Detachment Of Centrosomes Research Articles

Hybrid incompatibility emerges at the one-cell stage in interspecies Caenorhabditis embryos.

Intrinsic reproductive isolation occurs when genetic differences between populations disrupt the development of hybrid organisms, preventing gene flow and enforcing speciation. 1-4 While prior studies have examined the genetic origins of hybrid incompatibility, 5-18 the effects of incompatible factors on development remain poorly understood. Here, we investigate the mechanistic basis of hybrid incompatibility in Caenorhabditis nematodes by capitalizing on the ability of C. brenneri females to produce embryos after mating with males from several other species. Contrary to expectations, hybrid incompatibility was evident immediately after fertilization, suggesting that post-fertilization barriers to hybridization originate from physical incompatibility between sperm and oocyte-derived factors rather than from zygotic transcription, which starts after the 4-cell stage. 19-22 Sperm deliver chromatin, which expands to form a pronucleus, and a pair of centrioles, which form centrosomes that attach to the sperm-derived pronucleus and signal to establish the embryo's anterior-posterior axis. 23,24 In C. brenneri oocytes fertilized with C. elegans sperm, sperm pronuclear expansion was compromised, frequent centrosome detachment was observed, and cortical polarity was disrupted. Live imaging revealed that defective polar body extrusion contributes to defects in mitotic spindle morphology. C. brenneri oocytes fertilized with C. remanei or C. sp. 48 sperm showed similar defects, and their severity and frequency increased with phylogenetic distance. Defective expansion of the sperm-derived pronucleus and unreliable polar body extrusion immediately after fertilization generally underlie the inviability of hybrid embryos in this clade. These results indicate that physical mismatches between sperm and oocyte-derived structures may be a primary mechanism of hybrid incompatibility.

NuMA deficiency causes micronuclei via checkpoint-insensitive k-fiber minus-end detachment from mitotic spindle poles

Micronuclei resulting from improper chromosome segregation foster chromosome rearrangements.1,2 Toprevent micronuclei formation in mitosis, the dynamic plus ends of bundled kinetochore microtubules (k-fibers) must establish bipolar attachment with all sister kinetochores on chromosomes,3 whereas k-fiber minus ends must be clustered at the two opposing spindle poles, which are normally connected with centrosomes.4 The establishment of chromosome biorientation via k-fiber plus ends is carefully monitored by the spindle assembly checkpoint (SAC).5 However, how k-fiber minus-end clustering near centrosomes is maintained and monitored remains poorly understood. Here, we show that degradation of NuMA by auxin-inducible degron technologies results in micronuclei formation through k-fiber minus-end detachment from spindle poles during metaphase in HCT116 colon cancer cells. Importantly, k-fiber minus-end detachment from one pole creates misaligned chromosomes that maintain chromosome biorientation and satisfy the SAC, resulting in abnormal chromosome segregation. NuMA depletion also causes minus-end clustering defects in non-transformed Rpe1 cells, but it additionally induces centrosome detachment from partially focused poles, resulting in highly disorganized anaphase. Moreover, we find that NuMA depletion causes centrosome clustering defects in tetraploid-like cells, leading to an increased frequency of multipolar divisions. Together, our data indicate that NuMA is required for faithful chromosome segregation in human mitotic cells, generally by maintaining k-fiber minus-end clustering but also by promoting spindle pole-centrosome or centrosome-centrosome connection in specific cell types or contexts. Similar to erroneous merotelic kinetochore attachments,6 detachment of k-fiber minus ends from spindle poles evades spindle checkpoint surveillance and may therefore be a source of genomic instability in dividing cells.

Lymphocyte Polarization During Immune Synapse Assembly: Centrosomal Actin Joins the Game.

Interactions among immune cells are essential for the development of adaptive immune responses. The immunological synapse (IS) provides a specialized platform for integration of signals and intercellular communication between T lymphocytes and antigen presenting cells (APCs). In the T cell the reorganization of surface molecules at the synaptic interface is initiated by T cell receptor binding to a cognate peptide-major histocompatibility complex on the APC surface and is accompanied by a polarized remodelling of the cytoskeleton and centrosome reorientation to a subsynaptic position. Although there is a general agreement on polarizing signals and mechanisms driving centrosome reorientation during IS assembly, the primary events that prepare for centrosome repositioning remain largely unexplored. It has been recently shown that in resting lymphocytes a local polymerization of filamentous actin (F-actin) at the centrosome contributes to anchoring this organelle to the nucleus. During early stages of IS formation centrosomal F-actin undergoes depletion, allowing for centrosome detachment from the nucleus and its polarization towards the synaptic membrane. We recently demonstrated that in CD4+ T cells the reduction in centrosomal F-actin relies on the activity of a centrosome-associated proteasome and implicated the ciliopathy-related Bardet-Biedl syndrome 1 protein in the dynein-dependent recruitment of the proteasome 19S regulatory subunit to the centrosome. In this short review we will feature our recent findings that collectively provide a new function for BBS proteins and the proteasome in actin dynamics, centrosome polarization and T cell activation.

