Expression and analysis of properties of septin CDCrel-1 in exocytosis.
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Animals↗
Biomedical subjects
X R Peng
Publications and source records attributed to X R Peng.
Focal exocytosis of VAMP3-containing vesicles at sites of phagosome formation.
Phagocytosis involves the receptor-mediated extension of plasmalemmal protrusions, called pseudopods, which fuse at their tip to engulf a particle. Actin polymerizes under the nascent phagosome and may propel the protrusion of pseudopods. Alternatively, membrane extension could result from the localized insertion of intracellular membranes into the plasmalemma next to the particle. Here we show focal accumulation of VAMP3-containing vesicles, likely derived from recycling endosomes, in the vicinity of the nascent phagosome. Using green fluorescent protein (GFP) as both a fluorescent indicator and an exofacial epitope tag, we show that polarized fusion of VAMP3 vesicles precedes phagosome sealing. It is therefore likely that targeted delivery of endomembranes contributes to the elongation of pseudopods. In addition to mediating pseudopod formation, receptor-triggered focal secretion of endosomes may contribute to polarized membrane extension in processes such as lamellipodial elongation or chemotaxis.
Animals↗
The diabetes autoantigen ICA69 and its Caenorhabditis elegans homologue, ric-19, are conserved regulators of neuroendocrine secretion.
ICA69 is a diabetes autoantigen with no homologue of known function. Given that most diabetes autoantigens are associated with neuroendocrine secretory vesicles, we sought to determine if this is also the case for ICA69 and whether this protein participates in the process of neuroendocrine secretion. Western blot analysis of ICA69 tissue distribution in the mouse revealed a correlation between expression levels and secretory activity, with the highest expression levels in brain, pancreas, and stomach mucosa. Subcellular fractionation of mouse brain revealed that although most of the ICA69 pool is cytosolic and soluble, a subpopulation is membrane-bound and coenriched with synaptic vesicles. We used immunostaining in the HIT insulin-secreting beta-cell line to show that ICA69 localizes in a punctate manner distinct from the insulin granules, suggesting an association with the synaptic-like microvesicles found in these cells. To pursue functional studies on ICA69, we chose to use the model organism Caenorhabditis elegans, for which a homologue of ICA69 exists. We show that the promoter of the C. elegans ICA69 homologue is specifically expressed in all neurons and specialized secretory cells. A deletion mutant was isolated and found to exhibit resistance to the drug aldicarb (an inhibitor of acetylcholinesterase), suggesting defective neurotransmitter secretion in the mutant. On the basis of the aldicarb resistance phenotype, we named the gene ric-19 (resistance to inhibitors of cholinesterase-19). The resistance to aldicarb was rescued by introducing a ric-19 transgene into the ric-19 mutant background. This is the first study aimed at dissecting ICA69 function, and our results are consistent with the interpretation that ICA69/RIC-19 is an evolutionarily conserved cytosolic protein participating in the process of neuroendocrine secretion via association with certain secretory vesicles.
Amino Acid Sequence↗
VAMP2, but not VAMP3/cellubrevin, mediates insulin-dependent incorporation of GLUT4 into the plasma membrane of L6 myoblasts.
Like neuronal synaptic vesicles, intracellular GLUT4-containing vesicles must dock and fuse with the plasma membrane, thereby facilitating insulin-regulated glucose uptake into muscle and fat cells. GLUT4 colocalizes in part with the vesicle SNAREs VAMP2 and VAMP3. In this study, we used a single-cell fluorescence-based assay to compare the functional involvement of VAMP2 and VAMP3 in GLUT4 translocation. Transient transfection of proteolytically active tetanus toxin light chain cleaved both VAMP2 and VAMP3 proteins in L6 myoblasts stably expressing exofacially myc-tagged GLUT4 protein and inhibited insulin-stimulated GLUT4 translocation. Tetanus toxin also caused accumulation of the remaining C-terminal VAMP2 and VAMP3 portions in Golgi elements. This behavior was exclusive to these proteins, because the localization of intracellular myc-tagged GLUT4 protein was not affected by the toxin. Upon cotransfection of tetanus toxin with individual vesicle SNARE constructs, only toxin-resistant VAMP2 rescued the inhibition of insulin-dependent GLUT4 translocation by tetanus toxin. Moreover, insulin caused a cortical actin filament reorganization in which GLUT4 and VAMP2, but not VAMP3, were clustered. We propose that VAMP2 is a resident protein of the insulin-sensitive GLUT4 compartment and that the integrity of this protein is required for GLUT4 vesicle incorporation into the cell surface in response to insulin.
