Pericentriolar Material Assembly Research Articles

Multivalent coiled-coil interactions enable full-scale centrosome assembly and strength.

The outermost layer of centrosomes, called pericentriolar material (PCM), organizes microtubules for mitotic spindle assembly. The molecular interactions that enable PCM to assemble and resist external forces are poorly understood. Here, we use crosslinking mass spectrometry (XL-MS) to analyze PLK-1-potentiated multimerization of SPD-5, the main PCM scaffold protein in C. elegans. In the unassembled state, SPD-5 exhibits numerous intramolecular crosslinks that are eliminated after phosphorylation by PLK-1. Thus, phosphorylation induces a structural opening of SPD-5 that primes it for assembly. Multimerization of SPD-5 is driven by interactions between multiple dispersed coiled-coil domains. Structural analyses of a phosphorylated region (PReM) in SPD-5 revealed a helical hairpin that dimerizes to form a tetrameric coiled-coil. Mutations within this structure and other interacting regions cause PCM assembly defects that are partly rescued by eliminating microtubule-mediated forces, revealing that PCM assembly and strength are interdependent. We propose that PCM size and strength emerge from specific, multivalent coiled-coil interactions between SPD-5 proteins.

Self-assembly of pericentriolar material in interphase cells lacking centrioles.

The major microtubule-organizing center (MTOC) in animal cells, the centrosome, comprises a pair of centrioles surrounded by pericentriolar material (PCM), which nucleates and anchors microtubules. Centrosome assembly depends on PCM binding to centrioles, PCM self-association and dynein-mediated PCM transport, but the self-assembly properties of PCM components in interphase cells are poorly understood. Here, we used experiments and modeling to study centriole-independent features of interphase PCM assembly. We showed that when centrioles are lost due to PLK4 depletion or inhibition, dynein-based transport and self-clustering of PCM proteins are sufficient to form a single compact MTOC, which generates a dense radial microtubule array. Interphase self-assembly of PCM components depends on γ-tubulin, pericentrin, CDK5RAP2 and ninein, but not NEDD1, CEP152, or CEP192. Formation of a compact acentriolar MTOC is inhibited by AKAP450-dependent PCM recruitment to the Golgi or by randomly organized CAMSAP2-stabilized microtubules, which keep PCM mobile and prevent its coalescence. Linking of CAMSAP2 to a minus-end-directed motor leads to the formation of an MTOC, but MTOC compaction requires cooperation with pericentrin-containing self-clustering PCM. Our data reveal that interphase PCM contains a set of components that can self-assemble into a compact structure and organize microtubules, but PCM self-organization is sensitive to motor- and microtubule-based rearrangement.

PCMD-1 bridges the centrioles and the pericentriolar material scaffold in C. elegans.

Correct cell division relies on the formation of a bipolar spindle. In animal cells, microtubule nucleation at the spindle poles is facilitated by the pericentriolar material (PCM), which assembles around a pair of centrioles. Although centrioles are essential for PCM assembly, the proteins that anchor the PCM to the centrioles are less known. Here, we investigate the molecular function of PCMD-1 in bridging the PCM and the centrioles in Caenorhabditis elegans. We demonstrate that the centrosomal recruitment of PCMD-1 is dependent on the outer centriolar protein SAS-7. The most C-terminal part of PCMD-1 is sufficient to target it to the centrosome, and the coiled-coil domain promotes its accumulation by facilitating self-interaction. We reveal that PCMD-1 interacts with the PCM scaffold protein SPD-5, the mitotic kinase PLK-1 and the centriolar protein SAS-4. Using an ectopic translocation assay, we show that PCMD-1 can selectively recruit downstream PCM scaffold components to an ectopic location in the cell, indicating that PCMD-1 is able to anchor the PCM scaffold proteins at the centrioles. Our work suggests that PCMD-1 is an essential functional bridge between the centrioles and the PCM.

