bims-micesi 2024-07-07 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2024–07–07
nine papers selected by
Valentina Piano, Uniklinik Köln

Proximity-based activation of AURORA A by MPS1 potentiates error correction.
The cell cycle oscillator and spindle length set the speed of chromosome separation in Drosophila embryos.
A force-sensitive mutation reveals a non-canonical role for dynein in anaphase progression.
Structural basis for Mis18 complex assembly and its implications for centromere maintenance.
Non-homologous end joining shapes the genomic rearrangement landscape of chromothripsis from mitotic errors.
Nucleolin acute degradation reveals novel functions in cell cycle progression and division in TNBC.
Phosphorylation of the nuclear poly(A) binding protein (PABPN1) during mitosis protects mRNA from hyperadenylation and maintains transcriptome dynamics.
Genetic Variations Affect Chemotherapy Outcomes: A Role of the Spindle-assembly Checkpoint.
The DNA Damage Checkpoint Targets the Kinetochore for Relocation of Collapsed Forks to the Periphery.

bioRxiv. 2024 Jun 13. pii: 2024.06.11.598300. [Epub ahead of print]

Proximity-based activation of AURORA A by MPS1 potentiates error correction.

Nelson Leça, Francisca Barbosa, Sergi Rodriguez-Calado, Margarida Moura, Paulo D Pedroso, Inês Pinto, Arianna E Verza, Tanja Bange, Claudio E Sunkel, Marin Barisic, Thomas J Maresca, Carlos Conde.

Faithfull cell division relies on mitotic chromosomes becoming bioriented with each pair of sister kinetochores bound to microtubules oriented toward opposing spindle poles. Erroneous kinetochore-microtubule attachments often form during early mitosis, but are destabilized through the phosphorylation of outer kinetochore proteins by centromeric AURORA B kinase (ABK) and centrosomal AURORA A kinase (AAK), thus allowing for re-establishment of attachments until biorientation is achieved. MPS1-mediated phosphorylation of NDC80 has also been shown to directly weaken the kinetochore-microtubule interface in yeast. In human cells, MPS1 has been proposed to transiently accumulate at end-on attached kinetochores and phosphorylate SKA3 to promote microtubule release. Whether MPS1 directly targets NDC80 and/or promotes the activity of AURORA kinases in metazoans remains unclear. Here, we report a novel mechanism involving communication between kinetochores and centrosomes, wherein MPS1 acts upstream of AAK to promote error correction. MPS1 on pole-proximal kinetochores phosphorylates the C-lobe of AAK thereby increasing its activation at centrosomes. This proximity-based activation ensures the establishment of a robust AAK activity gradient that locally destabilizes mal-oriented kinetochores near spindle poles. Accordingly, MPS1 depletion from Drosophila cells causes severe chromosome misalignment and erroneous kinetochore-microtubule attachments, which can be rescued by tethering either MPS1 or constitutively active AAK mutants to centrosomes. Proximity-based activation of AAK by MPS1 also occurs in human cells to promote AAK-mediated phosphorylation of the NDC80 N-terminal tail. These findings uncover an MPS1-AAK cross-talk that is required for efficient error correction, showcasing the ability of kinetochores to modulate centrosome outputs to ensure proper chromosome segregation.

DOI: https://doi.org/10.1101/2024.06.11.598300
bioRxiv. 2024 Jun 18. pii: 2024.06.17.598879. [Epub ahead of print]

The cell cycle oscillator and spindle length set the speed of chromosome separation in Drosophila embryos.

Yitong Xu, Anna Chao, Melissa Rinaldin, Alison Kickuth, Jan Brugués, Stefano Di Talia.