PP2A-B55/SUR-6 collaborates with the nuclear lamina for centrosome separation during mitotic entry.

Across most sexually reproducing animals, centrosomes are provided to the oocyte through fertilization and must be positioned properly to establish the zygotic mitotic spindle. How centrosomes are positioned in space and time through the concerted action of key mitotic entry biochemical regulators, including protein phosphatase 2A (PP2A-B55/SUR-6), biophysical regulators, including dynein, and the nuclear lamina is unclear. Here, we uncover a role for PP2A-B55/SUR-6 in regulating centrosome separation. Mechanistically, PP2A-B55/SUR-6 regulates nuclear size before mitotic entry, in turn affecting nuclear envelope–based dynein density and motor capacity. Computational simulations predicted the requirement of PP2A-B55/SUR-6 regulation of nuclear size and nuclear-envelope dynein density for proper centrosome separation. Conversely, compromising nuclear lamina integrity led to centrosome detachment from the nuclear envelope and migration defects. Removal of PP2A-B55/SUR-6 and the nuclear lamina simultaneously further disrupted centrosome separation, leading to unseparated centrosome pairs dissociated from the nuclear envelope. Taking these combined results into consideration, we propose a model in which centrosomes migrate and are positioned through the concerted action of PP2A-B55/SUR-6–regulated nuclear envelope–based dynein pulling forces and centrosome–nuclear envelope tethering. Our results add critical precision to models of centrosome separation relative to the nucleus during spindle formation in cell division.

Loss of function of the Drosophila Ninein-related centrosomal protein Bsg25D causes mitotic defects and impairs embryonic development.

ABSTRACTThe centrosome-associated proteins Ninein (Nin) and Ninein-like protein (Nlp) play significant roles in microtubule stability, nucleation and anchoring at the centrosome in mammalian cells. Here, we investigate Blastoderm specific gene 25D (Bsg25D), which encodes the only Drosophila protein that is closely related to Nin and Nlp. In early embryos, we find that Bsg25D mRNA and Bsg25D protein are closely associated with centrosomes and astral microtubules. We show that sequences within the coding region and 3′UTR of Bsg25D mRNAs are important for proper localization of this transcript in oogenesis and embryogenesis. Ectopic expression of eGFP-Bsg25D from an unlocalized mRNA disrupts microtubule polarity in mid-oogenesis and compromises the distribution of the axis polarity determinant Gurken. Using total internal reflection fluorescence microscopy, we show that an N-terminal fragment of Bsg25D can bind microtubules in vitro and can move along them, predominantly toward minus-ends. While flies homozygous for a Bsg25D null mutation are viable and fertile, 70% of embryos lacking maternal and zygotic Bsg25D do not hatch and exhibit chromosome segregation defects, as well as detachment of centrosomes from mitotic spindles. We conclude that Bsg25D is a centrosomal protein that, while dispensable for viability, nevertheless helps ensure the integrity of mitotic divisions in Drosophila.

A CEP215–HSET complex links centrosomes with spindle poles and drives centrosome clustering in cancer

Numerical centrosome aberrations underlie certain developmental abnormalities and may promote cancer. A cell maintains normal centrosome numbers by coupling centrosome duplication with segregation, which is achieved through sustained association of each centrosome with a mitotic spindle pole. Although the microcephaly- and primordial dwarfism-linked centrosomal protein CEP215 has been implicated in this process, the molecular mechanism responsible remains unclear. Here, using proteomic profiling, we identify the minus end-directed microtubule motor protein HSET as a direct binding partner of CEP215. Targeted deletion of the HSET-binding domain of CEP215 in vertebrate cells causes centrosome detachment and results in HSET depletion at centrosomes, a phenotype also observed in CEP215-deficient patient-derived cells. Moreover, in cancer cells with centrosome amplification, the CEP215–HSET complex promotes the clustering of extra centrosomes into pseudo-bipolar spindles, thereby ensuring viable cell division. Therefore, stabilization of the centrosome–spindle pole interface by the CEP215–HSET complex could promote survival of cancer cells containing supernumerary centrosomes.

