Regulation of centrosome size by the cell-cycle oscillator in Drosophila embryos.

Mitotic centrosomes assemble when centrioles recruit large amounts of pericentriolar material (PCM) around themselves. In early C. elegans embryos, mitotic centrosome size appears to be set by the limiting amount of a key component. In Drosophila syncytial embryos, thousands of mitotic centrosomes are assembled as the embryo proceeds through 13 rounds of rapid nuclear division, driven by a core cell cycle oscillator. These divisions slow during nuclear cycles 11-13, and we find that centrosomes respond by reciprocally decreasing their growth rate, but increasing their growth period-so that they grow to a relatively consistent size at each cycle. At the start of each cycle, moderate CCO activity initially promotes centrosome growth, in part by stimulating Polo/PLK1 recruitment to centrosomes. Later in each cycle, high CCO activity inhibits centrosome growth by suppressing the centrosomal recruitment and/or maintenance of centrosome proteins. Thus, in fly embryos, mitotic centrosome size appears to be regulated predominantly by the core cell cycle oscillator, rather than by the depletion of a limiting component.

A simple Turing reaction-diffusion model explains how PLK4 breaks symmetry during centriole duplication and assembly.

Centrioles duplicate when a mother centriole gives birth to a daughter that grows from its side. Polo-like-kinase 4 (PLK4), the master regulator of centriole duplication, is recruited symmetrically around the mother centriole, but it then concentrates at a single focus that defines the daughter centriole assembly site. How PLK4 breaks symmetry is unclear. Here, we propose that phosphorylated and unphosphorylated species of PLK4 form the 2 components of a classical Turing reaction-diffusion system. These 2 components bind to/unbind from the surface of the mother centriole at different rates, allowing a slow-diffusing activator species of PLK4 to accumulate at a single site on the mother, while a fast-diffusing inhibitor species of PLK4 suppresses activator accumulation around the rest of the centriole. This "short-range activation/long-range inhibition," inherent to Turing systems, can drive PLK4 symmetry breaking on a either a continuous or compartmentalised Plk4-binding surface, with PLK4 overexpression producing multiple PLK4 foci and PLK4 kinase inhibition leading to a lack of symmetry-breaking and PLK4 accumulation-as observed experimentally.

Cell cycle: Making waves to coordinate the entry into mitosis.

How do very large cells coordinate their entry into mitosis? A new study shows that the bistability of the Cdk/Cyclin system allows cells to generate either 'trigger waves' or 'sweep waves' that drive cells into mitosis in different ways with distinct consequences.

Centriole growth is limited by the Cdk/Cyclin-dependent phosphorylation of Ana2/STIL.

Centrioles duplicate once per cell cycle, but it is unclear how daughter centrioles assemble at the right time and place and grow to the right size. Here, we show that in Drosophila embryos the cytoplasmic concentrations of the key centriole assembly proteins Asl, Plk4, Ana2, Sas-6, and Sas-4 are low, but remain constant throughout the assembly process-indicating that none of them are limiting for centriole assembly. The cytoplasmic diffusion rate of Ana2/STIL, however, increased significantly toward the end of S-phase as Cdk/Cyclin activity in the embryo increased. A mutant form of Ana2 that cannot be phosphorylated by Cdk/Cyclins did not exhibit this diffusion change and allowed daughter centrioles to grow for an extended period. Thus, the Cdk/Cyclin-dependent phosphorylation of Ana2 seems to reduce the efficiency of daughter centriole assembly toward the end of S-phase. This helps to ensure that daughter centrioles stop growing at the correct time, and presumably also helps to explain why centrioles cannot duplicate during mitosis.

Centriole distal-end proteins CP110 and Cep97 influence centriole cartwheel growth at the proximal end.

