scholarly journals Biochemical dissection of AP-1 recruitment onto Golgi membranes.

1993 ◽  
Vol 123 (3) ◽  
pp. 561-573 ◽  
Author(s):  
L M Traub ◽  
J A Ostrom ◽  
S Kornfeld
Keyword(s):  
Brefeldin A ◽  
In Vitro Assay ◽  
Coated Vesicle ◽  
Temperature Dependent ◽  
Fungal Metabolite ◽  
Vitro Assay ◽  
Golgi Membranes ◽  
Lag Period ◽  
Gamma S

Recruitment of the Golgi-specific AP-1 adaptor complex onto Golgi membranes is thought to be a prerequisite for clathrin coat assembly on the TGN. We have used an in vitro assay to examine the translocation of cytosolic AP-1 onto purified Golgi membranes. Association of AP-1 with the membranes required GTP or GTP analogues and was inhibited by the fungal metabolite, brefeldin A. In the presence of GTP gamma S, binding of AP-1 to Golgi membranes was strictly dependent on the concentration of cytosol added to the assay. AP-1 recruitment was also found to be temperature dependent, and relatively rapid at 37 degrees C, following a lag period of 3 to 4 min. Using only an adaptor-enriched fraction from cytosol, purified myristoylated ARF1, and Golgi membranes, the GTP gamma S-dependent recruitment of AP-1 could be reconstituted. Our results show that the association of the AP-1 complex with Golgi membranes, like the coatomer complex, requires ARF, which accounts for the sensitivity of both to brefeldin A. In addition, they provide the basis for a model for the early biochemical events that lead to clathrin-coated vesicle formation on the TGN.

scholarly journals Microtubule translocation properties of intact and proteolytically digested dyneins from Tetrahymena cilia.

1989 ◽  
Vol 108 (6) ◽  
pp. 2327-2334 ◽  
Author(s):  
R D Vale ◽  
Y Y Toyoshima
Keyword(s):  
Force Production ◽  
In Vitro Assay ◽  
Regulatory Function ◽  
Vitro Assay ◽  
Ciliary Beating ◽  
Glass Surfaces ◽  
Triton X ◽  
Gamma S ◽  
Motile Activity

Tetrahymena cilia contain a three-headed 22S (outer arm) dynein and a single-headed 14S dynein. In this study, we have employed an in vitro assay of microtubule translocation along dynein-coated glass surfaces to characterize the motile properties of 14S dynein, 22S dynein, and proteolytic fragments of 22S dynein. Microtubule translocation produced by intact 22S dynein and 14S dynein differ in a number of respects including (a) the maximal velocities of movement; (b) the ability of 22S dynein but not 14S dynein to utilize ATP gamma S to induce movement; (c) the optimal pH and ionic conditions for movement; and (d) the effects of Triton X-100 on the velocity of movement. These results indicate that 22S and 14S dyneins have distinct microtubule translocating properties and suggest that these dyneins may have specialized roles in ciliary beating. We have also explored the function of the multiple ATPase heads of 22S dynein by preparing one- and two-headed proteolytic fragments of this three-headed molecule and examining their motile activity in vitro. Unlike the single-headed 14S dynein, the single-headed fragment of 22S dynein did not induce movement, even though it was capable of binding to microtubules. The two-headed fragment, on the other hand, translocated microtubules at velocities similar to those measured for intact 22S dynein (10 microns/sec). This finding indicates that the intact three-headed structure of 22S dynein is not essential for generating microtubule movement, which raises the possibility that multiple heads may serve some regulatory function or may be required for maximal force production in the beating cilium.

scholarly journals Targeting and mistargeting of plasma membrane adaptors in vitro.

1993 ◽  
Vol 123 (5) ◽  
pp. 1093-1105 ◽  
Author(s):  
M N Seaman ◽  
C L Ball ◽  
M S Robinson
Keyword(s):  
Plasma Membrane ◽  
Brefeldin A ◽  
Coated Vesicle ◽  
Double Labeling ◽  
In Vitro System ◽  
Specific Antibodies ◽  
Excess Calcium ◽  
Gamma S ◽  
Intracellular Sequestration

