scholarly journals Evidence for a Role of CLIP-170 in the Establishment of Metaphase Chromosome Alignment

1998 ◽  
Vol 141 (4) ◽  
pp. 849-862 ◽  
Author(s):  
Denis Dujardin ◽  
U. Irene Wacker ◽  
Anne Moreau ◽  
Trina A. Schroer ◽  
Janet E. Rickard ◽  
...  
Keyword(s):  
Metaphase Plate ◽  
Polar Regions ◽  
Static Interaction ◽  
Transport Vesicles ◽  
Vitro Binding ◽  
Chromosome Alignment ◽  
Dynactin Complex

CLIPs (cytoplasmic linker proteins) are a class of proteins believed to mediate the initial, static interaction of organelles with microtubules. CLIP-170, the CLIP best characterized to date, is required for in vitro binding of endocytic transport vesicles to microtubules. We report here that CLIP-170 transiently associates with prometaphase chromosome kinetochores and codistributes with dynein and dynactin at kinetochores, but not polar regions, during mitosis. Like dynein and dynactin, a fraction of the total CLIP-170 pool can be detected on kinetochores of unattached chromosomes but not on those that have become aligned at the metaphase plate. The COOH-terminal domain of CLIP-170, when transiently overexpressed, localizes to kinetochores and causes endogenous full-length CLIP-170 to be lost from the kinetochores, resulting in a delay in prometaphase. Overexpression of the dynactin subunit, dynamitin, strongly reduces the amount of CLIP-170 at kinetochores suggesting that CLIP-170 targeting may involve the dynein/dynactin complex. Thus, CLIP-170 may be a linker for cargo in mitosis as well as interphase. However, dynein and dynactin staining at kinetochores are unaffected by this treatment and further overexpression studies indicate that neither CLIP-170 nor dynein and dynactin are required for the formation of kinetochore fibers. Nevertheless, these results strongly suggest that CLIP-170 contributes in some way to kinetochore function in vivo.

scholarly journals Ran Is Required before Metaphase for Spindle Assembly and Chromosome Alignment and after Metaphase for Chromosome Segregation and Spindle Midbody Organization

2006 ◽  
Vol 17 (4) ◽  
pp. 2069-2080 ◽  
Author(s):  
Rosalind V. Silverman-Gavrila ◽  
Andrew Wilde
Keyword(s):  
Chromosome Segregation ◽  
Metaphase Plate ◽  
Spindle Assembly ◽  
Aurora A ◽  
Microtubule Organization ◽  
Chromosome Alignment ◽  
Production Organization ◽  
Drosophila Embryos

The Ran pathway has been shown to have a role in spindle assembly. However, the extent of the role of the Ran pathway in mitosis in vivo is unclear. We report that perturbation of the Ran pathway disrupted multiple steps of mitosis in syncytial Drosophila embryos and uncovered new mitotic processes that are regulated by Ran. During the onset of mitosis, the Ran pathway is required for the production, organization, and targeting of centrosomally nucleated microtubules to chromosomes. However, the role of Ran is not restricted to microtubule organization, because Ran is also required for the alignment of chromosomes at the metaphase plate. In addition, the Ran pathway is required for postmetaphase events, including chromosome segregation and the assembly of the microtubule midbody. The Ran pathway mediates these mitotic events, in part, by facilitating the correct targeting of the kinase Aurora A and the kinesins KLP61F and KLP3A to spindles.

Alpha-COP can discriminate between distinct, functional di-lysine signals in vitro and regulates access into retrograde transport

1998 ◽  
Vol 111 (23) ◽  
pp. 3459-3470 ◽  
Author(s):  
S. Schroder-Kohne ◽  
F. Letourneur ◽  
H. Riezman
Keyword(s):  
Transmembrane Protein ◽  
Retrograde Transport ◽  
Special Role ◽  
Golgi Localization ◽  
Vitro Binding ◽  
Gradient Fractionation ◽  
New Alleles