Centrioles are composed of a central cartwheel tethered to nine-fold symmetric microtubule (MT) blades. The centriole cartwheel and MTs are thought to grow from opposite ends of these organelles, so it is unclear how they coordinate their assembly. We previously showed that in Drosophila embryos an oscillation of Polo-like kinase 4 (Plk4) helps to initiate and time the growth of the cartwheel at the proximal end. Here, in the same model, we show that CP110 and Cep97 form a complex close to the distal-end of the centriole MTs whose levels rise and fall as the new centriole MTs grow, in a manner that appears to be entrained by the core cyclin-dependent kinase (Cdk)-Cyclin oscillator that drives the nuclear divisions in these embryos. These CP110 and Cep97 dynamics, however, do not appear to time the period of centriole MT growth directly. Instead, we find that changing the levels of CP110 and Cep97 appears to alter the Plk4 oscillation and the growth of the cartwheel at the proximal end. These findings reveal an unexpected potential crosstalk between factors normally concentrated at opposite ends of the growing centrioles, which might help to coordinate centriole growth. This article has an associated First Person interview with the first authors of the paper.

Centrioles generate a local pulse of Polo/PLK1 activity to initiate mitotic centrosome assembly.

Mitotic centrosomes are formed when centrioles start to recruit large amounts of pericentriolar material (PCM) around themselves in preparation for mitosis. This centrosome "maturation" requires the centrioles and also Polo/PLK1 protein kinase. The PCM comprises several hundred proteins and, in Drosophila, Polo cooperates with the conserved centrosome proteins Spd-2/CEP192 and Cnn/CDK5RAP2 to assemble a PCM scaffold around the mother centriole that then recruits other PCM client proteins. We show here that in Drosophila syncytial blastoderm embryos, centrosomal Polo levels rise and fall during the assembly process-peaking, and then starting to decline, even as levels of the PCM scaffold continue to rise and plateau. Experiments and mathematical modelling indicate that a centriolar pulse of Polo activity, potentially generated by the interaction between Polo and its centriole receptor Ana1 (CEP295 in humans), could explain these unexpected scaffold assembly dynamics. We propose that centrioles generate a local pulse of Polo activity prior to mitotic entry to initiate centrosome maturation, explaining why centrioles and Polo/PLK1 are normally essential for this process.

Ana1 helps recruit Polo to centrioles to promote mitotic PCM assembly and centriole elongation.

Polo kinase (PLK1 in mammals) is a master cell cycle regulator that is recruited to various subcellular structures, often by its polo-box domain (PBD), which binds to phosphorylated S-pS/pT motifs. Polo/PLK1 kinases have multiple functions at centrioles and centrosomes, and we have previously shown that in Drosophila phosphorylated Sas-4 initiates Polo recruitment to newly formed centrioles, while phosphorylated Spd-2 recruits Polo to the pericentriolar material (PCM) that assembles around mother centrioles in mitosis. Here, we show that Ana1 (Cep295 in humans) also helps to recruit Polo to mother centrioles in Drosophila. If Ana1-dependent Polo recruitment is impaired, mother centrioles can still duplicate, disengage from their daughters and form functional cilia, but they can no longer efficiently assemble mitotic PCM or elongate during G2. We conclude that Ana1 helps recruit Polo to mother centrioles to specifically promote mitotic centrosome assembly and centriole elongation in G2, but not centriole duplication, centriole disengagement or cilia assembly. This article has an associated First Person interview with the first author of the paper.

An Autonomous Oscillation Times and Executes Centriole Biogenesis.

The accurate timing and execution of organelle biogenesis is crucial for cell physiology. Centriole biogenesis is regulated by Polo-like kinase 4 (Plk4) and initiates in S-phase when a daughter centriole grows from the side of a pre-existing mother. Here, we show that a Plk4 oscillation at the base of the growing centriole initiates and times centriole biogenesis to ensure that centrioles grow at the right time and to the right size. The Plk4 oscillation is normally entrained to the cell-cycle oscillator but can run autonomously of it-potentially explaining why centrioles can duplicate independently of cell-cycle progression. Mathematical modeling indicates that the Plk4 oscillation can be generated by a time-delayed negative feedback loop in which Plk4 inactivates the interaction with its centriolar receptor through multiple rounds of phosphorylation. We hypothesize that similar organelle-specific oscillations could regulate the timing and execution of organelle biogenesis more generally.

Drosophila Sas-6, Ana2 and Sas-4 self-organise into macromolecular structures that can be used to probe centriole and centrosome assembly.

