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 Cell cycle expression of RNA duplex unwindase activity in mammalian cells.

1988 ◽  
Vol 8 (2) ◽  
pp. 770-777 ◽  
Author(s):  
R W Wagner ◽  
K Nishikura
Keyword(s):  
Cell Cycle ◽  
Burkitt Lymphoma ◽  
Mammalian Cells ◽  
Somatic Cells ◽  
Mouse Fibroblast ◽  
In Vitro Assay ◽  
3T3 Cells ◽  
Vitro Assay ◽  
Gel Retardation Assay

An RNA duplex unwindase activity has been found by using an in vitro assay with various types of mammalian, somatic cells, including HeLa, mouse plasmacytoma, and Burkitt lymphoma. The unwindase activity is very low in mouse fibroblast 3T3 cells arrested into quiescence, but increases when the cells are released into renewed growth by serum. In addition, a gel retardation assay proved to be specific and sensitive for detection of RNA duplex-unwindase complexes.

Cell cycle expression of RNA duplex unwindase activity in mammalian cells

1988 ◽  
Vol 8 (2) ◽  
pp. 770-777
Author(s):  
R W Wagner ◽  
K Nishikura
Keyword(s):  
Cell Cycle ◽  
Burkitt Lymphoma ◽  
Mammalian Cells ◽  
Somatic Cells ◽  
Mouse Fibroblast ◽  
In Vitro Assay ◽  
3T3 Cells ◽  
Vitro Assay ◽  
Gel Retardation Assay

An RNA duplex unwindase activity has been found by using an in vitro assay with various types of mammalian, somatic cells, including HeLa, mouse plasmacytoma, and Burkitt lymphoma. The unwindase activity is very low in mouse fibroblast 3T3 cells arrested into quiescence, but increases when the cells are released into renewed growth by serum. In addition, a gel retardation assay proved to be specific and sensitive for detection of RNA duplex-unwindase complexes.

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.

Growth factors and pattern formation

Development ◽  
1981 ◽  
Vol 65 (Supplement) ◽  
pp. 187-207
Author(s):  
J. C. Smith
Keyword(s):  
Cell Cycle ◽  
Growth Factors ◽  
Pattern Formation ◽  
Positional Information ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Produce Cell ◽  
Morphogenetic Factors ◽  
Positional Value

Growth and pattern formation occur simultaneously in many epimorphic fields and it has been suggested that specification of positional information is somehow linked to cell division. It is possible, therefore, that boundary regions responsible for the specification of positional information produce cell growth factors. In this paper I review the properties of some known growth factors, describe their effects on the cell cycle and discuss how they might act. In developing a convenient in vitro assay for morphogenetic factors it will be much easier to measure incorporation of [3H]thymidine into responding cells than to estimate changes in positional value.

In vitro binding of Candida albicans yeast cells to human fibronectin

1984 ◽  
Vol 30 (2) ◽  
pp. 221-227 ◽  
Author(s):  
Katherine G. Skerl ◽  
Richard A. Calderone ◽  
Esther Segal ◽  
T. Sreevalsan ◽  
W. Michael Scheld
Keyword(s):  
Candida Albicans ◽  
Epithelial Cells ◽  
Mammalian Cells ◽  
Fluorescent Antibody ◽  
In Vitro Assay ◽  
Yeast Cells ◽  
Vitro Assay ◽  
Vaginal Epithelial Cells ◽  
Human Fibronectin

The binding of Candida albicans yeast cells to human fibronectin (Fn), a major glycoprotein of mammalian cells, was studied using an in vitro assay. Adherence was quantitated in microtiter dishes coated with Fn to which radiolabeled yeast cells were added. Under optimum conditions of the assay, i.e., 1 mM CaCl2 and 70 μg Fn protein, approximately 40% of the radiolabeled yeast cells adhered to the Fn. Adherence to Fn was greater at 30 °C than at 4 °C and was greater with viable yeast cells than with heat-killed cells. Candida albicans (two strains) and C. tropicalis adhered to Fn to a greater extent than C. pseudotropicalis, C. krusei, or Saccharomyces cerevisiae. Pretreatment of C. albicans with chymotrypsin, pronase, or papain, but not pepsin, decreased adherence to Fn. Blocking experiments using mannan, sugars, or amino sugars were carried out by preabsorbing the Fn with each of the above-mentioned compounds. Candida mannan blocked adherence of C. albicans to Fn. The mannan effect was dose dependent. However, adherence of C. albicans to Fn was not significantly reduced by mannose, glucose, or several other sugars. The role of FN as a receptor for the binding of C. albicans yeast cells to buccal and vaginal epithelial cells was investigated also using an in vitro assay. We determined, using indirect fluorescent antibody techniques, that both buccal and vaginal epithelial cells possessed Fn. In addition, yeast cells, when pretreated with Fn, showed reduced adherence with buccal and vaginal cells when compared with nontreated cells. These studies may indicate a role for Fn in the adherence of C. albicans to buccal and vaginal epithelial cells.

scholarly journals Mena–GRASP65 interaction couples actin polymerization to Golgi ribbon linking

2016 ◽  
Vol 27 (1) ◽  
pp. 137-152 ◽  
Author(s):  
Danming Tang ◽  
Xiaoyan Zhang ◽  
Shijiao Huang ◽  
Hebao Yuan ◽  
Jie Li ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Mammalian Cells ◽  
Actin Polymerization ◽  
Binding Protein ◽  
Elongation Factor ◽  
Golgi Membrane ◽  
Golgi Membranes ◽  
Golgi Fragmentation ◽  
Golgi Ribbon

In mammalian cells, the Golgi reassembly stacking protein 65 (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers through the N-terminal GRASP domain. Because the GRASP domain is globular and relatively small, but the gaps between stacks are large and heterogeneous, it remains puzzling how GRASP65 physically links Golgi stacks into a ribbon. To explore the possibility that other proteins may help GRASP65 in ribbon linking, we used biochemical methods and identified the actin elongation factor Mena as a novel GRASP65-binding protein. Mena is recruited onto the Golgi membranes through interaction with GRASP65. Depleting Mena or disrupting actin polymerization resulted in Golgi fragmentation. In cells, Mena and actin were required for Golgi ribbon formation after nocodazole washout; in vitro, Mena and microfilaments enhanced GRASP65 oligomerization and Golgi membrane fusion. Thus Mena interacts with GRASP65 to promote local actin polymerization, which facilitates Golgi ribbon linking.

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
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