Polewards chromosome movement driven by microtubule depolymerization in vitro

Nature ◽  
1988 ◽  
Vol 331 (6156) ◽  
pp. 499-504 ◽  
Author(s):  
Douglas E. Koshland ◽  
T. J. Mitchison ◽  
Marc W. Kirschner
Keyword(s):  
Chromosome Movement ◽  
Microtubule Depolymerization

scholarly journals The Ndc80 complex uses a tripartite attachment point to couple microtubule depolymerization to chromosome movement

2011 ◽  
Vol 22 (8) ◽  
pp. 1217-1226 ◽  
Author(s):  
John G. Tooley ◽  
Stephanie A. Miller ◽  
P. Todd Stukenberg
Keyword(s):  
Point Mutations ◽  
Chromosome Movement ◽  
Microtubule Depolymerization ◽  
Attachment Point ◽  
Binding Interface ◽  
Binding Data ◽  
Ndc80 Complex ◽  
Positively Charged ◽  
In Cells

In kinetochores, the Ndc80 complex couples the energy in a depolymerizing microtubule to perform the work of moving chromosomes. The complex directly binds microtubules using an unstructured, positively charged N-terminal tail located on Hec1/Ndc80. Hec1/Ndc80 also contains a calponin homology domain (CHD) that increases its affinity for microtubules in vitro, yet whether it is required in cells and how the tail and CHD work together are critical unanswered questions. Human kinetochores containing Hec1/Ndc80 with point mutations in the CHD fail to align chromosomes or form productive microtubule attachments. Kinetochore architecture and spindle checkpoint protein recruitment are unaffected in these mutants, and the loss of CHD function cannot be rescued by removing Aurora B sites from the tail. The interaction between the Hec1/Ndc80 CHD and a microtubule is facilitated by positively charged amino acids on two separate regions of the CHD, and both are required for kinetochores to make stable attachments to microtubules. Chromosome congression in cells also requires positive charge on the Hec1 tail to facilitate microtubule contact. In vitro binding data suggest that charge on the tail regulates attachment by directly increasing microtubule affinity as well as driving cooperative binding of the CHD. These data argue that in vertebrates there is a tripartite attachment point facilitating the interaction between Hec1/Ndc80 and microtubules. We discuss how such a complex microtubule-binding interface may facilitate the coupling of depolymerization to chromosome movement.

scholarly journals Microtubule depolymerization promotes particle and chromosome movement in vitro.

1991 ◽  
Vol 112 (6) ◽  
pp. 1165-1175 ◽  
Author(s):  
M Coue ◽  
V A Lombillo ◽  
J R McIntosh
Keyword(s):  
Atp Hydrolysis ◽  
Living Cells ◽  
Chromosome Movement ◽  
Sodium Orthovanadate ◽  
Microtubule Depolymerization ◽  
Glass Coverslip ◽  
Perfusion Chamber ◽  
Nucleotide Triphosphates ◽  
The Mean

We have developed a system for studying the motions of cellular objects attached to depolymerizing microtubules in vitro. Radial arrays of microtubules were grown from lysed and extracted Tetrahymena cells attached to a glass coverslip that formed the top of a light microscope perfusion chamber. A preparation of chromosomes, which also contained vesicles, was then perfused into the chamber and allowed to bind to the microtubule array. The concentration of tubulin was then reduced by perfusing buffer that lacked both tubulin and nucleotide triphosphates, and the resulting microtubule depolymerization was observed by light microscopy. A fraction of the bound objects detached in the flow and washed away, while others stabilized the microtubules to which they were bound. Some of the particles and chromosomes, however, moved in toward the Tetrahymena ghost as their associated microtubules shortened. The mean speeds for particles and chromosomes were 26 +/- 20 and 15 +/- 12 microns/min, respectively. These motions occurred when nucleotide triphosphate levels were very low, as a result of either dilution or by the action of apyrase. Furthermore, the motions were unaffected by 100 microM sodium orthovanadate, suggesting that these forces are not the result of ATP hydrolysis by a minus end-directed mechanoenzyme. We conclude that microtubule depolymerization provided the free energy for the motions observed. All the objects that we studied in detail moved against a stream of buffer flowing at approximately 100 microns/s, so that the force being developed was at least 10(-7) dynes. This force is large enough to contribute to some forms of motility in living cells.

