Centrosomes are critical microtubule (MT) nucleators and organizers in animal cells (Alberts et al., 2002
). Centrioles form the centrosome core and are surrounded by pericentriolar material (PCM) containing MT nucleating factors like γ-tubulin (γtub; Delattre and Gonczy, 2004
). Centrosomes play key roles in many processes, including organizing mitotic spindle poles (Kellogg et al., 1994
In animal cells, centrosome duplication occurs by a conserved cycle (Alberts et al., 2002
). It begins with centriole disengagement in late mitosis (Kuriyama and Borisy, 1981
), followed by procentriole assembly along the wall of each centriole in S phase. By G2, cells contain two mother/daughter centriole pairs that remain in proximity until mitosis. Both centriole pairs form functional centrosomes, maturing synchronously before mitotic entry, by recruiting PCM and acting as MT organizing centers (MTOCs; in contrast, there is a 10-min delay in activating the second yeast MTOC; Shaw et al., 1997
). The centrosomes then move to opposite sides of the nucleus to organize spindle poles and asters that position the spindle with respect to cortical cues. The essential role of centrosomes in animal cells was called into question by the fact that flies lacking functional centrosomes, or lacking centrioles entirely, live to adulthood (Megraw et al., 2001
; Basto et al., 2006
). However, not all is well: these animals have defects in divisions of larval neural stem/progenitor cells, the central brain neuroblasts (NBs).
Adult tissue stem cells play key roles in tissue maintenance/repair (Nystul and Spradling, 2006
). In each division, the daughters differ in fate: one retains stem cell character and the other differentiates. Drosophila melanogaster
central brain NBs are a superb model for asymmetric divisions of postembryonic tissue stem cells (Savoian and Rieder, 2002
; Siller et al., 2005
). Both embryonic and larval NBs are polarized cells exhibiting strict division patterns crucial for their roles as stem cells. Unlike the precise relationship between the embryonic NB division axis and adjacent epithelium, larval central brain NBs () do not appear to divide with specific orientations relative to the brain as a whole (). However, each NB creates a simpler microenvironment (): the NB and its ganglion mother cell (GMC) daughters. NBs divide asymmetrically, and the NB daughter retains stem cell character, whereas the GMC daughter goes on to differentiate. NBs divide according to strict local rules; each GMC is born adjacent to the previous GMC ( and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200612140/DC1
; Akong et al., 2002
), creating a GMC cap on one side of the NB ().
Figure 1. NB MTOCs form asynchronously. (A) Cartoon. (B–E) NB/GMCs in central brain (B), close-up (C and E), and cartoon (D). (B and C) Phalloidin. (E) Actin-GFP for two cell cycles; positions of successive GMCs are indicated (colored). The yellow dot represents (more ...)
Although differential fate allocation is critical in stem cells, we have much to learn about how a stereotyped division axis is established. NBs must coordinate cortical and spindle polarity so that neural determinants are packaged into the differentiating daughter (Yu et al., 2006
). Mutations affecting polarity or astral MT cortical interactions result in asymmetric division defects (Yu et al., 2006
). The importance of a properly aligned spindle is also suggested by spindle alignment defects in the absence of centrioles (14% symmetric divisions; Basto et al., 2006
) or in mutants that lack PCM (asterless
] or centrosomin
]) and have few or no astral MTs (Giansanti et al., 2001
; Megraw et al., 2001
). Thus, proper interactions between the spindle, astral MTs, and cortical polarity cues help maintain a constant division axis. Previous analyses revealed that NB spindles form at prophase already roughly aligned with the ultimate division axis (Siller et al., 2006
) but did not define how the initial axis forms.
Here, we address how this model stem cell maintains a persistent division axis. D. melanogaster
male germline stem cells also have a persistent division axis. It was proposed that one centrosome is cortically anchored by MT–adherens junction interactions (Yamashita et al., 2003
). To test whether a similar mechanism exists in NBs, we analyzed the centrosome cycle using 4D or 5D spinning disk confocal microscopy on brains prepared with no physical distortion (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200612140/DC1
), maintaining NB shape to replicate normal mitosis.