RESUMO
BACKGROUND: Prostate cancer (PrCa) is the most frequently diagnosed cancer in men. Variants in known moderate- to high-penetrance genes explain less than 5% of the cases arising at early-onset (< 56 years) and/or with familial aggregation of the disease. Considering that BubR1 is an essential component of the mitotic spindle assembly checkpoint, we hypothesized that monoallelic BUB1B variants could be sufficient to fuel chromosomal instability (CIN), potentially triggering (prostate) carcinogenesis. METHODS: To unveil BUB1B as a new PrCa predisposing gene, we performed targeted next-generation sequencing in germline DNA from 462 early-onset/familial PrCa patients and 1,416 cancer patients fulfilling criteria for genetic testing for other hereditary cancer syndromes. To explore the pan-cancer role of BUB1B, we used in silico BubR1 molecular modeling, in vitro gene-editing, and ex vivo patients' tumors and peripheral blood lymphocytes. RESULTS: Rare BUB1B variants were found in ~ 1.9% of the early-onset/familial PrCa cases and in ~ 0.6% of other cancer patients fulfilling criteria for hereditary disease. We further show that BUB1B variants lead to decreased BubR1 expression and/or stability, which promotes increased premature chromatid separation and, consequently, triggers CIN, driving resistance to Taxol-based therapies. CONCLUSIONS: Our study shows that different BUB1B variants may uncover a trigger for CIN-driven carcinogenesis, supporting the role of BUB1B as a (pan)-cancer predisposing gene with potential impact on genetic counseling and treatment decision-making.
Assuntos
Instabilidade Cromossômica , Predisposição Genética para Doença , Neoplasias da Próstata , Proteínas Serina-Treonina Quinases , Humanos , Masculino , Neoplasias da Próstata/genética , Proteínas Serina-Treonina Quinases/genética , Pessoa de Meia-Idade , Mutação em Linhagem Germinativa , Adulto , Proteínas de Ciclo CelularRESUMO
Enduring chromosome segregation errors represent potential threats to genomic stability due to eventual chromosome copy number alterations (aneuploidy) and formation of micronuclei-key intermediates of a rapid mutational process known as chromothripsis that is found in cancer and congenital disorders. The spindle assembly checkpoint (SAC) has been viewed as the sole surveillance mechanism that prevents chromosome segregation errors during mitosis and meiosis. However, different types of chromosome segregation errors stemming from incorrect kinetochore-microtubule attachments satisfy the SAC and are more frequent than previously anticipated. Remarkably, recent works have unveiled that most of these errors are corrected during anaphase and only rarely result in aneuploidy or formation of micronuclei. Here, we discuss recent progress in our understanding of the origin and fate of chromosome segregation errors that satisfy the SAC and shed light on the surveillance, correction, and clearance mechanisms that prevent their transmission, to preserve genomic stability.
Assuntos
Aneuploidia , Segregação de Cromossomos , Instabilidade Genômica , Humanos , Anáfase , Cinetocoros , Microtúbulos , Mitose , Fuso AcromáticoRESUMO
DNA damage response (DDR) during interphase involves active signaling and repair to ensure genomic stability. However, how mitotic cells respond to DNA damage remains poorly understood. Supported by correlative live-/fixed-cell microscopy, it was found that mitotic cells exposed to several cancer chemotherapy compounds acquire and signal DNA damage, regardless of how they interact with DNA. In-depth analysis upon DNA damage during mitosis revealed a spindle assembly checkpoint (SAC)-dependent, but ataxia telangiectasia mutated-independent, mitotic delay. This delay was due to the presence of misaligned chromosomes that ultimately satisfy the SAC and missegregate, leading to micronuclei formation. Mechanistically, it is shown that mitotic DNA damage causes missegregation of polar chromosomes due to the action of arm-ejection forces by chromokinesins. Importantly, with the exception of DNA damage induced by etoposide-a topoisomerase II inhibitor-this outcome was independent of a general effect on kinetochore microtubule stability. Colony formation assays in pan-cancer cell line models revealed that mitotic DNA damage causes distinct cytotoxic effects, depending on the nature and extent of the damage. Overall, these findings unveil and raise awareness that therapeutic DNA damage regimens may contribute to genomic instability through a surprising link with chromokinesin-mediated missegregation of polar chromosomes in cancer cells.
