The conformation and activation of Fyn kinase in the oocyte determine its localisation to the spindle poles and cleavage furrow

2011 ◽  
Vol 23 (7) ◽  
pp. 846 ◽  
Author(s):  
Mattan Levi ◽  
Bernard Maro ◽  
Ruth Shalgi
Keyword(s):  
Cell Cycle Control ◽  
Polar Body ◽  
Active Form ◽  
Spindle Pole ◽  
Confocal Imaging ◽  
Cleavage Furrow ◽  
Membrane Area ◽  
Spindle Poles ◽  
N Terminus ◽  
And Function

Several lines of evidence imply the involvement of Fyn, a Src family kinase, in cell-cycle control and cytoskeleton organisation in somatic cells. By live cell confocal imaging of immunostained or cRNA-microinjected mouse oocytes at metaphase of the second meiotic division, membrane localisation of active and non-active Fyn was demonstrated. However, Fyn with a disrupted membrane-binding domain at its N-terminus was targeted to the cytoplasm and spindle in its non-active form and concentrated at the spindle poles when active. During metaphase exit, the amount of phosphorylated Fyn and of spindle-poles Fyn decreased and it started appearing at the membrane area of the cleavage furrow surrounding the spindle midzone, either asymmetrically during polar body II extrusion or symmetrically during mitosis. These results demonstrate that post-translational modifications of Fyn, probably palmitoylation, determine its localisation and function; localisation of de-palmitoylated active Fyn to the spindle poles is involved in spindle pole integrity during metaphase, whereas the localisation of N-terminus palmitoylated Fyn at the membrane near the cleavage furrow indicates its participation in furrow ingression during cytokinesis.

scholarly journals Fyn kinase is involved in cleavage furrow ingression during meiosis and mitosis

Reproduction ◽  
2010 ◽  
Vol 140 (6) ◽  
pp. 827-834 ◽  
Author(s):  
Mattan Levi ◽  
Bernard Maro ◽  
Ruth Shalgi
Keyword(s):  
Cell Cycle Control ◽  
Polar Body ◽  
Mitotic Division ◽  
Dominant Negative ◽  
Confocal Imaging ◽  
Cleavage Furrow ◽  
Filamentous Actin ◽  
Fyn Kinase ◽  
Second Polar Body ◽  
First Time

Fertilization of mammalian oocytes triggers their exit from the second meiotic division metaphase arrest. The extrusion of the second polar body (PBII) that marks the completion of meiosis is followed by the first mitotic cleavage of the zygote. Several lines of evidence in somatic cells imply the involvement of Fyn, an Src family kinase (SFK), in cell cycle control and actin functions. In this study, we demonstrate, using live cell confocal imaging and microinjection of Fyn cRNAs, the recruitment of Fyn to the oocyte's cortical area overlying the chromosomes and its colocalization with filamentous actin (F-actin) during exit from the meiotic metaphase. Fyn concentrated asymmetrically at the cortical site designated for ingression of the PBII cleavage furrow, where F-actin had already been accumulated, and then redispersed throughout the entire cortex only to be recruited again to the cleavage furrow during the first mitotic division. Although microinjection of dominant negative Fyn did not affect initiation of the cleavage furrow, it prolonged the average duration of ingression, decreased the rates of PB extrusion and of the first cleavage, and led to the formation of bigger PBs and longer spindles. Extrusion of the PBII was blocked in oocytes exposed to SU6656, an SFK inhibitor. Our results demonstrate, for the first time, a continuous colocalization of Fyn and F-actin during meiosis and imply a role for the SFKs, in general, and for Fyn, in particular, in regulating pathways that involve actin cytoskeleton, during ingression of the meiotic and mitotic cleavage furrows.

scholarly journals CDK-1 inhibits meiotic spindle shortening and dynein-dependent spindle rotation in C. elegans