Actins↗
Prolactin regulation of tissue type plasminogen activator and plasminogen activator inhibitor type-I gene expression in eCG-primed rat granulosa cells in culture.
The present study was designed to investigate the effect of prolactin (PRL) on plasminogen activator inhibitor-I (PAI-I) and tissue type plasminogen activator (tPA) gene expression in eCG-primed granulosa cells in vitro. At 46 h after the hormone treatment, ovaries were removed, and granulosa cells were prepared for culture. Cells were incubated for various times in serum-free medium in the presence or absence of LH and PRL alone or in combination. tPA and PAI-I activities in the media were assayed by fibrin overlay and reverse fibrin autograph, respectively. Cytoplasmic RNA from granulosa cells was prepared using the NP-40 method and was assayed for PAI-I and tPA mRNA levels. We demonstrated the following. 1) PRL increased PAI-I mRNA production in cultured granulosa cells. Inclusion of LH with PRL had a synergistic effect on increasing PAI-I mRNA levels. After 48-h culture, 3-fold increases in PAI-I mRNA levels were seen with LH in combination with PRL as compared with PRL alone. The synergistic increase in PAI-I mRNA levels occurred in a dose- and time-dependent manner. 2) The increase in PAI-I mRNA synthesis by PRL alone, or by PRL in combination with LH, was well correlated with the changes in PAI-I activity and antigen levels in the conditioned media. 3) PRL in the culture also dramatically decreased LH-induced tPA mRNA and activity in a dose- and time-dependent fashion. The decrease in the tPA activity by PRL was also correlated with an increase in the amount of PA-PAI-I complexes in the cell-conditioned media. 4) In situ hybridization of tPA and PAI-I mRNAs in the cultured granulosa cells also showed that PRL was capable of enhancing PAI-I mRNA while diminishing tPA mRNA production induced by LH. This suggests that the dose- and time-dependent decrease in the gonadotropin-induced tPA activity in the culture by the presence of PRL may be due to decreasing tPA mRNA synthesis on one hand and to neutralization of the tPA activity by the increased PAI-I activity on the other.
Animals↗
Effect of mutations in vesicle-associated membrane protein (VAMP) on the assembly of multimeric protein complexes.
The assembly of multimeric protein complexes that include vesicle-associated membrane protein 2 (VAMP-2) and the plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) are thought to reflect the biochemical correlates of synaptic vesicle targeting, priming, or fusion. Using a variety of protein-protein interaction assays and a series of deletion and point mutations, we have investigated the domains of VAMP-2 required for the formation of binary complexes with either syntaxin 1A or SNAP-25 and ternary complexes with both syntaxin 1A and SNAP-25. Deletions within the central conserved domain of VAMP-2 eliminated binding to either syntaxin 1A or both syntaxin 1A and SNAP-25. Although all of the deletion mutants were able to form ternary complexes, only some of these complexes were resistant to denaturation in sodium dodecyl sulfate. These results demonstrate that cooperative interactions result in the formation of at least two biochemically distinct classes of ternary complex. Two point mutations previously shown to have effects on the intracellular trafficking of VAMP-2 (M46A, reduced endocytosis and sorting to synaptic vesicles; N49A, enhanced sorting to synaptic vesicles) lie within a domain required for both syntaxin 1A and SNAP-25 binding. Syntaxin 1A and SNAP-25 binding was reduced by the M46A mutation and enhanced by the N49A mutation, suggesting that a correlation exists between the membrane-trafficking phenotype of the two VAMP-2 point mutants and their competence to form complexes with either syntaxin 1A or SNAP-25.