Assuntos
Neoplasias , Proteínas Nucleares , Proteínas Nucleares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dano ao DNA , Cromossomos/metabolismo , Neoplasias/genéticaRESUMO
CLASPs (cytoplasmic linker-associated proteins) are ubiquitous stabilizers of microtubule dynamics, but their molecular targets at the microtubule plus-end are not understood. Using DNA origami-based reconstructions, we show that clusters of human CLASP2 form a load-bearing bond with terminal non-GTP tubulins at the stabilized microtubule tip. This activity relies on the unconventional TOG2 domain of CLASP2, which releases its high-affinity bond with non-GTP dimers upon their conversion into polymerization-competent GTP-tubulins. The ability of CLASP2 to recognize nucleotide-specific tubulin conformation and stabilize the catastrophe-promoting non-GTP tubulins intertwines with the previously underappreciated exchange between GDP and GTP at terminal tubulins. We propose that TOG2-dependent stabilization of sporadically occurring non-GTP tubulins represents a distinct molecular mechanism to suppress catastrophe at the freely assembling microtubule ends and to promote persistent tubulin assembly at the load-bearing tethered ends, such as at the kinetochores in dividing cells.
Assuntos
Proteínas Associadas aos Microtúbulos , Tubulina (Proteína) , Humanos , Tubulina (Proteína)/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Nucleotídeos/metabolismo , Microtúbulos/metabolismo , Polímeros/metabolismoRESUMO
Here, we take advantage of the low chromosome number (2N=6) and distinctively large kinetochores of female Indian muntjac cells to investigate the molecular mechanism underlying k-fiber maturation. We describe steps for monitoring kinetochore-microtubule dynamics over time. Specifically, we detail the combination of live-cell super-resolution CH-STED microscopy of microtubule growth events within individual k-fibers and a laser-mediated k-fiber injury/repair assay. These tools provide a direct assessment of microtubule amplification mechanisms within k-fibers in metazoans. For complete details on the use and execution of this protocol, please refer to Almeida et al. (2022).1.
Assuntos
Cinetocoros , Cervo Muntjac , Animais , Feminino , Microtúbulos , MicroscopiaRESUMO
α/ß-Tubulin posttranslational modifications (PTMs) generate microtubule diversity, but whether they account for cancer cell resistance to microtubule-targeting drugs remains unknown. Here, we performed a pilot dissection of the "cancer tubulin code" using the NCI-60 cancer cell panel. We found that acetylated, detyrosinated, and ∆2-α-tubulin that typically accumulate on stable microtubules were uncoupled in many cancer cells. Acetylated α-tubulin did not affect microtubule dynamics, whereas its levels correlated with, but were not required for, taxol-induced cytotoxicity. In contrast, experimental increase of α-tubulin detyrosination, and/or depletion of the detyrosination-sensitive microtubule-depolymerizing enzyme MCAK, enhanced taxol-induced cytotoxicity by promoting cell death in mitosis and the subsequent interphase, without causing a cumulative effect. Interestingly, only increased detyrosinated α-tubulin aggravated taxol-induced spindle multipolarity. Overall, we identified high α-tubulin acetylation as a potential biomarker for cancer cell response to taxol and uncovered a mechanistic link between α-tubulin detyrosination and the suppression of MCAK activity in taxol-induced cytotoxicity, likely by promoting chromosome missegregation, regardless of spindle defects.