Anaphase is tightly controlled in space and time to ensure proper separation of chromosomes. The mitotic spindle, the self-organized microtubule structure driving chromosome segregation, scales in size with the available cytoplasm. Yet, the relationship between spindle size and chromosome movement remains poorly understood. Here, we address how the movement of chromosomes changes during the cleavage divisions of the Drosophila blastoderm. We show that the speed of chromosome separation gradually decreases during the 4 nuclear divisions of the blastoderm. This reduction in speed is accompanied by a similar reduction in the length of the spindle, thus ensuring that these two quantities are tightly linked. Using a combination of genetic and quantitative imaging approaches, we find that two processes contribute to controlling the speed at which chromosomes move at mitotic exit: the activity of molecular motors important for microtubule depolymerization and the cell cycle oscillator. Specifically, we found that the levels of Klp10A, Klp67A, and Klp59C, three kinesin-like proteins important for microtubule depolymerization, contribute to setting the speed of chromosome separation. This observation is supported by quantification of microtubule dynamics indicating that poleward flux rate scales with the length of the spindle. Perturbations of the cell cycle oscillator using heterozygous mutants of mitotic kinases and phosphatases revealed that the duration of anaphase increases during the blastoderm cycles and is the major regulator of chromosome velocity. Thus, our work suggests a potential link between the biochemical rate of mitotic exit and the forces exerted by the spindle. Collectively, we propose that the cell cycle oscillator and spindle length set the speed of chromosome separation in anaphase.

DOI: https://doi.org/10.1101/2024.06.17.598879
J Cell Biol. 2024 Oct 07. pii: e202310022. [Epub ahead of print]223(10):

A force-sensitive mutation reveals a non-canonical role for dynein in anaphase progression.

David Salvador-Garcia, Li Jin, Andrew Hensley, Mert Gölcük, Emmanuel Gallaud, Sami Chaaban, Fillip Port, Alessio Vagnoni, Vicente José Planelles-Herrero, Mark A McClintock, Emmanuel Derivery, Andrew P Carter, Régis Giet, Mert Gür, Ahmet Yildiz, Simon L Bullock.

The diverse roles of the dynein motor in shaping microtubule networks and cargo transport complicate in vivo analysis of its functions significantly. To address this issue, we have generated a series of missense mutations in Drosophila Dynein heavy chain. We show that mutations associated with human neurological disease cause a range of defects, including impaired cargo trafficking in neurons. We also describe a novel microtubule-binding domain mutation that specifically blocks the metaphase-anaphase transition during mitosis in the embryo. This effect is independent from dynein's canonical role in silencing the spindle assembly checkpoint. Optical trapping of purified dynein complexes reveals that this mutation only compromises motor performance under load, a finding rationalized by the results of all-atom molecular dynamics simulations. We propose that dynein has a novel function in anaphase progression that depends on it operating in a specific load regime. More broadly, our work illustrates how in vivo functions of motors can be dissected by manipulating their mechanical properties.

DOI: https://doi.org/10.1083/jcb.202310022
EMBO Rep. 2024 Jul 01.

Structural basis for Mis18 complex assembly and its implications for centromere maintenance.

Reshma Thamkachy, Bethan Medina-Pritchard, Sang Ho Park, Carla G Chiodi, Juan Zou, Maria de la Torre-Barranco, Kazuma Shimanaka, Maria Alba Abad, Cristina Gallego Páramo, Regina Feederle, Emilija Ruksenaite, Patrick Heun, Owen R Davies, Juri Rappsilber, Dina Schneidman-Duhovny, Uhn-Soo Cho, A Arockia Jeyaprakash.

  The centromere, defined by the enrichment of CENP-A (a Histone H3 variant) containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. To preserve centromere identity, CENP-A levels must be maintained through active CENP-A loading during the cell cycle. A central player mediating this process is the Mis18 complex (Mis18α, Mis18β and Mis18BP1), which recruits the CENP-A-specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multi-pronged approach, we characterise the structure of the Mis18 complex and show that multiple hetero- and homo-oligomeric interfaces facilitate the hetero-octameric Mis18 complex assembly composed of 4 Mis18α, 2 Mis18β and 2 Mis18BP1. Evaluation of structure-guided/separation-of-function mutants reveals structural determinants essential for cell cycle controlled Mis18 complex assembly and centromere maintenance. Our results provide new mechanistic insights on centromere maintenance, highlighting that while Mis18α can associate with centromeres and deposit CENP-A independently of Mis18β, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.

Keywords:  CENP-A; Centromere; Centromere Inheritance; Integrative Structural Analysis; Mis18 Complex

DOI:  https://doi.org/10.1038/s44319-024-00183-w
Nat Commun. 2024 Jul 04. 15(1): 5611

Non-homologous end joining shapes the genomic rearrangement landscape of chromothripsis from mitotic errors.

