Cell cycle analysis and synchronization of the Xenopus laevis XL2 cell line: Study of the kinesin related protein XIEg5

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 rep lication, 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 hey cell cycle regulators like p34(cdc2) and cdk2 have been discovered and characterized using those extracts, but their regulation during somatic cell cycles have only been st udied in mammalian cultured cells. In this paper, we describe optimized con ditions to obtain cell cycle arrested Xenopus XL2 cultured cells. Synchroni zation of XL2 cells at different stages of the cell cycle was achieved by s erum starvation and drug treatments such as aphidicolin, nocodazole, and AL LN. The degree of synchronization was assessed by indirect fluorescence mic roscopy and FACS analysis. This method was used to study the cell cycle exp ression of the Xenopus kinesin-related protein, XIEg5, 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 mini mum in G(1), which correlated with the expression of its messenger RNA. XL2 cultured cells were also used to analyze the ultrastructural sub-cellular localization of XIEg5. During mitosis, the protein was found around the cen trosome in prophase, on the spindle microtubules in metaphase, and, interes tingly, around the minus end of the midbody microtubules in telophase. (C) 1999 Wiley-Liss, Inc.