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 Taxol binds to cellular microtubules.

1982 ◽  
Vol 94 (3) ◽  
pp. 688-696 ◽  
Author(s):  
J J Manfredi ◽  
J Parness ◽  
S B Horwitz
Keyword(s):  
Specific Binding ◽  
Microtubule Assembly ◽  
Scatchard Analysis ◽  
Cellular Mechanisms ◽  
Drug Concentrations ◽  
Binding Data ◽  
Direct Binding ◽  
Tubulin Immunofluorescence ◽  
In Cells

Taxol is a low molecular weight plant derivative which enhances microtubule assembly in vitro and has the unique ability to promote the formation of discrete microtubule bundles in cells. Tritium-labeled taxol binds directly to microtubules in vitro with a stoichiometry approaching one (Parness, J., and S. B. Horwitz, 1981, J. Cell Biol. 91:479-487). We now report studies in cells on the binding of [3H]taxol and the formation of microtubule bundles. [3H]Taxol binds to the macrophagelike cell line, J774.2, in a specific and saturable manner. Scatchard analysis of the specific binding data demonstrates a single set of high affinity binding sites. Maximal binding occurs at drug concentrations which produce maximal growth inhibition. Conditions which depolymerize microtubules in intact and extracted cells as determined by tubulin immunofluorescence inhibit the binding of [3H]taxol. This strongly suggests that taxol binds specifically to cellular microtubules. Extraction with 0.1% Nonidet P-40 or depletion of cellular ATP by treatment with 10 mM NaN3 prevents the characteristic taxol-induced bundle formation. The binding of [3H]taxol, however, is retained under these conditions. Thus, there formation. The binding of [3H]taxol, however, is retained under these conditions. Thus, there must be specific cellular mechanisms which are required for bundle formation, in addition to the direct binding of taxol to cytoplasmic microtubules.

scholarly journals Cyclin B1 scaffolds MAD1 at the corona to activate the spindle assembly checkpoint

2019 ◽  
Author(s):  
Lindsey A Allan ◽  
Magda Reis ◽  
Yahui Liu ◽  
Pim Huis in ’t Veld ◽  
Geert JPL Kops ◽  
...  
Keyword(s):  
Genome Stability ◽  
Spindle Assembly Checkpoint ◽  
Point Mutations ◽  
Spindle Assembly ◽  
Cyclin B1 ◽  
Cyclin B ◽  
Mitotic Progression ◽  
Mitotic Kinase ◽  
Binding Interface

ABSTRACTThe Cyclin B:CDK1 kinase complex is the master regulator of mitosis that phosphorylates hundreds of proteins to coordinate mitotic progression. We show here that, in addition to these kinase functions, Cyclin B also scaffolds a localised signalling pathway to help preserve genome stability. Cyclin B1 localises to an expanded region of the outer kinetochore, known as the corona, where it scaffolds the spindle assembly checkpoint (SAC) machinery by binding directly to MAD1. In vitro reconstitutions map the key binding interface to a few acidic residues in the N-terminus of MAD1, and point mutations in this region remove corona MAD1 and weaken the SAC. Therefore, Cyclin B1 is the long-sought-after scaffold that links MAD1 to the corona and this specific pool of MAD1 is needed to generate a robust SAC response. Robustness, in this context, arises because Cyclin B1-MAD1 localisation becomes MPS1-independent after the corona has been established. We demonstrate that this allows corona-MAD1 to persist at kinetochores when MPS1 activity falls, ensuring that it can still be phosphorylated on a key C-terminal catalytic site by MPS1. Therefore, this study explains how corona MAD1 generates a robust SAC signal and why stripping of this pool by dynein is essential for SAC silencing. It also reveals that the key mitotic kinase, Cyclin B1-Cdk1, scaffolds the pathway that inhibits its own degradation.

