scholarly journals Plk4 and Aurora A cooperate in the initiation of acentriolar spindle assembly in mammalian oocytes

2017 ◽  
Vol 216 (11) ◽  
pp. 3571-3590 ◽  
Author(s):  
Leah Bury ◽  
Paula A. Coelho ◽  
Angela Simeone ◽  
Samantha Ferries ◽  
Claire E. Eyers ◽  
...  
Keyword(s):  
Subsequent Development ◽  
Meiotic Spindle ◽  
Limiting Factors ◽  
Drug Resistant ◽  
Aurora A ◽  
Microtubule Nucleation ◽  
Microtubule Growth ◽  
Chemical Inhibition ◽  
Rate Limiting

Establishing the bipolar spindle in mammalian oocytes after their prolonged arrest is crucial for meiotic fidelity and subsequent development. In contrast to somatic cells, the first meiotic spindle assembles in the absence of centriole-containing centrosomes. Ran-GTP can promote microtubule nucleation near chromatin, but additional unidentified factors are postulated for the activity of multiple acentriolar microtubule organizing centers in the oocyte. We now demonstrate that partially overlapping, nonredundant functions of Aurora A and Plk4 kinases contribute to initiate acentriolar meiosis I spindle formation. Loss of microtubule nucleation after simultaneous chemical inhibition of both kinases can be significantly rescued by drug-resistant Aurora A alone. Drug-resistant Plk4 can enhance Aurora A–mediated rescue, and, accordingly, Plk4 can phosphorylate and potentiate the activity of Aurora A in vitro. Both kinases function distinctly from Ran, which amplifies microtubule growth. We conclude that Aurora A and Plk4 are rate-limiting factors contributing to microtubule growth as the acentriolar oocyte resumes meiosis.

scholarly journals Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells

2003 ◽  
Vol 162 (5) ◽  
pp. 757-764 ◽  
Author(s):  
Yasuhiko Terada ◽  
Yumi Uetake ◽  
Ryoko Kuriyama
Keyword(s):  
Mammalian Cells ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Mitotic Spindles ◽  
Microtubule Nucleation ◽  
Microtubule Organization ◽  
Centrosomal Protein ◽  
Spindle Poles

A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. Here, we show that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of γ-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with γ-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring γ-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.

scholarly journals Rapid Elimination of Blood Alcohol Using Erythrocytes: Mathematical Modeling and In Vitro Study

2017 ◽  
Vol 2017 ◽  
pp. 1-14 ◽  
Author(s):  
Yuliya G. Alexandrovich ◽  
Elena A. Kosenko ◽  
Elena I. Sinauridze ◽  
Sergey I. Obydennyi ◽  
Igor I. Kireev ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
Ethanol Oxidation ◽  
Alcohol Intoxication ◽  
Limiting Factors ◽  
Blood Alcohol ◽  
Cell Recovery ◽  
Optimal Method ◽  
Metabolic System ◽  
Rate Limiting

Erythrocytes (RBCs) loaded with alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALD) can metabolize plasma ethanol and acetaldehyde but with low efficiency. We investigated the rate-limiting factors in ethanol oxidation by these enzymes loaded into RBCs. Mathematical modeling and in vitro experiments on human RBCs loaded simultaneously with ADH and ALD (by hypoosmotic dialysis) were performed. The simulation showed that the rate of nicotinamide-adenine dinucleotide (NAD+) generation in RBC glycolysis, but not the activities of the loaded enzymes, is the rate-limiting step in external ethanol oxidation. The rate of oxidation could be increased if RBCs are supplemented by NAD+ and pyruvate. Our experimental data verified this theoretical conclusion. RBCs loaded with the complete system of ADH, ALD, NAD+, and pyruvate metabolized ethanol 20–40 times faster than reported in previous studies. The one-step procedure of hypoosmotic dialysis is the optimal method to encapsulate ADH and ALD in RBCs after cell recovery, encapsulation yield, osmotic resistance, and RBC-indexes. Consequently, transfusion of the RBCs loaded with the complete metabolic system, including ADH, ALD, pyruvate, and NAD+ in the patients with alcohol intoxication, may be a promising method for rapid detoxification of blood alcohol based on metabolism.

scholarly journals Stathmin and microtubules regulate mitotic entry in HeLa cells by controlling activation of both Aurora kinase A and Plk1

2013 ◽  
Vol 24 (24) ◽  
pp. 3819-3831 ◽  
Author(s):  
Victoria C. Silva ◽  
Lynne Cassimeris
Keyword(s):  
Hela Cells ◽  
Fluorescent Protein ◽  
Aurora Kinase ◽  
In Vitro Assays ◽  
Aurora A ◽  
Mitotic Entry ◽  
Cell Cycle Delay ◽  
Chemical Inhibition ◽  
Protein Recruitment

