Spontaneous slow replication fork progression elicits mitosis alterations in homologous recombination-deficient mammalian cells

Homologous recombination deficient (HR⁻) mammalian cells spontaneously display reduced replication fork (RF) movement and mitotic extra centrosomes. We show here that these cells present a complex mitotic phenotype, including prolonged metaphase arrest, anaphase bridges, and multipolar segregations. We then asked whether the replication and the mitotic phenotypes are interdependent. First, we determined low doses of hydroxyurea that did not affect the cell cycle distribution or activate CHK1 phosphorylation but did slow the replication fork movement of wild-type cells to the same level than in HR⁻ cells. Remarkably, these low hydroxyurea doses generated the same mitotic defects (and to the same extent) in wild-type cells as observed in unchallenged HR⁻ cells. Reciprocally, supplying nucleotide precursors to HR⁻ cells suppressed both their replication deceleration and mitotic extra centrosome phenotypes. Therefore, subtle replication stress that escapes to surveillance pathways and, thus, fails to prevent cells from entering mitosis alters metaphase progression and centrosome number, resulting in multipolar mitosis. Importantly, multipolar mitosis results in global unbalanced chromosome segregation involving the whole genome, even fully replicated chromosomes. These data highlight the cross-talk between chromosome replication and segregation, and the importance of HR at the interface of these two processes for protection against general genome instability.DNA is continuously subjected to injury by exogenous and endogenous sources. The faithful transmission of genetic material relies on the DNA damage response (DDR), which coordinates a network of pathways, including DNA replication-repair-recombination, the cell cycle checkpoint, and chromosome segregation. A defect in any of these pathways causes genetic instability and cancer predisposition. Strikingly, both spontaneous DDR activation as a consequence of endogenous replication stress and centrosome abnormalities, which cause uneven chromosome segregation, have been reported in precancerous and early-stage malignancies (1–10). Therefore, endogenous stresses must play a key role in spontaneous chromosome instability and in cancer etiology.Homologous recombination (HR) is an evolutionarily conserved process that controls the balance between genetic stability and diversity. Specifically, HR plays a pivotal role in the reactivation of replication forks that have been blocked, contributing to DNA replication accuracy (11–16). Replication forks are routinely inactivated by endogenous stress (17, 18); therefore, HR should play an essential role to protect cells against these types of stresses, and HR deficiency should reveal endogenous replication stress. Remarkably, unchallenged HR-deficient (HR⁻) cells display both a genome-wide decrease in replication fork speed (19) and a spontaneous increase in the frequency of cells containing extra centrosomes (20–28). Two hypotheses may account for these two phenotypes. First, replication stress leads to chromosome alteration at incomplete replicated regions and chromosome rearrangements (29). However, centrosomes do not contain DNA, and if extra centrosomes at mitosis [mitotic extra centrosome (MEC)] are active, unbalanced chromosome segregation should lead to global chromosome instability, even for fully replicated chromosomes. Second, HR proteins are associated with supernumerary centrosomes; therefore, centrosome duplication defects may directly result from HR misregulation (30, 31).In this study, we addressed whether spontaneous MECs result from slow replication fork movement in HR⁻ cells. The data presented here underline the importance of HR at the molecular interface between replication and chromosome segregation to protect against spontaneous genomic instability.     