Animals↗
Association of syntaxin 3 and vesicle-associated membrane protein (VAMP) with H+/K(+)-ATPase-containing tubulovesicles in gastric parietal cells.
H+/K(+)-ATPase is the proton pump in the gastric parietal cell that is responsible for gastric acid secretion. Stimulation of acid secretion is associated with a reorganization of the parietal cells resulting in the incorporation of H+/K(+)-ATPase from a cytoplasmic membrane pool, the tubulovesicle compartment, into the apical canalicular membrane. To better characterize the role of membrane trafficking events in the morphological and physiological changes associated with acid secretion from parietal cells, we have characterized the expression and localization of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in these cells. Each of the six different SNARE proteins examined [syntaxins 1 through 4 of 25-kDa synaptosome-associated protein, and vesicle-associated membrane protein] were found to be expressed in parietal cells. Furthermore, two of these SNAREs, vesicle-associated membrane protein and syntaxin 3, were associated with H+/K(+)-ATPase-containing tubulovesicles while the remainder were excluded from this compartment. The expression of syntaxin 1 and synaptosome-associated protein of 25 kDa in parietal cells, two SNAREs previously thought to be restricted to neuroendocrine tissues, suggests that parietal cells may utilize membrane trafficking machinery that is similar to that utilized for regulated exocytosis in neurons. Furthermore, the localization of syntaxin 3, a putative target membrane SNARE, to the tubulovesicle compartment indicates that syntaxin 3 may have an alternative function. These observations support a role for intracellular membrane trafficking events in the regulated recruitment of H+/K(+)-ATPase to the plasma membrane after parietal cell stimulation.
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Prolactin delays gonadotrophin-induced ovulation and down-regulates expression of plasminogen-activator system in ovary.
This study was conducted to determine whether prolactin (PRL) suppresses gonadotrophin-induced ovulation and disturbs the co-ordinated gene expression of tissue type plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAI-1) in rat ovary. Immature female rats were injected with 10 IU pregnant mare's serum gonadotrophin to stimulate follicle growth, and 48 h received different doses of prolactin followed by 7 IU human chorionic gonadotrophin (HCG). The oviducts were examined for the presence of ova, and the amounts of tPA and PAI-1 mRNA present in the ovary were measured at various times after the hormone treatment. PRL had no significant effect on ovarian weight but caused a dose-dependent decrease in ovulation number. In the control animals receiving HCG alone, 13.3 +/- 1.3 (mean +/- SEM) ova/oviduct were found; while in animals receiving HCG plus 50, 100 or 200 microg PRL, the ovulation number was dose-dependently suppressed by 53.6, 66.9 and 76% respectively at 18 h after treatment. PRL suppression of HCG-induced ovulation was time-dependent. By 24 h after treatment, the number of ova in the oviducts in HCG- and HCG plus PRL-treated groups was not significantly different. PRL also suppressed HCG-induced tPA gene expression in a dose- and time-dependent manner. At all time points examined, tPA mRNA content of whole ovaries and granulosa cells (GC) in PRL-treated groups was lower than in the HCG-treated controls. The activities of PAI-1 in ovarian extracellular fluid (OEF) and PAI-1 mRNA in the theca-interstitial cells (TI) in the PRL-treated groups were higher than in the HCG-treated controls. The highest stimulation by PRL of PAI-1 activity in OEF and of PAI-1 mRNA in TI was observed at 9 h and 6 h after HCG treatment respectively. The localization of tPA and PAI-1 antigens in the ovaries was consistent with changes in the mRNA and activity levels. These data suggest that PRL temporarily delays, but does not completely inhibit, HCG-induced ovulation, which may be caused by a suppression of PA-mediated proteolysis.
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Ovulation efficiency is reduced in mice that lack plasminogen activator gene function: functional redundancy among physiological plasminogen activators.
