Absence Of Centrosomes Research Articles

Spindle assembly checkpoint-dependent mitotic delay is required for cell division in absence of centrosomes.

The spindle assembly checkpoint (SAC) temporally regulates mitosis by preventing progression from metaphase to anaphase until all chromosomes are correctly attached to the mitotic spindle. Centrosomes refine the spatial organization of the mitotic spindle at the spindle poles. However, centrosome loss leads to elongated mitosis, suggesting that centrosomes also inform the temporal organization of mitosis in mammalian cells. Here, we find that the mitotic delay in acentrosomal cells is enforced by the SAC in a MPS1-dependent manner, and that a SAC-dependent mitotic delay is required for bipolar cell division to occur in acentrosomal cells. Although acentrosomal cells become polyploid, polyploidy is not sufficient to cause dependency on a SAC-mediated delay to complete cell division. Rather, the division failure in absence of MPS1 activity results from mitotic exit occurring before acentrosomal spindles can become bipolar. Furthermore, prevention of centrosome separation suffices to make cell division reliant on a SAC-dependent mitotic delay. Thus, centrosomes and their definition of two spindle poles early in mitosis provide a 'timely two-ness' that allows cell division to occur in absence of a SAC-dependent mitotic delay.

A Brief History of Drosophila (Female) Meiosis.

Drosophila has been a model system for meiosis since the discovery of nondisjunction. Subsequent studies have determined that crossing over is required for chromosome segregation, and identified proteins required for the pairing of chromosomes, initiating meiotic recombination, producing crossover events, and building a spindle to segregate the chromosomes. With a variety of genetic and cytological tools, Drosophila remains a model organism for the study of meiosis. This review focusses on meiosis in females because in male meiosis, the use of chiasmata to link homologous chromosomes has been replaced by a recombination-independent mechanism. Drosophila oocytes are also a good model for mammalian meiosis because of biological similarities such as long pauses between meiotic stages and the absence of centrosomes during the meiotic divisions.

TFEB- and TFE3-dependent autophagy activation supports cancer proliferation in the absence of centrosomes

ABSTRACT Centrosome amplification is a phenomenon frequently observed in human cancers, so centrosome depletion has been proposed as a therapeutic strategy. However, despite being afflicted with a lack of centrosomes, many cancer cells can still proliferate, implying there are impediments to adopting centrosome depletion as a treatment strategy. Here, we show that TFEB- and TFE3-dependent autophagy activation contributes to acentrosomal cancer proliferation. Our biochemical analyses uncover that both TFEB and TFE3 are novel PLK4 (polo like kinase 4) substrates. Centrosome depletion inactivates PLK4, resulting in TFEB and TFE3 dephosphorylation and subsequent promotion of TFEB and TFE3 nuclear translocation and transcriptional activation of autophagy- and lysosome-related genes. A combination of centrosome depletion and inhibition of the TFEB-TFE3 autophagy-lysosome pathway induced strongly anti-proliferative effects in cancer cells. Thus, our findings point to a new strategy for combating cancer. Abbreviations: AdCre: adenoviral Cre recombinase; AdLuc: adenoviral luciferase; ATG5: autophagy related 5; CQ: chloroquine; DAPI: 4’,6-diamidino-2-phenylindole; DKO: double knockout; GFP: green fluorescent protein; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; LTR: LysoTracker Red; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MITF: melanocyte inducing transcription factor; PLK4: polo like kinase 4; RFP: red fluorescent protein; SASS6: SAS-6 centriolar assembly protein; STIL: STIL centriolar assembly protein; TFEB: transcription factor EB; TFEBΔNLS: TFEB lacking a nuclear localization signal; TFE3: transcription factor binding to IGHM enhancer 3; TP53/p53: tumor protein p53

Multiple motors cooperate to establish and maintain acentrosomal spindle bipolarity in C. elegans oocyte meiosis.

While centrosomes organize spindle poles during mitosis, oocyte meiosis can occur in their absence. Spindles in human oocytes frequently fail to maintain bipolarity and consequently undergo chromosome segregation errors, making it important to understand the mechanisms that promote acentrosomal spindle stability. To this end, we have optimized the auxin-inducible degron system in Caenorhabditis elegans to remove the factors from pre-formed oocyte spindles within minutes and assess the effects on spindle structure. This approach revealed that dynein is required to maintain the integrity of acentrosomal poles; removal of dynein from bipolar spindles caused pole splaying, and when coupled with a monopolar spindle induced by depletion of the kinesin-12 motor KLP-18, dynein depletion led to a complete dissolution of the monopole. Surprisingly, we went on to discover that following monopole disruption, individual chromosomes were able to reorganize local microtubules and re-establish a miniature bipolar spindle that mediated chromosome segregation. This revealed the existence of redundant microtubule sorting forces that are undetectable when KLP-18 and dynein are active. We found that the kinesin-5 family motor BMK-1 provides this force, uncovering the first evidence that kinesin-5 contributes to C. elegans meiotic spindle organization. Altogether, our studies have revealed how multiple motors are working synchronously to establish and maintain bipolarity in the absence of centrosomes.

