Cytoplasmic division cycles without the nucleus and mitotic CDK/cyclin complexes.

Cytoplasmic divisions are thought to rely on nuclear divisions and mitotic signals. We demonstrate in Drosophila embryos that cytoplasm can divide repeatedly without nuclei and mitotic CDK/cyclin complexes. Cdk1 normally slows an otherwise faster cytoplasmic division cycle, coupling it with nuclear divisions, and when uncoupled, cytoplasm starts dividing before mitosis. In developing embryos where CDK/cyclin activity can license mitotic microtubule (MT) organizers like the spindle, cytoplasmic divisions can occur without the centrosome, a principal organizer of interphase MTs. However, centrosomes become essential in the absence of CDK/cyclin activity, implying that the cytoplasm can employ either the centrosome-based interphase or CDK/cyclin-dependent mitotic MTs to facilitate its divisions. Finally, we present evidence that autonomous cytoplasmic divisions occur during unperturbed fly embryogenesis and that they may help extrude mitotically stalled nuclei during blastoderm formation. We postulate that cytoplasmic divisions occur in cycles governed by a yet-to-be-uncovered clock mechanism autonomous from CDK/cyclin complexes.

Mouse oocytes develop in cysts with the help of nurse cells.

Mouse germline cysts, on average, develop into six oocytes supported by 24 nurse cells that transfer cytoplasm and organelles to generate a Balbiani body. We showed that between E14.5 and P5, cysts periodically activate some nurse cells to begin cytoplasmic transfer, which causes them to shrink and turnover within 2 days. Nurse cells die by a programmed cell death (PCD) pathway involving acidification, similar to Drosophila nurse cells, and only infrequently by apoptosis. Prior to initiating transfer, nurse cells co-cluster by scRNA-seq with their pro-oocyte sisters, but during their final 2 days, they cluster separately. The genes promoting oocyte development and nurse cell PCD are upregulated, whereas the genes that repress transfer, such as Tex14, and oocyte factors, such as Nobox and Lhx8, are under-expressed. The transferred nurse cell centrosomes build a cytocentrum that establishes a large microtubule aster in the primordial oocyte that organizes the Balbiani body, defining the earliest oocyte polarity.

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.

Self-Organization of Cellular Units.

The purpose of this review is to explore self-organizing mechanisms that pattern microtubules (MTs) and spatially organize animal cell cytoplasm, inspired by recent experiments in frog egg extract. We start by reviewing conceptual distinctions between self-organizing and templating mechanisms for subcellular organization. We then discuss self-organizing mechanisms that generate radial MT arrays and cell centers in the absence of centrosomes. These include autocatalytic MT nucleation, transport of minus ends, and nucleation from organelles such as melanosomes and Golgi vesicles that are also dynein cargoes. We then discuss mechanisms that partition the cytoplasm in syncytia, in which multiple nuclei share a common cytoplasm, starting with cytokinesis, when all metazoan cells are transiently syncytial. The cytoplasm of frog eggs is partitioned prior to cytokinesis by two self-organizing modules, protein regulator of cytokinesis 1 (PRC1)-kinesin family member 4A (KIF4A) and chromosome passenger complex (CPC)-KIF20A. Similar modules may partition longer-lasting syncytia, such as early Drosophila embryos. We end by discussing shared mechanisms and principles for the MT-based self-organization of cellular units.

Biophysical and Quantitative Principles of Centrosome Biogenesis and Structure.

The centrosome is a main orchestrator of the animal cellular microtubule cytoskeleton. Dissecting its structure and assembly mechanisms has been a goal of cell biologists for over a century. In the last two decades, a good understanding of the molecular constituents of centrosomes has been achieved. Moreover, recent breakthroughs in electron and light microscopy techniques have enabled the inspection of the centrosome and the mapping of its components with unprecedented detail. However, we now need a profound and dynamic understanding of how these constituents interact in space and time. Here, we review the latest findings on the structural and molecular architecture of the centrosome and how its biogenesis is regulated, highlighting how biophysical techniques and principles as well as quantitative modeling are changing our understanding of this enigmatic cellular organelle.

Mechanism and Regulation of Centriole and Cilium Biogenesis.

The centriole is an ancient microtubule-based organelle with a conserved nine-fold symmetry. Centrioles form the core of centrosomes, which organize the interphase microtubule cytoskeleton of most animal cells and form the poles of the mitotic spindle. Centrioles can also be modified to form basal bodies, which template the formation of cilia and play central roles in cellular signaling, fluid movement, and locomotion. In this review, we discuss developments in our understanding of the biogenesis of centrioles and cilia and the regulatory controls that govern their structure and number. We also discuss how defects in these processes contribute to a spectrum of human diseases and how new technologies have expanded our understanding of centriole and cilium biology, revealing exciting avenues for future exploration.

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.

The Centrosome Is a Selective Condensate that Nucleates Microtubules by Concentrating Tubulin.

Centrosomes are non-membrane-bound compartments that nucleate microtubule arrays. They consist of nanometer-scale centrioles surrounded by a micron-scale, dynamic assembly of protein called the pericentriolar material (PCM). To study how PCM forms a spherical compartment that nucleates microtubules, we reconstituted PCM-dependent microtubule nucleation in vitro using recombinant C. elegans proteins. We found that macromolecular crowding drives assembly of the key PCM scaffold protein SPD-5 into spherical condensates that morphologically and dynamically resemble in vivo PCM. These SPD-5 condensates recruited the microtubule polymerase ZYG-9 (XMAP215 homolog) and the microtubule-stabilizing protein TPXL-1 (TPX2 homolog). Together, these three proteins concentrated tubulin ∼4-fold over background, which was sufficient to reconstitute nucleation of microtubule asters in vitro. Our results suggest that in vivo PCM is a selective phase that organizes microtubule arrays through localized concentration of tubulin by microtubule effector proteins.

Microtubule-Organizing Centers.

The organization of microtubule networks is crucial for controlling chromosome segregation during cell division, for positioning and transport of different organelles, and for cell polarity and morphogenesis. The geometry of microtubule arrays strongly depends on the localization and activity of the sites where microtubules are nucleated and where their minus ends are anchored. Such sites are often clustered into structures known as microtubule-organizing centers, which include the centrosomes in animals and spindle pole bodies in fungi. In addition, other microtubules, as well as membrane compartments such as the cell nucleus, the Golgi apparatus, and the cell cortex, can nucleate, stabilize, and tether microtubule minus ends. These activities depend on microtubule-nucleating factors, such as γ-tubulin-containing complexes and their activators and receptors, and microtubule minus end-stabilizing proteins with their binding partners. Here, we provide an overview of the current knowledge on how such factors work together to control microtubule organization in different systems.

ATF5 Connects the Pericentriolar Materials to the Proximal End of the Mother Centriole.

Although it is known that the centrioles play instructive roles in pericentriolar material (PCM) assembly and that the PCM is essential for proper centriole formation, the mechanism that governs centriole-PCM interaction is poorly understood. Here, we show that ATF5 forms a characteristic 9-fold symmetrical ring structure in the inner layer of the PCM outfitting the proximal end of the mother centriole. ATF5 controls the centriole-PCM interaction in a cell-cycle- and centriole-age-dependent manner. Interaction of ATF5 with polyglutamylated tubulin (PGT) on the mother centriole and with PCNT in the PCM renders ATF5 as a required molecule in mother centriole-directed PCM accumulation and in PCM-dependent centriole formation. ATF5 depletion blocks PCM accumulation at the centrosome and causes fragmentation of centrioles, leading to the formation of multi-polar mitotic spindles and genomic instability. These data show that ATF5 is an essential structural protein that is required for the interaction between the mother centriole and the PCM.

Time is of the essence: the molecular mechanisms of primary microcephaly.

Primary microcephaly is a brain growth disorder characterized by a severe reduction of brain size and thinning of the cerebral cortex. Many primary microcephaly mutations occur in genes that encode centrosome proteins, highlighting an important role for centrosomes in cortical development. Centrosomes are microtubule organizing centers that participate in several processes, including controlling polarity, catalyzing spindle assembly in mitosis, and building primary cilia. Understanding which of these processes are altered and how these disruptions contribute to microcephaly pathogenesis is a central unresolved question. In this review, we revisit the different models that have been proposed to explain how centrosome dysfunction impairs cortical development. We review the evidence supporting a unified model in which centrosome defects reduce cell proliferation in the developing cortex by prolonging mitosis and activating a mitotic surveillance pathway. Finally, we also extend our discussion to centrosome-independent microcephaly mutations, such as those involved in DNA replication and repair.

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.

CEP192 localises mitotic Aurora-A activity by priming its interaction with TPX2.

