a dumbbell-shaped plasmodium was treated with Ca ionophore, X-537A, and its effect on the motive force for endoplasmic streaming and the distribution of Ca was investigated. The motive force was increased by X-537A, but the period of shuttle streaming was not changed. X-537A also induced a significant increase in the number of the cytoplasmic Ca precipitates in the X-537A-treated contracting half-mass, so that the asymmetry of the distribution of cytoplasmic Ca precipitates was enhanced. A large portion of the vacuoles were empty in the contracting half-mass, and Ca-containing in the relaxing one as in the case of the untreated plasmodium.

Calcium↗

Cytoplasmic organization during pattern formation in the alga Micrasterias.

Using an improved fixation for electron microscopy, we have observed an ordered cytoplasmic organization during pattern formation in the alga Micrasterias. At the earliest stages of morphogenesis (the bulge to 3-lobe stage), as observed previously, electron-dense vesicles and endoplasmic reticulum were found towards the cell periphery, while mitochondria and Golgi material were located internally. In contrast to previous studies, which found no organization in the cytoplasm of growing lobes, we observed a single cluster of electron-dense vesicles at the tip of each lobe at or beyond the 5-lobe stage. In lobes about to branch, two separate clusters of electron-dense vesicles were present in each lobe, apparently foreshadowing the sites of new lobe outgrowth. Individual lobes also possessed an ordered distribution of organelles. Immediately basal to the lobe tip, the concentration of electron-dense vesicles decreased and rough endoplasmic reticulum was more prevalent, followed more deeply in the cytoplasm by increased concentrations of mitochondria and then Golgi material. This cytoplasmic organization in individual lobes is characteristic of tip growth, thus suggesting that individual lobes elongate by this process. We have also investigated the effect of the ionophore A23187 on this cytoplasmic organization. The polarization of rough endoplasmic reticulum, mitochondria and Golgi material was destroyed by ionophore treatment in the presence of 2 mM-MgCl2 or 2 mM-CaCl2. The tip-associated clusters of electron-dense vesicles were dispersed by A23187 in 2 mM-CaCl2 but not significantly affected by A23187 in 2 mM-MgCl2. The latter observation indicates that the vesicle pattern can be disrupted by high calcium, but not high magnesium concentrations.

Calcimycin↗

Cytoplasmic domain phosphorylation of heparin-binding EGF-like growth factor.

Heparin-binding EGF-like growth factor (HB-EGF) is synthesized as a transmembrane precursor protein that is anchored to the plasma membrane. The extracellular EGF-like domain acts as a mitogen and motogen upon ectodomain shedding, but the functional roles of the transmembrane and cytoplasmic domains are largely unknown. We demonstrate here that cytoplasmic domain of HB-EGF is phosphorylated by external stimuli, and that the phosphorylation site is involved in HB-EGF-dependent tumorigenesis. Treatment of Vero cells overexpressing human HB-EGF with 12-O-tetradecanoylphorbol-13-acetate (TPA) caused ectodomain shedding of HB-EGF and generated two carboxyl (C)-terminal fragments with distinct electrophoretic mobilities. Mutation analysis showed that Ser207 in the cytoplasmic domain of HB-EGF is phosphorylated upon TPA stimulation, generating two C-terminal fragments with distinct phosphorylation states. Treatment of cells with lysophosphatidic acid, anisomycin, and calcium ionophore, all of which are known to induce ectodomain shedding, also caused phosphorylation of HB-EGF. Although ectodomain shedding and phosphorylation of HB-EGF occurred coordinately, Ala substitution of Ser207 had no effect on TPA-induced or constitutive ectodomain shedding. Injection of cells overexpressing HB-EGF into nude mice showed that Ala substitution of Ser207 reduced the tumorigenic activity of HB-EGF, even though the cell surface level and ectodomain shedding of HB-EGF were not affected by the mutation. Moreover, we found that the cytoplasmic domain of another EGFR ligand, transforming growth factor-alpha, is phosphorylated upon TPA stimulation. Thus, the present results suggest a novel role for the cytoplasmic domain of HB-EGF and other EGF family growth factors that is regulated by phosphorylation.

Alanine↗

QTL analysis of fertility-restoration against cytoplasmic male sterility in wheat.

