A fundamental question in cell biology is how cell geometry is established and maintained [1
]. Cell geometry refers to the characteristic positioning of organelles within the cell body in order for a cell to be able to carry out its specified function. Despite the importance of cell geometry in tissue organization and cell function, the mechanis-tic origins of cell geometry remain a mystery. Further compounding the mystery is the fact that, as demonstrated by the classic experiments of Beisson and Sonneborn [5
], cell organization can be propagated through cell division, alleviating the need for cells to re-establish their infrastructure after each round of mitosis, and potentially allowing a coherent organization to be maintained across developing tissue during proliferative growth. Many organelles take part in this elaborate cellular patterning. One organelle that is often found in specific subcellular locations is the centriole.
Centrioles are non–membrane-bound organelles composed of nine triplet microtubule blades arranged around a central cartwheel structure. Centrioles are found as a pair, composed of a mother and a daughter, which is duplicated during each cell cycle. Mother centrioles are so-called because they were assembled in a previous cell cycle to the daughter centriole. Mother centrioles have unique ultrastructural modifications [6
] and are decorated with a number of molecules not found on daughter centrioles.
Centrioles have two main functions in the cell. First, centrioles together with pericentriolar material comprise the centrosome, the major microtubule-organizing center of the cell. Indeed, centrioles are the highly stable, core nucleating centers for the centrosome, providing it with persisting structural integrity [7
] and attaching it to cytoplasmic microtubules during G1 [8
]. Second, centrioles serve as basal bodies to nucleate the assembly of cilia. In order to carry out these functions in the cell, centrioles often need to be specifically localized.
Although originally named for their centralized location, centrioles are repositioned to more peripheral sites during cell-state transitions such as wound healing, cell migration, and cell growth [9
]. The importance of centriole positioning for development and physiology is perhaps most clearly illustrated in situations involving cilia, which are assembled from centrioles. The problem of ciliary positioning is 2-fold. First, centrioles must migrate to the proper region on the cell surface where they will dock and assemble cilia. Second, once centrioles reach the cell surface, they must become properly oriented so as to create a proper directional stroke in the case of motile cilia, or so they are oriented to participate in signaling as in the case of a primary cilium. Perturbation in either step of ciliary positioning has severely deleterious effects in humans [12
]. For example, inability of centrioles to properly migrate prior to ciliary assembly has recently been linked to Meckel-Gruber syndrome [13
]. Additionally, proper orientation of cilia via centriole positioning towards the posterior of embryonic node cells is critical for establishing left–right asymmetry during mammalian development [14
]. Centrioles must also be properly positioned when they serve as basal bodies in multiciliated cells such as in the tracheal epithelium. Centriole orientation, and the resulting proper alignment of respiratory cilia, is required for effective mucus clearing in the airway [15
]. In all cases in which cilia act either to drive fluid flow or act as sensors, it is important that they be placed on the appropriate region of the cell surface; for example, in cells lining a duct, the cilia would have to face the lumen of the duct, which requires specific positioning of centrioles on a limited patch of cell surface.
It is clear that centriole positioning is critical in many aspects of cell behavior, especially in placing a cilium that will interact with the extracellular environment. Centriole position may also serve a function in intracellular events. As centrioles are anchored to the cytoskeleton during G1, they may act as a set of stable “handles” by which the centrosome can be repositioned to orient the cytoskeleton, cilia, and perhaps, other cellular structures as well. Moreover, the process of centriole duplication provides an ideal mechanism to transmit cell geometry across generations. Although both planar cell polarity [16
] and apical/basal cues [18
] can influence centriole position, the mechanism by which centrioles are positioned, and the degree to which their positioning is self-propagating, is currently unknown.
The unicellular alga Chlamydomonas reinhardtii
provides an ideal genetic system in which to study centriole positioning. Each pair of centrioles, composed of a mother and a daughter, must relocate from the apical cell surface to the spindle poles during mitosis. After division, centrioles return to the apical pole where they nucleate the assembly of two cilia (called flagella in this organism). Chlamydomonas
centrioles and cilia are structurally similar to those of vertebrates, with the vast majority of centriolar and ciliary proteins conserved between humans and Chlamydomonas
cells also have reproducible chiral cell geometry with many characteristically positioned structures [20
] (illustrated in A and B), facilitating quantification of geometric relationships within the cell. Given the importance of cilia positioning in animal tissues, and the high conservation of the ciliary apparatus components between Chlamydomonas
and animals, we feel that this unicellular alga is an excellent gene-discovery platform for analyzing cilia-placement mechanisms that may turn out to be important in human ciliary diseases.
Identification and Quantification of Defects in asq Mutants
Using Chlamydomonas cells, we identified mutants with defects in centriole positioning. Combining genetic analysis, three-dimensional (3D) imaging, and a novel algorithm for quantifying cellular geometry, we demonstrate that the mother centriole guides the daughter centriole to the proper subcellular location. Specifically, in mutants in which mother and daughter centrioles are separated, only mother centrioles localize properly. We further show that in mutants in which the centrioles are detached from the nucleus, the nucleus becomes randomly positioned, whereas the mother centrioles retain correct positioning, indicating that normally, the mother centriole plays a role in properly positioning the nucleus and not vice versa. These data indicate that the mother centriole may act as a node to coordinate the positioning of many subcellular structures.