Centriolar satellites are membraneless granules that localize and move around centrosomes and cilia. Once referred to as structures with no obvious function, research in the past decade has identified satellites as key regulators of a wide range of cellular and organismal processes. Importantly, these studies have revealed a substantial overlap between functions, proteomes, and disease links of satellites with centrosomes and cilia. Therefore, satellites are now accepted as the “third component” of the vertebrate centrosome/cilium complex, which profoundly changes the way we think about the assembly, maintenance, and remodeling of the complex at the cellular and organismal levels. In this perspective, we first provide an overview of the cellular and structural complexities of centriolar satellites. We then describe the progress in the identification of the satellite interactome, which have paved the way to a molecular understanding of their mechanism of action and assembly mechanisms. After exploring current insights into their functions as recently described by loss-of-function studies and comparative evolutionary approaches, we discuss major unanswered questions regarding their functional and compositional diversity and their functions outside centrosomes and cilia.

The primary cilium is a microtubule-based organelle that serves as a hub for many signaling pathways. It functions as part of the centrosome/cilium complex, which also contains the basal body and the centriolar satellites. Little is known about the mechanisms by which the microtubule-based ciliary axoneme is assembled with proper length and structure, particularly in terms of the activity of microtubule-associated proteins (MAPs) and the crosstalk between the different compartments of the centrosome/cilium complex. Here, we analyzed CCDC66, a MAP implicated in cilium biogenesis and ciliopathies. Live-cell imaging revealed that CCDC66 compartmentalizes between centrosomes, centriolar satellites, and ciliary axoneme and tip during cilium biogenesis. CCDC66 depletion in human cells causes defects in cilium assembly, length and morphology. Notably, CCDC66 interacts with the ciliopathy-linked MAPs CEP104 and CSPP1 and regulates axonemal length and Hedgehog pathway activation. Moreover, CCDC66 is required for the basal body recruitment of transition zone proteins and IFT-B machinery. Overall, our results establish CCDC66 as a multifaceted regulator of the primary cilium and propose insight into how ciliary MAPs and subcompartments cooperate to ensure assembly of functional cilia.

Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division and signaling via the primary cilium in quiescent cells. Microtubule-associated proteins (MAPs) govern assembly, maintenance, and remodeling of diverse microtubule arrays. While a set of conserved MAPs are only active during cell division, an emerging group of MAPs acts as dual regulators in dividing and nondividing cells. Here, we elucidated the nonciliary functions and molecular mechanism of action of the ciliopathy-linked protein CCDC66, which we previously characterized as a regulator of ciliogenesis in quiescent cells. We showed that CCDC66 dynamically localizes to the centrosomes, the bipolar spindle, the spindle midzone, the central spindle, and the midbody in dividing cells and interacts with the core machinery of centrosome maturation and MAPs involved in cell division. Loss-of-function experiments revealed its functions during mitotic progression and cytokinesis. Specifically, CCDC66 depletion resulted in defective spindle assembly and orientation, kinetochore fiber stability, chromosome alignment in metaphase as well as central spindle and midbody assembly and organization in anaphase and cytokinesis. Notably, CCDC66 regulates mitotic microtubule nucleation via noncentrosomal and centrosomal pathways via recruitment of gamma-tubulin to the centrosomes and the spindle. Additionally, CCDC66 bundles microtubules in vitro and in cells by its C-terminal microtubule-binding domain. Phenotypic rescue experiments showed that the microtubule and centrosome-associated pools of CCDC66 individually or cooperatively mediate its mitotic and cytokinetic functions. Collectively, our findings identify CCDC66 as a multifaceted regulator of the nucleation and organization of the diverse mitotic and cytokinetic microtubule arrays and provide new insight into nonciliary defects that underlie ciliopathies.

Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division and signaling via the primary cilium in quiescent cells. Microtubule-associated proteins (MAPs) govern assembly, maintenance, and remodeling of diverse microtubule arrays. While a set of conserved MAPs are only active during cell division, an emerging group of MAPs acts as dual regulators in dividing and non-dividing cells. Here, we elucidated the nonciliary functions and molecular mechanism of action of the ciliopathy-linked protein CCDC66, which we previously characterized as a regulator of ciliogenesis in quiescent cells. We showed that CCDC66 dynamically localizes to the spindle poles, the bipolar spindle, the spindle midzone, the central spindle and the midbody in dividing cells and interacts with the core machinery of centrosome maturation and MAPs involved in cell division. Loss-of-function experiments revealed its functions during mitotic progression and cytokinesis. Specifically, CCDC66 depletion resulted in defective spindle assembly and positioning, kinetochore fiber stability, chromosome alignment in metaphase as well as central spindle and midbody assembly and organization in anaphase and cytokinesis. Notably, CCDC66 regulates mitotic microtubule nucleation via noncentrosomal and centrosomal pathways via recruitment of gamma-tubulin to the spindle poles and the spindle. Additionally, CCDC66 bundles microtubules in vitro and in cells by its C-terminal microtubule-binding domain. Phenotypic rescue experiments showed that the microtubule and centrosome-associated pools of CCDC66 individually or cooperatively mediate its mitotic and cytokinetic functions. Collectively, our findings identify CCDC66 as a multifaceted regulator of the nucleation and organization of the diverse mitotic and cytokinetic microtubule arrays and provides new insight into nonciliary defects that underlie ciliopathies.

The primary cilium is a conserved microtubule-based organelle that serves as a hub for many signaling pathways. It functions as part of the centrosome/cilium complex, which also contains the basal body and the centriolar satellites. Little is known about the mechanisms by which the microtubule-based axoneme of the cilium is assembled with proper length and structure, particularly in terms of the activity of microtubule-associated proteins (MAPs) and the crosstalk between the different compartments of the centrosome/cilium complex. Here, we analyzed CCDC66, a MAP implicated in cilium biogenesis and ciliopathies affecting eye and brain. Live-cell imaging revealed that CCDC66 compartmentalizes between centrosomes, centriolar satellites, and the ciliary axoneme and tip during cilium assembly and disassembly. CCDC66 loss-of-function in human cells causes defects in cilium assembly, length and morphology. Notably, CCDC66 interacts with the MAPs and ciliopathy proteins CEP104 and CSPP1 at the cilia and cooperates with them during axonemal microtubule polymerization. Moreover, CCDC66 interacts with the transition zone protein CEP290 selectively at the centriolar satellites. Its loss disrupts basal body recruitment of transition zone proteins and IFT-B machinery and causes defective Hedgehog signaling. Overall, our results establish CCDC66 as a multifaceted regulator of the primary cilium, and propose a mechanistic insight into how the cooperation of ciliary MAPs as well as subcompartments ensures assembly of a functional cilium.