The cytoplasm of Triticum timopheevi causes cytoplasmic male sterility (CMS) in common wheat (T. aestivum) cv. 'Chinese Spring' (CS), and that of Aegilops kotschyi causes CMS in spelt wheat (T. spelta) var. duhamelianum (Sp). CS has fertility-restoring (Rf) genes against the latter cytoplasm and Sp has the ones against the former. To know the genetic system concerning to CMS, we crossed 66 F8 recombinant inbred lines (RILs) derived from a cross between CS and Sp as males to the alloplasmic lines of CS and Sp having the cytoplasms of T. timopheevi and Ae. kotschyi, respectively. The fertilities of respective F1 plants derived from the crosses were examined for QTL analysis. The major QTLs detected in both systems were located on the short arm of chromosome 1B. One minor QTL on chromosome 2B was also commonly detected in both of the systems, while other minor QTLs against T timopheevi cytoplasm were distributed on the chromosomes 2A, 4B, and 6A.

Animals↗

Cytoplasmic staining of TTF-1 in the differential diagnosis of hepatocellular carcinoma vs cholangiocarcinoma and metastatic carcinoma of the liver.

The cytoplasmic staining of thyroid transcription factor (TTF)-1 was analyzed in 86 liver resection specimens, including 40 hepatocellular carcinoma (HCC), 4 metastatic HCC, 20 cholangiocarcinoma, 2 combined hepatocellular-cholangiocarcinoma (CHC), and 20 metastatic carcinoma (MC) specimens with immunohistochemical stains to TTF-1, cytokeratin (CK)19, hepatocyte paraffin 1, alpha-fetoprotein, polyclonal carcinoembryonic antigen, CK7, and CK20. TTF-1 cytoplasmic staining was identified in 93% of HCCs (37/40), 100% of metastatic HCCs (4/4), 10% of cholangiocarcinomas (2/20), and 5% of MCs (1/20). CK19 was positive in all cholangiocarcinomas and MCs but only in 5% of HCCs (2/40) and none of the metastatic HCCs (0/4). TTF-1 cytoplasmic staining positively correlates with differentiation and the trabecular growth pattern of HCC. The results suggest TTF-1 cytoplasmic staining, together with CK19, might serve as a useful marker for the diagnosis of primary and metastatic HCC and for the differential diagnosis of HCC from cholangiocarcinoma and MC. The mechanism of TTF-1 cytoplasmic staining is explored.

Adult↗

Investigation of the association between the presence of cytoplasmic residues on the human sperm midpiece and defective sperm function.

Defective sperm function has been identified as one of the most common causes of human infertility. The aim of this investigation was to identify whether the presence of retained cytoplasm on the human sperm midpiece is associated with defective sperm function. Statistical analysis of data demonstrated a strong negative correlation between the presence of residual cytoplasm on the midpiece of spermatozoa in the inseminate and fertilization rate during IVF. Significant negative correlations were also identified between the percentage of spermatozoa in the ejaculate bearing cytoplasmic residues and (i) spermatozoa having membrane integrity and (ii) sperm concentration. A highly significant positive correlation was also revealed between the percentage of spermatozoa in the ejaculate with membrane integrity and the percentage of motile spermatozoa. These correlations suggest that retained cytoplasm is a cause of subfertility. Measurements of the percentage of spermatozoa bearing residual cytoplasm in the IVF inseminate could provide the basis for a simple predictive test before IVF.

Cytoplasm↗

Effect of teosinte cytoplasmic genomes on maize phenotype.

Determining the contribution of organelle genes to plant phenotype is hampered by several factors, including the paucity of variation in the plastid and mitochondrial genomes. To circumvent this problem, evolutionary divergence between maize (Zea mays ssp. mays) and the teosintes, its closest relatives, was utilized as a source of cytoplasmic genetic variation. Maize lines in which the maize organelle genomes were replaced through serial backcrossing by those representing the entire genus, yielding alloplasmic sublines, or cytolines were created. To avoid the confounding effects of segregating nuclear alleles, an inbred maize line was utilized. Cytolines with Z. mays teosinte cytoplasms were generally indistinguishable from maize. However, cytolines with cytoplasm from the more distantly related Z. luxurians, Z. diploperennis, or Z. perennis exhibited a plethora of differences in growth, development, morphology, and function. Significant differences were observed for 56 of the 58 characters studied. Each cytoline was significantly different from the inbred line for most characters. For a given character, variation was often greater among cytolines having cytoplasms from the same species than among those from different species. The characters differed largely independently of each other. These results suggest that the cytoplasm contributes significantly to a large proportion of plant traits and that many of the organelle genes are phenotypically important.

