Possible involvement of Class III phosphatidylinositol-3-kinase in meiotic progression of porcine oocytes beyond germinal vesicle stage

2011 ◽  
Vol 75 (5) ◽  
pp. 940-950 ◽  
Author(s):  
Myung Rae Park ◽  
Mukesh Kumar Gupta ◽  
Hye Ran Lee ◽  
Ziban Chandra Das ◽  
Sang Jun Uhm ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Meiotic Progression ◽  
Phosphatidylinositol 3

265 INHIBITION OF CLASS III PHOSPHATIDYLINOSITOL-3-KINASE, BY 3-METHYLADENINE, REVERSIBLY ARRESTS PORCINE OOCYTES AT GERMINAL VESICLE STAGE

2011 ◽  
Vol 23 (1) ◽  
pp. 230
Author(s):  
M. R. Park ◽  
M. K. Gupta ◽  
S. J. Uhm ◽  
S. T. Shin ◽  
Y. M. Han ◽  
...  
Keyword(s):  
Specific Inhibitor ◽  
Indirect Evidence ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Class Iii ◽  
Cleavage Rate ◽  
Chi Square ◽  
Meiotic Progression ◽  
Phosphatidylinositol 3

Phosphatidylinositol-3-kinases (PI3K) play pivotal roles in the meiotic progression of oocytes from metaphase I to metaphase II stage. This study evaluated the effect of 3-methyladenine (3MA), a specific inhibitor of Class III PI3K, on the meiotic progression of porcine oocytes. Immature porcine oocytes (n = 4744) retrieved from abattoir-derived oocytes were cultured in the absence or presence (10 mM) of 3MA for 22 h and evaluated for meiotic progression by florescent Hoechst 33342 staining. Data were analysed by chi-square test or ANOVA using SPSS software, and differences at P < 0.05 were considered significant. Results showed that 3MA treatment arrested the immature porcine oocytes at germinal vesicle (GV) stage in the presence (99.2 ± 0.8 v. 54.0 ± 10.1%) or absence (96.5 ± 1.8 v. 41.0 ± 17.6%) of cumulus cells. Furthermore, a significantly high proportion (60.9 ± 13.8%) of 3MA-treated oocytes acquired a nucleolus completely surrounded by a rim of highly condensed chromatin (GV-II stage). When immature oocytes, arrested at GV stage for 22 h by 3MA, were further cultured for 22 h in the absence of 3MA, 96.1 ± 1.5% of oocytes reached metaphase II stage at 42 h of in vitro maturation and did not differ (P > 0.05) from nontreated control oocytes with respect to their ability to fertilize, cleave (74.1 ± 1.2 v. 72.7 ± 2.8%), and form blastocyst (15.4 ± 1.5 v. 12.7 ± 0.6%) upon IVF or parthenogenetic activation (cleavage rate: 89.8 ± 1.7 v. 84.6 ± 5.1%; blastocyst rate: 44.3 ± 12.4 v. 45.1 ± 7.6%). These data suggest that 3MA reversibly blocks and synchronizes the meiotic progression of porcine oocytes at GV stage without affecting their ooplasmic maturation in terms of post-fertilization/activation in vitro embryonic development. Our data also provide indirect evidence for the likely participation of Class III PI3K in meiotic maturation of porcine oocytes beyond GV stage. This work was supported by grants (Code #200901FHT010305191 and #20070401034017) from BioGreen 21 program of RDA, Republic of Korea.

scholarly journals Beclin 1 Forms Two Distinct Phosphatidylinositol 3-Kinase Complexes with Mammalian Atg14 and UVRAG

2008 ◽  
Vol 19 (12) ◽  
pp. 5360-5372 ◽  
Author(s):  
Eisuke Itakura ◽  
Chieko Kishi ◽  
Kinji Inoue ◽  
Noboru Mizushima
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Pi3 Kinase ◽  
Beclin 1 ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Interacting Protein ◽  
Vacuolar Protein ◽  
Phosphatidylinositol 3

