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Spindle assembly occurs through three, often coexisting, pathways – the centrosome-mediated pathway, the chromatin-mediated pathway, and the microtubule-mediated pathway – collectively contributing to form a robust spindle apparatus.
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As cells progress into mitosis, the nuclear envelope breaks down, and the condensed chromosomes are exposed to the array of bipolar microtubules of the mitotic spindle. The kinetochore, a large, disc-shaped protein complex, is present at the centromere region of the sister chromatids and acts as a binding site for the microtubules.  Usually, the plus-end of a single microtubule is embedded within the kinetochore. However, some kinetochores first establish lateral contact with the side-wall...
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The spindle assembly checkpoint is a molecular surveillance mechanism ensuring the fidelity of chromosome segregation during anaphase. The checkpoint monitors the completion of all the prerequisite steps before chromosome segregation to determine whether the segregation process should proceed or be delayed.
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Forces Acting on Chromosomes02:11

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During mitosis, chromosome movements occur through the interplay of multiple piconewton level forces. In prometaphase, these forces help in chromosome assembly or congression at the equatorial plane, eventually leading to their alignment at the metaphase plate. The forces acting on the chromosomes are space and time-dependent; therefore, they vary with the position of the chromosomes as the cell progresses through mitosis. 
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The Mitotic Spindle02:27

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The mitotic spindle—or spindle apparatus—is a eukaryotic, cytoskeletal structure made up of long protein fibers called microtubules. Formed during cell division, the spindle separates sister chromatids and moves them to opposite ends of a parental cell, where the now individual chromosomes are distributed to two daughter cell nuclei.
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Microtubules form through the end-to-end polymerization of tubulin heterodimers. Kinetochore microtubules originate from the spindle poles, and their plus-ends connect with the kinetochores on sister-chromatids. Ndc80 protein complexes, present on the kinetochore, form low-affinity links with the plus end of these kinetochore microtubules.
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Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets
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In Vitro Kinetochore Assembly.

Matthew D D Miell1, Aaron F Straight2

  • 1Department of Biochemistry, Stanford University School of Medicine, Beckman 409A, 404 Stanford Serra Mall, Stanford, CA, 94305, USA.

Methods in Molecular Biology (Clifton, N.J.)
|May 20, 2016
PubMed
Summary
This summary is machine-generated.

Researchers developed a cell-free system to assemble centromeres and functional kinetochores. This method allows for in vitro manipulation and analysis of kinetochore protein structure and function, crucial for understanding chromosome segregation.

Keywords:
CentromereChromatinIn vitro assemblyKinetochoreXenopus egg extract

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • The kinetochore is essential for chromosome segregation, mediating interactions between chromosomes and spindle microtubules during mitosis.
  • Kinetochores comprise over 100 proteins that assemble at centromeres, epigenetically defined chromosomal regions.
  • Proper kinetochore assembly and function are critical for preventing aneuploidy and cell death, making their study vital.

Purpose of the Study:

  • To present a novel cell-free approach for assembling centromeres and recruiting functional kinetochores.
  • To enable in vitro manipulation and detailed analysis of kinetochore protein structure, function, and regulation.
  • To provide a valuable system for probing the biochemical activities of kinetochores outside of cellular constraints.

Main Methods:

  • Development of a cell-free system for centromere assembly.
  • Recruitment of functional kinetochores onto assembled centromeres in vitro.
  • Enabling manipulation and analysis of kinetochore components and their interactions.

Main Results:

  • Successfully demonstrated the assembly of centromeres and recruitment of functional kinetochores in a cell-free environment.
  • Established an in vitro system that allows for the manipulation of kinetochore protein structure and function.
  • Provided a platform for detailed biochemical analysis of kinetochore activities.

Conclusions:

  • The developed cell-free approach offers a powerful tool for dissecting kinetochore assembly and function.
  • This system overcomes limitations of in-cell studies, allowing unprecedented manipulation and analysis of kinetochore components.
  • The findings contribute to a deeper understanding of chromosome segregation fidelity and the molecular mechanisms underlying kinetochore function.