Assuntos
Cinesinas , Microtúbulos , Paclitaxel , Tubulina (Proteína) , Mitose , Paclitaxel/farmacologia , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/metabolismo , Humanos , Linhagem Celular Tumoral , Cinesinas/metabolismoRESUMO
The synaptonemal complex (SC) is a proteinaceous scaffold that is assembled between paired homologous chromosomes during the onset of meiosis. Timely expression of SC coding genes is essential for SC assembly and successful meiosis. However, SC components have an intrinsic tendency to self-organize into abnormal repetitive structures, which are not assembled between the paired homologs and whose formation is potentially deleterious for meiosis and gametogenesis. This creates an interesting conundrum, where SC genes need to be robustly expressed during meiosis, but their expression must be carefully regulated to prevent the formation of anomalous SC structures. In this manuscript, we show that the Polycomb group protein Sfmbt, the Drosophila ortholog of human MBTD1 and L3MBTL2, is required to avoid excessive expression of SC genes during prophase I. Although SC assembly is normal after Sfmbt depletion, SC disassembly is abnormal with the formation of multiple synaptonemal complexes (polycomplexes) within the oocyte. Overexpression of the SC gene corona and depletion of other Polycomb group proteins are similarly associated with polycomplex formation during SC disassembly. These polycomplexes are highly dynamic and have a well-defined periodic structure. Further confirming the importance of Sfmbt, germ line depletion of this protein is associated with significant metaphase I defects and a reduction in female fertility. Since transcription of SC genes mostly occurs during early prophase I, our results suggest a role of Sfmbt and other Polycomb group proteins in downregulating the expression of these and other early prophase I genes during later stages of meiosis.
Assuntos
Meiose , Complexo Sinaptonêmico , Proteínas Cromossômicas não Histona/genética , Pareamento Cromossômico , Feminino , Humanos , Prófase Meiótica I , Proteínas do Grupo Polycomb/genética , Complexo Sinaptonêmico/genéticaRESUMO
As cells prepare to divide, they must ensure that enough space is available to assemble the mitotic machinery without perturbing tissue homeostasis. To do so, cells undergo a series of biochemical reactions regulated by cyclin B1-CDK1 that trigger cytoskeletal reorganization and ensure the coordination of cytoplasmic and nuclear events. Along with the biochemical events that control mitotic entry, mechanical forces have recently emerged as important players in cell-cycle regulation. However, the exact link between mechanical forces and the biochemical pathways that control mitotic progression remains unknown. Here, we identify a tension-dependent signal on the nucleus that sets the time for nuclear envelope permeabilization (NEP) and mitotic entry. This signal relies on actomyosin contractility, which unfolds the nucleus during the G2-M transition, activating the stretch-sensitive cPLA2 on the nuclear envelope and regulating the nuclear translocation of cyclin B1. Our data demonstrate how nuclear tension during the G2-M transition contributes to timely and efficient mitotic spindle assembly and prevents chromosomal instability.
Assuntos
Transporte Ativo do Núcleo Celular , Ciclina B1 , Mitose , Actomiosina/metabolismo , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Núcleo Celular/metabolismo , Instabilidade Cromossômica , Ciclina B1/genética , Ciclina B1/metabolismo , Membrana Nuclear/metabolismo , Fosfolipases A2 Citosólicas/metabolismo , Fuso Acromático/metabolismoRESUMO
Chromosome alignment to the spindle equator is a hallmark of mitosis thought to promote chromosome segregation fidelity in metazoans. Yet chromosome alignment is only indirectly supervised by the spindle assembly checkpoint (SAC) as a byproduct of chromosome bi-orientation, and the consequences of defective chromosome alignment remain unclear. Here, we investigated how human cells respond to chromosome alignment defects of distinct molecular nature by following the fate of live HeLa cells after RNAi-mediated depletion of 125 proteins previously implicated in chromosome alignment. We confirmed chromosome alignment defects upon depletion of 108/125 proteins. Surprisingly, in all confirmed cases, depleted cells frequently entered anaphase after a delay with misaligned chromosomes. Using depletion of prototype proteins resulting in defective chromosome alignment, we show that misaligned chromosomes often satisfy the SAC and directly missegregate without lagging behind in anaphase. In-depth analysis of specific molecular perturbations that prevent proper kinetochore-microtubule attachments revealed that misaligned chromosomes that missegregate frequently result in micronuclei. Higher-resolution live-cell imaging indicated that, contrary to most anaphase lagging chromosomes that correct and reintegrate the main nuclei, misaligned chromosomes are a strong predictor of micronuclei formation in a cancer cell model of chromosomal instability, but not in non-transformed near-diploid cells. We provide evidence supporting that intrinsic differences in kinetochore-microtubule attachment stability on misaligned chromosomes account for this distinct outcome. Thus, misaligned chromosomes that satisfy the SAC may represent a previously overlooked mechanism driving chromosomal/genomic instability during cancer cell division, and we unveil genetic conditions predisposing for these events.
Assuntos
Cinetocoros , Neoplasias , Segregação de Cromossomos , Cromossomos , Células HeLa , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Mitose , Neoplasias/metabolismo , Fuso Acromático/metabolismoRESUMO
Chromosome segregation in mammals relies on the maturation of a thick bundle of kinetochore-attached microtubules known as k-fiber. How k-fibers mature from initial kinetochore microtubule attachments remains a fundamental question. By combining molecular perturbations and phenotypic analyses in Indian muntjac fibroblasts containing the lowest known diploid chromosome number in mammals (2N = 6) and distinctively large kinetochores, with fixed/live-cell super-resolution coherent-hybrid stimulated emission depletion (CH-STED) nanoscopy and laser microsurgery, we demonstrate a key role for augmin in kinetochore microtubule self-organization and maturation, regardless of pioneer centrosomal microtubules. In doing so, augmin promotes kinetochore and interpolar microtubule turnover and poleward flux. Tracking of microtubule growth events within individual k-fibers reveals a wide angular dispersion, consistent with augmin-mediated branched microtubule nucleation. Augmin depletion reduces the frequency of kinetochore microtubule growth events and hampers efficient repair after acute k-fiber injury by laser microsurgery. Together, these findings underscore the contribution of augmin-mediated microtubule amplification for k-fiber self-organization and maturation in mammals.
Assuntos
Cinetocoros , Fuso Acromático , Animais , Segregação de Cromossomos , Mamíferos/genética , Microtúbulos , Mitose , Fuso Acromático/genéticaRESUMO
The mechanism of chromosome biorientation during mitotic spindle assembly remains a century-old mystery. In contrast to the stochastic models that have dominated the field for decades, a new study now proposes that chromosome biorientation is instead deterministic and driven by microtubule self-organization at kinetochores.
Assuntos
Cinetocoros , Fuso Acromático , Segregação de Cromossomos , Microtúbulos , MitoseRESUMO
Micronuclei are a hallmark of cancer and several other human disorders. Recently, micronuclei were implicated in chromothripsis, a series of massive genomic rearrangements that may drive tumor evolution and progression. Here, we show that Aurora B kinase mediates a surveillance mechanism that integrates error correction during anaphase with spatial control of nuclear envelope reassembly to prevent micronuclei formation. Using high-resolution live-cell imaging of human cancer and non-cancer cells, we uncover that anaphase lagging chromosomes are more frequent than previously anticipated, yet they rarely form micronuclei. Micronuclei formation from anaphase lagging chromosomes is prevented by a midzone-based Aurora B phosphorylation gradient that stabilizes kinetochore-microtubule attachments and assists spindle forces required for anaphase error correction while delaying nuclear envelope reassembly on lagging chromosomes, independently of microtubule density. We propose that a midzone-based Aurora B phosphorylation gradient actively monitors and corrects frequent chromosome segregation errors to prevent micronuclei formation during human cell division.
Assuntos
Anáfase , Aurora Quinase B/metabolismo , Segregação de Cromossomos , Cinetocoros/enzimologia , Micronúcleos com Defeito Cromossômico , Membrana Nuclear/enzimologia , Fuso Acromático/enzimologia , Células HeLa , Humanos , Mecanotransdução Celular , Membrana Nuclear/genética , Fosforilação , Fuso Acromático/genética , Fatores de TempoRESUMO
The establishment of a metaphase plate in which all chromosomes are attached to mitotic spindle microtubules and aligned at the cell equator is required for faithful chromosome segregation in metazoans. The achievement of this configuration relies on the precise coordination between several concurrent mechanisms that start upon nuclear envelope breakdown, mediate chromosome capture at their kinetochores during mitotic spindle assembly and culminate with the congression of all chromosomes to the spindle equator. This period is called 'prometaphase'. Because the nature of chromosome capture by mitotic spindle microtubules is error prone, the cell is provided of error correction mechanisms that sense and correct most erroneous kinetochore-microtubule attachments before committing to separate sister chromatids in anaphase. In this review, aimed for newcomers in the field, more than providing an exhaustive mechanistic coverage of each and every concurrent mechanism taking place during prometaphase, we provide an integrative overview of these processes that ultimately promote the subsequent faithful segregation of chromosomes during mitosis.
Assuntos
Mitose/fisiologia , Prometáfase/fisiologia , Humanos , Fuso Acromático/metabolismoRESUMO
Microtubule attachments to kinetochores cause their deformation - a murky phenomenon known as intra-kinetochore stretching. A new study proposes that intra-kinetochore stretching is independent of microtubule-pulling forces and mediates efficient spindle assembly checkpoint silencing to prevent chromosomal instability.
Assuntos
Instabilidade Cromossômica , HumanosRESUMO
CLASPs are key modulators of microtubule dynamics throughout the cell cycle. During mitosis, CLASPs independently associate with growing microtubule plus-ends and kinetochores and play essential roles in chromosome segregation. In a proteomic survey for human CLASP1-interacting proteins during mitosis, we have previously identified SOGA1 and SOGA2/MTCL1, whose mitotic roles remained uncharacterized. Here we performed an initial functional characterization of human SOGA1 and SOGA2/MTCL1 during mitosis. Using specific polyclonal antibodies raised against SOGA proteins, we confirmed their expression and reciprocal interaction with CLASP1 and CLASP2 during mitosis. In addition, we found that both SOGA1 and SOGA2/MTCL1 are phospho-regulated during mitosis by CDK1. Immunofluorescence analysis revealed that SOGA2/MTCL1 co-localizes with mitotic spindle microtubules and spindle poles throughout mitosis and both SOGA proteins are enriched at the midbody during mitotic exit/cytokinesis. GFP-tagging of SOGA2/MTCL1 further revealed a microtubule-independent localization at kinetochores. Live-cell imaging after siRNA-mediated knockdown of SOGA1 and SOGA2/MTCL1 showed that they are independently required for distinct aspects of chromosome segregation. Thus, SOGA1 and SOGA2/MTCL1 are bona fide CLASP-interacting proteins during mitosis required for faithful chromosome segregation in human cells.
Assuntos
Segregação de Cromossomos , Proteômica , Humanos , Cinetocoros , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos , Fuso AcromáticoRESUMO
Mitotic spindle microtubules (MTs) undergo continuous poleward flux, whose driving force and function in humans remain unclear. Here, we combined loss-of-function screenings with analysis of MT-dynamics in human cells to investigate the molecular mechanisms underlying MT-flux. We report that kinesin-7/CENP-E at kinetochores (KTs) is the predominant driver of MT-flux in early prometaphase, while kinesin-4/KIF4A on chromosome arms facilitates MT-flux during late prometaphase and metaphase. Both these activities work in coordination with kinesin-5/EG5 and kinesin-12/KIF15, and our data suggest that the MT-flux driving force is transmitted from non-KT-MTs to KT-MTs by the MT couplers HSET and NuMA. Additionally, we found that the MT-flux rate correlates with spindle length, and this correlation depends on the establishment of stable end-on KT-MT attachments. Strikingly, we find that MT-flux is required to regulate spindle length by counteracting kinesin 13/MCAK-dependent MT-depolymerization. Thus, our study unveils the long-sought mechanism of MT-flux in human cells as relying on the coordinated action of four kinesins to compensate for MT-depolymerization and regulate spindle length.