Epidermal development,growth control,and homeostasis in the face of centrosome amplification

As nucleators of the mitotic spindle and primary cilium, centrosomes play crucial roles in equal segregation of DNA content to daughter cells, coordination of growth and differentiation, and transduction of homeostatic cues. Whereas the majority of mammalian cells carry no more than two centrosomes per cell, exceptions to this rule apply in certain specialized tissues and in select disease states, including cancer. Centrosome amplification, or the condition of having more than two centrosomes per cell, has been suggested to contribute to instability of chromosomes, imbalance in asymmetric divisions, and reorganization of tissue architecture; however, the degree to which these conditions are a direct cause of or simply a consequence of human disease is poorly understood. Here we addressed this issue by generating a mouse model inducing centrosome amplification in a naturally proliferative epithelial tissue by elevating Polo-like kinase 4 (Plk4) expression in the skin epidermis. By altering centrosome numbers, we observed multiciliated cells, spindle orientation errors, and chromosome segregation defects within developing epidermis. None of these defects was sufficient to impart a proliferative advantage within the tissue, however. Rather, impaired mitoses led to p53-mediated cell death and contributed to defective growth and stratification. Despite these abnormalities, mice remained viable and healthy, although epidermal cells with centrosome amplification were still appreciable. Moreover, these abnormalities were insufficient to disrupt homeostasis and initiate or enhance tumorigenesis, underscoring the powerful surveillance mechanisms in the skin.Centrosomes play crucial functions within the cell by organizing microtubules and by participating in the assembly of the primary cilium, an antenna-like structure that senses the cellular environment and transmits signaling cues. On a structural basis, each centrosome consists of orthogonally positioned centrioles and its surrounding protein-rich pericentriolar material (PCM). The majority of mammalian cells contain one centrosome throughout interphase (G1), and then replicate during S-phase of the cell cycle in preparation for mitosis (1). Centrosomes generally template their own duplication, and they keep their numbers in check through tight posttranslational regulation of the duplication process itself. Through licensing mechanisms, centrosomal reduplication is prevented, and by virtue of their role in nucleating a bipolar mitotic spindle, centrosomes are faithfully partitioned into each daughter cell at the end of mitosis (2, 3).Mutations and misregulation of centrosomal proteins have been associated with various human disorders, including ciliopathies, obesity, neurologic disorders, and miscarriages (4–6). Numerical aberrations in centrosome numbers have been demonstrated to alter ciliary signaling and have been proposed to be the underlying cause for certain defects in tissue organization (7, 8). In addition, recent genetic studies on the master regulator of centrosome number, polo-like kinase 4 (PLK4), have shown that, whether too few or too many, perturbations in centrosome numbers can directly impede brain development, leading to microcephaly both in mice and in humans (9, 10). PLK4 mutations also have been associated with mitotic-origin aneuploidy in human embryos, suggestive of a possible link among PLK4, aneuploidy, and pregnancy loss (6).Centrosome amplification, or the condition of having more than a cell’s customary pair of such structures, has garnered attention for more than a century (11). Given the enhanced apoptosis resulting from centrosome amplification in the brain, it is intriguing that centrosome amplification was originally noted for its presence in cancer cells (9). Indeed, increased centrosome number is a hallmark of many cancers, and it correlates with poor clinical prognoses in some malignancies, including those of epithelial origin (12, 13). In flies, centrosomal alterations have been found to expand the pool of proliferative progenitors in serial neuroblast transplantation assays, a phenomenon attributed to an imbalance in asymmetric divisions (14, 15). In mammals, however, despite the strong correlation with hyperproliferative disorders, whether centrosomal abnormalities are the cause, the consequence, or a neutral bystander of cancer remains unclear (16, 17).Based on the assumption that centrosome amplification results in multipolar mitoses, initial research efforts focused on drawing mechanistic links to chromosomal instability (18–20). Surprisingly, however, at least in various cancer cell lines examined in vitro, multipolar divisions turned out to be rare and typically inviable (21). Instead, these cells seemed to have developed strategies to cope with extra centrosomes, including clustering them together such that a bipolar spindle could still form (22–25). That said, even within a bipolar spindle network, chromosome segregation errors involving merotelic attachment have been observed, and these can contribute to chromosomal instability if the mitotic checkpoint is bypassed (21, 26). Taken together, these studies underscore the importance of delving more deeply into the physiological relevance of centrosome amplification in additional mammalian tissues in vivo, and to parse out the centrosomal contribution to tissue function.Mammalian epidermis offers an excellent opportunity to evaluate the various proposed cellular mechanisms in which centrosome amplification affects tissue development, homeostasis, and tumorigenesis. During embryogenesis, it begins as a single layer of proliferative progenitors, which divide laterally to accommodate embryonic growth, and also perpendicularly to give rise to a stratified, differentiating tissue (27). Only the innermost basal layer retains progenitor status, which relies on integrin-mediated attachment to an underlying basement membrane rich in extracellular matrix. Perpendicular divisions are asymmetric, involving differential Notch and ciliary signaling for proper morphogenesis (28–30). The epidermis matures shortly before birth, and at this stage proliferative basal cells give rise to spinous, granular, and surface stratum corneum cells, which are sloughed and continually replaced by inner cells differentiating upward to maintain homeostasis. In the adult, the epidermis is exposed to a variety of environmental assaults and must undergo frequent turnover to maintain the body’s protective barrier. These features contribute to the skin accounting for the most common cancers worldwide.In the present work, we evaluated centrosome dysfunction in the context of centrosome amplification in mouse epidermis. To do so, we generated mice that induce PLK4 overexpression in the basal layer. PLK4 is the key to initiating centriole duplication, and at elevated levels, this kinase is capable of replicating more than a single centriole on the existing one (31–34). After establishing that the epidermis acquires an excess of centrosomes, we examined the consequences on growth and differentiation, cilia and Notch signaling, mitotic spindle formation and orientation, mitotic error-induced DNA damage response/aneuploidy, and p53-mediated apoptosis. Finally, because our mice remained viable, we could evaluate the effects of sustained centrosome amplification on tissue integrity. Our findings reveal a remarkable resilience of the skin epidermis in coping with abnormalities induced by centrosome amplification. Moreover, despite the longevity of animals that overexpress PLK4 in the skin epidermis, this did not lead to an increased propensity of these mice to initiate or promote tumorigenesis in the skin.     