2011 ◽  
Vol 193 (7) ◽  
pp. 1229-1244 ◽  
Author(s):  
Marina L. Ellefson ◽  
Francis J. McNally
Keyword(s):  
Polar Body ◽  
Spindle Pole ◽  
Cyclin B ◽  
Meiotic Spindle ◽  
Anaphase Promoting Complex ◽  
C Elegans ◽  
Perpendicular Orientation ◽  
Spindle Poles ◽  
Spindle Rotation ◽  
Chromosome Separation

In animals, the female meiotic spindle is positioned at the egg cortex in a perpendicular orientation to facilitate the disposal of half of the chromosomes into a polar body. In Caenorhabditis elegans, the metaphase spindle lies parallel to the cortex, dynein is dispersed on the spindle, and the dynein activators ASPM-1 and LIN-5 are concentrated at spindle poles. Anaphase-promoting complex (APC) activation results in dynein accumulation at spindle poles and dynein-dependent rotation of one spindle pole to the cortex, resulting in perpendicular orientation. To test whether the APC initiates spindle rotation through cyclin B–CDK-1 inactivation, separase activation, or degradation of an unknown dynein inhibitor, CDK-1 was inhibited with purvalanol A in metaphase-I–arrested, APC-depleted embryos. CDK-1 inhibition resulted in the accumulation of dynein at spindle poles and dynein-dependent spindle rotation without chromosome separation. These results suggest that CDK-1 blocks rotation by inhibiting dynein association with microtubules and with LIN-5–ASPM-1 at meiotic spindle poles and that the APC promotes spindle rotation by inhibiting CDK-1.

scholarly journals Centrosomes as signalling centres

2014 ◽  
Vol 369 (1650) ◽  
pp. 20130464 ◽  
Author(s):  
Christian Arquint ◽  
Anna-Maria Gabryjonczyk ◽  
Erich A. Nigg
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Cell Cycle Control ◽  
Spindle Pole ◽  
Signalling Pathways ◽  
Spindle Poles ◽  
Spindle Pole Bodies ◽  
Damage Response ◽  
The Subject

Centrosomes—as well as the related spindle pole bodies (SPBs) of yeast—have been extensively studied from the perspective of their microtubule-organizing roles. Moreover, the biogenesis and duplication of these organelles have been the subject of much attention, and the importance of centrosomes and the centriole–ciliary apparatus for human disease is well recognized. Much less developed is our understanding of another facet of centrosomes and SPBs, namely their possible role as signalling centres. Yet, many signalling components, including kinases and phosphatases, have been associated with centrosomes and spindle poles, giving rise to the hypothesis that these organelles might serve as hubs for the integration and coordination of signalling pathways. In this review, we discuss a number of selected studies that bear on this notion. We cover different processes (cell cycle control, development, DNA damage response) and organisms (yeast, invertebrates and vertebrates), but have made no attempt to be comprehensive. This field is still young and although the concept of centrosomes and SPBs as signalling centres is attractive, it remains primarily a concept—in need of further scrutiny. We hope that this review will stimulate thought and experimentation.

scholarly journals Cell Cycle-dependent Dynamics and Regulation of Mitotic Kinesins in Drosophila S2 Cells

2005 ◽  
Vol 16 (8) ◽  
pp. 3896-3907 ◽  
Author(s):  
Gohta Goshima ◽  
Ronald D. Vale
Keyword(s):  
Cell Cycle ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Active Form ◽  
Nuclear Export Signal ◽  
Nuclear Envelope Breakdown ◽  
Spindle Poles ◽  
Cycle Dependent ◽  
Green Fluorescent ◽  
And Function