Class III phosphatidylinositol 3-kinase (PI3-kinase) regulates multiple membrane trafficking. In yeast, two distinct PI3-kinase complexes are known: complex I (Vps34, Vps15, Vps30/Atg6, and Atg14) is involved in autophagy, and complex II (Vps34, Vps15, Vps30/Atg6, and Vps38) functions in the vacuolar protein sorting pathway. Atg14 and Vps38 are important in inducing both complexes to exert distinct functions. In mammals, the counterparts of Vps34, Vps15, and Vps30/Atg6 have been identified as Vps34, p150, and Beclin 1, respectively. However, orthologues of Atg14 and Vps38 remain unknown. We identified putative mammalian homologues of Atg14 and Vps38. The Vps38 candidate is identical to UV irradiation resistance-associated gene (UVRAG), which has been reported as a Beclin 1-interacting protein. Although both human Atg14 and UVRAG interact with Beclin 1 and Vps34, Atg14, and UVRAG are not present in the same complex. Although Atg14 is present on autophagic isolation membranes, UVRAG primarily associates with Rab9-positive endosomes. Silencing of human Atg14 in HeLa cells suppresses autophagosome formation. The coiled-coil region of Atg14 required for binding with Vps34 and Beclin 1 is essential for autophagy. These results suggest that mammalian cells have at least two distinct class III PI3-kinase complexes, which may function in different membrane trafficking pathways.

scholarly journals Ca2+-regulated Pool of Phosphatidylinositol-3-phosphate Produced by Phosphatidylinositol 3-Kinase C2α on Neurosecretory Vesicles

2008 ◽  
Vol 19 (12) ◽  
pp. 5593-5603 ◽  
Author(s):  
Peter J. Wen ◽  
Shona L. Osborne ◽  
Isabel C. Morrow ◽  
Robert G. Parton ◽  
Jan Domin ◽  
...  
Keyword(s):  
Critical Role ◽  
Neurosecretory Cells ◽  
Secretory Vesicles ◽  
Endosomal Trafficking ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Product ◽  
Phosphatidylinositol 3

Phosphatidylinositol-3-phosphate [PtdIns(3)P] is a key player in early endosomal trafficking and is mainly produced by class III phosphatidylinositol 3-kinase (PI3K). In neurosecretory cells, class II PI3K-C2α and its lipid product PtdIns(3)P have recently been shown to play a critical role during neuroexocytosis, suggesting that two distinct pools of PtdIns(3)P might coexist in these cells. However, the precise characterization of this additional pool of PtdIns(3)P remains to be established. Using a selective PtdIns(3)P probe, we have identified a novel PtdIns(3)P-positive pool localized on secretory vesicles, sensitive to PI3K-C2α knockdown and relatively resistant to wortmannin treatment. In neurosecretory cells, stimulation of exocytosis promoted a transient albeit large increase in PtdIns(3)P production localized on secretory vesicles sensitive to PI3K-C2α knockdown and expression of PI3K-C2α catalytically inactive mutant. Using purified chromaffin granules, we found that PtdIns(3)P production is controlled by Ca2+. We confirmed that PtdIns(3)P production from recombinantly expressed PI3K-C2α is indeed regulated by Ca2+. We provide evidence that a dynamic pool of PtdIns(3)P synthesized by PI3K-C2α occurs on secretory vesicles in neurosecretory cells, demonstrating that the activity of a member of the PI3K family is regulated by Ca2+ in vitro and in living neurosecretory cells.

Modulation of phosphatidylinositol 3-kinase activity during in vitro oocyte maturation increases the production of bovine blastocysts

Zygote ◽  
2020 ◽  
Vol 28 (5) ◽  
pp. 371-376
Author(s):  
Janaína Leite Pereira ◽  
Alinne Glória Curcio ◽  
Laura Mathias Barroso ◽  
Edgar Mauricio Mogollón-Waltero ◽  
Helga Fernandes Gomes ◽  
...  
Keyword(s):  
Phosphatidylinositol 3 Kinase ◽  
Nuclear Maturation ◽  
Meiotic Progression ◽  
Pi3k Activity ◽  
Maturation Rate ◽  
Blastocyst Rate ◽  
20 Nm ◽  
Kinetics Of ◽  
Phosphatidylinositol 3