Hierarchical recruitment of Plk4 and regulation of centriole biogenesis by two centrosomal scaffolds,Cep192 and Cep152

Centrosomes play an important role in various cellular processes, including spindle formation and chromosome segregation. They are composed of two orthogonally arranged centrioles, whose duplication occurs only once per cell cycle. Accurate control of centriole numbers is essential for the maintenance of genomic integrity. Although it is well appreciated that polo-like kinase 4 (Plk4) plays a central role in centriole biogenesis, how it is recruited to centrosomes and whether this step is necessary for centriole biogenesis remain largely elusive. Here we showed that Plk4 localizes to distinct subcentrosomal regions in a temporally and spatially regulated manner, and that Cep192 and Cep152 serve as two distinct scaffolds that recruit Plk4 to centrosomes in a hierarchical order. Interestingly, Cep192 and Cep152 competitively interacted with the cryptic polo box of Plk4 through their homologous N-terminal sequences containing acidic-α-helix and N/Q-rich motifs. Consistent with these observations, the expression of either one of these N-terminal fragments was sufficient to delocalize Plk4 from centrosomes. Furthermore, loss of the Cep192- or Cep152-dependent interaction with Plk4 resulted in impaired centriole duplication that led to delayed cell proliferation. Thus, the spatiotemporal regulation of Plk4 localization by two hierarchical scaffolds, Cep192 and Cep152, is critical for centriole biogenesis.The centrosome is the main microtubule-organizing center in mammalian cells that plays a central role in spindle formation and chromosome segregation during mitosis. Centrosomes are composed of two orthogonally arranged centrioles surrounded by an amorphous mass of electron-dense pericentriolar material (PCM). Centrioles duplicate precisely once per cell cycle and serve as platforms for the assembly of centrosomes, primary cilia, and flagella (1–4).Centriole duplication is initiated by the assembly of a procentriole in early S phase. In Caenorhabditis elegans, a centrosomal scaffold protein, called Spd-2, is required for proper recruitment of a Ser/Thr kinase, Zyg-1 (5), to centrosomes, and this step in turn allows the recruitment of Sas-6, Sas-5, and Sas-4 to the site of procentriole assembly (6, 7). Sas6 plays a pivotal role in self-assembling a cartwheel-like structure at this site of the procentriole with Sas5 and Sas4 (8–12). In Drosophila, the overexpression of polo-like kinase 4 (Plk4; also called Sak), the Zyg-1 ortholog, is sufficient to induce centriole amplification, whereas the depletion of Plk4 disrupts centriole duplication (12, 13). Interestingly, however, Drosophila Spd-2 is dispensable for Plk4-mediated centriole duplication (14). Instead, another scaffold, Asterless, has been suggested to play a critical role in targeting Plk4 to centrosomes (15), hinting that the mechanism underlying Plk4 recruitment is distinct in different organisms.Accumulated evidence in humans suggests that Plk4 is a functional ortholog of C. elegans Zyg-1 and Drosophila Plk4, and that it plays a key role in centriole duplication (16, 17). When overexpressed, Plk4 can induce multiple centriole precursors surrounding a single parental centriole, and centrosomally localized Plk4 appears to be required for this event (16). The cryptic polo box (CPB) present at the upstream of the C-terminal polo box (PB) (18) is necessary and sufficient for targeting Plk4 to centrosomes (16, 19). Interestingly, the CPB comprises two structurally related motifs and forms a homodimer (19) to interact with its binding targets. However, the molecular basis of how Plk4 binds to its targets and localizes to centrosomes remains largely elusive.Studies have shown that Cep152, a human ortholog of Drosophila Asterless, interacts with Plk4 through the CPB (20, 21). However, the depletion of Cep152 does not significantly decrease the level of Plk4 at centrosomes. Recently, Sonnen et al. have shown that a C. elegans Spd-2 ortholog, Cep192, interacts with Plk4 and promotes the recruitment of Plk4 to centrosomes (22). Moreover, Cep192 binds to Cep152, and the depletion of both enhances the Plk4 localization defect (22). Based on these observations, Sonnen et al. proposed that Cep192 cooperates with Cep152 to properly recruit Plk4 to centrosomes and to promote centriole duplication (22).In this study, we demonstrated that disrupting either the Cep192–Plk4 interaction or the Cep152–Plk4 interaction was sufficient to impair centriole duplication. We further showed that Plk4 dynamically localizes to different subcentrosomal regions in a cell cycle-specific manner, and that Cep192 functions at a point upstream of Cep152 to regulate Plk4 localization. Thus, we propose that the spatiotemporal regulation of Plk4 localization by two hierarchical scaffolds, Cep192 and Cep152, is critical for Plk4-dependent centriole biogenesis.     