scholarly journals Synthesis of New Simplified and Racemic Hemiasterlin Derivatives with Extremely High Cytotoxicity

2018 ◽  
Vol 13 (12) ◽  
pp. 1934578X1801301
Author(s):  
Pham The Chinh ◽  
Đang Thi Tuyet Anh ◽  
Duong Huong Quynh ◽  
Le Nhat Thuy Giang ◽  
Nguyen Ha Thanh ◽  
...  
Keyword(s):  
Cytotoxic Activity ◽  
Cell Lines ◽  
Human Cancer ◽  
Microtubule Depolymerization ◽  
Antimitotic Agent ◽  
Human Cancer Cell Lines ◽  
Weak Activity ◽  
High Cytotoxicity ◽  
Chemistry Community

Hemiasterlin is a potent antimitotic agent acting through inhibition of microtubule depolymerization. For this reason, the synthesis of new hemiasterlin derivatives has attracted a lot of interest in the organic chemistry community recently. In this paper, the synthesis and evaluation of the cytotoxicity of new simplified and racemic hemiasterlin derivatives were reported. All of the synthesized analogues were evaluated in vitro for cytotoxic activity against four human cell lines (KB, Hep-G2, LU and MCF7). Most of these analogues possess a strong cytotoxic activity on two human cancer cell lines (KB and Hep-G2) and very weak activity on LU and MCF7 cell lines.

scholarly journals A permeabilized cell model for studying cell division: a comparison of anaphase chromosome movement and cleavage furrow constriction in lysed PtK1 cells.

1981 ◽  
Vol 88 (3) ◽  
pp. 618-629 ◽  
Author(s):  
W Z Cande ◽  
K McDonald ◽  
R L Meeusen
Keyword(s):  
Cell Model ◽  
Chromosome Movement ◽  
Microtubule Depolymerization ◽  
Mitotic Apparatus ◽  
Permeabilized Cell ◽  
Culture Cells ◽  
Brij 58 ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Chromosome Movements

After lysis in a Brij 58-polyethylene glycol medium, PtK1 cells are permeable to small molecules, such as erythrosin B, and to proteins, such as rhodamine-labeled FAB, myosin subfragment-1, and tubulin. Holes are present in the plasma membrane, and the mitochondria are swollen and distorted, but other membrane-bounded organelles of the lysed cell model are not noticeably altered. After lysis, the mitotic apparatus is functional; chromosomes move poleward and the spindle elongates. Cells lysed while in cytokinesis will continue to divide for several minutes. Addition of crude tubulin extracts, MAP-free tubulin, or taxol to the lysis medium retards anaphase chromosome movements but does not affect cleavage. On the other hand, N-ethylmaleimide-modified myosin subfragment-1, phalloidin, and cytochalasin B inhibit cleavage but have no effect on anaphase chromosome movements under identical lysis conditions. These results suggest that actomyosin plays no functional role in anaphase chromosome movement in mammalian tissue culture cells and that microtubule depolymerization is a rate-limiting step for chromosome-to-pole movements.

scholarly journals Evidence that myosin does not contribute to force production in chromosome movement.