SummaryThis study aimed to evaluate the effect of regulating phosphatidylinositol 3-kinase (PI3K) activity on the kinetics of oocyte nuclear maturation and the blastocyst rate. To evaluate oocyte viability, nuclear maturation rate and in vitro embryo production, cumulus–oocyte complexes (COCs) were maintained for 0, 10 min, 6 h or 22 h in TCM 199 medium supplemented with 20 nM wortmannin, an inhibitor of PI3K. After each period, COCs were transferred to the same medium without wortmannin and kept under the same conditions until completion of 22 h of in vitro maturation (IVM). To evaluate the effect of time on progression of nuclear maturation, COCs cultivated with 20 nM wortmannin was maintained for 22, 28 or 34 h of IVM. To determine the effect of wortmannin on the activity of maturation-promoting factor (MPF), COCs were kept under IVM conditions in the presence of the inhibitor for 0, 1, 3, 6, or 8 h. Exposure of COCs to wortmannin decreased (P < 0.05) the percentage of oocytes that reached metaphase II (MII) up to 22 h, MPF activity and reduced PI3K activity by 30%. However, after 28 and 34 h, 70% of oocytes reached the MII stage in the presence of inhibitor Moreover, COCs matured in the presence of wortmannin showed an increase (P < 0.05) in the blastocyst rate. These findings suggested that the regulation of the PI3K activity during IVM of bovine COCs interfered with the meiotic progression due to control of MPF activity, positively affecting the blastocyst rate.

scholarly journals Dynamics and architecture of the NRBF2-containing phosphatidylinositol 3-kinase complex I of autophagy

2016 ◽  
Vol 113 (29) ◽  
pp. 8224-8229 ◽  
Author(s):  
Lindsey N. Young ◽  
Kelvin Cho ◽  
Rosalie Lawrence ◽  
Roberto Zoncu ◽  
James H. Hurley
Keyword(s):  
Complex I ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Negative Stain ◽  
Binding Factor ◽  
Hydrogen Deuterium ◽  
Negative Stain Electron Microscopy ◽  
Vacuolar Protein ◽  
Phosphatidylinositol 3

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is central to autophagy initiation. We previously reported the V-shaped architecture of the four-subunit version of PI3KC3-C1 consisting of VPS (vacuolar protein sorting) 34, VPS15, BECN1 (Beclin 1), and ATG (autophagy-related) 14. Here we show that a putative fifth subunit, nuclear receptor binding factor 2 (NRBF2), is a tightly bound component of the complex that profoundly affects its activity and architecture. NRBF2 enhances the lipid kinase activity of the catalytic subunit, VPS34, by roughly 10-fold. We used hydrogen–deuterium exchange coupled to mass spectrometry and negative-stain electron microscopy to map NRBF2 to the base of the V-shaped complex. NRBF2 interacts primarily with the N termini of ATG14 and BECN1. We show that NRBF2 is a homodimer and drives the dimerization of the larger PI3KC3-C1 complex, with implications for the higher-order organization of the preautophagosomal structure.

scholarly journals Phosphatidylinositol 3,5-bisphosphate plays a role in the activation and subcellular localization of mechanistic target of rapamycin 1

2012 ◽  
Vol 23 (15) ◽  
pp. 2955-2962 ◽  
Author(s):  
Dave Bridges ◽  
Jing-Tyan Ma ◽  
Sujin Park ◽  
Ken Inoki ◽  
Lois S. Weisman ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Target Of Rapamycin ◽  
Class Iii ◽  
Cell Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Mechanistic Target Of Rapamycin ◽  
Cell Type Specific ◽  
Stepwise Formation ◽  
Phosphatidylinositol 3

The kinase complex mechanistic target of rapamycin 1 (mTORC1) plays an important role in controlling growth and metabolism. We report here that the stepwise formation of phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) regulates the cell type–specific activation and localization of mTORC1. PI(3)P formation depends on the class II phosphatidylinositol 3-kinase (PI3K) PI3K-C2α, as well as the class III PI3K Vps34, while PI(3,5)P2 requires the phosphatidylinositol-3-phosphate-5-kinase PIKFYVE. In this paper, we show that PIKFYVE and PI3K-C2α are necessary for activation of mTORC1 and its translocation to the plasma membrane in 3T3-L1 adipocytes. Furthermore, the mTORC1 component Raptor directly interacts with PI(3,5)P2. Together these results suggest that PI(3,5)P2 is an essential mTORC1 regulator that defines the localization of the complex.

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