Constructing a mitotic spindle requires the coordinated actions of several kinesin motor proteins. Here, we have visualized the dynamics of five green fluorescent protein (GFP)-tagged mitotic kinesins (class 5, 6, 8, 13, and 14) in live Drosophila Schneider cell line (S2), after first demonstrating that the GFP-tag does not interfere with the mitotic functions of these kinesins using an RNA interference (RNAi)-based rescue strategy. Class 8 (Klp67A) and class 14 (Ncd) kinesin are sequestered in an active form in the nucleus during interphase and engage their microtubule targets upon nuclear envelope breakdown (NEB). Relocalization of Klp67A to the cytoplasm using a nuclear export signal resulted in the disassembly of the interphase microtubule array, providing support for the hypothesis that this kinesin class possesses microtubule-destabilizing activity. The interactions of Kinesin-5 (Klp61F) and -6 (Pavarotti) with microtubules, on the other hand, are activated and inactivated by Cdc2 phosphorylation, respectively, as shown by examining localization after mutating Cdc2 consensus sites. The actions of microtubule-destabilizing kinesins (class 8 and 13 [Klp10A]) seem to be controlled by cell cycle-dependent changes in their localizations. Klp10A, concentrated on microtubule plus ends in interphase and prophase, relocalizes to centromeres and spindle poles upon NEB and remains at these sites throughout anaphase. Consistent with this localization, RNAi analysis showed that this kinesin contributes to chromosome-to-pole movement during anaphase A. Klp67A also becomes kinetochore associated upon NEB, but the majority of the population relocalizes to the central spindle by the timing of anaphase A onset, consistent with our RNAi result showing no effect of depleting this motor on anaphase A. These results reveal a diverse spectrum of regulatory mechanisms for controlling the localization and function of five mitotic kinesins at different stages of the cell cycle.

scholarly journals A Genetic Analysis of Interactions with Spc110p Reveals Distinct Functions of Spc97p and Spc98p, Components of the Yeast γ-Tubulin Complex

1998 ◽  
Vol 9 (8) ◽  
pp. 2201-2216 ◽  
Author(s):  
Thu Nguyen ◽  
Dani B.N. Vinh ◽  
Douglas K. Crawford ◽  
Trisha N. Davis
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Amino Terminus ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Synthetic Lethal ◽  
Lethal Phenotype ◽  
Amino Terminal ◽  
N Terminus ◽  
Two Hybrid

The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the microtubule-organizing center. Spc110p is an essential structural component of the SPB and spans between the central and inner plaques of this multilamellar organelle. The amino terminus of Spc110p faces the inner plaque, the substructure from which spindle microtubules radiate. We have undertaken a synthetic lethal screen to identify mutations that enhance the phenotype of the temperature-sensitive spc110–221 allele, which encodes mutations in the amino terminus. The screen identified mutations inSPC97 and SPC98, two genes encoding components of the Tub4p complex in yeast. The spc98–63allele is synthetic lethal only with spc110 alleles that encode mutations in the N terminus of Spc110p. In contrast, thespc97 alleles are synthetic lethal withspc110 alleles that encode mutations in either the N terminus or the C terminus. Using the two-hybrid assay, we show that the interactions of Spc110p with Spc97p and Spc98p are not equivalent. The N terminus of Spc110p displays a robust interaction with Spc98p in two different two-hybrid assays, while the interaction between Spc97p and Spc110p is not detectable in one strain and gives a weak signal in the other. Extra copies of SPC98 enhance the interaction between Spc97p and Spc110p, while extra copies of SPC97interfere with the interaction between Spc98p and Spc110p. By testing the interactions between mutant proteins, we show that the lethal phenotype in spc98–63 spc110–221 cells is caused by the failure of Spc98–63p to interact with Spc110–221p. In contrast, the lethal phenotype in spc97–62 spc110–221 cells can be attributed to a decreased interaction between Spc97–62p and Spc98p. Together, these studies provide evidence that Spc110p directly links the Tub4p complex to the SPB. Moreover, an interaction between Spc98p and the amino-terminal region of Spc110p is a critical component of the linkage, whereas the interaction between Spc97p and Spc110p is dependent on Spc98p.

scholarly journals Interaction between Poly(ADP-ribose) and NuMA Contributes to Mitotic Spindle Pole Assembly