1982 ◽  
Vol 94 (1) ◽  
pp. 165-178 ◽  
Author(s):  
D P Kiehart ◽  
I Mabuchi ◽  
S Inoué
Keyword(s):  
Gamma Globulin ◽  
Meiotic Chromosome ◽  
Force Production ◽  
Chromosome Movement ◽  
Actomyosin Interaction ◽  
Dividing Cells ◽  
Spindle Elongation ◽  
Chromosome Movements

Antibody against cytoplasmic myosin, when microinjected into actively dividing cells, provides a physiological test for the role of actin and myosin in chromosome movement. Anti-Asterias egg myosin, characterized by Mabuchi and Okuno (1977, J. Cell Biol., 74:251), completely and specifically inhibits the actin activated Mg++ -ATPase of myosin in vitro and, when microinjected, inhibits cytokinesis in vivo. Here, we demonstrate that microinjected antibody has no observable effect on the rate or extent of anaphase chromosome movements. Neither central spindle elongation nor chromosomal fiber shortening is affected by doses up to eightfold higher than those require to uniformly inhibit cytokinesis in all injected cells. We calculate that such doses are sufficient to completely inhibit myosin ATPase activity in these cells. Cells injected with buffer alone, with myosin-absorbed antibody, or with nonimmune gamma-globulin, proceed normally through both mitosis and cytokinesis. Control gamma-globulin, labeled with fluorescein, diffuses to homogeneity throughout the cytoplasm in 2-4 min and remains uniformly distributed. Antibody is not excluded from the spindle region. Prometaphase chromosome movements, fertilization, pronuclear migration, and pronuclear fusion are also unaffected by microinjected antimyosin. These experiments demonstrate that antimyosin blocks the actomyosin interaction thought to be responsible for force production in cytokinesis but has no effect on mitotic or meiotic chromosome motion. They provide direct physiological evidence that myosin is not involved in force production for chromosome movement.

scholarly journals Serum- and Glucocorticoid-Inducible Kinase 1 (SGK1) Increases Neurite Formation through Microtubule Depolymerization by SGK1 and by SGK1 Phosphorylation oftau

2006 ◽  
Vol 26 (22) ◽  
pp. 8357-8370 ◽  
Author(s):  
Ying C. Yang ◽  
Cheng H. Lin ◽  
Eminy H. Y. Lee
Keyword(s):  
Hippocampal Neurons ◽  
Kinase Assay ◽  
Phosphorylated Tau ◽  
Microtubule Depolymerization ◽  
Independent Manner ◽  
Cell Functions ◽  
Neurite Formation ◽  
Cultured Hippocampal Neurons

ABSTRACT Serum- and glucocorticoid-inducible kinase 1 (SGK1) is a member of the Ser/Thr protein kinase family that regulates a variety of cell functions. Recently, SGK1 was shown to increase dendritic growth but the mechanism underlying the increase is unknown. Here we demonstrated that SGK1 increased the neurite formation of cultured hippocampal neurons through microtubule (MT) depolymerization via two distinct mechanisms. First, SGK1 directly depolymerized MTs. In vitro MT depolymerization experiments revealed that SGK1, especially N-truncated SGK1, directly disassembled self-polymerized MTs and taxol-stabilized MTs in a dose-dependent and ATP-independent manner. The transfection of sgk1 to HeLa cells also inhibited MT assembly in vivo. Second, SGK1 indirectly depolymerized MTs through the phosphorylation of tau at Ser214. An in vitro kinase assay revealed that active SGK1 phosphorylated tau Ser214 specifically. In vivo transfection of sgk1 also phosphorylated tau Ser214 in HEK293T cells and hippocampal neurons. Further, sgk1 transfection significantly increased the number of primary neurites and shortened the length of the total process in cultured hippocampal neurons. These effects were antagonized by the cotransfection of the tauS214A mutant plasmid. Dexamethasone, a synthetic glucocorticoid, mimics the effect of sgk1 overexpression. Together, these results suggest that SGK1 enhances neurite formation through MT depolymerization by a direct action of SGK1 and by the SGK1 phosphorylation of tau.