Qing Hu, Jose Espejo Valle-Inclán, Rashmi Dahiya, Alison Guyer, Alice Mazzagatti, Elizabeth G Maurais, Justin L Engel, Huiming Lu, Anthony J Davis, Isidro Cortés-Ciriano, Peter Ly.

Mitotic errors generate micronuclei entrapping mis-segregated chromosomes, which are susceptible to catastrophic fragmentation through chromothripsis. The reassembly of fragmented chromosomes by error-prone DNA double-strand break (DSB) repair generates diverse genomic rearrangements associated with human diseases. How specific repair pathways recognize and process these lesions remains poorly understood. Here we use CRISPR/Cas9 to systematically inactivate distinct DSB repair pathways and interrogate the rearrangement landscape of fragmented chromosomes. Deletion of canonical non-homologous end joining (NHEJ) components substantially reduces complex rearrangements and shifts the rearrangement landscape toward simple alterations without the characteristic patterns of chromothripsis. Following reincorporation into the nucleus, fragmented chromosomes localize within sub-nuclear micronuclei bodies (MN bodies) and undergo ligation by NHEJ within a single cell cycle. In the absence of NHEJ, chromosome fragments are rarely engaged by alternative end-joining or recombination-based mechanisms, resulting in delayed repair kinetics, persistent 53BP1-labeled MN bodies, and cell cycle arrest. Thus, we provide evidence supporting NHEJ as the exclusive DSB repair pathway generating complex rearrangements from mitotic errors.

DOI: https://doi.org/10.1038/s41467-024-49985-5
bioRxiv. 2024 Jun 22. pii: 2024.06.17.599429. [Epub ahead of print]

Nucleolin acute degradation reveals novel functions in cell cycle progression and division in TNBC.

Joseph Mills, Anna Tessari, Vollter Anastas, Damu Sunil Kumar, Nastaran Samadi Rad, Saranya Lamba, Ilaria Cosentini, Ashley Reers, Zirui Zhu, Wayne O Miles, Vincenzo Coppola, Emanuele Cocucci, Thomas J Magliery, Heather Shive, Alexander E Davies, Lara Rizzotto, Carlo M Croce, Dario Palmieri.

Nucleoli are large nuclear sub-compartments where vital processes, such as ribosome assembly, take place. Technical obstacles still limit our understanding of the biological functions of nucleolar proteins in cell homeostasis and cancer pathogenesis. Since most nucleolar proteins are essential, their abrogation cannot be achieved through conventional approaches. Additionally, the biological activities of many nucleolar proteins are connected to their physiological concentration. Thus, artificial overexpression might not fully recapitulate their endogenous functions. Proteolysis-based approaches, such as the Auxin Inducible Degron (AID) system paired with CRISPR/Cas9 knock-in gene-editing, have the potential to overcome these limitations, providing unprecedented characterization of the biological activities of endogenous nucleolar proteins. We applied this system to endogenous nucleolin (NCL), one of the most abundant nucleolar proteins, and characterized the impact of its acute depletion on Triple-Negative Breast Cancer (TNBC) cell behavior. Abrogation of endogenous NCL reduced proliferation and caused defective cytokinesis, resulting in bi-nucleated tetraploid cells. Bioinformatic analysis of patient data, and quantitative proteomics using our experimental NCL-depleted model, indicated that NCL levels are correlated with the abundance of proteins involved in chromosomal segregation. In conjunction with its effects on sister chromatid dynamics, NCL abrogation enhanced the anti-proliferative effects of chemical inhibitors of mitotic modulators such as the Anaphase Promoting Complex. In summary, using the AID system in combination with CRISPR/Cas9 for endogenous gene editing, our findings indicate a novel role for NCL in supporting the completion of the cell division in TNBC models, and that its abrogation could enhance the therapeutic activity of mitotic-progression inhibitors.

DOI: https://doi.org/10.1101/2024.06.17.599429
Nucleic Acids Res. 2024 Jun 29. pii: gkae562. [Epub ahead of print]

Phosphorylation of the nuclear poly(A) binding protein (PABPN1) during mitosis protects mRNA from hyperadenylation and maintains transcriptome dynamics.