Assuntos
Cinesinas/genética , Cinesinas/metabolismo , Microtúbulos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromossomos , Humanos , Metáfase/fisiologia , Mitose , Fuso Acromático/fisiologiaRESUMO
The "tubulin code" combines different α/ß-tubulin isotypes with several post-translational modifications (PTMs) to generate microtubule diversity in cells. During cell division, specific microtubule populations in the mitotic spindle are differentially modified, but only recently, the functional significance of the tubulin code, with particular emphasis on the role specified by tubulin PTMs, started to be elucidated. This is the case of α-tubulin detyrosination, which was shown to guide chromosomes during congression to the metaphase plate and allow the discrimination of mitotic errors, whose correction is required to prevent chromosomal instability-a hallmark of human cancers implicated in tumor evolution and metastasis. Although alterations in the expression of certain tubulin isotypes and associated PTMs have been reported in human cancers, it remains unclear whether and how the tubulin code has any functional implications for cancer cell properties. Here, we review the role of the tubulin code in chromosome segregation during mitosis and how it impacts cancer cell properties. In this context, we discuss the existence of an emerging "cancer tubulin code" and the respective implications for diagnostic, prognostic and therapeutic purposes.
Assuntos
Mitose , Neoplasias/etiologia , Neoplasias/metabolismo , Tubulina (Proteína)/metabolismo , Movimento Celular/genética , Centrossomo/metabolismo , Instabilidade Cromossômica , Citocinese , Suscetibilidade a Doenças , Humanos , Microtúbulos/metabolismo , Mitose/genética , Invasividade Neoplásica , Neoplasias/patologia , Isoformas de Proteínas , Processamento de Proteína Pós-Traducional , Fuso Acromático/metabolismo , Tubulina (Proteína)/genéticaRESUMO
Incorrect kinetochore-microtubule attachments during mitosis can lead to chromosomal instability, a hallmark of human cancers. Mitotic error correction relies on the kinesin-13 MCAK, a microtubule depolymerase whose activity in vitro is suppressed by α-tubulin detyrosination-a posttranslational modification enriched on long-lived microtubules. However, whether and how MCAK activity required for mitotic error correction is regulated by α-tubulin detyrosination remains unknown. Here we found that detyrosinated α-tubulin accumulates on correct, more stable, kinetochore-microtubule attachments. Experimental manipulation of tubulin tyrosine ligase (TTL) or carboxypeptidase (Vasohibins-SVBP) activities to constitutively increase α-tubulin detyrosination near kinetochores compromised efficient error correction, without affecting overall kinetochore microtubule stability. Rescue experiments indicate that MCAK centromeric activity was required and sufficient to correct the mitotic errors caused by excessive α-tubulin detyrosination independently of its global impact on microtubule dynamics. Thus, microtubules are not just passive elements during mitotic error correction, and the extent of α-tubulin detyrosination allows centromeric MCAK to discriminate correct vs. incorrect kinetochore-microtubule attachments, thereby promoting mitotic fidelity.
Assuntos
Centrômero/metabolismo , Cinesinas/metabolismo , Mitose , Tubulina (Proteína)/metabolismo , Linhagem Celular Tumoral , Humanos , Microtúbulos/metabolismoRESUMO
During the initial stages of cell division, the cytoskeleton is extensively reorganized so that a bipolar mitotic spindle can be correctly assembled. This process occurs through the action of molecular motors, cytoskeletal networks, and the nucleus. How the combined activity of these different components is spatiotemporally regulated to ensure efficient spindle assembly remains unclear. To investigate how cell shape, cytoskeletal organization, and molecular motors cross-talk to regulate initial spindle assembly, we use a combination of micropatterning with high-resolution imaging and 3D cellular reconstruction. We show that during prophase, centrosomes and nucleus reorient so that centrosomes are positioned on the shortest nuclear axis at nuclear envelope (NE) breakdown. We also find that this orientation depends on a combination of centrosome movement controlled by Arp2/3-mediated regulation of microtubule dynamics and Dynein-generated forces on the NE that regulate nuclear reorientation. Finally, we observe this centrosome configuration favors the establishment of an initial bipolar spindle scaffold, facilitating chromosome capture and accurate segregation, without compromising division plane orientation.