scholarly journals Spatiotemporal organization of branched microtubule networks

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Akanksha Thawani ◽  
Howard A Stone ◽  
Joshua W Shaevitz ◽  
Sabine Petry
Keyword(s):  
Single Molecule ◽  
Reaction Model ◽  
Sequential Reaction ◽  
Spatiotemporal Organization ◽  
Nucleation Sites ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Microtubule Networks ◽  
Xenopus Egg Extracts ◽  
Rate Limiting

To understand how chromosomes are segregated, it is necessary to explain the precise spatiotemporal organization of microtubules (MTs) in the mitotic spindle. We use Xenopus egg extracts to study the nucleation and dynamics of MTs in branched networks, a process that is critical for spindle assembly. Surprisingly, new branched MTs preferentially originate near the minus-ends of pre-existing MTs. A sequential reaction model, consisting of deposition of nucleation sites on an existing MT, followed by rate-limiting nucleation of branches, reproduces the measured spatial profile of nucleation, the distribution of MT plus-ends and tubulin intensity. By regulating the availability of the branching effectors TPX2, augmin and γ-TuRC, combined with single-molecule observations, we show that first TPX2 is deposited on pre-existing MTs, followed by binding of augmin/γ-TuRC to result in the nucleation of branched MTs. In sum, regulating the localization and kinetics of nucleation effectors governs the architecture of branched MT networks.

scholarly journals Spatiotemporal organization of branched microtubule networks

2019 ◽  
Author(s):  
Akanksha Thawani ◽  
Howard A Stone ◽  
Joshua W Shaevitz ◽  
Sabine Petry
Keyword(s):  
Single Molecule ◽  
Reaction Pathway ◽  
Reaction Model ◽  
Sequential Reaction ◽  
Spatiotemporal Organization ◽  
Nucleation Sites ◽  
Egg Extracts ◽  
Microtubule Networks ◽  
Rate Limiting ◽  
Kinetics Of

AbstractTo understand how chromosomes are segregated, it is necessary to explain the precise spatiotemporal organization of microtubules (MTs) in the mitotic spindle. We useXenopusegg extracts to study the nucleation and dynamics of MTs in branched networks, a process that is critical for spindle assembly. Surprisingly, new branched MTs preferentially originate near the minus-ends of pre-existing MTs. A sequential reaction model, consisting of deposition of nucleation sites on an existing MT, followed by rate-limiting nucleation of branches, reproduces the measured spatial profile of nucleation, the distribution of MT plus-ends and tubulin intensity. By regulating the availability of the branching effectors TPX2, augmin and γ-TuRC, combined with single-molecule observations, we show that first TPX2 is deposited on pre-existing MTs, followed by binding of augmin/γ-TuRC to result in the nucleation of branched MTs. In sum, regulating the localization and kinetics of nucleation effectors governs the architecture of branched MT networks.Impact StatementA sequential reaction pathway involving TPX2, augmin and γ-TuRC governs the assembly and architecture of branched microtubule networks.

scholarly journals Single Molecule Studies of Eukaryotic Replisomes in Xenopus Egg Extracts

2010 ◽  
Vol 98 (3) ◽  
pp. 437a
Author(s):  
Hasan Yardimci ◽  
Anna B. Kochaniak ◽  
Satoshi Habuchi ◽  
Courtney G. Havens ◽  
Johannes C. Walter ◽  
...  
Keyword(s):  
Single Molecule ◽  
Egg Extracts ◽  
Single Molecule Studies ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

scholarly journals Single-molecule analysis of DNA replication in Xenopus egg extracts

Methods ◽  
2012 ◽  
Vol 57 (2) ◽  
pp. 179-186 ◽  
Author(s):  
Hasan Yardimci ◽  
Anna B. Loveland ◽  
Antoine M. van Oijen ◽  
Johannes C. Walter
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Single Molecule Analysis

scholarly journals Studying chromosome biology with single-molecule resolution in Xenopus laevis egg extracts

2021 ◽  
Author(s):  
George Cameron ◽  
Hasan Yardimci
Keyword(s):  
Xenopus Laevis ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Structural Rearrangement ◽  
Alternative Approach ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Exciting Development

Abstract Cell-free extracts from Xenopus laevis eggs are a model system for studying chromosome biology. Xenopus egg extracts can be synchronised in different cell cycle stages, making them useful for studying DNA replication, DNA repair and chromosome organisation. Combining single-molecule approaches with egg extracts is an exciting development being used to reveal molecular mechanisms that are difficult to study using conventional approaches. Fluorescence-based single-molecule imaging of surface-tethered DNAs has been used to visualise labelled protein movements on stretched DNA, the dynamics of DNA–protein complexes and extract-dependent structural rearrangement of stained DNA. Force-based single-molecule techniques are an alternative approach to measure mechanics of DNA and proteins. In this essay, the details of these single-molecule techniques, and the insights into chromosome biology they provide, will be discussed.

scholarly journals The bulk of unpolymerized actin in Xenopus egg extracts is ATP-bound.

1995 ◽  
Vol 6 (2) ◽  
pp. 227-236 ◽  
Author(s):  
J Rosenblatt ◽  
P Peluso ◽  
T J Mitchison
Keyword(s):  
Actin Polymerization ◽  
Critical Concentration ◽  
Large Fraction ◽  
Nucleotide Exchange ◽  
Chromatography Analysis ◽  
Thymosin Beta 4 ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Non-muscle cells contain 15-500 microM actin, a large fraction of which is unpolymerized. Thus, the concentration of unpolymerized actin is well above the critical concentration for polymerization in vitro (0.2 microM). This fraction of actin could be prevented from polymerization by being ADP bound (therefore less favored to polymerize) or by being ATP bound and sequestered by a protein such as thymosin beta 4, or both. We isolated the unpolymerized actin from Xenopus egg extracts using immobilized DNase 1 and assayed the bound nucleotide. High-pressure liquid chromatography analysis showed that the bulk of soluble actin is ATP bound. Analysis of actin-bound nucleotide exchange rates suggested the existence of two pools of unpolymerized actin, one of which exchanges nucleotide relatively rapidly and another that apparently does not exchange. Native gel electrophoresis of Xenopus egg extracts demonstrated that most of the soluble actin exists in complexes with other proteins, one of which might be thymosin beta 4. These results are consistent with actin polymerization being controlled by the sequestration and release of ATP-bound actin, and argue against nucleotide exchange playing a major role in regulating actin polymerization.

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