Aurora-A is an essential cell-cycle kinase with critical roles in mitotic entry and spindle dynamics. These functions require binding partners such as CEP192 and TPX2, which modulate both kinase activity and localisation of Aurora-A. Here we investigate the structure and role of the centrosomal Aurora-A:CEP192 complex in the wider molecular network. We find that CEP192 wraps around Aurora-A, occupies the binding sites for mitotic spindle-associated partners, and thus competes with them. Comparison of two different Aurora-A conformations reveals how CEP192 modifies kinase activity through the site used for TPX2-mediated activation. Deleting the Aurora-A-binding interface in CEP192 prevents centrosomal accumulation of Aurora-A, curtails its activation-loop phosphorylation, and reduces spindle-bound TPX2:Aurora-A complexes, resulting in error-prone mitosis. Thus, by supplying the pool of phosphorylated Aurora-A necessary for TPX2 binding, CEP192:Aurora-A complexes regulate spindle function. We propose an evolutionarily conserved spatial hierarchy, which protects genome integrity through fine-tuning and correctly localising Aurora-A activity.

Systematic Discovery of Short Linear Motifs Decodes Calcineurin Phosphatase Signaling.

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.

Centrosome linker diversity and its function in centrosome clustering and mitotic spindle formation.

The centrosome linker joins the two interphase centrosomes of a cell into one microtubule organizing center. Despite increasing knowledge on linker components, linker diversity in different cell types and their role in cells with supernumerary centrosomes remained unexplored. Here, we identified Ninein as a C-Nap1-anchored centrosome linker component that provides linker function in RPE1 cells while in HCT116 and U2OS cells, Ninein and Rootletin link centrosomes together. In interphase, overamplified centrosomes use the linker for centrosome clustering, where Rootletin gains centrosome linker function in RPE1 cells. Surprisingly, in cells with centrosome overamplification, C-Nap1 loss prolongs metaphase through persistent activation of the spindle assembly checkpoint indicated by BUB1 and MAD1 accumulation at kinetochores. In cells lacking C-Nap1, the reduction of microtubule nucleation at centrosomes and the delay in nuclear envelop rupture in prophase probably cause mitotic defects like multipolar spindle formation and chromosome mis-segregation. These defects are enhanced when the kinesin HSET, which normally clusters multiple centrosomes in mitosis, is partially inhibited indicating a functional interplay between C-Nap1 and centrosome clustering in mitosis.

Extra centrosomes induce PIDD1-mediated inflammation and immunosurveillance.

Unscheduled increases in ploidy underlie defects in tissue function, premature aging, and malignancy. A concomitant event to polyploidization is the amplification of centrosomes, the main microtubule organization centers in animal cells. Supernumerary centrosomes are frequent in tumors, correlating with higher aggressiveness and poor prognosis. However, extra centrosomes initially also exert an onco-protective effect by activating p53-induced cell cycle arrest. If additional signaling events initiated by centrosomes help prevent pathology is unknown. Here, we report that extra centrosomes, arising during unscheduled polyploidization or aberrant centriole biogenesis, induce activation of NF-κB signaling and sterile inflammation. This signaling requires the NEMO-PIDDosome, a multi-protein complex composed of PIDD1, RIPK1, and NEMO/IKKγ. Remarkably, the presence of supernumerary centrosomes suffices to induce a paracrine chemokine and cytokine profile, able to polarize macrophages into a pro-inflammatory phenotype. Furthermore, extra centrosomes increase the immunogenicity of cancer cells and render them more susceptible to NK-cell attack. Hence, the PIDDosome acts as a dual effector, able to engage not only the p53 network for cell cycle control but also NF-κB signaling to instruct innate immunity.

Centrosome amplification fine tunes tubulin acetylation to differentially control intracellular organization.

Intracellular organelle organization is conserved in eukaryotic cells and is primarily achieved through active transport by motor proteins along the microtubule cytoskeleton. Microtubule post-translational modifications (PTMs) can contribute to microtubule diversity and differentially regulate motor-mediated transport. Here, we show that centrosome amplification, commonly observed in cancer and shown to promote aneuploidy and invasion, induces a global change in organelle positioning towards the cell periphery and facilitates nuclear migration through confined spaces. This reorganization requires kinesin-1 and is analogous to the loss of dynein. Cells with amplified centrosomes display increased levels of acetylated tubulin, a PTM that could enhance kinesin-1-mediated transport. Depletion of α-tubulin acetyltransferase 1 (αTAT1) to block tubulin acetylation rescues the displacement of centrosomes, mitochondria, and vimentin but not Golgi or endosomes. Analyses of the distribution of total and acetylated microtubules indicate that the polarized distribution of modified microtubules, rather than levels alone, plays an important role in the positioning of specific organelles, such as the centrosome. We propose that increased tubulin acetylation differentially impacts kinesin-1-mediated organelle displacement to regulate intracellular organization.