Abstract P351: The Post-transcriptional Regulations Of Centrosomal Plp Mrna In Drosophila

Centrosomes, functioning as microtubule organizing centers, are composed of a proteinaceous matrix of pericentriolar material (PCM) that surrounds a pair of centrioles. Drosophila Pericentrin (Pcnt)-like protein (PLP) is a key component of the centrosome that serves as a scaffold for PCM assembly. The disruption of plp in Drosophila results in embryonic lethality, while the deregulation of Pcnt in humans is associated with MOPD II and Trisomy 21.We recently found plp mRNA localizes to Drosophila embryonic centrosomes. While RNA is known to associate with centrosomes in diverse cell types, the elements required for plp mRNA localization to centrosomes remains completely unknown. Additionally, how plp translation is regulated to accommodate rapid cell divisions during early embryogenesis is unclear. RNA localization coupled with translational control is a conserved mechanism that functions in diverse cellular processes. Control of mRNA localization and translation is mediated by RNA-binding proteins (RBPs). We find PLP protein expression is specifically promoted by an RNA-binding protein, Orb, during embryogenesis; moreover, plp mRNA interacts with Orb. Importantly, we find overexpression of full-length PLP can rescue cell division defects and embryonic lethality caused by orb depletion. We aim to uncover the mechanisms underlying embryonic plp mRNA localization and function and how Orb regulates plp translation.

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

ABSTRACTPolo 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.

Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly.

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. Here, we show the cooperative roles of the centriole and PCM scaffold proteins, pericentrin and CDK5RAP2, in the recruitment of CEP192 to spindle poles during mitosis. Systematic depletion of PCM proteins revealed that CEP192, but not pericentrin and/or CDK5RAP2, was crucial for bipolar spindle assembly in HeLa, RPE1, and A549 cells with centrioles. Upon double depletion of pericentrin and CDK5RAP2, CEP192 that remained at centriole walls was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the perturbation of PLK1, a critical kinase for PCM assembly, efficiently suppressed bipolar spindle formation in mitotic cells with one centrosome. Overall, these data suggest that the centriole and PCM scaffold proteins cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation.

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

ABSTRACTCentriole 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.

Pericentrin-mediated SAS-6 recruitment promotes centriole assembly.

The centrosome is composed of two centrioles surrounded by a microtubule-nucleating pericentriolar material (PCM). Although centrioles are known to regulate PCM assembly, it is less known whether and how the PCM contributes to centriole assembly. Here we investigate the interaction between centriole components and the PCM by taking advantage of fission yeast, which has a centriole-free, PCM-containing centrosome, the SPB. Surprisingly, we observed that several ectopically-expressed animal centriole components such as SAS-6 are recruited to the SPB. We revealed that a conserved PCM component, Pcp1/pericentrin, interacts with and recruits SAS-6. This interaction is conserved and important for centriole assembly, particularly its elongation. We further explored how yeasts kept this interaction even after centriole loss and showed that the conserved calmodulin-binding region of Pcp1/pericentrin is critical for SAS-6 interaction. Our work suggests that the PCM not only recruits and concentrates microtubule-nucleators, but also the centriole assembly machinery, promoting biogenesis close by.

Centriolar SAS-7 acts upstream of SPD-2 to regulate centriole assembly and pericentriolar material formation

The centriole/basal body is a eukaryotic organelle that plays essential roles in cell division and signaling. Among five known core centriole proteins, SPD-2/Cep192 is the first recruited to the site of daughter centriole formation and regulates the centriolar localization of the other components in C. elegans and in humans. However, the molecular basis for SPD-2 centriolar localization remains unknown. Here, we describe a new centriole component, the coiled-coil protein SAS-7, as a regulator of centriole duplication, assembly and elongation. Intriguingly, our genetic data suggest that SAS-7 is required for daughter centrioles to become competent for duplication, and for mother centrioles to maintain this competence. We also show that SAS-7 binds SPD-2 and regulates SPD-2 centriolar recruitment, while SAS-7 centriolar localization is SPD-2-independent. Furthermore, pericentriolar material (PCM) formation is abnormal in sas-7 mutants, and the PCM-dependent induction of cell polarity that defines the anterior-posterior body axis frequently fails. We conclude that SAS-7 functions at the earliest step in centriole duplication yet identified and plays important roles in the orchestration of centriole and PCM assembly.