Several lines of indirect evidence suggest that plasminogen activation plays a crucial role in degradation of the follicular wall during ovulation. However, single-deficient mice lacking tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), or PA inhibitor type 1(PAI-1) gene function were recently found to have normal reproduction, although mice with a combined deficiency of tPA and uPA were significantly less fertile. To investigate whether the reduced fertility of mice lacking PA gene function is due to a reduced ovulation mechanism, we have determined the ovulation efficiency in 25-day-old mice during gonadotropin-induced ovulation. Our results reveal that ovulation efficiency is normal in mice with a single deficiency of tPA or uPA but reduced by 26% in mice lacking both physiological PAs. This result suggests that plasminogen activation plays a role in ovulatory response, although neither tPA nor uPA individually or in combination is obligatory for ovulation. The loss of an individual PA seems to be functionally complemented by the remaining PA but this compensation does not appear to involve any compensatory up-regulation. Our data imply that a functionally redundant mechanism for plasmin formation operates during gonadotropin-induced ovulation and that PAs together with other proteases generate the proteolytic activity required for follicular wall degradation.
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Coordinated regulation of tissue type plasminogen activator and plasminogen activator inhibitor type-1 gene expression in hypophysectomized rat ovaries during GnRHa-induced ovulation.
In this study we have demonstrated that both granulosa and theca-interstitial cells of hypophysectomized rat ovaries are capable of synthesizing tPA and PAI-1. Injection of a GnRH agonist can markedly induce these gene expressions in the ovary in a cell-specific and time-coordinated manner, so that a surge of tPA mRNA and its activity in both granulosa and theca-interstitial cells was obtained just prior to ovulation. Theca-interstitial cells make PAI-1 become the most active in the ovary. Both the amount PAI-1 mRNA and its activity in the cells reach the maximum level 6 h before the tPA peak. By contrast, granulosa cells produce only a little amount of PAI-1 (most increase tPA activity), and both PAI-1 mRNA and activity in the cells reach the maximum after ovulation. The coordinated regulation of tPA and PAI-1 in the ovary may fine-tune the peak of tPA activity which may be important for the regulation of the ovulatory process. The changes of tPA and PAI-1 in the ovarian cells of hypophysectomized rats during GnRHa-induced ovulation are similar to that in intact rats during hCG-induced ovulation, suggesting that the ovulatory process can be modulated by different regulatory signals mediated by influencing the coordinated expression of both tPA and PAI-1.
Animals↗
Secretion of plasminogen activator inhibitor type 1 by cultured ovarian cells obtained from gonadotropin-treated immature rats.
It is demonstrated that i) theca-interstitial compartment synthesizes the majority of plasminogen activator inhibitor type 1 (PAI-1) in the ovary before ovulation, and the follicular wall may therefore serve as a specific barrier with the presence of PAI-1 activity to prevent the secretion of tPA into the extrafollicular compartments; ii) granulosa cells secrete only a small amount of ovarian PAI-1, but synthesize the most of tissue-type plasminogen activator tPA involved in the processes leading to ovulation; iii) since only matured cumulus-oocyte complexes secrete a large amount of tPA and PAI-1, both tPA and PAI-1 activity in the conditioned medium may be used as reliable markers for evaluating oocyte quality for in vitro fertilization.
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Hormonal regulation of expression of tissue type plasminogen activator and plasminogen activator inhibitor type 1 in cultured rat granulosa cells.
In the present study, gonadotropin and gonadotropin-releasing hormone (GnRH) regulation of tPA and PAI-1 expression in PMSG-primed granulosa cells has been investigated. (i) Addition of gonadotropins (FSH and LH) and GnRH agonist (GnRHa) or PMA to the culture increases tPA activity; FSH (or LH) plus GnRHa (or PMA) in the culture further enhances the enzyme production to such an extent that a more obvious effect than the additive effect caused by these hormones used alone has been observed; (ii) in contrast, FSH and LH decrease PAI-1 activity, whereas GnRHa and PMA alone markedly increase PAI-1 mRNA level and PAI-1 activity. Because FSH and LH stimulate tPA production and have no significant effect on PAI-1 mRNA induction, the observed inhibition of PAI-1 activity by gonadotropins may be due to the occurrence of neutralization of PA and PAI-1 proteins in the conditioned media by the formation of complexes between PA and PAI-1; (iii) increases in PAI-1 mRNA level and activity by GnRH and PMA are completely inhibited by the co-addition of FSH or LH to the culture. It is, therefore, suggested that the mechanism of gonadotropin and GnRHa regulation of tPA and PAI-1 expression in granulosa cells is different.
