JCB logo
R&D Systems: New Poster Available
  Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents
Published online 18 September 2000. doi:10.1083/jcb.150.6.1233
This Article
Right arrow Full Text
Right arrow Full Text (PDF, 1413K)
Right arrow PPT slides of all figures
Right arrow Supplemental Material Index
Right arrow Alert me when this article is cited
Right arrow Citation Map
Services
Right arrow Email this article
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new content in the JCB
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Howell, B.J.
Right arrow Articles by Salmon, E.D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Howell, B.J.
Right arrow Articles by Salmon, E.D.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Related Collections
Right arrowRelated Article
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Facebook   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

© The Rockefeller University Press, 0021-9525/2000//1233 $5.00
The Journal of Cell Biology, Volume 150, Number 6, , 2000 1233-1250

Original Article

Visualization of Mad2 Dynamics at Kinetochores, along Spindle Fibers, and at Spindle Poles in Living Cells



B.J. Howella, D.B. Hoffmana, G. Fangb, A.W. Murrayc, and E.D. Salmona

a Department of Biology, CB#3280, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
b Department of Biological Sciences, Stanford University, Stanford, California 94305-5020
c Department of Physiology, University of California at San Francisco, San Francisco, California 94143-0444
Department of Biology, CB#3280, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.(919) 962-1625(919) 962-2354

The spindle checkpoint prevents errors in chromosome segregation by inhibiting anaphase onset until all chromosomes have aligned at the spindle equator through attachment of their sister kinetochores to microtubules from opposite spindle poles. A key checkpoint component is the mitotic arrest–deficient protein 2 (Mad2), which localizes to unattached kinetochores and inhibits activation of the anaphase-promoting complex (APC) through an interaction with Cdc20. Recent studies have suggested a catalytic model for kinetochore function where unattached kinetochores provide sites for assembling and releasing Mad2–Cdc20 complexes, which sequester Cdc20 and prevent it from activating the APC. To test this model, we examined Mad2 dynamics in living PtK1 cells that were either injected with fluorescently labeled Alexa 488-XMad2 or transfected with GFP-hMAD2. Real-time, digital imaging revealed fluorescent Mad2 localized to unattached kinetochores, spindle poles, and spindle fibers depending on the stage of mitosis. FRAP measurements showed that Mad2 is a transient component of unattached kinetochores, as predicted by the catalytic model, with a t1/2 of ~24–28 s. Cells entered anaphase ~10 min after Mad2 was no longer detectable on the kinetochores of the last chromosome to congress to the metaphase plate. Several observations indicate that Mad2 binding sites are translocated from kinetochores to spindle poles along microtubules. First, Mad2 that bound to sites on a kinetochore was dynamically stretched in both directions upon microtubule interactions, and Mad2 particles moved from kinetochores toward the poles. Second, spindle fiber and pole fluorescence disappeared upon Mad2 disappearance at the kinetochores. Third, ATP depletion resulted in microtubule-dependent depletion of Mad2 fluorescence at kinetochores and increased fluorescence at spindle poles. Finally, in normal cells, the half-life of Mad2 turnover at poles, 23 s, was similar to kinetochores. Thus, kinetochore-derived sites along spindle fibers and at spindle poles may also catalyze Mad2 inhibitory complex formation.

Key Words: cell cycle • mitosis • spindle checkpoint • chromosome • microtubule

© 2000 The Rockefeller University Press

The online version of this article contains supplemental material.

Abbreviations used in this paper: APC, anaphase-promoting complex; Bub, budding uninhibited by benzimidazole; GFP, green fluorescent protein; hMad2, human Mad2; Mad2, mitotic arrest–deficient protein 2; XMad2, Xenopus Mad2.


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Facebook Facebook   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?

Related Article


J. Cell Biol. 2000 150: 0-2. [Full Text] [PDF]



This article has been cited by other articles:



  Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents