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 Localization of the Kinesin-like Protein Xklp2 to Spindle Poles Requires a Leucine Zipper, a Microtubule-associated Protein, and Dynein

1998 ◽  
Vol 143 (3) ◽  
pp. 673-685 ◽  
Author(s):  
Torsten Wittmann ◽  
Haralabia Boleti ◽  
Claude Antony ◽  
Eric Karsenti ◽  
Isabelle Vernos
Keyword(s):  
Leucine Zipper ◽  
Molecular Mechanisms ◽  
Single Point ◽  
Spindle Pole ◽  
Single Point Mutation ◽  
Spindle Poles ◽  
Centrosome Separation ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Xklp2 is a plus end–directed Xenopus kinesin-like protein localized at spindle poles and required for centrosome separation during spindle assembly in Xenopus egg extracts. A glutathione-S-transferase fusion protein containing the COOH-terminal domain of Xklp2 (GST-Xklp2-Tail) was previously found to localize to spindle poles (Boleti, H., E. Karsenti, and I. Vernos. 1996. Cell. 84:49–59). Now, we have examined the mechanism of localization of GST-Xklp2-Tail. Immunofluorescence and electron microscopy showed that Xklp2 and GST-Xklp2-Tail localize specifically to the minus ends of spindle pole and aster microtubules in mitotic, but not in interphase, Xenopus egg extracts. We found that dimerization and a COOH-terminal leucine zipper are required for this localization: a single point mutation in the leucine zipper prevented targeting. The mechanism of localization is complex and two additional factors in mitotic egg extracts are required for the targeting of GST-Xklp2-Tail to microtubule minus ends: (a) a novel 100-kD microtubule-associated protein that we named TPX2 (Targeting protein for Xklp2) that mediates the binding of GST-Xklp2-Tail to microtubules and (b) the dynein–dynactin complex that is required for the accumulation of GST-Xklp2-Tail at microtubule minus ends. We propose two molecular mechanisms that could account for the localization of Xklp2 to microtubule minus ends.

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 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 DNA content contributes to nuclear size control in Xenopus laevis

2020 ◽  
Vol 31 (24) ◽  
pp. 2703-2717
Author(s):  
Hiroko Heijo ◽  
Sora Shimogama ◽  
Shuichi Nakano ◽  
Anna Miyata ◽  
Yasuhiro Iwao ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Physical Properties ◽  
Chromatin Structure ◽  
Dna Content ◽  
Size Control ◽  
Nuclear Size ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Nuclear Expansion

Physical properties of DNA, including quantity and chromatin structure within the nucleus, contribute to nuclear expansion dynamics and final nuclear size in Xenopus egg extracts and embryos.

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