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Hormonal regulation of the fibrinolytic components in the ovary.
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Animals↗
Transient and cell-specific expression of tissue-type plasminogen activator and plasminogen-activator-inhibitor type 1 results in controlled and directed proteolysis during gonadotropin-induced ovulation.
Proteolytic activity generated by the plasminogen-activator system (PA system) is associated with many biological processes. However, it is not known how the proteolytic activity is regulated in vivo in order to obtain directed proteolysis while, at the same time, protecting unrestrained tissue destruction. Using gonadotropin-induced ovulation as a model, we have studied how two components of the PA system, tissue-type plasminogen activator (tPA) and plasminogen-activator-inhibitor type 1 (PAI-1), are regulated temporally and spatially by gonadotropins, leading to the initiation and termination of a well-directed proteolytic process. In-situ hybridization and in-situ zymography were used to analyze the expression of tPA and PAI-1 mRNA and PA-activity in specific ovarian cell types. Both tPA and PAI-1 were found to be regulated and to have a distinct expression pattern in different ovarian compartments. tPA was expressed in both granulosa and thecal-interstitial cells; the highest levels of tPA mRNA were found in the granulosa cells of preovulatory follicles, just prior to ovulation. Consistent with a role for luteinizing hormone/chorionic gonadotropin (LH/CG) in triggering ovulation, the cells and follicles that actively expressed tPA also contained high levels of LH-receptor mRNA while cumulus cells that contain undetectable amounts of tPA mRNA were devoid of LH-receptor expression. The highest levels of PAI-1 mRNA were found about 6 h before ovulation and mainly in the thecal-interstitial cells and ovarian stroma tissue which encapsulate the follicle. Preovulatory follicles, protruding onto the surface of the ovary with less surrounding stroma tissue, expressed less PAI-1 compared to small non-ovulatory follicles embedded in inner part of the ovary. In-situ zymography also revealed that the PA activity was colocalized to the surface of the ovary just prior to ovulation. Our studies suggest that a proteolytic activity provided by tPA and modulated by PAI-1 is responsible for a controlled and directed proteolysis leading to rupture of selected follicles during ovulation.
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Tissue-specific and time-coordinated hormone regulation of plasminogen-activator-inhibitor type I and tissue-type plasminogen activator in the rat ovary during gonadotropin-induced ovulation.
The plasminogen-activator system provides proteolytic activity in many biological processes. The regulation of plasminogen activation may occur at many levels including the synthesis and secretion of plasminogen activators (PA) and the specific inhibition of PA activity by inhibitors. PA-inhibitor type-1 (PAI-1) is an efficient inhibitor of tissue-type PA (tPA) and urokinase-type PA (uPA) that may therefore be instrumental for the control of plasminogen activation. To investigate if coordinated regulation of PA and PA inhibitors take place in vivo in response to physiological signals, we have examined the regulation of PAI-1 and tPA in the ovary during gonadotropin-induced ovulation. We found that PAI-1, as well as tPA activity and mRNA levels, were coordinately regulated by gonadotropins in a time-dependent and cell-specific manner, such that a surge of PA-activity was obtained just prior to ovulation. Both theca-interstitial and granulosa cells synthesized PAI-1, but their maximal PAI-1 expression occurred at different times during the periovulatory period, ensuring inhibition of proteolytic activity in ovarian extra cellular compartments both before and after ovulation. The coordinated regulation of tPA and PAI-1 in the ovary may fine-tune the peak of PA activity which may be important for the regulation of the ovulatory process.