Crosses, Genetic↗

Nuclear and cytoplasmic contributions to intraspecific divergence in an annual legume.

The genetic architecture of trait differentiation was evaluated between two ecologically distinct populations of Chamaecrista fasciculata. Individuals from Maryland and Illinois populations were crossed to create 10 types of seed: Maryland and Illinois parents, reciprocal F1 and F2 hybrids, and backcrosses to Maryland and to Illinois on reciprocal F1 hybrids. Reciprocal crosses created hybrid generation seeds with both Maryland and Illinois cytoplasmic backgrounds. Experimental individuals were grown in a common garden near the site of the Maryland population. In the garden, plants from the Illinois population flowered, set fruit, and died earlier than those from Maryland, likely reflecting adaptations to differences in growing season length between the two populations. Although reproductive components at the flower and whole plant level differed between the two populations, reproductive output as measured by fruit and seed production was similar. Cytoplasmic genes had a subtle but pervasive effect on population differentiation; hybrids with Maryland cytoplasm were significantly differentiated from those with Illinois cytoplasm when all characters were evaluated jointly. The nuclear genetic architecture of population differentiation was evaluated with joint scaling tests. Depending on the trait, both additive and nonadditive genetic effects contributed to population differentiation. Intraspecific genetic differentiation in this wild plant species appears to reflect a complex genetic architecture that includes the contribution of additive, dominance, and epistatic components in addition to subtle cytoplasmic effects.

Biological Evolution↗

Dose-related effects of non-steroidal antioestrogens nad oestrogens on the measurement of cytoplasmic oestrogen receptors in the rat and mouse uterus.

The dose-related effects of non-steroidal antioestrogens and oestrogens on the measurement of cytoplasmic oestrogen receptors in the rat uterus have been determined. The simultaneous administration of tamoxifen or monohydroxytamoxifen and oestradiol on three consecutive days resulted in dose-dependent decreases in both the wet weight of the uterus and the number of available cytoplasmic oestrogen receptors. The oestrogenic triphenylethylenes ICI 47 699 and ICI 3188 both produced dose-dependent decreases in the number of available cytoplasmic oestrogen receptors. Increasing doses of ICI 47 699 resulted in increasing concentrations of oestrogen receptors within the nucleus. The effects of tamoxifen and oestradiol-17 beta were compared in the ovariectomized mouse; replenishment of uterine oestrogen receptors was less evident in tamoxifen-treated animals than in animals receiving oestradiol, although increases in uterine weight were similar. A single large dose of tamoxifen (50 microgram) produced a prolonged depletion of cytoplasmic oestrogen receptors whilst stimulating rises in uterine weight and DNA and protein content. The results demonstrate that the depletion of the uterine cytoplasmic oestrogen receptor pool is a function of the dose administered for any compound with the ability to translocate oestrogen receptors to the nucleus and as such is not an exclusive characteristic of non-steroidal antioestrogens.

Animals↗

Electron microscope study on the bat testicular interstitial cell with special reference to the cytoplasmic crystalloid.

The testicular interstitial cell of the bat, Myotis schreibersi (Temmink), captured late in August was electron microscopically investigated. The cytoplasm of Myotis interstitial cells is packed by strikingly numerous mitochondria with many electron dense small matrix granules, endoplasmic reticulum and variable numbers of osmiophilic lipid droplets. The Myotis interstitial cell is characterized by alternate development of the SER and RER: In some Leydig cells the major part of the cytoplasm is packed by tubular SER, whereas in others this apparently is replaced by abundant free ribosomes containing sparse cisternae of the RER. These changes in the amounts of ser and RER are likely reversible and probably reflect functional alterations of Leydig cells of the bat, a hibernator. The Myotis interstitial cell possesses small cytoplasmic crystalloids composed of regularly oriented, compressed tubules, 20-28 m mu thick, which are continuous to the tubular SER (SER derived crystalloids). Type I and type II crystalloids are classified, which show, in suitable planes of section, honeycomb-like and fabric-like patterns, respectively. The Myotis Leydig cell extends cytoplasmic processes of irregular shape and long microvilli, the latter often conglomerates on the cell body or cytoplasmic processes. The entire surface of the cell including the processes is completely surrounded by a basal lamina.