Cep295 is a conserved scaffold protein required for generation of a bona fide mother centriole.

Centrioles surrounded by pericentriolar material (PCM) serve as the core structure of the centrosome. A newly formed daughter centriole grows into a functional mother centriole. However, the underlying mechanisms remain poorly understood. Here we show that Cep295, an evolutionarily conserved protein, is required for generation of a bona fide mother centriole organizing a functional centrosome. We find that Cep295 is recruited to the proximal centriole wall in the early stages of procentriole assembly. Cep295 then acts as a scaffold for the proper assembly of the daughter centriole. We also find that Cep295 binds directly to and recruits Cep192 onto the daughter centriole wall, which presumably endows the function of the new mother centriole for PCM assembly, microtubule-organizing centre activity and the ability for centriole formation. These findings led us to propose that Cep295 acts upstream of the conserved pathway for centriole formation and promotes the daughter-to-mother centriole conversion.

Re-examining the role of Drosophila Sas-4 in centrosome assembly using two-colour-3D-SIM FRAP.

Centrosomes have many important functions and comprise a 'mother' and 'daughter' centriole surrounded by pericentriolar material (PCM). The mother centriole recruits and organises the PCM and templates the formation of the daughter centriole. It has been reported that several important Drosophila PCM-organising proteins are recruited to centrioles from the cytosol as part of large cytoplasmic 'S-CAP' complexes that contain the centriole protein Sas-4. In a previous paper (Conduit et al., 2014b) we showed that one of these proteins, Cnn, and another key PCM-organising protein, Spd-2, are recruited around the mother centriole before spreading outwards to form a scaffold that supports mitotic PCM assembly; the recruitment of Cnn and Spd-2 is dependent on another S-CAP protein, Asl. We show here, however, that Cnn, Spd-2 and Asl are not recruited to the mother centriole as part of a complex with Sas-4. Thus, PCM recruitment in fly embryos does not appear to require cytosolic S-CAP complexes.

The Drosophila Pericentrin-like-protein (PLP) cooperates with Cnn to maintain the integrity of the outer PCM.

ABSTRACTCentrosomes comprise a pair of centrioles surrounded by a matrix of pericentriolar material (PCM). In vertebrate cells, Pericentrin plays an important part in mitotic PCM assembly, but the Drosophila Pericentrin-like protein (PLP) appears to have a more minor role in mitotic fly cells. Here we investigate the function of PLP during the rapid mitotic cycles of the early Drosophila embryo. Unexpectedly, we find that PLP is specifically enriched in the outer-most regions of the PCM, where it largely co-localizes with the PCM scaffold protein Cnn. In the absence of PLP the outer PCM appears to be structurally weakened, and it rapidly disperses along the centrosomal microtubules (MTs). As a result, centrosomal MTs are subtly disorganized in embryos lacking PLP, although mitosis is largely unperturbed and these embryos develop and hatch at near-normal rates. Y2H analysis reveals that PLP can potentially form multiple interactions with itself and with the PCM recruiting proteins Asl, Spd-2 and Cnn. A deletion analysis suggests that PLP participates in a complex network of interactions that ultimately help to strengthen the PCM.

Centrosomes. Regulated assembly of a supramolecular centrosome scaffold in vitro.

