The microtubule (MT) cytoskeleton is essential for organizing the cytoplasm, polarity establishment, cell motility, morphogenesis, and cell division. Polarized arrays of MTs are nucleated by the centrosome, an organelle consisting of a pair of “mother-daughter” centrioles that recruit and organize a surrounding matrix of pericentriolar material (PCM; Bornens, 2002
). Proteomic analyses of centrosomes identified several hundred different proteins, some of which are unique to centrioles (e.g., the structural subunits SAS-4, SAS-6, and SPD-2) or the PCM (e.g., pericentrin; Andersen et al., 2003
; Keller et al., 2005
; Leidel and Gonczy, 2005a
). Within the PCM, pericentrin and other coiled-coil proteins assemble into a scaffold that docks γ-tubulin ring complexes, which nucleate MT growth (Moritz and Agard, 2001
). By manipulating centrosome number and position, cells exert precise control over the MT arrays needed for a variety of critical MT-dependent processes.
Centrosomes adhere to a cycle of duplication and function that is intimately coupled to the cell cycle (Tsou and Stearns, 2006
). Centrosomes act as dominant MT-organizing and nucleating centers (MTOCs; Schiebel, 2000
); however, this activity is modulated in a cell cycle–dependent manner. During interphase, cells possess two paired centrioles that form a single centrosome and organize the interphase MT array. Before mitotic entry the centrosome duplicates once; the two centrosomes then separate from one another and undergo “maturation,” recruiting more PCM to nucleate additional MT growth and facilitate spindle assembly (Glover, 2005
). On mitotic exit, each daughter cell receives a single centrosome that reduces its amount of associated PCM (Dictenberg et al., 1998
), but remains active as an MTOC. γ-tubulin is thought to be the primary source of microtubule-nucleating activity from the centrosome (Wiese and Zheng, 2006
), although it is likely not the only protein capable of this task. For example, developing Drosophila
ovaries that harbor homozygous double mutations in both γ-tubulin genes (γTub23C and γTub37C) organize abnormal spindles within mitotic germ cells but are still competent to nucleate MT growth (Tavosanis and Gonzalez, 2003
). Likewise, RNA interference (RNAi) of the single γ-tubulin gene in Caenorhabditis elegans
leads to defects in spindle bipolarity but does not abolish MT nucleation (Strome et al., 2001
; Hannak et al., 2002
). These studies suggest that alternative γ-tubulin-independent MT-assembly pathways exist in cells.
The importance and functional versatility of centrosomes is illustrated by their capacity to build several different MT-based machines including cilia, flagella, and mitotic spindles (Rieder et al., 2001
). This suggests that centrosomes should be essential for cell function. A recent study tested this hypothesis using Drosophila sas-4
mutants that fail to form centrioles, and therefore centrosomes (Basto et al., 2006
). Surprisingly, zygotic mutant embryos developed into viable adults with near normal timing and morphology. Although centrosomes usually organize mitotic spindle poles and were thought to be important for high-fidelity chromosome transmission, dividing sas-4
mutant cells displayed few errors in chromosome segregation, because their chromosomes induced spindle assembly via an acentrosomal pathway (Basto et al., 2006
). Similarly, mutant flies that lack Centrosomin, a PCM component essential for centrosome function, undergo normal zygotic development to form viable adults (Megraw et al., 2001
). Importantly, these studies raised new questions: how do interphase Drosophila
cells nucleate MT growth, organize their cytoplasm, and survive without the polarized MT arrays that centrosomes provide?
Much of our understanding of the cycle of centrosome function in Drosophila
is derived from work in early embryos and asymmetrically dividing larval neuroblasts. During the rapid mitotic divisions of the early syncytial embryo, gap phases of the cell cycle (G1 and G2) are absent. Nonetheless, centrosome duplication and function largely follow the canonical cycle observed in most animal cells, as the centrosomes act as MTOCs throughout this cell cycle (Callaini and Riparbelli, 1990
). Larval neuroblasts, however, display a novel twist to the classic cycle that is used to more precisely position the mitotic spindle (Rebollo et al., 2007
; Rusan and Peifer, 2007
). After asymmetric division in these stem cells, the centriole pair separates as expected, but only one centriole (the “dominant centriole”) retains its PCM and MTOC activity during the intervening interphase, whereas the other sheds its PCM and is inactive with respect to MT nucleation. Before entering the next division, the inactive centriole moves to the opposite side of the neuroblast, matures into a functional centrosome and is eventually segregated into the smaller ganglion mother cell. Although these two specialized cell types possess functional centrosomes throughout their respective cell cycles, it is not clear how other Drosophila
cells govern their centrosome cycles.
Here, we examine the Drosophila cycle of centrosome function both in cultured cells and within developing animals. Contrary to models of the conventional cycle, we find that Drosophila cells typically utilize centrosomes as MTOCs exclusively during mitosis. As cells exit mitosis, centrosomes disassemble both in cycling cells and within interphase-arrested cells. Furthermore, the generation and arrangement of interphase MTs appears to be independent of centrioles and is not disrupted by γ-tubulin RNAi at steady state. However, interphase MT regrowth assays reveal a “fast” MT-assembly pathway that uses not only γ-tubulin but Mini-spindles, CLIP-190, EB1, and dynein. Our results suggest that Drosophila cells utilize a distinctive “canonical” cycle of centrosome function, in which interphase centrosomes are inactivated, being replaced by an alternative mechanism of organizing the interphase MT array. This provides a mechanistic explanation for the survival of centrosome-deficient flies.