Centriole assembly requires a small number of conserved proteins. The precise pathway of centriole assembly has been difficult to study, as the lack of any one of the core assembly proteins [Plk4, Ana2 (the homologue of mammalian STIL), Sas-6, Sas-4 (mammalian CPAP) or Asl (mammalian Cep152)] leads to the absence of centrioles. Here, we use Sas-6 and Ana2 particles (SAPs) as a new model to probe the pathway of centriole and centrosome assembly. SAPs form in Drosophila eggs or embryos when Sas-6 and Ana2 are overexpressed. SAP assembly requires Sas-4, but not Plk4, whereas Asl helps to initiate SAP assembly but is not required for SAP growth. Although not centrioles, SAPs recruit and organise many centriole and centrosome components, nucleate microtubules, organise actin structures and compete with endogenous centrosomes to form mitotic spindle poles. SAPs require Asl to efficiently recruit pericentriolar material (PCM), but Spd-2 (the homologue of mammalian Cep192) can promote some PCM assembly independently of Asl. These observations provide new insights into the pathways of centriole and centrosome assembly.

Evidence that a positive feedback loop drives centrosome maturation in fly embryos.

Centrosomes are formed when mother centrioles recruit pericentriolar material (PCM) around themselves. The PCM expands dramatically as cells prepare to enter mitosis (a process termed centrosome maturation), but it is unclear how this expansion is achieved. In flies, Spd-2 and Cnn are thought to form a scaffold around the mother centriole that recruits other components of the mitotic PCM, and the Polo-dependent phosphorylation of Cnn at the centrosome is crucial for scaffold assembly. Here, we show that, like Cnn, Spd-2 is specifically phosphorylated at centrosomes. This phosphorylation appears to create multiple phosphorylated S-S/T(p) motifs that allow Spd-2 to recruit Polo to the expanding scaffold. If the ability of Spd-2 to recruit Polo is impaired, the scaffold is initially assembled around the mother centriole, but it cannot expand outwards, and centrosome maturation fails. Our findings suggest that interactions between Spd-2, Polo and Cnn form a positive feedback loop that drives the dramatic expansion of the mitotic PCM in fly embryos.

Phase Separation and the Centrosome: A Fait Accompli?

There is currently intense interest in the idea that many membraneless organelles might assemble through phase separation of their constituent molecules into biomolecular 'condensates' that have liquid-like properties. This idea is intuitively appealing, especially for complex organelles such as centrosomes, where a liquid-like structure would allow the many constituent molecules to diffuse and interact with one another efficiently. I discuss here recent studies that either support the concept of a liquid-like centrosome or suggest that centrosomes are assembled upon a more solid, stable scaffold. I suggest that it may be difficult to distinguish between these possibilities. I argue that the concept of biomolecular condensates is an important advance in cell biology, with potentially wide-ranging implications, but it seems premature to conclude that centrosomes, and perhaps other membraneless organelles, are necessarily best described as liquid-like phase-separated condensates.

A homeostatic clock sets daughter centriole size in flies.

Centrioles are highly structured organelles whose size is remarkably consistent within any given cell type. New centrioles are born when Polo-like kinase 4 (Plk4) recruits Ana2/STIL and Sas-6 to the side of an existing "mother" centriole. These two proteins then assemble into a cartwheel, which grows outwards to form the structural core of a new daughter. Here, we show that in early Drosophila melanogaster embryos, daughter centrioles grow at a linear rate during early S-phase and abruptly stop growing when they reach their correct size in mid- to late S-phase. Unexpectedly, the cartwheel grows from its proximal end, and Plk4 determines both the rate and period of centriole growth: the more active the centriolar Plk4, the faster centrioles grow, but the faster centriolar Plk4 is inactivated and growth ceases. Thus, Plk4 functions as a homeostatic clock, establishing an inverse relationship between growth rate and period to ensure that daughter centrioles grow to the correct size.

Drosophila PLP assembles pericentriolar clouds that promote centriole stability, cohesion and MT nucleation.

