Polarized Distribution of the Trans-Golgi Network Marker TGN38 During the In Vitro Development of Neocortical Neurons: Effects of Nocodazole and Brefeldin A

1994 ◽  
Vol 6 (9) ◽  
pp. 1453-1465 ◽  
Author(s):  
P. R. Lowenstein ◽  
E. E. Morrison ◽  
D. Bain ◽  
A. F. Shering ◽  
G. Banting ◽  
...  
Keyword(s):  
Brefeldin A ◽  
In Vitro Development ◽  
Trans Golgi Network ◽  
Neocortical Neurons ◽  
Golgi Network ◽  
Vitro Development

scholarly journals Clathrin-dependent Association of CVAK104 with Endosomes and the Trans-Golgi Network

2006 ◽  
Vol 17 (10) ◽  
pp. 4513-4525 ◽  
Author(s):  
Michael Düwel ◽  
Ernst J. Ungewickell
Keyword(s):  
Fluorescent Protein ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Terminal Segment ◽  
Coated Vesicle ◽  
Membrane Association ◽  
Permeabilized Cells ◽  
Trans Golgi Network ◽  
Golgi Network

CVAK104 is a novel coated vesicle-associated protein with a serine/threonine kinase homology domain that was recently shown to phosphorylate the β2-subunit of the adaptor protein (AP) complex AP2 in vitro. Here, we demonstrate that a C-terminal segment of CVAK104 interacts with the N-terminal domain of clathrin and with the α-appendage of AP2. CVAK104 localizes predominantly to the perinuclear region of HeLa and COS-7 cells, but it is also present on peripheral vesicular structures that are accessible to endocytosed transferrin. The distribution of CVAK104 overlaps extensively with that of AP1, AP3, the mannose 6-phosphate receptor, and clathrin but not at all with its putative phosphorylation target AP2. RNA interference-mediated clathrin knockdown reduced the membrane association of CVAK104. Recruitment of CVAK104 to perinuclear membranes of permeabilized cells is enhanced by guanosine 5′-O-(3-thio)triphosphate, and brefeldin A redistributes CVAK104 in cells. Both observations suggest a direct or indirect requirement for GTP-binding proteins in the membrane association of CVAK104. Live-cell imaging showed colocalization of green fluorescent protein-CVAK104 with endocytosed transferrin and with red fluorescent protein-clathrin on rapidly moving endosomes. Like AP1-depleted COS-7 cells, CVAK104-depleted cells missort the lysosomal hydrolase cathepsin D. Together, our data suggest a function for CVAK104 in clathrin-dependent pathways between the trans-Golgi network and the endosomal system.

Interaction of amphiphysins with AP-1 clathrin adaptors at the membrane

2013 ◽  
Vol 450 (1) ◽  
pp. 73-83 ◽  
Author(s):  
Sonja Huser ◽  
Gregor Suri ◽  
Pascal Crottet ◽  
Martin Spiess
Keyword(s):  
Plasma Membrane ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Trans Golgi Network ◽  
Src Homology 3 ◽  
Amphiphysin 1 ◽  
Src Homology ◽  
Golgi Network

The assembly of clathrin/AP (adaptor protein)-1-coated vesicles on the trans-Golgi network and endosomes is much less studied than that of clathrin/AP-2 vesicles at the plasma membrane for endocytosis. In vitro, the association of AP-1 with protein-free liposomes had been shown to require phosphoinositides, Arf1 (ADP-ribosylation factor 1)–GTP and additional cytosolic factor(s). We have purified an active fraction from brain cytosol and found it to contain amphiphysin 1 and 2 and endophilin A1, three proteins known to be involved in the formation of AP-2/clathrin coats at the plasma membrane. Assays with bacterially expressed and purified proteins showed that AP-1 stabilization on liposomes depends on amphiphysin 2 or the amphiphysin 1/2 heterodimer. Activity is independent of the SH3 (Src homology 3) domain, but requires interaction of the WDLW motif with γ-adaptin. Endogenous amphiphysin in neurons and transfected protein in cell lines co-localize perinuclearly with AP-1 at the trans-Golgi network. This localization depends on interaction of clathrin and the adaptor sequence in the amphiphysins and is sensitive to brefeldin A, which inhibits Arf1-dependent AP-1 recruitment. Interaction between AP-1 and amphiphysin 1/2 in vivo was demonstrated by co-immunoprecipitation after cross-linking. These results suggest an involvement of amphiphysins not only with AP-2 at the plasma membrane, but also in AP-1/clathrin coat formation at the trans-Golgi network.

In vitro development of human triploid zygotes reconstructed by pronuclear transfer

2002 ◽  
Vol 78 ◽  
pp. S180-S181
Author(s):  
John Zhang ◽  
Yi Ming Shu ◽  
Lewis C Krey ◽  
Hui Liu ◽  
Guang Lun Zhuang ◽  
...  
Keyword(s):  
In Vitro Development ◽  
Pronuclear Transfer ◽  
Vitro Development

Gallic acid promotes the in vitro development of sheep secondary isolated follicles involving the phosphatidylinositol 3-kinase pathway

2021 ◽  
pp. 106767
Author(s):  
Gizele A.L. Silva ◽  
Luana B. Araújo ◽  
Larissa C.R. Silva ◽  
Bruna B. Gouveia ◽  
Ricássio S. Barberino ◽  
...  
Keyword(s):  
Gallic Acid ◽  
Phosphatidylinositol 3 Kinase ◽  
In Vitro Development ◽  
Vitro Development ◽  
Kinase Pathway ◽  
Phosphatidylinositol 3

scholarly journals Necrostatin-1 Supplementation to Islet Tissue Culture Enhances the In-Vitro Development and Graft Function of Young Porcine Islets

2021 ◽  
Vol 22 (16) ◽  
pp. 8367
Author(s):  
Hien Lau ◽  
Shiri Li ◽  
Nicole Corrales ◽  
Samuel Rodriguez ◽  
Mohammadreza Mohammadi ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Oxygen Consumption ◽  
Insulin Secretion ◽  
Oxygen Consumption Rate ◽  
Consumption Rate ◽  
In Vitro Development ◽  
Porcine Islets ◽  
Vitro Development

Pre-weaned porcine islets (PPIs) represent an unlimited source for islet transplantation but are functionally immature. We previously showed that necrostatin-1 (Nec-1) immediately after islet isolation enhanced the in vitro development of PPIs. Here, we examined the impact of Nec-1 on the in vivo function of PPIs after transplantation in diabetic mice. PPIs were isolated from pancreata of 8–15-day-old, pre-weaned pigs and cultured in media alone, or supplemented with Nec-1 (100 µM) on day 0 or on day 3 of culture (n = 5 for each group). On day 7, islet recovery, viability, oxygen consumption rate, insulin content, cellular composition, insulin secretion capacity, and transplant outcomes were evaluated. While islet viability and oxygen consumption rate remained high throughout 7-day tissue culture, Nec-1 supplementation on day 3 significantly improved islet recovery, insulin content, endocrine composition, GLUT2 expression, differentiation potential, proliferation capacity of endocrine cells, and insulin secretion. Adding Nec-1 on day 3 of tissue culture enhanced the islet recovery, proportion of delta cells, beta-cell differentiation and proliferation, and stimulation index. In vivo, this leads to shorter times to normoglycemia, better glycemic control, and higher circulating insulin. Our findings identify the novel time-dependent effects of Nec-1 supplementation on porcine islet quantity and quality prior to transplantation.

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