The centrosome organizes microtubule arrays within animal cells and comprises two centrioles surrounded by an amorphous protein mass called the pericentriolar material (PCM). Despite the importance of centrosomes as microtubule-organizing centers, the mechanism and regulation of PCM assembly are not well understood. In Caenorhabditis elegans, PCM assembly requires the coiled-coil protein SPD-5. We found that recombinant SPD-5 could polymerize to form micrometer-sized porous networks in vitro. Network assembly was accelerated by two conserved regulators that control PCM assembly in vivo, Polo-like kinase-1 and SPD-2/Cep192. Only the assembled SPD-5 networks, and not unassembled SPD-5 protein, functioned as a scaffold for other PCM proteins. Thus, PCM size and binding capacity emerge from the regulated polymerization of one coiled-coil protein to form a porous network.

Method: In vitro analysis of pericentriolar material assembly.

Centrosomes are major microtubule-organizing centers in eukaryotic cells and play a critical role in embryonic development and asymmetric cell division. Centrosomes comprise a pair of centrioles surrounded by an amorphous proteinaceous meshwork called the pericentriolar material (PCM). Robust deposition of PCM around the centrioles is essential for a centrosome to achieve full microtubule nucleating potential. Despite the wealth of information on PCM composition and function, the mechanism and regulation of PCM assembly have been difficult to ascertain, due in part to the lack of an in vitro system. Here, we describe methods to establish an in vitro system to study PCM assembly in Caenorhabditis elegans. Specifically, we describe (1) how to express and purify the C. elegans PCM proteins SPD-5, SPD-2, and PLK-1 from baculovirus-infected insect cells, (2) how to assemble these proteins into PCM-like structures in vitro, and (3) how to quantify this assembly process in a semiautomated fashion.

A molecular mechanism of mitotic centrosome assembly in Drosophila.

Centrosomes comprise a pair of centrioles surrounded by pericentriolar material (PCM). The PCM expands dramatically as cells enter mitosis, but it is unclear how this occurs. In this study, we show that the centriole protein Asl initiates the recruitment of DSpd-2 and Cnn to mother centrioles; both proteins then assemble into co-dependent scaffold-like structures that spread outwards from the mother centriole and recruit most, if not all, other PCM components. In the absence of either DSpd-2 or Cnn, mitotic PCM assembly is diminished; in the absence of both proteins, it appears to be abolished. We show that DSpd-2 helps incorporate Cnn into the PCM and that Cnn then helps maintain DSpd-2 within the PCM, creating a positive feedback loop that promotes robust PCM expansion around the mother centriole during mitosis. These observations suggest a surprisingly simple mechanism of mitotic PCM assembly in flies.

The Caenorhabditis elegans pericentriolar material components SPD-2 and SPD-5 are monomeric in the cytoplasm before incorporation into the PCM matrix.

Centrosomes are the main microtubule-organizing centers in animal cells. Centrosomes consist of a pair of centrioles surrounded by a matrix of pericentriolar material (PCM) that assembles from cytoplasmic components. In Caenorhabditis elegans embryos, interactions between the coiled-coil proteins SPD-5 and SPD-2 and the kinase PLK-1 are critical for PCM assembly. However, it is not known whether these interactions promote the formation of cytoplasmic complexes that are added to the PCM or whether the components interact only during incorporation into the PCM matrix. Here we address this problem by using a combination of live-cell fluorescence correlation spectroscopy, mass spectrometry, and hydrodynamic techniques to investigate the native state of PCM components in the cytoplasm. We show that SPD-2 is monomeric, and neither SPD-2 nor SPD-5 exists in complex with PLK-1. SPD-5 exists mostly as a monomer but also forms complexes with the PP2A-regulatory proteins RSA-1 and RSA-2, which are required for microtubule organization at centrosomes. These results suggest that the interactions between SPD-2, SPD-5, and PLK-1 do not result in formation of cytoplasmic complexes, but instead occur in the context of PCM assembly.