2009 ◽  
Vol 20 (21) ◽  
pp. 4575-4585 ◽  
Author(s):  
Paul Chang ◽  
Margaret Coughlin ◽  
Timothy J. Mitchison
Keyword(s):  
Binding Proteins ◽  
Magnetic Beads ◽  
Spindle Pole ◽  
Covalent Modification ◽  
Mitotic Spindles ◽  
Spindle Poles ◽  
Tankyrase 1 ◽  
Mitotic Spindle Pole

Poly(ADP-ribose) (pADPr), made by PARP-5a/tankyrase-1, localizes to the poles of mitotic spindles and is required for bipolar spindle assembly, but its molecular function in the spindle is poorly understood. To investigate this, we localized pADPr at spindle poles by immuno-EM. We then developed a concentrated mitotic lysate system from HeLa cells to probe spindle pole assembly in vitro. Microtubule asters assembled in response to centrosomes and Ran-GTP in this system. Magnetic beads coated with pADPr, extended from PARP-5a, also triggered aster assembly, suggesting a functional role of the pADPr in spindle pole assembly. We found that PARP-5a is much more active in mitosis than interphase. We used mitotic PARP-5a, self-modified with pADPr chains, to capture mitosis-specific pADPr-binding proteins. Candidate binding proteins included the spindle pole protein NuMA previously shown to bind to PARP-5a directly. The rod domain of NuMA, expressed in bacteria, bound directly to pADPr. We propose that pADPr provides a dynamic cross-linking function at spindle poles by extending from covalent modification sites on PARP-5a and NuMA and binding noncovalently to NuMA and that this function helps promote assembly of exactly two poles.

scholarly journals Role of Ubiquitin Ligases and the Proteasome in Oncogenesis: Novel Targets for Anticancer Therapies

2013 ◽  
Vol 31 (9) ◽  
pp. 1231-1238 ◽  
Author(s):  
Lindsey N. Micel ◽  
John J. Tentler ◽  
Peter G. Smith ◽  
Gail S. Eckhardt
Keyword(s):  
Cell Cycle ◽  
Anticancer Agents ◽  
Cell Cycle Control ◽  
Ubiquitin Proteasome System ◽  
Cellular Regulation ◽  
Ubiquitin Chain ◽  
Key Enzyme ◽  
Ubiquitin Proteasome ◽  
Enzyme Families ◽  
And Function

The ubiquitin proteasome system (UPS) regulates the ubiquitination, and thus degradation and turnover, of many proteins vital to cellular regulation and function. The UPS comprises a sequential series of enzymatic processes using four key enzyme families: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-carrier proteins), E3 (ubiquitin-protein ligases), and E4 (ubiquitin chain assembly factors). Because the UPS is a crucial regulator of the cell cycle, and abnormal cell-cycle control can lead to oncogenesis, aberrancies within the UPS pathway can result in a malignant cellular phenotype and thus has become an attractive target for novel anticancer agents. This article will provide an overall review of the mechanics of the UPS, describe aberrancies leading to cancer, and give an overview of current drug therapies selectively targeting the UPS.

scholarly journals Cytoplasmic dynein participates in meiotic checkpoint inactivation in mouse oocytes by transporting cytoplasmic mitotic arrest-deficient (Mad) proteins from kinetochores to spindle poles

Reproduction ◽  
2007 ◽  
Vol 133 (4) ◽  
pp. 685-695 ◽  
Author(s):  
Dong Zhang ◽  
Shen Yin ◽  
Man-Xi Jiang ◽  
Wei Ma ◽  
Yi Hou ◽  
...  
Keyword(s):  
Spindle Checkpoint ◽  
Germinal Vesicle ◽  
Cytoplasmic Dynein ◽  
Mitotic Arrest ◽  
Checkpoint Proteins ◽  
Meiotic Progression ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Oocyte Meiosis ◽  
And Function