An Asp–CaM complex is required for centrosome–pole cohesion and centrosome inheritance in neural stem cells

The interaction between centrosomes and mitotic spindle poles is important for efficient spindle formation, orientation, and cell polarity. However, our understanding of the dynamics of this relationship and implications for tissue homeostasis remains poorly understood. Here we report that Drosophila melanogaster calmodulin (CaM) regulates the ability of the microcephaly-associated protein, abnormal spindle (Asp), to cross-link spindle microtubules. Both proteins colocalize on spindles and move toward spindle poles, suggesting that they form a complex. Our binding and structure-function analysis support this hypothesis. Disruption of the Asp-CaM interaction alone leads to unfocused spindle poles and centrosome detachment. This behavior leads to randomly inherited centrosomes after neuroblast division. We further show that spindle polarity is maintained in neuroblasts despite centrosome detachment, with the poles remaining stably associated with the cell cortex. Finally, we provide evidence that CaM is required for Asp's spindle function; however, it is completely dispensable for Asp's role in microcephaly suppression.

CENP-32 is required to maintain centrosomal dominance in bipolar spindle assembly.

Centrosomes nucleate spindle formation, direct spindle pole positioning, and are important for proper chromosome segregation during mitosis in most animal cells. We previously reported that centromere protein 32 (CENP-32) is required for centrosome association with spindle poles during metaphase. In this study, we show that CENP-32 depletion seems to release centrosomes from bipolar spindles whose assembly they had previously initiated. Remarkably, the resulting anastral spindles function normally, aligning the chromosomes to a metaphase plate and entering anaphase without detectable interference from the free centrosomes, which appear to behave as free asters in these cells. The free asters, which contain reduced but significant levels of CDK5RAP2, show weak interactions with spindle microtubules but do not seem to make productive attachments to kinetochores. Thus CENP-32 appears to be required for centrosomes to integrate into a fully functional spindle that not only nucleates astral microtubules, but also is able to nucleate and bind to kinetochore and central spindle microtubules. Additional data suggest that NuMA tethers microtubules at the anastral spindle poles and that augmin is required for centrosome detachment after CENP-32 depletion, possibly due to an imbalance of forces within the spindle.

Drosophila Larp associates with poly(A)-binding protein and is required for male fertility and syncytial embryo development

As the influence of mRNA translation upon cell cycle regulation becomes clearer, we searched for genes that might specify such control in Drosophila. A maternal-effect lethal screen identified mutants in the Drosophila gene for Larp (La-related protein) which displayed maternal-effect lethality and male sterility. A role for La protein has already been implicated in mRNA translation whereas Larp has been proposed to regulate mRNA stability. Here we demonstrate that Larp exists in a physical complex with, and also interacts genetically with, the translation regulator poly(A)-binding protein (PABP). Most mutant alleles of pAbp are embryonic lethal. However hypomorphic pAbp alleles show similar meiotic defects to larp mutants. We find that larp mutant-derived syncytial embryos show a range of mitotic phenotypes, including failure of centrosomes to migrate around the nuclear envelope, detachment of centrosomes from spindle poles, the formation of multipolar spindle arrays and cytokinetic defects. We discuss why the syncytial mitotic cycles and male meiosis should have a particularly sensitive requirement for Larp proteins in regulating not only transcript stability but also potentially the translation of mRNAs.

Centrosome attachment to the C. elegans male pronucleus is dependent on the surface area of the nuclear envelope

A close association must be maintained between the male pronucleus and the centrosomes during pronuclear migration. In C. elegans, simultaneous depletion of inner nuclear membrane LEM proteins EMR-1 and LEM-2, depletion of the nuclear lamina proteins LMN-1 or BAF-1, or the depletion of nuclear import components leads to embryonic lethality with small pronuclei. Here, a novel centrosome detachment phenotype in C. elegans zygotes is described. Zygotes with defects in the nuclear envelope had small pronuclei with a single centrosome detached from the male pronucleus. ZYG-12, SUN-1, and LIS-1, which function at the nuclear envelope with dynein to attach centrosomes, were observed at normal concentrations on the nuclear envelope of pronuclei with detached centrosomes. Analysis of time-lapse images showed that as mutant pronuclei grew in surface area, they captured detached centrosomes. Larger tetraploid or smaller histone::mCherry pronuclei suppressed or enhanced the centrosome detachment phenotype respectively. In embryos fertilized with anucleated sperm, only one centrosome was captured by small female pronuclei, suggesting the mechanism of capture is dependent on the surface area of the outer nuclear membrane available to interact with aster microtubules. We propose that the limiting factor for centrosome attachment to the surface of abnormally small pronuclei is dynein.