Targeting and recruitment of the plasma membrane (PM) clathrin-coated vesicle adaptor complexes has been studied using an in vitro system based on permeabilized acceptor cells and donor cytosol. Through the use of species- and/or tissue-specific antibodies, only newly recruited exogenous PM adaptors are visualized. Targeting of PM adaptors can be switched from the plasma membrane to a perinuclear compartment by GTP gamma S or excess calcium. Prior treatment with brefeldin A prevents GTP gamma S-induced mistargeting. Double-labeling immunofluorescence and immunogold EM indicate that the perinuclear PM adaptor binding compartment is late endosomal. We propose that receptors for PM adaptors cycle between the plasma membrane and an endosomal storage compartment. Normally the receptors would be switched on only at the plasma membrane, but both GTP gamma S and calcium are capable of reversing this switch. Intracellular sequestration of PM adaptor receptors may provide the cell with a mechanism for up-regulating endocytosis following a burst of exocytosis.

scholarly journals The localization and phosphorylation of p47 are important for Golgi disassembly–assembly during the cell cycle

2003 ◽  
Vol 161 (6) ◽  
pp. 1067-1079 ◽  
Author(s):  
Keiji Uchiyama ◽  
Eija Jokitalo ◽  
Mervi Lindman ◽  
Mark Jackman ◽  
Fumi Kano ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
In Vitro Assay ◽  
Inhibitory Mechanism ◽  
Vitro Assay ◽  
Daughter Cells ◽  
Golgi Membranes ◽  
Golgi Fragmentation ◽  
Mitotic Phosphorylation

In mammalian cells, the Golgi apparatus is disassembled at the onset of mitosis and reassembled at the end of mitosis. This disassembly–reassembly is generally believed to be essential for the equal partitioning of Golgi into two daughter cells. For Golgi disassembly, membrane fusion, which is mediated by NSF and p97, needs to be blocked. For the NSF pathway, the tethering of p115-GM130 is disrupted by the mitotic phosphorylation of GM130, resulting in the inhibition of NSF-mediated fusion. In contrast, the p97/p47 pathway does not require p115-GM130 tethering, and its mitotic inhibitory mechanism has been unclear. Now, we have found that p47, which mainly localizes to the nucleus during interphase, is phosphorylated on Serine-140 by Cdc2 at mitosis. The phosphorylated p47 does not bind to Golgi membranes. An in vitro assay shows that this phosphorylation is required for Golgi disassembly. Microinjection of p47(S140A), which is unable to be phosphorylated, allows the cell to keep Golgi stacks during mitosis and has no effect on the equal partitioning of Golgi into two daughter cells, suggesting that Golgi fragmentation-dispersion may not be obligatory for equal partitioning even in mammalian cells.

scholarly journals Tubulation of Golgi membranes in vivo and in vitro in the absence of brefeldin A.

1993 ◽  
Vol 120 (1) ◽  
pp. 15-24 ◽  
Author(s):  
E B Cluett ◽  
S A Wood ◽  
M Banta ◽  
W J Brown
Keyword(s):  
Brefeldin A ◽  
Reciprocal Relationship ◽  
In Vivo Studies ◽  
Coat Proteins ◽  
Fungal Metabolite ◽  
Golgi Membranes ◽  
Golgi Network ◽  
Cytosolic Factors

Recent in vivo studies with the fungal metabolite, brefeldin A (BFA), have shown that in the absence of vesicle formation, membranes of the Golgi complex and the trans-Golgi network (TGN) are nevertheless able to extend long tubules which fuse with selected target organelles. We report here that the ability to form tubules (> 7 microns long) could be reproduced in vitro by treatment of isolated, intact Golgi membranes with BFA under certain conditions. Surprisingly, an even more impressive degree of tubulation could be achieved by incubating Golgi stacks with an ATP-reduced cytosolic fraction, without any BFA at all. Similarly, tubulation of Golgi membranes in vivo occurred after treatment of cells with intermediate levels of NaN3 and 2-deoxyglucose. The formation of tubules in vitro, either by BFA treatment or low-ATP cytosol, correlated precisely with a loss of the vesicle-associated coat protein beta-COP from Golgi membranes. After removal of BFA or addition of ATP, membrane tubules served as substrates for the rebinding of beta-COP and for the formation of vesicles in vitro. These results provide support for the idea that a reciprocal relationship exists between tubulation and vesiculation (Klausner, R. D., J. G. Donaldson, and J. Lippincott-Schwartz. 1992. J. Cell Biol. 116:1071-1080). Moreover, they show that tubulation is an inherent property of Golgi membranes, since it occurs without the aid of microtubules or BFA treatment. Finally the results indicate the presence of cytosolic factors, independent of vesicle-associated coat proteins, that mediate the budding/tubulation of Golgi membranes.