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.

Wwc2 Is a Novel Cell Division Regulator During Preimplantation Mouse Embryo Lineage Formation and Oogenesis.

Formation of the hatching mouse blastocyst marks the end of preimplantation development, whereby previous cell cleavages culminate in the formation of three distinct cell lineages (trophectoderm, primitive endoderm and epiblast). We report that dysregulated expression of Wwc2, a genetic paralog of Kibra/Wwc1 (a known activator of Hippo-signaling, a key pathway during preimplantation development), is specifically associated with cell autonomous deficits in embryo cell number and cell division abnormalities. Division phenotypes are also observed during mouse oocyte meiotic maturation, as Wwc2 dysregulation blocks progression to the stage of meiosis II metaphase (MII) arrest and is associated with spindle defects and failed Aurora-A kinase (AURKA) activation. Oocyte and embryo cell division defects, each occurring in the absence of centrosomes, are fully reversible by expression of recombinant HA-epitope tagged WWC2, restoring activated oocyte AURKA levels. Additionally, clonal embryonic dysregulation implicates Wwc2 in maintaining the pluripotent epiblast lineage. Thus, Wwc2 is a novel regulator of meiotic and early mitotic cell divisions, and mouse blastocyst cell fate.

Targeting TRIM37-driven centrosome dysfunction in 17q23-amplified breast cancer

Genomic instability is a hallmark of cancer, and has a central role in the initiation and development of breast cancer1,2. The success of poly-ADP ribose polymerase inhibitors in the treatment of breast cancers that are deficient in homologous recombination exemplifies the utility of synthetically lethal genetic interactions in the treatment of breast cancers that are driven by genomic instability3. Given that defects in homologous recombination are present in only a subset of breast cancers, there is a need to identify additional driver mechanisms for genomic instability and targeted strategies to exploit these defects in the treatment of cancer. Here we show that centrosome depletion induces synthetic lethality in cancer cells that contain the 17q23 amplicon, a recurrent copy number aberration that defines about 9% of all primary breast cancer tumours and is associated with high levels of genomic instability4-6. Specifically, inhibition of polo-like kinase4 (PLK4) using small molecules leads to centrosome depletion, which triggers mitotic catastrophe in cells that exhibit amplicon-directed overexpression of TRIM37. To explain this effect, we identify TRIM37 as a negative regulator of centrosomal pericentriolar material. In 17q23-amplified cells that lack centrosomes, increased levels of TRIM37 block the formation of foci that comprise pericentriolar material-these foci are structures with a microtubule-nucleating capacity that are required for successful cell division in the absence of centrosomes. Finally, we find thatthe overexpression of TRIM37 causes genomic instability by delaying centrosome maturation and separation at mitotic entry, and thereby increases the frequency of mitotic errors. Collectively, these findings highlight TRIM37-dependent genomic instability as a putative driver event in 17q23-amplified breast cancer and provide a rationale for the use of centrosome-targeting therapeutic agents in treating these cancers.

Cell Cycle-Dependent Dynamics of the Golgi-Centrosome Association in Motile Cells.

Here, we characterize spatial distribution of the Golgi complex in human cells. In contrast to the prevailing view that the Golgi compactly surrounds the centrosome throughout interphase, we observe characteristic differences in the morphology of Golgi ribbons and their association with the centrosome during various periods of the cell cycle. The compact Golgi complex is typical in G1; during S-phase, Golgi ribbons lose their association with the centrosome and extend along the nuclear envelope to largely encircle the nucleus in G2. Interestingly, pre-mitotic separation of duplicated centrosomes always occurs after dissociation from the Golgi. Shortly before the nuclear envelope breakdown, scattered Golgi ribbons reassociate with the separated centrosomes restoring two compact Golgi complexes. Transitions between the compact and distributed Golgi morphologies are microtubule-dependent. However, they occur even in the absence of centrosomes, which implies that Golgi reorganization is not driven by the centrosomal microtubule asters. Cells with different Golgi morphology exhibit distinct differences in the directional persistence and velocity of migration. These data suggest that changes in the radial distribution of the Golgi around the nucleus define the extent of cell polarization and regulate cell motility in a cell cycle-dependent manner.

Microtubule assembly and pole coalescence: early steps in Caenorhabditiselegans oocyte meiosis I spindle assembly.

ABSTRACTHow oocytes assemble bipolar meiotic spindles in the absence of centrosomes as microtubule organizing centers remains poorly understood. We have used live cell imaging in Caenorhabditis elegans to investigate requirements for the nuclear lamina and for conserved regulators of microtubule dynamics during oocyte meiosis I spindle assembly, assessing these requirements with respect to recently identified spindle assembly steps. We show that the nuclear lamina is required for microtubule bundles to form a peripheral cage-like structure that appears shortly after oocyte nuclear envelope breakdown and surrounds the oocyte chromosomes, although bipolar spindles still assembled in its absence. Although two conserved regulators of microtubule nucleation, RAN-1 and γ-tubulin, are not required for bipolar spindle assembly, both contribute to normal levels of spindle-associated microtubules and spindle assembly dynamics. Finally, the XMAP215 ortholog ZYG-9 and the nearly identical minus-end directed kinesins KLP-15/16 are required for proper assembly of the early cage-like structure of microtubule bundles, and for early spindle pole foci to coalesce into a bipolar structure. Our results provide a framework for assigning molecular mechanisms to recently described steps in C. elegans oocyte meiosis I spindle assembly.

Spindle assembly without spindle pole body insertion into the nuclear envelope in fission yeast meiosis.

Centrosomes represent the major microtubule organizing center (MTOC) in eukaryotic cells and are responsible for nucleation of the spindle, the vehicle of chromosome segregation. In human female meiosis, however, spindle assembly occurs in the absence of centrosomes or other MTOCs and microtubules are nucleated around chromosomes. In yeast, spindle formation in mitosis and meiosis depends on the activity of spindle pole bodies (SPBs), the functional equivalents of centrosomes; thus, SPBs and centrosomes use similar machineries to assemble spindles. Here, we develop a system to explore the molecular mechanisms supporting acentrosomal spindle formation using fission yeast meiosis as a model scenario. We achieve this situation by removing access of the SPBs to the nucleus after their duplication. Under these conditions, we observe self-assembly-based spindle formation in the nuclear environment, conferring an ability to segregate chromosomes independently of the SPBs. Our results open the possibility to utilize the experimental advantages of fission yeast for insights into the molecular basis of acentrosomal spindle formation in meiosis.

Assembly and Positioning of the Oocyte Meiotic Spindle.

Fertilizable eggs develop from diploid precursor cells termed oocytes. Once every menstrual cycle, an oocyte matures into a fertilizable egg in the ovary. To this end, the oocyte eliminates half of its chromosomes into a small cell termed a polar body. The egg is then released into the Fallopian tube, where it can be fertilized. Upon fertilization, the egg completes the second meiotic division, and the mitotic division of the embryo starts. This review highlights recent work that has shed light on the cytoskeletal structures that drive the meiotic divisions of the oocyte in mammals. In particular, we focus on how mammalian oocytes assemble a microtubule spindle in the absence of centrosomes, how they position the spindle in preparation for polar body extrusion, and how the spindle segregates the chromosomes. We primarily focus on mouse oocytes as a model system but also highlight recent insights from human oocytes.

Proteomic Profiling of Microtubule Self-organization in M-phase

Microtubules (MTs) and associated proteins can self-organize into complex structures such as the bipolar spindle, a process in which RanGTP plays a major role. Addition of RanGTP to M-phase Xenopus egg extracts promotes the nucleation and self-organization of MTs into asters and bipolar-like structures in the absence of centrosomes or chromosomes. We show here that the complex proteome of these RanGTP-induced MT assemblies is similar to that of mitotic spindles. Using proteomic profiling we show that MT self-organization in the M-phase cytoplasm involves the non-linear and non-stoichiometric recruitment of proteins from specific functional groups. Our study provides for the first time a temporal understanding of the protein dynamics driving MT self-organization in M-phase.