Pericentrin is a conserved centrosomal protein whose dysfunction has been linked to several human diseases. It has been implicated in many aspects of centrosome and cilia function, but its precise role is unclear. Here, we examine Drosophila Pericentrin-like-protein (PLP) function in vivo in tissues that form both centrosomes and cilia. Plp mutant centrioles exhibit four major defects: (1) They are short and have subtle structural abnormalities; (2) They disengage prematurely, and so overduplicate; (3) They organise fewer cytoplasmic MTs during interphase; (4) When forming cilia, they fail to establish and/or maintain a proper connection to the plasma membrane-although, surprisingly, they can still form an axoneme-like structure that can recruit transition zone (TZ) proteins. We show that PLP helps assemble "pericentriolar clouds" of electron-dense material that emanate from the central cartwheel spokes and spread outward to surround the mother centriole. We propose that the partial loss of these structures may largely explain the complex centriole, centrosome and cilium defects we observe in Plp mutant cells.

A combined 3D-SIM/SMLM approach allows centriole proteins to be localized with a precision of ∼4-5 nm.

Centrioles are small barrel-shaped structures that form centrosomes and cilia [1]. Centrioles assemble around a central cartwheel comprising the Sas-6 and Ana2/STIL proteins. The amino termini of nine Sas-6 dimers form a central hub of ∼12 nm radius from which nine dimer spokes radiate, placing the Sas-6 carboxyl termini at the outer edge of the ∼60 nm radius cartwheel [2]. Several centriole proteins are distributed in a toroid around the cartwheel, and super-resolution light microscopy studies have measured the average radii of these ∼100-200 nm radius toroids with a 'precision' - or standard deviation (s.d. or 1σ) - of ±âˆ¼10-40 nm. The organization of Ana2/STIL within the cartwheel, however, has not been resolvable. Here, we develop methods to calculate the average toroidal radius of centriolar proteins in the ∼20-60 nm range with a s.d. of just ±âˆ¼4-5 nm, revealing that the amino and carboxyl termini of Ana2 are located in the outer cartwheel region.

Structural Basis for Mitotic Centrosome Assembly in Flies.

In flies, Centrosomin (Cnn) forms a phosphorylation-dependent scaffold that recruits proteins to the mitotic centrosome, but how Cnn assembles into a scaffold is unclear. We show that scaffold assembly requires conserved leucine zipper (LZ) and Cnn-motif 2 (CM2) domains that co-assemble into a 2:2 complex in vitro. We solve the crystal structure of the LZ:CM2 complex, revealing that both proteins form helical dimers that assemble into an unusual tetramer. A slightly longer version of the LZ can form micron-scale structures with CM2, whose assembly is stimulated by Plk1 phosphorylation in vitro. Mutating individual residues that perturb LZ:CM2 tetramer assembly perturbs the formation of these micron-scale assemblies in vitro and Cnn-scaffold assembly in vivo. Thus, Cnn molecules have an intrinsic ability to form large, LZ:CM2-interaction-dependent assemblies that are critical for mitotic centrosome assembly. These studies provide the first atomic insight into a molecular interaction required for mitotic centrosome assembly.

A key centriole assembly interaction interface between human PLK4 and STIL appears to not be conserved in flies.

A small number of proteins form a conserved pathway of centriole duplication. In humans and flies, the binding of PLK4/Sak to STIL/Ana2 initiates daughter centriole assembly. In humans, this interaction is mediated by an interaction between the Polo-Box-3 (PB3) domain of PLK4 and the coiled-coil domain of STIL (HsCCD). We showed previously that the Drosophila Ana2 coiled-coil domain (DmCCD) is essential for centriole assembly, but it forms a tight parallel tetramer in vitro that likely precludes an interaction with PB3. Here, we show that the isolated HsCCD and HsPB3 domains form a mixture of homo-multimers in vitro, but these readily dissociate when mixed to form the previously described 1:1 HsCCD:HsPB3 complex. In contrast, although Drosophila PB3 (DmPB3) adopts a canonical polo-box fold, it does not detectably interact with DmCCD in vitro Thus, surprisingly, a key centriole assembly interaction interface appears to differ between humans and flies.

Centrosome Amplification and Cancer: A Question of Sufficiency.

Centrosome amplification is a common feature of many types of cancer, but whether it is a cause or consequence is hotly debated. In this issue of Developmental Cell, Levine et al. (2017) provide strong evidence that centrosome amplification is sufficient to initiate tumorigenesis in a mouse model.