Emp47p is a yeast Golgi transmembrane protein with a retrograde, Golgi to ER transport di-lysine signal in its cytoplasmic tail. Emp47p has previously been shown to recycle between the Golgi complex and the ER and to require its di-lysine signal for Golgi localization. In contrast to other proteins with di-lysine signals, the Golgi-localization of Emp47p has been shown to be preserved in ret1-1 cells expressing a mutant alpha-COP subunit of coatomer. Here we demonstrate by sucrose gradient fractionation and immunofluorescence analysis that recycling of Emp47p was unimpaired in ret1-1. Furthermore we have characterized three new alleles of ret1 and showed that Golgi localization of Emp47p was intact in cells with those mutant alleles. We could correlate the ongoing recycling of Emp47p in ret1-1 with preserved in vitro binding of coatomer from ret1-1 cells to immobilized GST-Emp47p-tail fusion protein. As previously reported, the di-lysine signal of Wbp1p was not recognized by ret1-1 mutant coatomer, suggesting a possible role for alpha-COP in the differential binding to distinct di-lysine signals. In contrast to results with alpha-COP mutants, we found that Emp47p was mislocalised to the vacuole in mutants affecting beta'-, gamma-, delta-, and zeta-COP subunits of coatomer and that the mutant coatomer bound neither to the Emp47p nor to the Wbp1p di-lysine signal in vitro. Therefore, the retrograde transport of Emp47p displayed a differential requirement for individual coatomer subunits and a special role of alpha-COP for a particular transport step in vivo.

O-171 Altered meiotic spindle morphology and composition in in vitro matured oocytes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
P Karamtzioti ◽  
G Tiscornia ◽  
D Garcia ◽  
A Rodriguez ◽  
I Vernos ◽  
...  
Keyword(s):  
Metaphase Plate ◽  
Spindle Pole ◽  
Meiotic Spindle ◽  
Developmental Potential ◽  
Software Analysis ◽  
Spindle Microtubules ◽  
Non Parametric

Abstract Study question How does the meiotic spindle tubulin PTMs of MII oocytes matured in vitro compare to that of MII oocytes matured in vivo? Summary answer MII cultured in vitro present detyrosinated tubulin in the spindle microtubules, while MII oocytes matured in vivo do not. What is known already A functional spindle is required for chromosomal segregation during meiosis, but the role of tubulin post-translational modifications (PTMs) in spindle meiotic dynamics remains poorly characterized. In contrast with GVs matured in vitro within the cumulus oophorous, in vitro maturation of denuded GVs to the MII stage (GV-MII) is associated with spindle abnormalities, chromosome misalignment and compromised developmental potential. Although aneuploidy rates in GV-MII are not higher than in vivo matured MII, disorganized chromosomes may contribute to compromised developmental potential. However, to date, spindle PTMs morphology of GV-MII has not been compared to that of in vivo cultured MII oocytes. Study design, size, duration GV (n = 125), and MII oocytes (n = 24) were retrieved from hormonally stimulated women, aged 20 to 35 years old. GVs were matured to the MII stage in vitro in G-2 PLUS medium for 30h; the maturation rate was 68,2%; the 46 GV-MII oocytes obtained were vitrified, stored, and warmed before fixing and subjecting to immunofluorescent analysis. In vivo matured MII oocytes donated to research were used as controls. Participants/materials, setting, methods Women were stimulated using a GnRH antagonist protocol, with GnRH agonist trigger. Trigger criterion was ≥2 follicles ≥18mm; oocytes were harvested 36h later. Spindle microtubules were incubated with antibodies against alpha tubulin and tubulin PTMs (acetylation, tyrosination, polyglutamylation, Δ2-tubulin, and detyrosination); chromosomes were stained with Hoechst 33342 and samples subjected to confocal immunofluorescence microscopy (ZEISS LSM780), with ImageJ software analysis. Differences in spindle morphometric parameters were assessed by non-parametric Kruskal–Wallis and Fisher’s exact tests. Main results and the role of chance Qualitatively, Δ2-tubulin, tyrosination and polyglutamylation were similar for both groups. Acetylation was also present in both groups, albeit in different patterns: while in vivo matured MII oocytes showed acetylation at the poles, GV-MII showed a symmetrical distribution of signal intensity, but discontinuous signal on individual microtubule tracts, suggesting apparent islands of acetylation. In contrast, detyrosination was detected in in vivo matured MII oocytes but was absent from GV-MII. Regarding spindle pole morphology, of the four possible phenotypes described in the literature (double flattened and double focused; flattened-focused, focused-flattened, with the first word characterizing the cortex side of the spindle), we observed double flat shaped spindle poles in 86% of GV-MII oocytes (25/29) as opposed to 40.5% (15/37) for the in vivo matured MII oocytes (p = 0.0004, Fisher’s exact test). Further morphometric analysis of the spindle size (maximum projection, major and minor axis length) and the metaphase plate position (proximal to distal ratio, angle) revealed decreased spindle size in GV-MII oocytes (p = 0.019, non parametric Kruskal- Wallis test). Limitations, reasons for caution Oocytes retrieved from hyperstimulation cycles could be intrinsically impaired since they failed to mature in vivo. Our conclusions should not be extrapolated to IVM in non-stimulated cycles, as in this model, the cumulus oophorus is a major factor in oocyte maturation and correlation with spindle dynamics has been inferred. Wider implications of the findings The metaphase II spindle stability compared to the mitotic or metaphase I meiotic one justifies the presence of PTMs such as acetylation and glutamylation, which are found in stable, long-lived microtubules. The significance of the absence of detyrosinated microtubules in the MII-GV group remains to be determined Trial registration number not applicable

scholarly journals Dynamics of Ankyrin-containing Complexes in Chicken Embryonic Erythroid Cells: Role of Phosphorylation

