Access

You are not currently logged in.

Access your personal account or get JSTOR access through your library or other institution:

login

Log in to your personal account or through your institution.

Regulation of the Microtubule Nucleating Activity of Centrosomes in Xenopus Egg Extracts: Role of Cyclin A-Associated Protein Kinase

Brigitte Buendia, G. Draetta and Eric Karsenti
The Journal of Cell Biology
Vol. 116, No. 6 (Mar., 1992), pp. 1431-1442
Stable URL: http://www.jstor.org/stable/1615004
Page Count: 12
  • More info
  • Cite this Item
Regulation of the Microtubule Nucleating Activity of Centrosomes in Xenopus Egg Extracts: Role of Cyclin A-Associated Protein Kinase
Preview not available

Abstract

Isolated centrosomes nucleate microtubules when incubated in pure tubulin solutions well below the critical concentration for spontaneous polymer assembly (∼15 μM instead of 60 μM). Treatment with urea (2-3 M) does not severely damage the centriole cylinders but inactivates their ability to nucleate microtubules even at high tubulin concentrations. Here we show that centrosomes inactivated by urea are functionally complemented in frog egg extracts. Centrosomes can then be reisolated on sucrose gradients and assayed in different concentrations of pure tubulin to quantify their nucleating activity. We show that the material that complements centrosomes is stored in a soluble form in the egg. Each frog egg contains enough material to complement >6,000 ureainactivated centrosomes. The material is heat inactivated above 56°C. One can use this in vitro system to study how the microtubule nucleating activity of centrosomes is regulated. Native centrosomes require ∼15 μM tubulin to begin nucleating microtubules, whereas centrosomes complemented in interphase extracts begin nucleating microtubules around 7-8 μM tubulin. Therefore, the critical tubulin concentration for polymer assembly off native centrosomes is higher than that observed for the centrosomes first denatured and then complemented in egg extracts. In vivo, the microtubule nucleating activity of centrosomes seems to be regulated by phosphorylation at the onset of mitosis (Centonze, V. E., and G. G. Borisy. 1990. J. Cell Sci. 95:405-411). Since cyclins are major regulators of mitosis, we tested the effect of adding bacterially produced cyclins to interphase egg extracts. Both cyclin A and B activate an H1 kinase in the extracts. Cyclin A-associated kinase causes an increase in the microtubule nucleating activity of centrosomes complemented in the extract but cyclin B does not. The critical tubulin concentration for polymer assembly off centrosomes complemented in cyclin A-treated extracts is similar to that observed for centrosomes complemented in interphase extracts. However, centrosomes complemented in cyclin A treated extracts nucleate much more microtubules at high tubulin concentration. We define this as the "capacity" of centrosomes to nucleate microtubules. It seems that the microtubule nucleating activity of centrosomes can be defined by two distinct parameters: (a) the critical tubulin concentration at which they begin to nucleate microtubules and (b) their capacity to nucleate microtubules at high tubulin concentrations, the latter being modulated by phosphorylation.

Page Thumbnails

  • Thumbnail: Page 
1431
    1431
  • Thumbnail: Page 
1432
    1432
  • Thumbnail: Page 
1433
    1433
  • Thumbnail: Page 
1434
    1434
  • Thumbnail: Page 
1435
    1435
  • Thumbnail: Page 
1436
    1436
  • Thumbnail: Page 
1437
    1437
  • Thumbnail: Page 
1438
    1438
  • Thumbnail: Page 
1439
    1439
  • Thumbnail: Page 
1440
    1440
  • Thumbnail: Page 
1441
    1441
  • Thumbnail: Page 
1442
    1442