The present study was designed to investigate the localization and function of cytoplasmic dynein (dynein) during mouse oocyte meiosis and its relationship with two major spindle checkpoint proteins, mitotic arrest-deficient (Mad) 1 and Mad2. Oocytes at various stages during the first meiosis were fixed and immunostained for dynein, Mad1, Mad2, kinetochores, microtubules, and chromosomes. Some oocytes were treated with nocodazole before examination. Anti-dynein antibody was injected into the oocytes at germinal vesicle (GV) stage before the examination of its effects on meiotic progression or Mad1 and Mad2 localization. Results showed that dynein was present in the oocytes at various stages from GV to metaphase II and the locations of Mad1 and Mad2 were associated with dynein’s movement. Both Mad1 and Mad2 had two existing states: one existed in the cytoplasm (cytoplasmic Mad1 or cytoplasmic Mad2), which did not bind to kinetochores, while the other bound to kinetochores (kinetochore Mad1 or kinetochore Mad2). The equilibrium between the two states varied during meiosis and/or in response to the changes of the connection between microtubules and kinetochores. Cytoplasmic Mad1 and Mad2 recruited to chromosomes when the connection between microtubules and chromosomes was destroyed. Inhibition of dynein interferes with cytoplasmic Mad1 and Mad2 transportation from chromosomes to spindle poles, thus inhibits checkpoint silence and delays anaphase onset. These results indicate that dynein may play a role in spindle checkpoint inactivation.

Abstract 425: Development of a Vascular Cell Adhesion Molecule-1 Targeted Esterase Nanofiber that Binds Activated Endothelial Cells

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Mazen S Albaghdadi ◽  
Charles Rupert Perez ◽  
Amanda Worthy ◽  
Edward S Bahnson ◽  
Clifford Carpenter ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Adhesion Molecule ◽  
Free Cholesterol ◽  
Confocal Imaging ◽  
Atherosclerotic Plaques ◽  
Atherosclerotic Cardiovascular Disease ◽  
Dependent Manner ◽  
Cholesterol Esters ◽  
Binding Peptide ◽  
N Terminus

Introduction: Novel therapies capable of directly targeting and regressing atherosclerotic plaques would provide tremendous benefit to patients with atherosclerotic cardiovascular disease. Molecular imaging studies have shown the feasibility of targeting vascular adhesion molecule (VCAM-1) for non-invasive atherosclerosis imaging. Histidine contains an imidazole ring with esterase activity that can convert cholesterol esters to free cholesterol. Thus, we hypothesize that a peptide amphiphile (PA) functionalized with a VCAM-1 binding peptide and histidine will spontaneously form a targeted nanofiber that binds activated endothelium and liberates free cholesterol from cholesterol esters. Methods: PAs were synthesized by solid phase methods and purified by reversed phase HPLC. Three different VCAM-1 binding peptide sequences were conjugated to the PAs (free N-terminus, free C-terminus, and cyclic). To assess 3-dimensional structure of the co-assembled PAs, cryogenic transmission electron microscopy (TEM) was used. To assess targeting of the VCAM-PAs (50μM), human umbilical endothelial cells (HUVECs) were stimulated with TNFα (10ng/ml). To assess the ability of the histidine-PA to liberate free cholesterol, a modified Amplex Red Cholesterol Assay was used. Results: Using TEM, we demonstrated that the co-assembled VCAM- and histidine-PAs formed characteristic nanofibers with diameters of 5-8nm and lengths ranging from 300nm to 1μm. TNFα-activated HUVECs were confirmed to express VCAM-1 protein by Western blot analysis. Confocal imaging revealed that the VCAM-1 binding peptide with a free N-terminus conjugated to the PA had the greatest binding affinity to the TNFα-activated HUVECs. Co-incubation of the histidine-PA with cholesterol esters demonstrated that free cholesterol was liberated from cholesterol esters in a concentration-dependent manner with a five-fold increase in free cholesterol release with 25μM of PA versus 1μM. Conclusion: We have designed and characterized a VCAM-1 targeted nanofiber that successfully binds activated endothelium and generates free cholesterol from cholesterol esters. This novel therapeutic nanotechnology has the capacity to target and regress atherosclerotic plaques.

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