A Switch in Microtubule Orientation during C. elegans Meiosis

In oocytes of many organisms, meiotic spindles form in the absence of centrosomes [1-5]. Such female meiotic spindles have a pointed appearance in metaphase with microtubules focused at acentrosomal spindle poles. At anaphase, the microtubules of acentrosomal spindles then transition to an inter-chromosomal array, while the spindle poles disappear. This transition is currently not understood. Previous studies have focused on this inter-chromosomal microtubule array and proposed a pushing model to drive chromosome segregation [6, 7]. This model includes an end-on orientation of microtubules with chromosomes. Alternatively, chromosomes were thought to associate along bundles of microtubules [8, 9]. Starting with metaphase, this second model proposed a pure lateral chromosome-to-microtubule association up to the final meiotic stages of anaphase. Here, we applied large-scale electron tomography [10] of staged C.elegans oocytes in meiosis to analyze the orientation of microtubules in respect to chromosomes. We show that microtubules at metaphase I are primarily oriented laterally to the chromosomes and that microtubules switch to an end-on orientation during progression through anaphase. We further show that this switch in microtubule orientation involves a kinesin-13 microtubule depolymerase, KLP-7, which removes laterally associated microtubules around chromosomes. From this, we conclude that both lateral and end-on modes of microtubule-to-chromosome orientations are successively used in C.elegans oocytes to segregate meiotic chromosomes.

A novel microtubule nucleation pathway for meiotic spindle assembly in oocytes.

The meiotic spindle in oocytes is assembled in the absence of centrosomes, the major microtubule nucleation sites in mitotic and male meiotic cells. A crucial, yet unresolved question in meiosis is how spindle microtubules are generated without centrosomes and only around chromosomes in the exceptionally large volume of oocytes. Here we report a novel oocyte-specific microtubule nucleation pathway that is essential for assembling most spindle microtubules complementarily with the Augmin pathway. This pathway is mediated by the kinesin-6 Subito/MKlp2, which recruits the γ-tubulin complex to the spindle equator to nucleate microtubules in Drosophila oocytes. Away from chromosomes, Subito interaction with the γ-tubulin complex is suppressed by its N-terminal region to prevent ectopic microtubule assembly in oocytes. We further demonstrate in vitro that the Subito complex from ovaries can nucleate microtubules from pure tubulin dimers. Collectively, microtubule nucleation regulated by Subito drives spatially restricted spindle assembly in oocytes.

Kinesin 6 Regulation in Drosophila Female Meiosis by the Non-conserved N- and C- Terminal Domains.

Bipolar spindle assembly occurs in the absence of centrosomes in the oocytes of most organisms. In the absence of centrosomes in Drosophila oocytes, we have proposed that the kinesin 6 Subito, a MKLP-2 homolog, is required for establishing spindle bipolarity and chromosome biorientation by assembling a robust central spindle during prometaphase I. Although the functions of the conserved motor domains of kinesins is well studied, less is known about the contribution of the poorly conserved N- and C- terminal domains to motor function. In this study, we have investigated the contribution of these domains to kinesin 6 functions in meiosis and early embryonic development. We found that the N-terminal domain has antagonistic elements that regulate localization of the motor to microtubules. Other parts of the N- and C-terminal domains are not required for microtubule localization but are required for motor function. Some of these elements of Subito are more important for either mitosis or meiosis, as revealed by separation-of-function mutants. One of the functions for both the N- and C-terminals domains is to restrict the CPC to the central spindle in a ring around the chromosomes. We also provide evidence that CDK1 phosphorylation of Subito regulates its activity associated with homolog bi-orientation. These results suggest the N- and C-terminal domains of Subito, while not required for localization to the central spindle microtubules, have important roles regulating Subito, by interacting with other spindle proteins and promoting activities such as bipolar spindle formation and homologous chromosome bi-orientation during meiosis.

Recent advances in understanding oogenesis: interactions with the cytoskeleton, microtubule organization, and meiotic spindle assembly in oocytes.

Maternal control of development begins with production of the oocyte during oogenesis. All of the factors necessary to complete oocyte maturation, meiosis, fertilization, and early development are produced in the transcriptionally active early oocyte. Active transcription of the maternal genome is a mechanism to ensure that the oocyte and development of the early embryo begin with all of the factors needed for successful embryonic development. To achieve the maximum maternal store, only one functional cell is produced from the meiotic divisions that produce the oocyte. The oocyte receives the bulk of the maternal cytoplasm and thus is significantly larger than its sister cells, the tiny polar bodies, which receive a copy of the maternal genome but essentially none of the maternal cytoplasm. This asymmetric division is accomplished by an enormous cell that is depleted of centrosomes in early oogenesis; thus, meiotic divisions in oocytes are distinct from those of mitotic cells. Therefore, these cells must partition the chromosomes faithfully to ensure euploidy by using mechanisms that do not rely on a conventional centrosome-based mitotic spindle. Several mechanisms that contribute to assembly and maintenance of the meiotic spindle in oocytes have been identified; however, none is fully understood. In recent years, there have been many exciting and significant advances in oogenesis, contributed by studies using a myriad of systems. Regrettably, I cannot adequately cover all of the important advances here and so I apologize to those whose beautiful work has not been included. This review focuses on a few of the most recent studies, conducted by several groups, using invertebrate and vertebrate systems, that have provided mechanistic insight into how microtubule assembly and meiotic spindle morphogenesis are controlled in the absence of centrosomes.

