Parental genome unification is highly error-prone in mammalian embryos.

Most human embryos are aneuploid. Aneuploidy frequently arises during the early mitotic divisions of the embryo, but its origin remains elusive. Human zygotes that cluster their nucleoli at the pronuclear interface are thought to be more likely to develop into healthy euploid embryos. Here, we show that the parental genomes cluster with nucleoli in each pronucleus within human and bovine zygotes, and clustering is required for the reliable unification of the parental genomes after fertilization. During migration of intact pronuclei, the parental genomes polarize toward each other in a process driven by centrosomes, dynein, microtubules, and nuclear pore complexes. The maternal and paternal chromosomes eventually cluster at the pronuclear interface, in direct proximity to each other, yet separated. Parental genome clustering ensures the rapid unification of the parental genomes on nuclear envelope breakdown. However, clustering often fails, leading to chromosome segregation errors and micronuclei, incompatible with healthy embryo development.

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.

Multiple Functions and Regulation of Mammalian Peroxiredoxins.

Peroxiredoxins (Prxs) constitute a major family of peroxidases, with mammalian cells expressing six Prx isoforms (PrxI to PrxVI). Cells produce hydrogen peroxide (H2O2) at various intracellular locations where it can serve as a signaling molecule. Given that Prxs are abundant and possess a structure that renders the cysteine (Cys) residue at the active site highly sensitive to oxidation by H2O2, the signaling function of this oxidant requires extensive and highly localized regulation. Recent findings on the reversible regulation of PrxI through phosphorylation at the centrosome and on the hyperoxidation of the Cys at the active site of PrxIII in mitochondria are described in this review as examples of such local regulation of H2O2 signaling. Moreover, their high affinity for and sensitivity to oxidation by H2O2 confer on Prxs the ability to serve as sensors and transducers of H2O2 signaling through transfer of their oxidation state to bound effector proteins.

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.

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.

Mechanisms of Mitotic Spindle Assembly.

Life depends on cell proliferation and the accurate segregation of chromosomes, which are mediated by the microtubule (MT)-based mitotic spindle and ∼200 essential MT-associated proteins. Yet, a mechanistic understanding of how the mitotic spindle is assembled and achieves chromosome segregation is still missing. This is mostly due to the density of MTs in the spindle, which presumably precludes their direct observation. Recent insight has been gained into the molecular building plan of the metaphase spindle using bulk and single-molecule measurements combined with computational modeling. MT nucleation was uncovered as a key principle of spindle assembly, and mechanistic details about MT nucleation pathways and their coordination are starting to be revealed. Lastly, advances in studying spindle assembly can be applied to address the molecular mechanisms of how the spindle segregates chromosomes.

Cell-Type-Specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex.

Alternative splicing is prevalent in the mammalian brain. To interrogate the functional role of alternative splicing in neural development, we analyzed purified neural progenitor cells (NPCs) and neurons from developing cerebral cortices, revealing hundreds of differentially spliced exons that preferentially alter key protein domains-especially in cytoskeletal proteins-and can harbor disease-causing mutations. We show that Ptbp1 and Rbfox proteins antagonistically govern the NPC-to-neuron transition by regulating neuron-specific exons. Whereas Ptbp1 maintains apical progenitors partly through suppressing a poison exon of Flna in NPCs, Rbfox proteins promote neuronal differentiation by switching Ninein from a centrosomal splice form in NPCs to a non-centrosomal isoform in neurons. We further uncover an intronic human mutation within a PTBP1-binding site that disrupts normal skipping of the FLNA poison exon in NPCs and causes a brain-specific malformation. Our study indicates that dynamic control of alternative splicing governs cell fate in cerebral cortical development.

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.

A Dynamic Protein Interaction Landscape of the Human Centrosome-Cilium Interface.