scholarly journals Neocentromere-mediated Chromosome Movement in Maize

1997 ◽  
Vol 139 (4) ◽  
pp. 831-840 ◽  
Author(s):  
Hong-Guo Yu ◽  
Evelyn N. Hiatt ◽  
Annette Chan ◽  
Mary Sweeney ◽  
R. Kelly Dawe
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequences ◽  
Average Rate ◽  
Meiotic Chromosome ◽  
Chromosome Movement ◽  
Microtubule Depolymerization ◽  
Chromosome 10 ◽  
Chromosome Congression ◽  
Or Genes

Neocentromere activity is a classic example of nonkinetochore chromosome movement. In maize, neocentromeres are induced by a gene or genes on Abnormal chromosome 10 (Ab10) which causes heterochromatic knobs to move poleward at meiotic anaphase. Here we describe experiments that test how neocentromere activity affects the function of linked centromere/kinetochores (kinetochores) and whether neocentromeres and kinetochores are mobilized on the spindle by the same mechanism. Using a newly developed system for observing meiotic chromosome congression and segregation in living maize cells, we show that neocentromeres are active from prometaphase through anaphase. During mid-anaphase, normal chromosomes move on the spindle at an average rate of 0.79 μm/min. The presence of Ab10 does not affect the rate of normal chromosome movement but propels neocentromeres poleward at rates as high as 1.4 μm/min. Kinetochore-mediated chromosome movement is only marginally affected by the activity of a linked neocentromere. Combined in situ hybridization/immunocytochemistry is used to demonstrate that unlike kinetochores, neocentromeres associate laterally with microtubules and that neocentromere movement is correlated with knob size. These data suggest that microtubule depolymerization is not required for neocentromere motility. We argue that neocentromeres are mobilized on microtubules by the activity of minus end–directed motor proteins that interact either directly or indirectly with knob DNA sequences.

scholarly journals Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells

1998 ◽  
Vol 142 (6) ◽  
pp. 1519-1532 ◽  
Author(s):  
Yasmina Saoudi ◽  
Rati Fotedar ◽  
Ariane Abrieu ◽  
Marcel Dorée ◽  
Jürgen Wehland ◽  
...  
Keyword(s):  
Model System ◽  
Microtubule Dynamics ◽  
In Vitro Assays ◽  
Microtubule Depolymerization ◽  
Dynamic Activity ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Cell Extracts

Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end–directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.

scholarly journals Xkid Is Degraded in a D-Box, KEN-Box, and A-Box-Independent Pathway

2003 ◽  
Vol 23 (12) ◽  
pp. 4126-4138 ◽  
Author(s):  
Anna Castro ◽  
Suzanne Vigneron ◽  
Cyril Bernis ◽  
Jean-Claude Labbé ◽  
Thierry Lorca
Keyword(s):  
Chromosome Segregation ◽  
Degradation Pathway ◽  
Cyclin B ◽  
Chromosome Movement ◽  
Regulatory Factor ◽  
Independent Manner ◽  
C Terminus ◽  
Chromosome Congression

ABSTRACT During mitosis, the Xenopus chromokinesin Kid (Xkid) provides the polar ejection forces needed at metaphase for chromosome congression, and its degradation is required at anaphase to induce chromosome segregation. Despite the fact that the degradation of Xkid at anaphase seems to be a key regulatory factor to induce chromosome movement to the poles, little is known about the mechanisms controlling this proteolysis. We investigated here the degradation pathway of Xkid. We demonstrate that Xkid is degraded both in vitro and in vivo by APC/Cdc20 and APC/Cdh1. We show that, despite the presence of five putative D-box motifs in its sequence, Xkid is proteolyzed in a D-box-independent manner. We identify a domain within the C terminus of this chromokinesin, with sequence GxEN, whose mutation completely stabilizes this protein by both APC/Cdc20 and APC/Cdh1. Moreover, we show that this degradation sequence acts as a transposable motif and induces the proteolysis of a GST-GXEN fusion protein. Finally, we demonstrate that both a D-box and a GXEN-containing peptides completely block APC-dependent degradation of cyclin B and Xkid, indicating that the GXEN domain might mediate the recognition and association of Xkid with the APC.

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