2001 ◽  
Vol 12 (12) ◽  
pp. 3864-3874 ◽  
Author(s):  
Sourav Ghosh ◽  
John V. Cox
Keyword(s):  
Complex Formation ◽  
Anion Exchanger ◽  
Erythroid Cells ◽  
Binding Studies ◽  
Phosphatase Inhibitors ◽  
In Vitro Binding ◽  
Vitro Binding

Chicken erythroid ankyrin undergoes a fairly rapid cycle of cytoskeletal association, dissociation, and turnover. In addition, the cytoskeletal association of ankyrin is regulated by phosphorylation. Treatment of erythroid cells with serine and threonine phosphatase inhibitors stimulated the hyperphosphorylation of the 225- and 205-kDa ankyrin isoforms, and dissociated the bulk of these isoforms from cytoskeletal spectrin. In vitro binding studies have shown that this dissociation of ankyrin from spectrin in vivo can be attributed to a reduced ability of hyperphosphorylated ankyrin to bind spectrin. Interestingly, a significant fraction of detergent insoluble ankyrin accumulates in a spectrin-independent pool. At least some of this spectrin-independent pool of ankyrin is complexed with the AE1 anion exchanger, and the solubility properties of this pool are also regulated by phosphorylation. Treatment of cells with serine and threonine phosphatase inhibitors had no effect on ankyrin/AE1 complex formation. However, these inhibitors were sufficient to shift ankyrin/AE1 complexes from the detergent insoluble to the soluble pool. These analyses, which are the first to document the in vivo consequences of ankyrin phosphorylation, indicate that erythroid ankyrin-containing complexes can undergo dynamic rearrangements in response to changes in phosphorylation.

scholarly journals p150Glued, the largest subunit of the dynactin complex, is nonessential in Neurospora but required for nuclear distribution.

1996 ◽  
Vol 7 (5) ◽  
pp. 731-742 ◽  
Author(s):  
J H Tinsley ◽  
P F Minke ◽  
K S Bruno ◽  
M Plamann
Keyword(s):  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Structural Features ◽  
Gene Encoding ◽  
Nuclear Distribution ◽  
Transport Vesicles ◽  
Dynactin Complex ◽  
Intermediate Chains

Dynactin is a multisubunit complex that is required for cytoplasmic dynein, a minus-end-directed, microtubule-associated motor, to efficiently transport vesicles along microtubules in vitro. p150Glued, the largest subunit of dynactin, has been identified in vertebrates and Drosophila and recently has been shown to interact with cytoplasmic dynein intermediate chains in vitro. The mechanism by which dynactin facilitates cytoplasmic dynein-dependent vesicle transport is unknown. We have devised a genetic screen for cytoplasmic dynein/dynactin mutants in the filamentous fungus Neurospora crassa. In this paper, we report that one of these mutants, ro-3, defines a gene encoding an apparent homologue of p150Glued, and we provide genetic evidence that cytoplasmic dynein and dynactin interact in vivo. The major structural features of vertebrate and Drosophila p150Glued, a microtubule-binding site at the N-terminus and two large alpha-helical coiled-coil regions contained within the distal two-thirds of the polypeptide, are conserved in Ro3. Drosophila p150Glued is essential for viability; however, ro-3 null mutants are viable, indicating that dynactin is not an essential complex in N. crassa. We show that N. crassa cytoplasmic dynein and dynactin mutants have abnormal nuclear distribution but retain the ability to organize cytoplasmic microtubules and actin in anucleate hyphae.

scholarly journals Role of Vma21p in Assembly and Transport of the Yeast Vacuolar ATPase

2004 ◽  
Vol 15 (11) ◽  
pp. 5075-5091 ◽  
Author(s):  
Per Malkus ◽  
Laurie A. Graham ◽  
Tom H. Stevens ◽  
Randy Schekman
Keyword(s):  
Atp Hydrolysis ◽  
Proton Translocation ◽  
Wild Type ◽  
Vitro Assay ◽  
Accessory Factor ◽  
Transport Vesicles ◽  
Er Export