Animals↗

Nucleo-cytoplasmic transport of proteins as a target for therapeutic drugs.

Recruitment of cytoplasmic signaling proteins into the nucleus is an essential step in the activation of gene expression in response to an extracellular signal. Nucleo-cytoplasmic transport of macromolecules is mediated by the transport receptors of an importin beta family. Post-translational modifications and masking/unmasking of specific signal sequences responsible for nuclear import and export are important for the coordinated control of the nucleo-cytoplasmic transport. Malfunctioning of the nucleo-cytoplasmic transport is profoundly involved in a number of diseases including cancer. Leptomycin B (LMB) is a Streptomyces metabolite that causes specific inhibition of the cell cycle of fission yeast and mammalian cells. The target molecule of LMB has been shown by genetic and biochemical analyses to be CRM1, a highly conserved protein in eukaryotes. CRM1 was shown to be a member of the importin beta family and a receptor for the nuclear export signal (NES) of proteins in both yeast and mammalian cells. LMB binds directly to CRM1, which results in dissociation of the NES from the nuclear export machinery containing CRM1. Thus, LMB serves as a potent tool for understanding the molecular mechanisms of nucleo-cytoplasmic transport of proteins and a potential therapeutic drug for diseases caused by mislocalization of regulatory proteins.

Active Transport, Cell Nucleus↗

Cytoplasmic genetic effects and growth of hybrid mice.

Variation in cellular biochemical functions controlled by cytoplasmic genes was studied in relation to phenotypic differences between progeny of reciprocal hybrid female mice. Least squares procedures were used to test for differences in mitochondrial respiratory metabolism and in capacity for ATP synthesis, and differences in growth of progeny of hybrid dams. Under identical nuclear influences, mitochondria of A/J and C57BL/6J cytoplasms differed (P less than .10 to P less than .01) from those of BALB/cJ cytoplasm in energy conservation. No differences were detected in mitochondrial efficiency between BALB/cJ cytoplasm evaluated in different nuclear environments. Three-way cross progeny of C57BL/6J x BALB/cJ reciprocal hybrid females mated to DBA/2J males differed (P less than .05) in litter weight at weaning and 1 wk and 2 wk postweaning. The F2 progeny of reciprocal C57BL/6J x BALB/cJ dams and F2 and three-way cross progeny of reciprocal A/J x BALB/cJ dams did not differ in weight at any age measured. Across all genotypes of dam, rank correlations of mitochondrial traits with F2 litter weights were nonsignificant. Observed variation in mitochondrial functions partially controlled by cytoplasmic genes did not limit mouse growth under these experimental conditions.

Adenosine Triphosphate↗

Stimulation of 36Cl- permeation in the in----out direction across the Dieters' neuron membrane by GABA on its cytoplasmic side: effect of different ionic conditions.

The permeation of labelled Cl- ions across single plasma membranes microdissected from rabbit Deiters' neuron was studied in a microchamber system. In particular, we studied 36Cl- in----out permeation and its stimulation by 10(-6) M GABA on the cytoplasmic side under different ionic conditions on both sides of the membrane. Three main results were found: a) The GABA effect turns up at a cytoplasmic side [Cl-], 21 mM, in the range of the normal steady state intracellular Cl- concentration. It disappears both at tracer level of Cl- on the cytoplasmic side and when [Cl-] is high there (140 mM). b) The increase in ionic strength due to the equinormal substitution of monovalent anions (Cl- and acetate) with the trivalent impermeant anion citrate on both sides of the membrane erases the GABA effect even if Cl- is at the optimal cytoplasmic side concentration, 21 mM. c) Citrate ions reduce to the limit of significance the GABA effect even when they are only on the membrane extracellular side and [Cl-] is at the optimal level on the other side. These results confirm that GABA stimulated Cl- permeation in the in----out direction is via Cl- channels exposing positive charges at their cytoplasmic mouth. In addition, they point out that such GABA activated channels are endowed with a partial electrical positivity at their extracellular exit.

Animals↗