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 Selective sorting of microRNAs into exosomes by phase-separated YBX1 condensates

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Xiao-Man Liu ◽  
Liang Ma ◽  
Randy Schekman
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Cultured Cells ◽  
Rna Binding Proteins ◽  
Point Mutations ◽  
Cell Communication ◽  
Mediate Cell ◽  
Local Enrichment ◽  
In Cells

Exosomes may mediate cell-to-cell communication by transporting various proteins and nucleic acids to neighboring cells. Some protein and RNA cargoes are significantly enriched in exosomes. How cells efficiently and selectively sort them into exosomes remains incompletely explored. Previously we reported that YBX1 is required in sorting of miR-223 into exosomes. Here we show that YBX1 undergoes liquid-liquid phase separation (LLPS) in vitro and in cells. YBX1 condensates selectively recruit miR-223 in vitro and into exosomes secreted by cultured cells. Point mutations that inhibit YBX1 phase separation impair the incorporation of YBX1 protein into biomolecular condensates formed in cells, and perturb miR-233 sorting into exosomes. We propose that phase separation-mediated local enrichment of cytosolic RNA binding proteins and their cognate RNAs enables their targeting and packaging by vesicles that bud into multivesicular bodies. This provides a possible mechanism for efficient and selective engulfment of cytosolic proteins and RNAs into intraluminal vesicles which are then secreted as exosomes from cells.

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 Structural basis for Scc3-dependent cohesin recruitment to chromatin

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Yan Li ◽  
Kyle W Muir ◽  
Matthew W Bowler ◽  
Jutta Metz ◽  
Christian H Haering ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Interaction ◽  
Structural Basis ◽  
Binding Interface ◽  
Double Stranded Dna ◽  
Damage Repair ◽  
Chromatid Cohesion ◽  
Ring Complex ◽  
Positively Charged

The cohesin ring complex is required for numerous chromosomal transactions including sister chromatid cohesion, DNA damage repair and transcriptional regulation. How cohesin engages its chromatin substrate has remained an unresolved question. We show here, by determining a crystal structure of the budding yeast cohesin HEAT-repeat subunit Scc3 bound to a fragment of the Scc1 kleisin subunit and DNA, that Scc3 and Scc1 form a composite DNA interaction module. The Scc3-Scc1 subcomplex engages double-stranded DNA through a conserved, positively charged surface. We demonstrate that this conserved domain is required for DNA binding by Scc3-Scc1 in vitro, as well as for the enrichment of cohesin on chromosomes and for cell viability. These findings suggest that the Scc3-Scc1 DNA-binding interface plays a central role in the recruitment of cohesin complexes to chromosomes and therefore for cohesin to faithfully execute its functions during cell division.

scholarly journals Selective sorting of microRNAs into exosomes by phase-separated YBX1 condensates

2021 ◽  
Author(s):  
Xiao-Man Liu ◽  
Liang Ma ◽  
Randy Schekman
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Cultured Cells ◽  
Rna Binding Proteins ◽  
Point Mutations ◽  
Cell Communication ◽  
Mediate Cell ◽  
Local Enrichment ◽  
In Cells

Exosomes may mediate cell-to-cell communication by transporting various proteins and nucleic acids to neighboring cells. Some protein and RNA cargoes are significantly enriched in exosomes. How cells efficiently and selectively sort them into exosomes remains incompletely explored. Previously we reported that YBX1 is required in sorting of miR-223 into exosomes. Here we show that YBX1 undergoes liquid-liquid phase separation (LLPS) in vitro and in cells. YBX1 condensates selectively recruit miR-223 in vitro and into exosomes secreted by cultured cells. Point mutations that inhibit YBX1 phase separation impair the incorporation of YBX1 protein into biomolecular condensates formed in cells, and perturb miR-233 sorting into exosomes. We propose that phase separation-mediated local enrichment of cytosolic RNA binding proteins and their cognate RNAs enables their targeting and packaging by vesicles that bud into multivesicular bodies. This provides a possible mechanism for efficient and selective engulfment of cytosolic proteins and RNAs into intraluminal vesicles which are then secreted as exosomes from cells.