Animals↗
Hormone regulation of tissue-type plasminogen activator gene expression and plasminogen activator-mediated proteolysis.
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Animals↗
Localization of luteinizing hormone receptor messenger ribonucleic acid expression in ovarian cell types during follicle development and ovulation.
The action of LH is mediated through specific plasma membrane receptors that are both up- and down-regulated in the ovary during the reproductive cycle. Using immature rats treated with PMSG and hCG as a model system, we have studied the regulation and distribution of LH receptor mRNA in different cell types during follicle development, ovulation, and luteinization by Northern blot and in situ hybridization. In untreated rats, LH receptor mRNA was below the detection level in granulosa cells, cumulus cells, and oocytes, while low levels of LH receptor mRNA were found in the thecal cells. After stimulation with PMSG, expression of LH receptor mRNA was enhanced in the thecal-interstitial cells, while a more dramatic increase in receptor mRNA abundance took place in granulosa cells of large tertiary follicles. In these follicles, the abundance of LH receptor mRNA varied among different subpopulations of granulosa cells, with mural granulosa cells close to the basement membrane exhibiting higher levels than granulosa cells located closer to the antrum, and cumulus cells and the oocyte lacking detectable hybridization signal. The uneven expression of LH receptor mRNA endows different ovarian cells with varying hormonal responsiveness. After an ovulatory dose of hCG, LH receptor mRNA levels were dramatically decreased, particularly in the granulosa cells of preovulatory follicles, to reach the lowest levels just before ovulation. During the transformation of ovulated follicles into corpora lutea, the expression of LH receptor message was again increased. Our results reveal that the previously documented regulation of the LH receptor-binding activity during ovarian development correlates with expression of the LH receptor transcripts, suggesting that the LH receptor gene is regulated in a complex manner during the periovulatory period to achieve cell-specific expression together with gonadotropin induction and suppression of receptor gene activity.
Animals↗
Tissue-type plasminogen activator in rat oocytes: expression during the periovulatory period, after fertilization, and during follicular atresia.
The regulation of tissue-type plasminogen activator (tPA) in rat oocytes during the periovulatory period, in early embryos, and in oocytes during induced follicular atresia was studied using a quantitative chromogenic substrate assay. Oocytes and early embryos were collected from three ovulation models: 1) intact immature female rats treated with PMSG, followed by hCG 48 h later; 2) hypophysectomized immature rats treated with PMSG, followed by a GnRH agonist (GnRHa) 56 h later; and 3) adult cyclic rats on the mornings of proestrus and estrus and up to 5 days after fertilization. In addition, follicular atresia was induced by either withdrawal of diethylstilbestrol (DES) for 2 days or injection of GnRHa for 2 days in hypophysectomized DES-implanted immature rats. Treatment with PMSG alone did not increase oocyte tPA content (5-20 microIU/oocyte) in either immature rat model, but treatment with either hCG or GnRHa induced meiotic maturation and ovulation and increased tPA activity to 80 and 140 microIU/oocyte 24 h after hCG and GnRHa treatment, respectively. Northern blot analysis of total RNA extracted from oocytes of PMSG-treated rats indicated the presence of a specific tPA message at 22S. tPA levels were low in preovulatory oocytes obtained on proestrus morning and increased in ovulated oocytes on estrus morning. After fertilization, tPA levels remained high in the embryos on days 1-4 of pregnancy, but dropped dramatically on day 5. Furthermore, oocytes from atretic follicles of hypophysectomized DES-implanted rats after either DES withdrawal or GnRHa treatment contained elevated levels of tPA, coincident with germinal vesicle breakdown (GVBD). Immunohistochemical staining revealed tPA antigen only in those oocytes that had undergone apparent meiotic maturation, as confirmed by GVBD. Thus, oocytes contain tPA mRNA and synthesize the active protease under a variety of stimuli which result in GVBD. The observed periovulatory increase in oocyte tPA activity, its maintenance until day 5 of pregnancy, and expression of tPA in nonovulatory oocytes of atretic follicles suggest diverse functions for the oocyte and embryo tPA.
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