The Saccharomyces cerevisiae vacuolar H+-ATPase (V-ATPase) is a multisubunit complex composed of a peripheral membrane sector (V1) responsible for ATP hydrolysis and an integral membrane sector (V0) required for proton translocation. Biogenesis of V0 requires an endoplasmic reticulum (ER)-localized accessory factor, Vma21p. We found that in vma21Δ cells, the major proteolipid subunit of V0 failed to interact with the 100-kDa V0 subunit, Vph1p, indicating that Vma21p is necessary for V0 assembly. Immunoprecipitation of Vma21p from wild-type membranes resulted in coimmunoprecipitation of all five V0 subunits. Analysis of vmaΔ strains showed that binding of V0 subunits to Vma21p was mediated by the proteolipid subunit Vma11p. Although Vma21p/proteolipid interactions were independent of Vph1p, Vma21p/Vph1p association was dependent on all other V0 subunits, indicating that assembly of V0 occurs in a defined sequence, with Vph1p recruitment into a Vma21p/proteolipid/Vma6p complex representing the final step. An in vitro assay for ER export was used to demonstrate preferential packaging of the fully assembled Vma21p/proteolipid/Vma6p/Vph1p complex into COPII-coated transport vesicles. Pulse-chase experiments showed that the interaction between Vma21p and V0 was transient and that Vma21p/V0 dissociation was concomitant with V0/V1 assembly. Blocking ER export in vivo stabilized the interaction between Vma21p and V0 and abrogated assembly of V0/V1. Although a Vma21p mutant lacking an ER-retrieval signal remained associated with V0 in the vacuole, this interaction did not affect the assembly of vacuolar V0/V1 complexes. We conclude that Vma21p is not involved in regulating the interaction between V0 and V1 sectors, but that it has a crucial role in coordinating the assembly of V0 subunits and in escorting the assembled V0 complex into ER-derived transport vesicles.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

Anti-obesity role of Aster glehni extract: in vivo and in vitro effects

Planta Medica ◽  
2012 ◽  
Vol 78 (11) ◽  
Author(s):  
HM Lee ◽  
TG Ahn ◽  
CW Kim ◽  
HJ An
Keyword(s):  
In Vitro Effects

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

In Vitro and in Vivo Comparison of Binding of 99m-Tc-Iabeled Anti-CEA MAb F33-104 with 99m-Tc-labeled Anti-CEA MAb BW431/26

1999 ◽  
Vol 38 (04) ◽  
pp. 115-119
Author(s):  
N. Oriuchi ◽  
S. Sugiyama ◽  
M. Kuroki ◽  
Y. Matsuoka ◽  
S. Tanada ◽  
...  
Keyword(s):  
Nude Mice ◽  
Binding Capacity ◽  
Colon Adenocarcinoma ◽  
Human Colon ◽  
Cell Binding ◽  
Vitro Binding ◽  
Labeling Method ◽  
Human Colon Adenocarcinoma

Summary Aim: The purpose of this study was to assess the potential for radioimmunodetection (RAID) of murine anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb) F33-104 labeled with technetium-99m (99m-Tc) by a reduction-mediated labeling method. Methods: The binding capacity of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA by means of in vitro procedures such as immunoradiometric assay and cell binding assay and the biodistribution of 99m-Tc-labeled anti-CEA MAb F33-104 in normal nude mice and nude mice bearing human colon adenocarcinoma LS180 tumor were investigated and compared with 99m-Tc-labeled anti-CEA MAb BW431/26. Results: The in vitro binding rate of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA in solution and attached to the cell membrane was significantly higher than 99m-Tclabeled anti-CEA MAb BW431/261 (31.4 ± 0.95% vs. 11.9 ± 0.55% at 100 ng/mL of soluble CEA, 83.5 ± 2.84% vs. 54.0 ± 2.54% at 107 of LS 180 cells). In vivo, accumulation of 99m-Tc-labeled anti-CEA MAb F33-104 was higher at 18 h postinjection than 99m-Tc-labeled anti-CEA MAb BW431/26 (20.1 ± 3.50% ID/g vs. 14.4 ± 3.30% ID/g). 99m-Tcactivity in the kidneys of nude mice bearing tumor was higher at 18 h postinjection than at 3 h (12.8 ± 2.10% ID/g vs. 8.01 ± 2.40% ID/g of 99m-Tc-labeled anti-CEA MAb F33-104, 10.7 ± 1.70% ID/g vs. 8.10 ± 1.75% ID/g of 99m-Tc-labeled anti-CEA MAb BW431/26). Conclusion: 99m-Tc-labeled anti-CEA MAb F33-104 is a potential novel agent for RAID of recurrent colorectal cancer.

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