The centrosome is the primary microtubule organizing center of the cells and templates the formation of cilia, thereby operating at a nexus of critical cellular functions. Here, we use proximity-dependent biotinylation (BioID) to map the centrosome-cilium interface; with 58 bait proteins we generate a protein topology network comprising >7,000 interactions. Analysis of interaction profiles coupled with high resolution phenotypic profiling implicates a number of protein modules in centriole duplication, ciliogenesis, and centriolar satellite biogenesis and highlights extensive interplay between these processes. By monitoring dynamic changes in the centrosome-cilium protein interaction landscape during ciliogenesis, we also identify satellite proteins that support cilia formation. Systematic profiling of proximity interactions combined with functional analysis thus provides a rich resource for better understanding human centrosome and cilia biology. Similar strategies may be applied to other complex biological structures or pathways.

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.

Formation of memory assemblies through the DNA-sensing TLR9 pathway.

As hippocampal neurons respond to diverse types of information1, a subset assembles into microcircuits representing a memory2. Those neurons typically undergo energy-intensive molecular adaptations, occasionally resulting in transient DNA damage3-5. Here we found discrete clusters of excitatory hippocampal CA1 neurons with persistent double-stranded DNA (dsDNA) breaks, nuclear envelope ruptures and perinuclear release of histone and dsDNA fragments hours after learning. Following these early events, some neurons acquired an inflammatory phenotype involving activation of TLR9 signalling and accumulation of centrosomal DNA damage repair complexes6. Neuron-specific knockdown of Tlr9 impaired memory while blunting contextual fear conditioning-induced changes of gene expression in specific clusters of excitatory CA1 neurons. Notably, TLR9 had an essential role in centrosome function, including DNA damage repair, ciliogenesis and build-up of perineuronal nets. We demonstrate a novel cascade of learning-induced molecular events in discrete neuronal clusters undergoing dsDNA damage and TLR9-mediated repair, resulting in their recruitment to memory circuits. With compromised TLR9 function, this fundamental memory mechanism becomes a gateway to genomic instability and cognitive impairments implicated in accelerated senescence, psychiatric disorders and neurodegenerative disorders. Maintaining the integrity of TLR9 inflammatory signalling thus emerges as a promising preventive strategy for neurocognitive deficits.

Mitotic spindle assembly in animal cells: a fine balancing act.

The mitotic spindle has a crucial role in ensuring the accurate segregation of chromosomes into the two daughter cells during cell division, which is paramount for maintaining genome integrity. It is a self-organized and dynamic macromolecular structure that is constructed from microtubules, microtubule-associated proteins and motor proteins. Thirty years of research have led to the identification of centrosome-, chromatin- and microtubule-mediated microtubule nucleation pathways that each contribute to mitotic spindle assembly. Far from being redundant pathways, data are now emerging regarding how they function together to ensure the timely completion of mitosis. We are also beginning to comprehend the multiple mechanisms by which cells regulate spindle scaling. Together, this research has increased our understanding of how cells coordinate hundreds of proteins to assemble the dynamic, precise and robust structure that is the mitotic spindle.

Cytokinesis failure triggers hippo tumor suppressor pathway activation.

Genetically unstable tetraploid cells can promote tumorigenesis. Recent estimates suggest that ∼37% of human tumors have undergone a genome-doubling event during their development. This potentially oncogenic effect of tetraploidy is countered by a p53-dependent barrier to proliferation. However, the cellular defects and corresponding signaling pathways that trigger growth suppression in tetraploid cells are not known. Here, we combine RNAi screening and in vitro evolution approaches to demonstrate that cytokinesis failure activates the Hippo tumor suppressor pathway in cultured cells, as well as in naturally occurring tetraploid cells in vivo. Induction of the Hippo pathway is triggered in part by extra centrosomes, which alter small G protein signaling and activate LATS2 kinase. LATS2 in turn stabilizes p53 and inhibits the transcriptional regulators YAP and TAZ. These findings define an important tumor suppression mechanism and uncover adaptive mechanisms potentially available to nascent tumor cells that bypass this inhibitory regulation.

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.

The Crk4-Cyc4 complex regulates G2/M transition in Toxoplasma gondii.