scholarly journals The NDC80 complex proteins Nuf2 and Hec1 make distinct contributions to kinetochore–microtubule attachment in mitosis

2011 ◽  
Vol 22 (6) ◽  
pp. 759-768 ◽  
Author(s):  
Lynsie J.R. Sundin ◽  
Geoffrey J. Guimaraes ◽  
Jennifer G. DeLuca
Keyword(s):  
Structural Studies ◽  
Tail Domain ◽  
Wild Type ◽  
Timely Manner ◽  
Microtubule Attachment ◽  
Ndc80 Complex ◽  
Spindle Microtubules ◽  
Mutant Cells ◽  
Positively Charged ◽  
In Cells

Successful mitosis requires that kinetochores stably attach to the plus ends of spindle microtubules. Central to generating these attachments is the NDC80 complex, made of the four proteins Spc24, Spc25, Nuf2, and Hec1/Ndc80. Structural studies have revealed that portions of both Hec1 and Nuf2 N termini fold into calponin homology (CH) domains, which are known to mediate microtubule binding in certain proteins. Hec1 also contains a basic, positively charged stretch of amino acids that precedes its CH domain, referred to as the “tail.” Here, using a gene silence and rescue approach in HeLa cells, we show that the CH domain of Hec1, the CH domain of Nuf2, and the Hec1 tail each contributes to kinetochore–microtubule attachment in distinct ways. The most severe defects in kinetochore–microtubule attachment were observed in cells rescued with a Hec1 CH domain mutant, followed by those rescued with a Hec1 tail domain mutant. Cells rescued with Nuf2 CH domain mutants, however, generated stable kinetochore–microtubule attachments but failed to generate wild-type interkinetochore tension and failed to enter anaphase in a timely manner. These data suggest that the CH and tail domains of Hec1 generate essential contacts between kinetochores and microtubules in cells, whereas the Nuf2 CH domain does not.

scholarly journals A novel compound that disrupts mitotic spindle poles in human cells

2020 ◽  
Author(s):  
Dilan Jaunky ◽  
Mathieu Husser ◽  
Kevin Larocque ◽  
Peter Liu ◽  
Sajinth Thampipillai ◽  
...  
Keyword(s):  
Spindle Pole ◽  
Cancer Therapies ◽  
Microtubule Depolymerization ◽  
Microtubule Polymerization ◽  
Spindle Poles ◽  
Multipolar Spindles ◽  
Unique Effect ◽  
Anti Cancer ◽  
In Cells

ABSTRACTWe characterize the mechanism of action of a new microtubule-targeting compound in cells. Microtubule-targeting drugs are used as successful anti-cancer therapies. We synthesized a family of compounds that share a common scaffold and have several functional groups amenable to modifications. We found that one of the active derivatives, C75, reduces cell viability and prevents microtubule polymerization in vitro. In this study, we explore the phenotypes caused by C75 in cells. It causes mitotic arrest and spindle phenotypes in several cancer cell lines in the nanomolar range. C75 can bind to the Colchicine-pocket on tubulin in vitro, but causes different effects on microtubules in cells. While Colchicine causes a decrease in microtubules and spindle pole collapse without re-growth, similar concentrations of C75 cause a rapid loss of microtubules and spindle pole fragmentation followed by microtubule re-growth to form multipolar spindles. In addition, C75 and Colchicine synergize for reduced viability and spindle phenotypes. Importantly, the phenotypes caused by C75 are similar to those caused by the depletion of ch-TOG, a microtubule polymerase, and tubulin and ch-TOG are displaced and oscillate in C75-treated cells. This suggests that C75 causes microtubule depolymerization in cells either directly or indirectly via inhibiting ch-TOG. This unique effect of C75 on microtubules warrants further exploration of its anti-cancer potential.

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