Analysis of centrosome function and microtubule dynamics by time-lapse microscopy in Xenopus egg extracts.
Centrosomes are essential organelles that organize the microtubule cytoskeleton during interphase and mitosis. Centrosomes are assembled from tens to hundreds of proteins, but how these proteins are organized into functional microtubule nucleating and organizing centers is not yet clear. An important step in understanding the role of individual proteins in centrosome function is to understand whether they are involved in forming, stabilizing, or anchoring microtubules. It is becoming increasingly clear that the analysis of fixed samples is inadequate for a true understanding of the dynamics that drive cell biological processes. In this chapter we focus on methods to analyze microtubule nucleation, organization, and dynamics using assays based on mitotic Xenopus egg extracts and in vitro reactions. These methods can easily be adapted to the study of interphase processes, or to the study of other cytoskeletal proteins and their dynamics.
Wiese
Christiane
C
Department of Biochemistry, University of Wisconsin - Madison, Madison, WI, USA.
Mayers
Jonathan R
JR
Albee
Alison J
AJ
D000818:Animals; D016466:CHO Cells; D002417:Cattle; D002457:Cell Extracts; D018385:Centrosome / Q000378:metabolism* / Q000648:ultrastructure; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D019793:Fluorescein / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D018715:Microscopy, Video; D008870:Microtubules / Q000502:physiology* / Q000648:ultrastructure; D010063:Ovum / Q000378:metabolism / Q000648:ultrastructure; D012235:Rhodamines / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism; D014966:Xanthenes / Q000378:metabolism; D014981:Xenopus / Q000378:metabolism*; D029867:Xenopus Proteins / Q000302:isolation & purification / Q000378:metabolism
D002457:Cell Extracts; D005456:Fluorescent Dyes; D012235:Rhodamines; D014404:Tubulin; D014966:Xanthenes; D029867:Xenopus Proteins; C034657:Texas red; D019793:Fluorescein