A method that allows the assembly of kinetochore components onto chromosomes condensed in clarified Xenopus egg extracts

Arshad Desai, Heather W. Deacon, Claire Walczak, Timothy J. Mitchison

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Kinetochores are complex macromolecular structures that link mitotic chromosomes to spindle microtubules. Although a small number of kinetochore components have been identified, including the kinesins CENP-E and XKCM1 as well as cytoplasmic dynein, neither how these and other proteins are organized to produce a kinetochore nor their exact functions within this structure are understood. For this reason, we have developed an assay that allows kinetochore components to assemble onto discrete foci on in vitro- condensed chromosomes. The source of the kinetochore components is a clarified cell extract from Xenopus eggs that can be fractionated or immunodepleted of individual proteins. Kinetochore assembly in these clarified extracts requires preincubating the substrate sperm nuclei in an extract under low ATP conditions. Immunodepletion of XKCM1 from the extracts prevents the localization of kinetochore-associated XKCM1 without affecting the targeting of CENP-E and cytoplasmic dynein or the binding of monomeric tubulin to the kinetochore. Extension of this assay for the analysis of other components should help to dissect the protein-protein interactions involved in kinetochore assembly and function.

Original languageEnglish (US)
Pages (from-to)12378-12383
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume94
Issue number23
DOIs
StatePublished - Nov 11 1997
Externally publishedYes

Fingerprint

Kinetochores
Xenopus
Ovum
Chromosomes
Cytoplasmic Dyneins
Proteins
Macromolecular Substances
Kinesin
Tubulin
Cell Extracts
Microtubules
Eggs
Spermatozoa
Adenosine Triphosphate

Keywords

  • CENP-E
  • Dynein
  • In vitro
  • Mitosis
  • XKCMI

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

A method that allows the assembly of kinetochore components onto chromosomes condensed in clarified Xenopus egg extracts. / Desai, Arshad; Deacon, Heather W.; Walczak, Claire; Mitchison, Timothy J.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 94, No. 23, 11.11.1997, p. 12378-12383.

Research output: Contribution to journalArticle

@article{d8b00bed8ce148dcada65e8ac95439b1,
title = "A method that allows the assembly of kinetochore components onto chromosomes condensed in clarified Xenopus egg extracts",
abstract = "Kinetochores are complex macromolecular structures that link mitotic chromosomes to spindle microtubules. Although a small number of kinetochore components have been identified, including the kinesins CENP-E and XKCM1 as well as cytoplasmic dynein, neither how these and other proteins are organized to produce a kinetochore nor their exact functions within this structure are understood. For this reason, we have developed an assay that allows kinetochore components to assemble onto discrete foci on in vitro- condensed chromosomes. The source of the kinetochore components is a clarified cell extract from Xenopus eggs that can be fractionated or immunodepleted of individual proteins. Kinetochore assembly in these clarified extracts requires preincubating the substrate sperm nuclei in an extract under low ATP conditions. Immunodepletion of XKCM1 from the extracts prevents the localization of kinetochore-associated XKCM1 without affecting the targeting of CENP-E and cytoplasmic dynein or the binding of monomeric tubulin to the kinetochore. Extension of this assay for the analysis of other components should help to dissect the protein-protein interactions involved in kinetochore assembly and function.",
keywords = "CENP-E, Dynein, In vitro, Mitosis, XKCMI",
author = "Arshad Desai and Deacon, {Heather W.} and Claire Walczak and Mitchison, {Timothy J.}",
year = "1997",
month = "11",
day = "11",
doi = "10.1073/pnas.94.23.12378",
language = "English (US)",
volume = "94",
pages = "12378--12383",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "23",

}

TY - JOUR

T1 - A method that allows the assembly of kinetochore components onto chromosomes condensed in clarified Xenopus egg extracts

AU - Desai, Arshad

AU - Deacon, Heather W.

AU - Walczak, Claire

AU - Mitchison, Timothy J.

PY - 1997/11/11

Y1 - 1997/11/11

N2 - Kinetochores are complex macromolecular structures that link mitotic chromosomes to spindle microtubules. Although a small number of kinetochore components have been identified, including the kinesins CENP-E and XKCM1 as well as cytoplasmic dynein, neither how these and other proteins are organized to produce a kinetochore nor their exact functions within this structure are understood. For this reason, we have developed an assay that allows kinetochore components to assemble onto discrete foci on in vitro- condensed chromosomes. The source of the kinetochore components is a clarified cell extract from Xenopus eggs that can be fractionated or immunodepleted of individual proteins. Kinetochore assembly in these clarified extracts requires preincubating the substrate sperm nuclei in an extract under low ATP conditions. Immunodepletion of XKCM1 from the extracts prevents the localization of kinetochore-associated XKCM1 without affecting the targeting of CENP-E and cytoplasmic dynein or the binding of monomeric tubulin to the kinetochore. Extension of this assay for the analysis of other components should help to dissect the protein-protein interactions involved in kinetochore assembly and function.

AB - Kinetochores are complex macromolecular structures that link mitotic chromosomes to spindle microtubules. Although a small number of kinetochore components have been identified, including the kinesins CENP-E and XKCM1 as well as cytoplasmic dynein, neither how these and other proteins are organized to produce a kinetochore nor their exact functions within this structure are understood. For this reason, we have developed an assay that allows kinetochore components to assemble onto discrete foci on in vitro- condensed chromosomes. The source of the kinetochore components is a clarified cell extract from Xenopus eggs that can be fractionated or immunodepleted of individual proteins. Kinetochore assembly in these clarified extracts requires preincubating the substrate sperm nuclei in an extract under low ATP conditions. Immunodepletion of XKCM1 from the extracts prevents the localization of kinetochore-associated XKCM1 without affecting the targeting of CENP-E and cytoplasmic dynein or the binding of monomeric tubulin to the kinetochore. Extension of this assay for the analysis of other components should help to dissect the protein-protein interactions involved in kinetochore assembly and function.

KW - CENP-E

KW - Dynein

KW - In vitro

KW - Mitosis

KW - XKCMI

UR - http://www.scopus.com/inward/record.url?scp=0030785518&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030785518&partnerID=8YFLogxK

U2 - 10.1073/pnas.94.23.12378

DO - 10.1073/pnas.94.23.12378

M3 - Article

VL - 94

SP - 12378

EP - 12383

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 23

ER -