Depletion of stathmin, a microtubule (MT) destabilizer, delays mitotic entry by ∼4 h in HeLa cells. Stathmin depletion reduced the activity of CDC25 and its upstream activators, Aurora A and Plk1. Chemical inhibition of both Aurora A and Plk1 was sufficient to delay mitotic entry by 4 h, while inhibiting either kinase alone did not cause a delay. Aurora A and Plk1 are likely regulated downstream of stathmin, because the combination of stathmin knockdown and inhibition of Aurora A and Plk1 was not additive and again delayed mitotic entry by 4 h. Aurora A localization to the centrosome required MTs, while stathmin depletion spread its localization beyond that of γ-tubulin, indicating an MT-dependent regulation of Aurora A activation. Plk1 was inhibited by excess stathmin, detected in in vitro assays and cells overexpressing stathmin–cyan fluorescent protein. Recruitment of Plk1 to the centrosome was delayed in stathmin-depleted cells, independent of MTs. It has been shown that depolymerizing MTs with nocodazole abrogates the stathmin-depletion induced cell cycle delay; in this study, depolymerization with nocodazole restored Plk1 activity to near normal levels, demonstrating that MTs also contribute to Plk1 activation. These data demonstrate that stathmin regulates mitotic entry, partially via MTs, to control localization and activation of both Aurora A and Plk1.

scholarly journals Aurora A phosphorylation of TACC3/maskin is required for centrosome-dependent microtubule assembly in mitosis

2005 ◽  
Vol 170 (7) ◽  
pp. 1047-1055 ◽  
Author(s):  
Kazuhisa Kinoshita ◽  
Tim L. Noetzel ◽  
Laurence Pelletier ◽  
Karl Mechtler ◽  
David N. Drechsel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Xenopus Laevis ◽  
Microtubule Assembly ◽  
Aurora A ◽  
Specific Mechanism ◽  
Microtubule Polymerization ◽  
Egg Extracts ◽  
Microtubule Growth

Centrosomes act as sites of microtubule growth, but little is known about how the number and stability of microtubules emanating from a centrosome are controlled during the cell cycle. We studied the role of the TACC3–XMAP215 complex in this process by using purified proteins and Xenopus laevis egg extracts. We show that TACC3 forms a one-to-one complex with and enhances the microtubule-stabilizing activity of XMAP215 in vitro. TACC3 enhances the number of microtubules emanating from mitotic centrosomes, and its targeting to centrosomes is regulated by Aurora A–dependent phosphorylation. We propose that Aurora A regulation of TACC3 activity defines a centrosome-specific mechanism for regulation of microtubule polymerization in mitosis.

scholarly journals A novel microtubule nucleation pathway for meiotic spindle assembly in oocytes

2018 ◽  
Vol 217 (10) ◽  
pp. 3431-3445 ◽  
Author(s):  
Pierre Romé ◽  
Hiroyuki Ohkura
Keyword(s):  
Large Volume ◽  
Spindle Assembly ◽  
Terminal Region ◽  
Microtubule Assembly ◽  
Meiotic Spindle ◽  
Microtubule Nucleation ◽  
Unresolved Question ◽  
Nucleation Sites ◽  
Spindle Microtubules

The meiotic spindle in oocytes is assembled in the absence of centrosomes, the major microtubule nucleation sites in mitotic and male meiotic cells. A crucial, yet unresolved question in meiosis is how spindle microtubules are generated without centrosomes and only around chromosomes in the exceptionally large volume of oocytes. Here we report a novel oocyte-specific microtubule nucleation pathway that is essential for assembling most spindle microtubules complementarily with the Augmin pathway. This pathway is mediated by the kinesin-6 Subito/MKlp2, which recruits the γ-tubulin complex to the spindle equator to nucleate microtubules in Drosophila oocytes. Away from chromosomes, Subito interaction with the γ-tubulin complex is suppressed by its N-terminal region to prevent ectopic microtubule assembly in oocytes. We further demonstrate in vitro that the Subito complex from ovaries can nucleate microtubules from pure tubulin dimers. Collectively, microtubule nucleation regulated by Subito drives spatially restricted spindle assembly in oocytes.

scholarly journals A novel microtubule nucleation pathway for meiotic spindle assembly in oocytes

2018 ◽  
Author(s):  
Pierre Romé ◽  
Hiroyuki Ohkura
Keyword(s):  
Large Volume ◽  
Spindle Assembly ◽  
Terminal Region ◽  
Microtubule Assembly ◽  
Meiotic Spindle ◽  
Microtubule Nucleation ◽  
Unresolved Question ◽  
Nucleation Sites ◽  
Spindle Microtubules