Genes involved in centrosome-independent mitotic spindle assembly in Drosophila S2 cells

Animal mitotic spindle assembly relies on centrosome-dependent and centrosome-independent mechanisms, but their relative contributions remain unknown. Here, we investigated the molecular basis of the centrosome-independent spindle assembly pathway by performing a whole-genome RNAi screen in Drosophila S2 cells lacking functional centrosomes. This screen identified 197 genes involved in acentrosomal spindle assembly, eight of which had no previously described mitotic phenotypes and produced defective and/or short spindles. All 197 genes also produced RNAi phenotypes when centrosomes were present, indicating that none were entirely selective for the acentrosomal pathway. However, a subset of genes produced a selective defect in pole focusing when centrosomes were absent, suggesting that centrosomes compensate for this shape defect. Another subset of genes was specifically associated with the formation of multipolar spindles only when centrosomes were present. We further show that the chromosomal passenger complex orchestrates multiple centrosome-independent processes required for mitotic spindle assembly/maintenance. On the other hand, despite the formation of a chromosome-enriched RanGTP gradient, S2 cells depleted of RCC1, the guanine-nucleotide exchange factor for Ran on chromosomes, established functional bipolar spindles. Finally, we show that cells without functional centrosomes have a delay in chromosome congression and anaphase onset, which can be explained by the lack of polar ejection forces. Overall, these findings establish the constitutive nature of a centrosome-independent spindle assembly program and how this program is adapted to the presence/absence of centrosomes in animal somatic cells.Chromosome segregation during mitosis/meiosis is mediated by a microtubule (MT)-based bipolar spindle structure. Mitotic spindle assembly in animal somatic cells was initially believed to rely exclusively on the presence of centrosomes, but it is now well established that centrosomes are not essential (1–6). Land plants and many animal oocytes are paradigmatic examples in which an MT-based spindle normally assembles without centrosomes (7, 8). More recently, it was shown that spindle assembly during somatic cell divisions in the early mouse embryo is also independent of centrosomes (9) and that centrosomes are fully dispensable in planarians throughout their development (10). Overall, these data support the existence of centrosome-independent mechanisms that mediate mitotic/meiotic spindle assembly in animals.Acentrosomal spindle assembly has been particularly well characterized in Xenopus laevis egg extracts, in which MTs form in the vicinity of mitotic chromatin due to a stabilizing effect imposed by a Ras-related nuclear protein in the GTP-bound state (RanGTP) gradient. RanGTP is present at highest concentrations around chromosomes, due to the localization of the Ran guanine nucleotide exchange factor regulator of chromosome condensation 1 (RCC1) on chromosomes (11). However, it remains controversial whether the gradient of RanGTP is required for spindle assembly in other systems (12, 13). Some of the downstream effectors of RanGTP include TPX2 and augmin, which promote MT assembly (14, 15). The chromosomal passenger complex (CPC) has also been implicated in acentrosomal spindle assembly/function in X. laevis egg extracts, as well as in Drosophila and mouse oocytes, and is believed to function independent of RanGTP (16–20). However, despite significant recent progress, a full picture of the molecular mechanisms behind acentrosomal spindle assembly in animal somatic cells is lacking. Moreover, it remains unknown whether the genes involved in acentrosomal spindle assembly are just a subset of those required when centrosomes are present or include specific genes that become essential only when centrosomes are compromised/absent.Here, we investigated the gene requirements for acentrosomal spindle assembly in Drosophila S2 cells by performing a whole-genome RNAi screen. We found that virtually the same set of genes is involved in spindle assembly either with or without centrosomes, although a small subset of genes exhibit a different specific phenotype in the presence or absence of centrosomes.     

Asymmetric partitioning of transfected DNA during mammalian cell division

Foreign DNA molecules and chromosomal fragments are generally eliminated from proliferating cells, but we know little about how mammalian cells prevent their propagation. Here, we show that dividing human and canine cells partition transfected plasmid DNA asymmetrically, preferentially into the daughter cell harboring the young centrosome. Independently of how they entered the cell, most plasmids clustered in the cytoplasm. Unlike polystyrene beads of similar size, these clusters remained relatively immobile and physically associated to endoplasmic reticulum-derived membranes, as revealed by live cell and electron microscopy imaging. At entry of mitosis, most clusters localized near the centrosomes. As the two centrosomes split to assemble the bipolar spindle, predominantly the old centrosome migrated away, biasing the partition of the plasmid cluster toward the young centrosome. Down-regulation of the centrosomal proteins Ninein and adenomatous polyposis coli abolished this bias. Thus, we suggest that DNA clustering, cluster immobilization through association to the endoplasmic reticulum membrane, initial proximity between the cluster and centrosomes, and subsequent differential behavior of the two centrosomes together bias the partition of plasmid DNA during mitosis. This process leads to their progressive elimination from the proliferating population and might apply to any kind of foreign DNA molecule in mammalian cells. Furthermore, the functional difference of the centrosomes might also promote the asymmetric partitioning of other cellular components in other mammalian and possibly stem cells.Generally, noncentromeric DNA molecules are mitotically instable in eukaryotes. This results in their apparent disappearance from an ever-increasing proportion of the progeny of an affected cell (e.g., 1–3). Endogenous sources of such DNA are recombination byproducts [double minutes, extrachromosomal ribosomal (r)DNA circles (ERCs) and other DNA circles (3–6)] or mitotic defects generating noncentromeric chromosomal fragments and cytoplasmic micronuclei (1, 7). Exogenous sources are DNA of pathogens or DNA, typically plasmids, artificially introduced into cells. For the latter, decades of work established that plasmid-born protein expression is transient, persisting only for a few cell cycles (8). This finding is consistent with plasmid DNA being somehow eliminated through divisions. Thus, some mechanisms seem to prevent the propagation of foreign DNA and extrachromosomal DNA in proliferating eukaryotic cells. However, how this is achieved is unclear.In animal cells, DNA sensors mediate the early detection of exogenous DNA, such as DNA of invading pathogens and artificially introduced DNA (9–11). Both in leukocytes and nonprofessional immune cells, these can trigger innate immune responses, such as cytokine production, autophagy, and apoptosis (9). However, what happens to the DNA molecules themselves over time is unclear. When microinjected into the nucleus, plasmid DNA clusters and is expelled into the cytoplasm at mitosis (12). In the cytoplasm, the amount of DNA decreased within a few hours without completely disappearing, suggesting a rapid degradation of a major fraction and the persistence of a minor fraction of the molecules (13). Within a few hours after introducing DNA into the cytoplasm, tubular membranes and Emerin, a protein synthesized in the endoplasmic reticulum (ER) and subsequently predominantly present in the inner nuclear membrane, appear in the cytoplasm (10, 11). However, the functional relevance of these observations is not clear. Furthermore, the destiny of the DNA molecules, especially during subsequent mitoses, is elusive.To better understand these phenomena and their functional relevance, we analyzed the fate of transfected plasmid DNA in dividing mammalian tissue culture cells.     