A versatile division of apicomplexan parasites and a dearth of conserved regulators have hindered the progress of apicomplexan cell cycle studies. While most apicomplexans divide in a multinuclear fashion, Toxoplasma gondii tachyzoites divide in the traditional binary mode. We previously identified five Toxoplasma CDK-related kinases (Crk). Here, we investigated TgCrk4 and its cyclin partner TgCyc4. We demonstrated that TgCrk4 regulates conventional G2 phase processes, such as repression of chromosome rereplication and centrosome reduplication, and acts upstream of the spindle assembly checkpoint. The spatial TgCyc4 dynamics supported the TgCrk4-TgCyc4 complex role in the coordination of chromosome and centrosome cycles. We also identified a dominant TgCrk4-TgCyc4 complex interactor, TgiRD1 protein, related to DNA replication licensing factor CDT1 but played no role in licensing DNA replication in the G1 phase. Our results showed that TgiRD1 also plays a role in controlling chromosome and centrosome reduplication. Global phosphoproteome analyses identified TgCrk4 substrates, including TgORC4, TgCdc20, TgGCP2, and TgPP2ACA. Importantly, the phylogenetic and structural studies suggest the Crk4-Cyc4 complex is limited to a minor group of the binary dividing apicomplexans.

Asymmetric inheritance of centrosome-associated primary cilium membrane directs ciliogenesis after cell division.

Primary cilia are key sensory organelles that are thought to be disassembled prior to mitosis. Inheritance of the mother centriole, which nucleates the primary cilium, in relation to asymmetric daughter cell behavior has previously been studied. However, the fate of the ciliary membrane upon cell division is unknown. Here, we followed the ciliary membrane in dividing embryonic neocortical stem cells and cultured cells. Ciliary membrane attached to the mother centriole was endocytosed at mitosis onset, persisted through mitosis at one spindle pole, and was asymmetrically inherited by one daughter cell, which retained stem cell character. This daughter re-established a primary cilium harboring an activated signal transducer earlier than the noninheriting daughter. Centrosomal association of ciliary membrane in dividing neural stem cells decreased at late neurogenesis when these cells differentiate. Our data imply that centrosome-associated ciliary membrane acts as a determinant for the temporal-spatial control of ciliogenesis.

Dynein recruitment to nuclear pores activates apical nuclear migration and mitotic entry in brain progenitor cells.

Radial glial progenitors (RGPs) are elongated epithelial cells that give rise to neurons, glia, and adult stem cells during brain development. RGP nuclei migrate basally during G1, apically using cytoplasmic dynein during G2, and undergo mitosis at the ventricular surface. By live imaging of in utero electroporated rat brain, we find that two distinct G2-specific mechanisms for dynein nuclear pore recruitment are essential for apical nuclear migration. The "RanBP2-BicD2" and "Nup133-CENP-F" pathways act sequentially, with Nup133 or CENP-F RNAi arresting nuclei close to the ventricular surface in a premitotic state. Forced targeting of dynein to the nuclear envelope rescues nuclear migration and cell-cycle progression, demonstrating that apical nuclear migration is not simply correlated with cell-cycle progression from G2 to mitosis, but rather, is a required event. These results reveal that cell-cycle control of apical nuclear migration occurs by motor protein recruitment and identify a role for nucleus- and centrosome-associated forces in mitotic entry. PAPERCLIP:

ER proteins decipher the tubulin code to regulate organelle distribution.

Organelles move along differentially modified microtubules to establish and maintain their proper distributions and functions1,2. However, how cells interpret these post-translational microtubule modification codes to selectively regulate organelle positioning remains largely unknown. The endoplasmic reticulum (ER) is an interconnected network of diverse morphologies that extends promiscuously throughout the cytoplasm3, forming abundant contacts with other organelles4. Dysregulation of endoplasmic reticulum morphology is tightly linked to neurologic disorders and cancer5,6. Here we demonstrate that three membrane-bound endoplasmic reticulum proteins preferentially interact with different microtubule populations, with CLIMP63 binding centrosome microtubules, kinectin (KTN1) binding perinuclear polyglutamylated microtubules, and p180 binding glutamylated microtubules. Knockout of these proteins or manipulation of microtubule populations and glutamylation status results in marked changes in endoplasmic reticulum positioning, leading to similar redistributions of other organelles. During nutrient starvation, cells modulate CLIMP63 protein levels and p180-microtubule binding to bidirectionally move endoplasmic reticulum and lysosomes for proper autophagic responses.