Big Lessons from Little Yeast: Budding and Fission Yeast Centrosome Structure, Duplication, and Function.

Centrosomes are a functionally conserved feature of eukaryotic cells that play an important role in cell division. The conserved γ-tubulin complex organizes spindle and astral microtubules, which, in turn, separate replicated chromosomes accurately into daughter cells. Like DNA, centrosomes are duplicated once each cell cycle. Although in some cell types it is possible for cell division to occur in the absence of centrosomes, these divisions typically result in defects in chromosome number and stability. In single-celled organisms such as fungi, centrosomes [known as spindle pole bodies (SPBs)] are essential for cell division. SPBs also must be inserted into the membrane because fungi undergo a closed mitosis in which the nuclear envelope (NE) remains intact. This poorly understood process involves events similar or identical to those needed for de novo nuclear pore complex assembly. Here, we review how analysis of fungal SPBs has advanced our understanding of centrosomes and NE events.

The Centrioles, Centrosomes, Basal Bodies, and Cilia of Drosophila melanogaster.

Centrioles play a key role in the development of the fly. They are needed for the correct formation of centrosomes, the organelles at the poles of the spindle that can persist as microtubule organizing centers (MTOCs) into interphase. The ability to nucleate cytoplasmic microtubules (MTs) is a property of the surrounding pericentriolar material (PCM). The centriole has a dual life, existing not only as the core of the centrosome but also as the basal body, the structure that templates the formation of cilia and flagellae. Thus the structure and functions of the centriole, the centrosome, and the basal body have an impact upon many aspects of development and physiology that can readily be modeled in Drosophila. Centrosomes are essential to give organization to the rapidly increasing numbers of nuclei in the syncytial embryo and for the spatially precise execution of cell division in numerous tissues, particularly during male meiosis. Although mitotic cell cycles can take place in the absence of centrosomes, this is an error-prone process that opens up the fly to developmental defects and the potential of tumor formation. Here, we review the structure and functions of the centriole, the centrosome, and the basal body in different tissues and cultured cells of Drosophila melanogaster, highlighting their contributions to different aspects of development and cell division.

The chromosomal basis of meiotic acentrosomal spindle assembly and function in oocytes.

Several aspects of meiosis are impacted by the absence of centrosomes in oocytes. Here, we review four aspects of meiosis I that are significantly affected by the absence of centrosomes in oocyte spindles. One, microtubules tend to assemble around the chromosomes. Two, the organization of these microtubules into a bipolar spindle is directed by the chromosomes. Three, chromosome bi-orientation and attachment to microtubules from the correct pole require modification of the mechanisms used in mitotic cells. Four, chromosome movement to the poles at anaphase cannot rely on polar anchoring of spindle microtubules by centrosomes. Overall, the chromosomes are more active participants during acentrosomal spindle assembly in oocytes, compared to mitotic and male meiotic divisions where centrosomes are present. The chromosomes are endowed with information that can direct the meiotic divisions and dictate their own behavior in oocytes. Processes beyond those known from mitosis appear to be required for their bi-orientation at meiosis I. As mitosis occurs without centrosomes in many systems other than oocytes, including all plants, the concepts discussed here may not be limited to oocytes. The study of meiosis in oocytes has revealed mechanisms that are operating in mitosis and will probably continue to do so.

Molecular Pathway of Microtubule Organization at the Golgi Apparatus

The Golgi apparatus controls the formation of non-centrosomal microtubule arrays important for Golgi organization, polarized transport, cell motility, and cell differentiation. Here, we show that CAMSAP2 stabilizes and attaches microtubule minus ends to the Golgi through a complex of AKAP450 and myomegalin. CLASPs stabilize CAMSAP2-decorated microtubules but are not required for their Golgi tethering. AKAP450 is also essential for Golgi microtubule nucleation, and myomegalin and CDK5RAP2 but not CAMSAP2 contribute to this function. In the absence of centrosomes, AKAP450- and CAMSAP2-dependent pathways of microtubule minus-end organization become dominant, and the presence of at least one of them is needed to maintain microtubule density. Strikingly, a compact Golgi can be assembled in the absence of both centrosomal and Golgi microtubules. However, CAMSAP2- and AKAP450-dependent Golgi microtubules facilitate Golgi reorientation and cell invasion in a 3D matrix. We propose that Golgi-anchored microtubules are important for polarized cell movement but not for coalescence of Golgi membranes.