Amorphous no more: subdiffraction view of the pericentriolar material architecture

The centrosome influences the shape, orientation and activity of the microtubule cytoskeleton. The pericentriolar material (PCM), determines this functionality by providing a dynamic platform for nucleating microtubules and acts as a nexus for molecular signaling. Although great strides have been made in understanding PCM activity, its diffraction-limited size and amorphous appearance on electron microscopy (EM) have limited analysis of its high-order organization. Here, we outline current knowledge of PCM architecture and assembly, emphasizing recent super-resolution imaging studies that revealed the PCM has a layered structure made of fibers and matrices conserved from flies to humans. Notably, these studies debunk the long-standing view of an amorphous PCM and provide a paradigm to dissect the supramolecular organization of organelles in cells.

Promoter hijack reveals pericentrin functions in mitosis and the DNA damage response

Centrosomes, the principal microtubule-organizing centers of animal somatic cells, consist of two centrioles embedded in the pericentriolar material (PCM). Pericentrin is a large PCM protein that is required for normal PCM assembly. Mutations in PCNT cause primordial dwarfism. Pericentrin has also been implicated in the control of DNA damage responses. To test how pericentrin is involved in cell cycle control after genotoxic stress, we disrupted the Pcnt locus in chicken DT40 cells. Pericentrin-deficient cells proceeded through mitosis more slowly, with a high level of monopolar spindles, and were more sensitive to spindle poisons than controls. Centriole structures appeared normal by light and electron microscopy, but the PCM did not recruit γ-tubulin efficiently. Cell cycle delays after ionizing radiation (IR) treatment were normal in pericentrin-deficient cells. However, pericentrin disruption in Mcph1−/− cells abrogated centrosome hyperamplification after IR. We conclude that pericentrin controls genomic stability by both ensuring appropriate mitotic spindle activity and centrosome regulation.

Tubulin nucleotide status controls Sas-4-dependent pericentriolar material recruitment

Regulated centrosome biogenesis is required for accurate cell division and for maintaining genome integrity1. Centrosomes consist of a centriole pair surrounded by a protein network known as pericentriolar material (PCM)1. PCM assembly is a tightly regulated, critical step that determines a centrosome’s size and capability2–4. Here, we report a role for tubulin in regulating PCM recruitment via the conserved centrosomal protein Sas-4. Tubulin directly binds to Sas-4; together they are components of cytoplasmic complexes of centrosomal proteins5,6. A Sas-4 mutant, which cannot bind tubulin, enhances centrosomal protein complex formation and has abnormally large centrosomes with excessive activity. These suggest that tubulin negatively regulates PCM recruitment. Whereas tubulin-GTP prevents Sas-4 from forming protein complexes, tubulin-GDP promotes it. Thus, tubulin’s regulation of PCM recruitment depends on its GTP/GDP-bound state. These results identify a role for tubulin in regulating PCM recruitment independent of its well-known role as a building block of microtubules7. Based on its guanine bound state, tubulin can act as a molecular switch in PCM recruitment.

Sas-4 provides a scaffold for cytoplasmic complexes and tethers them in a centrosome

Centrosomes are conserved organelles that are essential for accurate cell division and cilium formation. A centrosome consists of a pair of centrioles surrounded by a protein network of pericentriolar material (PCM) that is essential for the centrosome's function. In this study, we show that Sas-4 provides a scaffold for cytoplasmic complexes (named S-CAP), which include CNN, Asl and D-PLP, proteins that are all found in the centrosomes at the vicinity of the centriole. When Sas-4 is absent, nascent procentrioles are unstable and lack PCM, and functional centrosomes are not generated. When Sas-4 is mutated, so that it cannot form S-CAP complexes, centrosomes are present but with dramatically reduced levels of PCM. Finally, purified S-CAP complexes or recombinant Sas-4 can bind centrosomes stripped of PCM, whereas recombinant CNN or Asl cannot. In summary, PCM assembly begins in the cytosol where Sas-4 provides a scaffold for pre-assembled cytoplasmic complexes before tethering of the complexes in a centrosome.