Microcephalin coordinates mitosis in the syncytialDrosophilaembryo

Microcephalin (MCPH1) is mutated in primary microcephaly, an autosomal recessive human disorder of reduced brain size. It encodes a protein with three BRCT domains that has established roles in DNA damage signalling and the cell cycle, regulating chromosome condensation. Significant adaptive evolutionary changes in primate MCPH1 sequence suggest that changes in this gene could have contributed to the evolution of the human brain. To understand the developmental role of microcephalin we have studied its function in Drosophila. We report here that Drosophila MCPH1 is cyclically localised during the cell cycle, co-localising with DNA during interphase, but not with mitotic chromosomes. mcph1 mutant flies have a maternal effect lethal phenotype, due to mitotic arrest occurring in early syncytial cell cycles. Mitotic entry is slowed from the very first mitosis in such embryos, with prolonged prophase and metaphase stages; and frequent premature separation as well as detachment of centrosomes. As a consequence, centrosome and nuclear cycles become uncoordinated, resulting in arrested embryonic development. Phenotypic similarities with abnormal spindle (asp) and centrosomin (cnn) mutants (whose human orthologues are also mutated in primary microcephaly), suggest that further studies in the Drosophila embryo may establish a common developmental and cellular pathway underlying the human primary microcephaly phenotype.

Making Microtubules and Mitotic Spindles in Cells without Functional Centrosomes

Centrosomes are considered to be the major sites of microtubule nucleation in mitotic cells (reviewed in ), yet mitotic spindles can still form after laser ablation or disruption of centrosome function . Although kinetochores have been shown to nucleate microtubules, mechanisms for acentrosomal spindle formation remain unclear. Here, we performed live-cell microscopy of GFP-tubulin to examine spindle formation in Drosophila S2 cells after RNAi depletion of either gamma-tubulin, a microtubule nucleating protein, or centrosomin, a protein that recruits gamma-tubulin to the centrosome. In these RNAi-treated cells, we show that poorly focused bipolar spindles form through the self-organization of microtubules nucleated from chromosomes (a process involving gamma-tubulin), as well as from other potential sites, and through the incorporation of microtubules from the preceding interphase network. By tracking EB1-GFP (a microtubule-plus-end binding protein) in acentrosomal spindles, we also demonstrate that the spindle itself represents a source of new microtubule formation, as suggested by observations of numerous microtubule plus ends growing from acentrosomal poles toward the metaphase plate. We propose that the bipolar spindle propagates its own architecture by stimulating microtubule growth, thereby augmenting the well-described microtubule nucleation pathways that take place at centrosomes and chromosomes.

Spindle Pole Organization inDrosophilaS2 Cells by Dynein,Abnormal SpindleProtein (Asp), and KLP10A

Dynein is a critical mitotic motor whose inhibition causes defects in spindle pole organization and separation, chromosome congression or segregation, and anaphase spindle elongation, but results differ in different systems. We evaluated the functions of the dynein-dynactin complex by using RNA interference (RNAi)-mediated depletion of distinct subunits in Drosophila S2 cells. We observed a striking detachment of centrosomes from spindles, an increase in spindle length, and a loss of spindle pole focus. RNAi depletion of Ncd, another minus-end motor, produced disorganized spindles consisting of multiple disconnected mini-spindles, a different phenotype consistent with distinct pathways of spindle pole organization. Two candidate dynein-dependent spindle pole organizers also were investigated. RNAi depletion of the abnormal spindle protein, Asp, which localizes to focused poles of control spindles, produced a severe loss of spindle pole focus, whereas depletion of the pole-associated microtubule depolymerase KLP10A increased spindle microtubule density. Depletion of either protein produced long spindles. After RNAi depletion of dynein-dynactin, we observed subtle but significant mislocalization of KLP10A and Asp, suggesting that dynein-dynactin, Asp, and KLP10A have complex interdependent functions in spindle pole focusing and centrosome attachment. These results extend recent findings from Xenopus extracts to Drosophila cultured cells and suggest that common pathways contribute to spindle pole organization and length determination.