Centrosome duplication proceeds during mimosine-induced G1 cell cycle arrest.

The single interphase centrosome duplicates exactly once in a cell cycle dependent fashion, yielding two daughter centrosomes, each of which contributes to the assembly of a mitotic spindle pole (reviewed in Stearns, 2001; Delattre and Gonczy, 2004). The two initial events in the centrosome duplication cycle are commonly thought to be the “disorientation” or “disengagement” of the parental centriole pair (termed the diplosome), which prepares the centrosome for duplication, followed by the assembly of short daughter centrioles—called pro-centrioles—at right angles to the pre-existing centrioles (Kuriyama and Borisy, 1981; Kochanski and Borisy, 1990). Centrosome duplication is coordinated with nuclear events during cell cycle progression (reviewed in Hinchcliffe and Sluder, 2001a). This is important because centrosomes play a dominant role in spindle pole organization; there must be two and only two centrosomes present as the cell enters mitosis, otherwise the potential exists to assemble a multipolar spindle, resulting in an increased frequency of aneuploidy and tumor cell progression (reviewed in Brinkley, 2001; Fisk et al., 2002; Sluder and Nordberg, 2004). Understanding the cell cycle control of centrosome duplication has been difficult, primarily because there is no good marker to signify when duplication begins. By the time morphologically distinct centrioles have formed, and are recognizable in the electron microscope, the initial events of centrosome duplication may have already taken place (discussed in Hinchcliffe and Sluder, 1998). In order to examine the cell cycle regulation of centrosome duplication, several studies have relied on arresting cell cycle progression in a particular phase, and then assaying whether or not centrosomes are capable of duplicating one or more times (reviewed in Hinchcliffe and Sluder, 2001a). The results of this work have experimentally defined those stages that are capable of supporting centrosome duplication, and those that cannot. In cells that are driven into G0, the centrosome does not duplicate until serum-released (Tucker et al., 1979; Okuda et al., 2000). During S-phase arrest, centrosomes can clearly undergo repeated rounds of duplication in both zygotes and certain transformed somatic cells (Kuriyama et al., 1986; Sluder and Lewis, 1987; Raff and Glover, 1988; Balczon et al., 1995; Hinchcliffe et al., 1998). In cells arrested in G2 with topoisomerase inhibitors, the duplicated centrosome cannot undergo further rounds of duplication (Balczon et al., 1995). When mitosis is prolonged in a variety of cell types, centrosome duplication cannot proceed (Hinchcliffe et al., 1998; Vidwans et al., 1999), although the diplosome can undergo disengagement (Tsou and Stearns, 2006). While the aforementioned studies have provided information about which cell cycle stages can or cannot support centrosome duplication, there are conflicting reports about whether or not centrosome duplication can occur during G1. This phase of the cell cycle is particularly important, because centrosome duplication is said to normally occur at the G1/S phase transition (Robbins et al., 1968; Rattner and Phillips, 1973; Kuriyama and Borisy, 1981; Vorobjev and Chentsov, 1982; Alvey, 1985). The implication is that as the cell cycle proceeds into S-phase, the signals that initiate DNA replication also drive the duplication of the centrosome (discussed in Sluder and Rieder, 1996). However, it remains a formal possibility that centrosome duplication could be initiated without the activation of these signals, and then continues in parallel as the cell cycle transitions into S-phase. Several studies have suggested that centrosome duplication cannot occur until after the G1/S phase transition (Robbins et al., 1968; Vorobjev and Chentsov, 1982; Marshall et al., 2001), while other have revealed that it can proceed prior to entry into S-phase (Rattner and Phillips, 1973; Winey and Byers, 1993; Fukasawa et al., 1996; Schutz et al., 1997; Hinchcliffe et al., 1998; Uzawa et al., 2004). However, many of these studies have examined when centrosome duplication occurs in cycling cells, rather than examining the effect of prolonging the cell cycle in G1, and assaying for centrosome duplication. Put another way, centrosome duplication may begin in G1, and by the time morphologically distinct centrioles are recognizable in the electron microscope, the cell cycle has proceeded into S-phase. The consequences of prolonging G1 on the centrosome duplication cycle have not been experimentally tested in mammalian somatic cells. To address this, we use both transformed and non-transformed cultured Chinese hamster cells to directly test whether or not centriole reproduction can occur when the cell cycle is prolonged in G1.