Conserved TCP domain of Sas-4/CPAP is essential for pericentriolar material tethering during centrosome biogenesis

Pericentriolar material (PCM) recruitment to centrioles forms a key step in centrosome biogenesis. Deregulation of this process leads to centrosome aberrations causing disorders, one of which is autosomal recessive primary microcephaly (MCPH), a neurodevelopmental disorder where brain size is reduced. During PCM recruitment, the conserved centrosomal protein Sas-4/CPAP/MCPH6, known to play a role in centriole formation, acts as a scaffold for cytoplasmic PCM complexes to bind and then tethers them to centrioles to form functional centrosomes. To understand Sas-4’s tethering role, we determined the crystal structure of its T complex protein 10 (TCP) domain displaying a solvent-exposed single-layer of β-sheets fold. This unique feature of the TCP domain suggests that it could provide an “extended surface-like” platform to tether the Sas-4–PCM scaffold to a centriole. Functional studies in Drosophila, human cells, and human induced pluripotent stem cell-derived neural progenitor cells were used to test this hypothesis, where point mutations within the 9–10th β-strands (β9–10 mutants including a MCPH-associated mutation) perturbed PCM tethering while allowing Sas-4/CPAP to scaffold cytoplasmic PCM complexes. Specifically, the Sas-4 β9–10 mutants displayed perturbed interactions with Ana2, a centrosome duplication factor, and Bld-10, a centriole microtubule-binding protein, suggesting a role for the β9–10 surface in mediating protein–protein interactions for efficient Sas-4–PCM scaffold centriole tethering. Hence, we provide possible insights into how centrosomal protein defects result in human MCPH and how Sas-4 proteins act as a vehicle to tether PCM complexes to centrioles independent of its well-known role in centriole duplication.Centrosomes consist of a pair of centrioles surrounded by a protein network of pericentriolar material (PCM), the main sites for microtubule nucleation and anchoring and thus responsible for PCM’s role as the principle microtubule-organizing centers (MTOCs) of cells (1–4). When PCM is not recruited, centrioles are unstable, and thus no functional centrosomes are generated (5, 6). Although initial proteomic studies suggested PCM to be an amorphous cloud composed of more than a 100 different proteins (7), recent superresolution microscopy of fly and human centrosomes have indicated key centrosomal proteins essential for centrosome biogenesis to be organized into distinct spatial compartments before appearing as a PCM cloud surrounding the centriole (6, 8–11).Thus, there could be a protein providing an interface for mediating PCM tethering to a centriole, a suitable candidate of which is the conserved centrosomal protein Sas-4 (CPAP in human), forming a layer closely associated with the centriole wall and yet shown to interact with various PCM components (6, 12). Functional studies in various model organisms suggest that Sas-4 proteins are required for both centriole formation and PCM assembly (6, 12); in the absence of Sas-4, nascent centrioles form but fail to mature into centrosomes (6). Overexpression of Sas-4 in flies produces PCM-like structures (13), whereas reduced amounts of Sas-4 in worms result in centrosomes having proportionally less PCM (12). Thus, although it is clear that Sas-4 is essential for centrosome biogenesis, the mechanisms by which Sas-4 contributes to PCM assembly remains elusive.During the course of these studies, we and others have reported that Sas-4/CPAP, a protein essential for centriole formation was found to interact with several centrosomal and PCM proteins including Cnn, Asl, D-PLP, γ-TuRC, SIL, Cep135, Cep120, and tubulin dimers (5, 6, 14–16). In Drosophila, the N-terminal domain of Sas-4 provides a scaffolding site for cytoplasmic protein complexes (hereafter referred to as Sas-4–PCM scaffold) and tethers the components of Sas-4–PCM scaffold to a centrosome matrix via its C terminus (6).Interestingly, the C-terminal region of Sas-4 proteins contains a conserved TCP10c domain (Pfam: PF07202) (hereafter referred to as TCP for brevity) (Fig. 1A and SI Appendix, Fig. S1). An E1235V missense mutation within this domain in CPAP has been identified in patients with primary microcephaly (MCPH), resulting in a reduced interaction with STIL (Ana2 in Drosophila), a centriole duplication factor also implicated in MCPH (16–18). Accordingly, recent structural studies on CPAP-STIL complex revealed that CPAP-STIL interaction is required during centriole assembly (19, 20). The C-terminal domain of CPAP has also been shown to mediate an interaction with another MCPH protein Cep135 (Bld-10 in Drosophila) and that interaction is required for centriole assembly. Bld-10 is a core centriolar protein and is required to stabilize structural integrity of centrioles (21–23). Taken together, we therefore speculate that the TCP domain could mediate protein–protein interactions and might serve as a tethering site for Sas-4–PCM scaffold–centriole interactions.Open in a separate windowFig. 