Cell cycle analysis and synchronization of the Xenopus laevis XL2 cell line: study of the kinesin related protein XlEg5.
Cell free extracts prepared from Xenopus eggs are one of the most powerful in vitro systems to analyze cell cycle-regulated mechanisms such as DNA replication, nuclear assembly, chromosome condensation, or spindle formation. Xenopus embryos can complete several synchronous cell cycles in the absence of transcription, consequently Xenopus extracts are very helpful to study the molecular level of cellular mechanisms. Many key cell cycle regulators like p34cdc2 and cdk2 have been discovered and characterized using those extracts, but their regulation during somatic cell cycles have only been studied in mammalian cultured cells. In this paper, we describe optimized conditions to obtain cell cycle arrested Xenopus XL2 cultured cells. Synchronization of XL2 cells at different stages of the cell cycle was achieved by serum starvation and drug treatments such as aphidicolin, nocodazole, and ALLN. The degree of synchronization was assessed by indirect fluorescence microscopy and FACS analysis. This method was used to study the cell cycle expression of the Xenopus kinesin-related protein, XlEg5, a microtubule-based motor protein involved in movement and cell division in early development. We found that the expression of the protein was maximum in mitosis and minimum in G1, which correlated with the expression of its messenger RNA. XL2 cultured cells were also used to analyze the ultrastructural sub-cellular localization of XlEg5. During mitosis, the protein was found around the centrosome in prophase, on the spindle microtubules in metaphase, and, interestingly, around the minus end of the midbody microtubules in telophase.
Uzbekov
R
R
Laboratoire de Biologie et Génétique du Développement, Groupe Cycle Cellulaire, CNRS UPR 41, Faculté de Médecine de Rennes, France.
Prigent
C
C
Arlot-Bonnemains
Y
Y
D000818:Animals; D015153:Blotting, Western; D002453:Cell Cycle / Q000502:physiology*; D002460:Cell Line; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D018507:Gene Expression Regulation, Developmental; D007399:Interphase / Q000502:physiology; D016547:Kinesins / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D016196:S Phase / Q000502:physiology; D029867:Xenopus Proteins; D014982:Xenopus laevis
C088335:KIF11 protein, Xenopus; D008869:Microtubule-Associated Proteins; D012333:RNA, Messenger; D029867:Xenopus Proteins; D016547:Kinesins