The HSV-1 pUL37 protein promotes cell invasion by regulating the kinesin-1 motor.

Neurotropic alphaherpesviruses, including herpes simplex virus type 1 (HSV-1), recruit microtubule motor proteins to invade cells. The incoming viral particle traffics to nuclei in a two-step process. First, the particle uses the dynein-dynactin motor to sustain transport to the centrosome. In neurons, this step is responsible for long-distance retrograde axonal transport and is an important component of the neuroinvasive property shared by these viruses. Second, a kinesin-dependent mechanism redirects the particle from the centrosome to the nucleus. We have reported that the kinesin motor used during the second step of invasion is assimilated into nascent virions during the previous round of infection. Here, we report that the HSV-1 pUL37 tegument protein suppresses the assimilated kinesin-1 motor during retrograde axonal transport. Region 2 (R2) of pUL37 was required for suppression and functioned independently of the autoinhibitory mechanism native to kinesin-1. Furthermore, the motor domain and proximal coiled coil of kinesin-1 were sufficient for HSV-1 assimilation, pUL37 suppression, and nuclear trafficking. pUL37 localized to the centrosome, the site of assimilated kinesin-1 activation during infection, when expressed in cells in the absence of other viral proteins; however, pUL37 did not suppress kinesin-1 in this context. These results indicate that the pUL37 tegument protein spatially and temporally regulates kinesin-1 via the amino-terminal motor region in the context of the incoming viral particle.

Cep57 regulates human centrosomes through multivalent interactions.

Human Cep57 is a coiled-coil scaffold at the pericentriolar matrix (PCM), controlling centriole duplication and centrosome maturation for faithful cell division. Genetic truncation mutations of Cep57 are associated with the mosaic-variegated aneuploidy (MVA) syndrome. During interphase, Cep57 forms a complex with Cep63 and Cep152, serving as regulators for centrosome maturation. However, the molecular interplay of Cep57 with these essential scaffolding proteins remains unclear. Here, we demonstrate that Cep57 undergoes liquid-liquid phase separation (LLPS) driven by three critical domains (NTD, CTD, and polybasic LMN). In vitro Cep57 condensates catalyze microtubule nucleation via the LMN motif-mediated tubulin concentration. In cells, the LMN motif is required for centrosomal microtubule aster formation. Moreover, Cep63 restricts Cep57 assembly, expansion, and microtubule polymerization activity. Overexpression of competitive constructs for multivalent interactions, including an MVA mutation, leads to excessive centrosome duplication. In Cep57-depleted cells, self-assembly mutants failed to rescue centriole disengagement and PCM disorganization. Thus, Cep57's multivalent interactions are pivotal for maintaining the accurate structural and functional integrity of human centrosomes.

Centrosome function is critical during terminal erythroid differentiation.

Red blood cells are produced by terminal erythroid differentiation, which involves the dramatic morphological transformation of erythroblasts into enucleated reticulocytes. Microtubules are important for enucleation, but it is not known if the centrosome, a key microtubule-organizing center, is required as well. Mice lacking the conserved centrosome component, CDK5RAP2, are likely to have defective erythroid differentiation because they develop macrocytic anemia. Here, we show that fetal liver-derived, CDK5RAP2-deficient erythroid progenitors generate fewer and larger reticulocytes, hence recapitulating features of macrocytic anemia. In erythroblasts, but not in embryonic fibroblasts, loss of CDK5RAP2 or pharmacological depletion of centrosomes leads to highly aberrant spindle morphologies. Consistent with such cells exiting mitosis without chromosome segregation, tetraploidy is frequent in late-stage erythroblasts, thereby giving rise to fewer but larger reticulocytes than normal. Our results define a critical role for CDK5RAP2 and centrosomes in spindle formation specifically during blood production. We propose that disruption of centrosome and spindle function could contribute to the emergence of macrocytic anemias, for instance, due to nutritional deficiency or exposure to chemotherapy.