In Vitro Assay of Platelet Adhesiveness with Washed and Tanned Human Red Cells

1968 ◽  
Vol 20 (03/04) ◽  
pp. 384-396 ◽  
Author(s):  
G Zbinden ◽  
S Tomlin
Keyword(s):  
Platelet Adhesion ◽  
Sodium Citrate ◽  
Blood Platelets ◽  
In Vitro Assay ◽  
Red Cells ◽  
Platelet Adhesiveness ◽  
Vitro Assay ◽  
In Vitro System ◽  
Human Red Cells

SummaryAn in vitro system is described in which adhesion of blood platelets to washed and tannic acid-treated red cells was assayed quantitatively by microscopic observation. ADP, epinephrine and TAME produced a reversible increase in platelet adhesiveness which was antagonized by AMP. With Evans blue, polyanetholsulfonate, phthalanilide NSC 38280, thrombin and heparin at concentrations above 1-4 u/ml the increase was irreversible. The ADP-induced increase in adhesiveness was inhibited by sodium citrate, EDTA, AMP, ATP and N-ethylmaleimide. EDTA, AMP and the SH-blocker N-ethylmaleimide also reduced spontaneous platelet adhesion to red cells. No significant effects were observed with adenosine, phenprocoumon, 5-HT, phthalanilide NSC 57155, various estrogens, progestogens and fatty acids, acetylsalicylic acid and similarly acting agents, hydroxylamine, glucose and KCN. The method may be useful for the screening of thrombogenic and antithrombotic properties of drugs.

A comparative study on adhesion and recovery of potential probiotic strains ofLactobacillusspp. by in vitro assay and analysis of human colon biopsies

2009 ◽  
Vol 21 (2) ◽  
Author(s):  
Nadja Larsen ◽  
Kim F. Michaelsen ◽  
Anders Pærregaard ◽  
Finn K. Vogensen ◽  
Mogens Jakobsen
Keyword(s):  
Comparative Study ◽  
Human Colon ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Probiotic Strains

Angiogenic Effect of Pravastatin Alone and with Sera from Healthy and Complicated Pregnancies Studied by in vitro Vasculogenesis/Angiogenesis Assay

2021 ◽  
pp. 1-9
Author(s):  
Anita Virtanen ◽  
Outi Huttala ◽  
Kati Tihtonen ◽  
Tarja Toimela ◽  
Tuula Heinonen ◽  
...  
Keyword(s):  
Direct Effect ◽  
Late Onset ◽  
Maternal Serum ◽  
In Vitro Assay ◽  
Serum Samples ◽  
Therapeutic Concentration ◽  
Tubule Formation ◽  
Vitro Assay ◽  
Angiogenesis Assay

<b><i>Objective:</i></b> To determine the direct effect of pravastatin on angiogenesis and to study the interaction between pravastatin and maternal sera from women with early- or late-onset pre-eclampsia (PE), intrauterine growth restriction, or healthy pregnancy. <b><i>Methods:</i></b> We collected 5 maternal serum samples from each group. The effect of pravastatin on angiogenesis was assessed with and without maternal sera by quantifying tubule formation in a human-based in vitro assay. Pravastatin was added at 20, 1,000, and 8,000 ng/mL concentrations. Concentrations of angiogenic and inflammatory biomarkers in serum and in test medium after supplementation of serum alone and with pravastatin (1,000 ng/mL) were measured. <b><i>Results:</i></b> Therapeutic concentration of pravastatin (20 ng/mL) did not have significant direct effect on angiogenesis, but the highest concentrations inhibited angiogenesis. Pravastatin did not change the levels of biomarkers in the test media. There were no changes in angiogenesis when therapeutic dose of pravastatin was added with maternal sera, but there was a trend to wide individual variation towards enhanced angiogenesis, particularly in the early-onset PE group. <b><i>Conclusions:</i></b> At therapeutic concentration, pravastatin alone or with maternal sera has no significant effect on angiogenesis, but at high concentrations the effect seems to be anti-angiogenic estimated by in vitro assay.

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