AbstractThe meiotic spindle in oocytes is assembled in the absence of centrosomes, the major microtubule nucleation sites in mitotic and male meiotic cells. A crucial, yet unresolved question in meiosis is how spindle microtubules are generated without centrosomes and only around chromosomes in the large volume of oocytes. Here we report a novel oocyte-specific microtubule nucleation pathway that is essential for assembling most spindle microtubules complementarily with the Augmin pathway, and sufficient for triggering microtubule assembly in oocytes. This pathway is mediated by the kinesin-6 Subito/MKlp2, which recruits the γ-tubulin complex to the spindle equator to nucleate microtubules in Drosophila oocytes. Away from chromosomes, Subito interaction with the γ-tubulin complex is suppressed by its N-terminal region to prevent ectopic microtubule assembly in oocytes. We further demonstrate that the Subito complex from ovaries can nucleate microtubules from pure tubulin dimers in vitro. Taken together, microtubule nucleation regulated by Subito drives spatially restricted spindle assembly in oocytes.

Probing the ultrastructure of the mitotic and meiotic spindle in eggs: An expeditious approach based on semi-thin (0.25 μm) serial sections

1983 ◽  
Vol 41 ◽  
pp. 552-555
Author(s):  
Conly L. Rieder ◽  
S. Bowser ◽  
R. Nowogrodzki ◽  
K. Ross ◽  
G. Sluder
Keyword(s):  
Small Sample Size ◽  
Small Sample ◽  
Meiotic Spindle ◽  
Serial Sections ◽  
Mitotic Apparatus ◽  
Thin Sections ◽  
Cell Cycles ◽  
Ultrastructural Studies

Eggs have long been a favorite material for studying the mechanism of karyokinesis in-vivo and in-vitro. They can be obtained in great numbers and, when fertilized, divide synchronously over many cell cycles. However, they are not considered to be a practical system for ultrastructural studies on the mitotic apparatus (MA) for several reasons, the most obvious of which is that sectioning them is a formidable task: over 1000 ultra-thin sections need to be cut from a single 80-100 μm diameter egg and of these sections only a small percentage will contain the area or structure of interest. Thus it is difficult and time consuming to obtain reliable ultrastructural data concerning the MA of eggs; and when it is obtained it is necessarily based on a small sample size.We have recently developed a procedure which will facilitate many studies concerned with the ultrastructure of the MA in eggs. It is based on the availability of biological HVEM's and on the observation that 0.25 μm thick serial sections can be screened at high resolution for content (after mounting on slot grids and staining with uranyl and lead) by phase contrast light microscopy (LM; Figs 1-2).

Anti-Mycobacterial Peroxides: A New Class of Agents for Development Against Tuberculosis

2020 ◽  
Vol 16 (3) ◽  
pp. 392-402
Author(s):  
Christiaan W. van der Westhuyzen ◽  
Richard K. Haynes ◽  
Jenny-Lee Panayides ◽  
Ian Wiid ◽  
Christopher J. Parkinson
Keyword(s):  
Subsequent Development ◽  
Antitubercular Activity ◽  
Organic Peroxide ◽  
New Drugs ◽  
Skeletal Myoblast ◽  
Artemisinin Derivatives ◽  
Malaria Therapy ◽  
New Class ◽  
Alternative Approach

Background: With few exceptions, existing tuberculosis drugs were developed many years ago and resistance profiles have emerged. This has created a need for new drugs with discrete modes of action. There is evidence that tuberculosis (like other bacteria) is susceptible to oxidative pressure and this has yet to be properly utilised as a therapeutic approach in a manner similar to that which has proven highly successful in malaria therapy. Objective: To develop an alternative approach to the incorporation of bacterial siderophores that results in the creation of antitubercular peroxidic leads for subsequent development as novel agents against tuberculosis. Methods: Eight novel peroxides were prepared and the antitubercular activity (H37Rv) was compared to existing artemisinin derivatives in vitro. The potential for toxicity was evaluated against the L6 rat skeletal myoblast and HeLa cervical cancer lines in vitro. Results: The addition of a pyrimidinyl residue to an artemisinin or, preferably, a tetraoxane peroxidic structure results in antitubercular activity in vitro. The same effect is not observed in the absence of the pyrimidine or with other heteroaromatic substituents. Conclusion: The incorporation of a pyrimidinyl residue adjacent to the peroxidic function in an organic peroxide results in anti-tubercular activity in an otherwise inactive peroxidic compound. This will be a useful approach for creating oxidative drugs to target tuberculosis.

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