1.Crystal structure of Drosophila Sas-4–TCP domain. (A) Domain architecture of Drosophila Sas-4 and its human ortholog CPAP. The fragment used for crystallization is indicated by a black underline. (B) Cartoon view of the overall structure of Sas-4–TCP. The invisible part of β16–20 in the crystal structure is shown as dotted lines. (C) Side view of Sas-4–TCP along the longitudinal axis from the N to C termini. Twisting of the TCP β-strands is diagramed below. F_L, surface left to β1; F_R, surface right to β1. (D and E) Cross-strand ladder residues on F_L (D) and F_R (E) are shown in spheres and classified into different types by color (purple, positively charged residues; red, negatively charged residues; orange, polar residues; green, hydrophobic residues).Although it appears that Sas-4 plays pivotal roles in centriole formation, assembling protein complexes in the cytoplasm, and tethering them to a developing centrosome, the mechanisms by which Sas-4 accomplishes its tethering role have remained unclear. In this study, we therefore investigated the structural basis of Sas-4 and show that via its conserved C-terminal TCP domain, it could provide an “extended surface-like” platform by which Sas-4 could mediate the Sas-4–PCM scaffold–centriole interaction during centrosome biogenesis.     

Acentriolar mitosis activates a p53-dependent apoptosis pathway in the mouse embryo

Centrosomes are the microtubule-organizing centers of animal cells that organize interphase microtubules and mitotic spindles. Centrioles are the microtubule-based structures that organize centrosomes, and a defined set of proteins, including spindle assembly defective-4 (SAS4) (CPAP/CENPJ), is required for centriole biogenesis. The biological functions of centrioles and centrosomes vary among animals, and the functions of mammalian centrosomes have not been genetically defined. Here we use a null mutation in mouse Sas4 to define the cellular and developmental functions of mammalian centrioles in vivo. Sas4-null embryos lack centrosomes but survive until midgestation. As expected, Sas4^−/− mutants lack primary cilia and therefore cannot respond to Hedgehog signals, but other developmental signaling pathways are normal in the mutants. Unlike mutants that lack cilia, Sas4^−/− embryos show widespread apoptosis associated with global elevated expression of p53. Cell death is rescued in Sas4^−/− p53^−/− double-mutant embryos, demonstrating that mammalian centrioles prevent activation of a p53-dependent apoptotic pathway. Expression of p53 is not activated by abnormalities in bipolar spindle organization, chromosome segregation, cell-cycle profile, or DNA damage response, which are normal in Sas4^−/− mutants. Instead, live imaging shows that the duration of prometaphase is prolonged in the mutants while two acentriolar spindle poles are assembled. Independent experiments show that prolonging spindle assembly is sufficient to trigger p53-dependent apoptosis. We conclude that a short delay in the prometaphase caused by the absence of centrioles activates a previously undescribed p53-dependent cell death pathway in the rapidly dividing cells of the mouse embryo.Centrioles are cylinders of triplet microtubules that provide the template for cilia and nucleate the centrosomes that act as microtubule organizing centers (MTOCs) at spindle poles and during interphase (1, 2). Genetic analysis has demonstrated that the biological roles of centrioles differ widely among organisms: Caenorhabditis elegans embryos without centrioles arrest at the two-cell stage, whereas zygotic removal of centrioles in Drosophila allows survival to adult stages (3–5). In humans, mutations in centriolar and centrosomal proteins are associated with microcephaly or microcephaly in the context of dwarfism (6–10). Abnormal numbers of centrioles are associated with cancer, although it is not clear whether abnormal centrosome number is a cause or an effect of tumorigenesis (1, 11–13). Studies in cultured cell lines have given conflicting results on the roles of vertebrate centrioles in mitosis, chromosome segregation, DNA damage response, and intercellular signaling (14–19), but the precise functions of mammalian centrioles have not been defined genetically.A small number of core proteins have been shown to be required for centriole biogenesis in organisms ranging from Chlamydomonas reinhardtii to human cells. Spindle assembly defective-4 (SAS4), one of these core proteins, acts at an early step in the assembly pathway, when it is required for the addition of tubulin subunits to the forming procentrioles; it also is required for recruitment of the pericentriolar material (PCM) to form the centrosome (3, 20, 21). Mutations in Sas4 block centriole formation in Drosophila and C. elegans, and mutations in human SAS4 (CPAP/CENPJ) cause Seckel syndrome (dwarfism with microcephaly) (3–6). siRNA knockdown of SAS4 in cultured mammalian cells was reported to cause formation of multipolar spindles (14).Here we use null mutations in Sas4 to define the cellular and developmental functions of centrioles in the mouse embryo. As expected, Sas4 is essential for formation of centrioles, centrosomes, and cilia and for cilia-dependent Hedgehog (Hh) signaling. Unexpectedly, Sas4^−/− embryos arrest at an earlier stage than mutants that lack cilia and show widespread cell death associated with strong up-regulation of p53 in most cells in the embryo. Genetic removal of p53 rescues both the cell death and the early lethality of Sas4^−/− mutants. Cell death in the mutants is not associated with defects in the cell-cycle profile, DNA damage response, or chromosome segregation. The data indicate that in Sas4^−/− mouse embryos prolonged prometaphase, caused by a delay in spindle pole assembly, triggers a previously uncharacterized checkpoint that activates p53-dependent apoptosis in vivo.     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Microtubule nucleation remote from centrosomes may explain how asters span large cells

A major challenge in cell biology is to understand how nanometer-sized molecules can organize micrometer-sized cells in space and time. One solution in many animal cells is a radial array of microtubules called an aster, which is nucleated by a central organizing center and spans the entire cytoplasm. Frog (here Xenopus laevis) embryos are more than 1 mm in diameter and divide with a defined geometry every 30 min. Like smaller cells, they are organized by asters, which grow, interact, and move to precisely position the cleavage planes. It has been unclear whether asters grow to fill the enormous egg by the same mechanism used in smaller somatic cells, or whether special mechanisms are required. We addressed this question by imaging growing asters in a cell-free system derived from eggs, where asters grew to hundreds of microns in diameter. By tracking marks on the lattice, we found that microtubules could slide outward, but this was not essential for rapid aster growth. Polymer treadmilling did not occur. By measuring the number and positions of microtubule ends over time, we found that most microtubules were nucleated away from the centrosome and that interphase egg cytoplasm supported spontaneous nucleation after a time lag. We propose that aster growth is initiated by centrosomes but that asters grow by propagating a wave of microtubule nucleation stimulated by the presence of preexisting microtubules.The large cells in early vertebrate embryos are organized by radial arrays of microtubules called asters. This general organization was described by early cytologists (1) but is clearly illustrated by modern fixed immunofluorescence or live imaging. At the end of mitosis, a pair of asters is observed at the spindle poles but remains small in radius, presumably because cyclin-dependent kinase 1 (Cdk1) inhibits aster growth (2). Once the cell enters interphase, the asters grow at rates of 30 µm/min in Xenopus zygotes and 15 µm/min in zebrafish, while maintaining a high density of microtubules at their periphery (2–4). Paired asters interact at the cell’s midplane to form a specialized zone of microtubule overlaps, which in turn recruit cytokinesis factors to the cell cortex (5, 6). Cell-spanning dimensions are presumably required so that the microtubules can touch the cortex to accurately position the cleavage furrow according to cell geometry (3, 7, 8).In the standard model of aster growth, microtubules are nucleated with their minus-ends anchored at the centrosome (9) and polymerize outward with plus-ends undergoing dynamic instability (10). However, there are several issues in applying this model to a very large cytoplasm (11). Because of the radial geometry, the standard model implies a decrease in microtubule density with increasing radius. In contrast, microtubule density seems to be constant or even increase toward the aster periphery in frog and fish zygotes (3). Furthermore, this radial elongation model predicts that a subset of microtubules spans the entire aster radius, but it is unknown whether such long microtubules exist. We wondered whether additional mechanisms promoted aster growth in large cells, such as microtubule sliding, treadmilling, or nucleation remote from centrosomes.Previously we developed a cell-free system to reconstitute cleavage furrow signaling where growing asters interacted (5, 12). Here, we combine cell-free reconstitution and quantitative imaging to identify microtubule nucleation away from the centrosome as the key biophysical mechanism underlying aster growth. We propose that aster growth in large cells should be understood as a spatial propagation of microtubule-stimulated microtubule nucleation.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.