Jackson M Gordon, David V Phizicky, Leonard Schärfen, Courtney L Brown, Dahyana Arias Escayola, Jean Kanyo, TuKiet T Lam, Matthew D Simon, Karla M Neugebauer.

Polyadenylation controls mRNA biogenesis, nucleo-cytoplasmic export, translation and decay. These processes are interdependent and coordinately regulated by poly(A)-binding proteins (PABPs), yet how PABPs are themselves regulated is not fully understood. Here, we report the discovery that human nuclear PABPN1 is phosphorylated by mitotic kinases at four specific sites during mitosis, a time when nucleoplasm and cytoplasm mix. To understand the functional consequences of phosphorylation, we generated a panel of stable cell lines inducibly over-expressing PABPN1 with point mutations at these sites. Phospho-inhibitory mutations decreased cell proliferation, highlighting the importance of PABPN1 phosphorylation in cycling cells. Dynamic regulation of poly(A) tail length and RNA stability have emerged as important modes of gene regulation. We therefore employed long-read sequencing to determine how PABPN1 phospho-site mutants affected poly(A) tails lengths and TimeLapse-seq to monitor mRNA synthesis and decay. Widespread poly(A) tail lengthening was observed for phospho-inhibitory PABPN1 mutants. In contrast, expression of phospho-mimetic PABPN1 resulted in shorter poly(A) tails with increased non-A nucleotides, in addition to increased transcription and reduced stability of a distinct cohort of mRNAs. Taken together, PABPN1 phosphorylation remodels poly(A) tails and increases mRNA turnover, supporting the model that enhanced transcriptome dynamics reset gene expression programs across the cell cycle.

DOI: https://doi.org/10.1093/nar/gkae562
Indian J Public Health. 2024 Apr 01. 68(2): 314-317

Genetic Variations Affect Chemotherapy Outcomes: A Role of the Spindle-assembly Checkpoint.

Sinjini Sarkar, Ranita Pal, Trisha Choudhury, Manisha Vernekar, Partha Nath, Vilas D Nasare.

Cancer patients suffer from complicated chemotoxicity. Pharmacogenomics can help stratify patients by predicting their response to treatment and susceptibility toward severe side effects. The spindle-assembly checkpoint (SAC) is an important pathway that is activated by platinum and taxane compounds and plays a crucial role in their cytotoxic activity. This study investigated a SAC component, Budding Uninhibited by Benzimidazoles 3 (BUB3), its expression, and genetic variants in advanced ovarian cancer patients treated with paclitaxel-carboplatin chemotherapy. Among 80 patients, BUB3 expression correlated with chemosensitivity, suggesting its potential as a predictive marker for chemotherapy response. However, high BUB3 expression was associated with a higher risk of poor survival. In addition, genetic polymorphisms in BUB3 (rs11248416 and rs11248419) were significantly linked to chemotherapy-related toxicities, with rs11248416 showing a negative impact on the patient's physical quality of life.

DOI: https://doi.org/10.4103/ijph.ijph_809_23
bioRxiv. 2024 Jun 17. pii: 2024.06.17.599319. [Epub ahead of print]

The DNA Damage Checkpoint Targets the Kinetochore for Relocation of Collapsed Forks to the Periphery.

Tyler Maclay, Jenna Whalen, Matthew Johnson, Catherine H Freudenreich.

Hairpin forming expanded CAG/CTG repeats pose significant challenges to DNA replication which can lead to replication fork collapse. Long CAG/CTG repeat tracts relocate to the nuclear pore complex to maintain their integrity. Forks impeded by DNA structures are known to activate the DNA damage checkpoint, thus we asked whether checkpoint proteins play a role in relocation of collapsed forks to the nuclear periphery in S. cerevisiae . We show that relocation of a (CAG/CTG) 130 tract is dependent on activation of the Mrc1/Rad53 replication checkpoint. Further, checkpoint-mediated phosphorylation of the kinetochore protein Cep3 is required for relocation, implicating detachment of the centromere from the spindle pole body. Activation of this pathway leads to DNA damage-induced microtubule recruitment to the repeat. These data suggest a role for the DNA replication checkpoint in facilitating movement of collapsed replication forks to the nuclear periphery by centromere release and microtubule-directed motion.
Highlights: The DNA damage checkpoint is needed for relocation of a structure-forming CAG repeat tract to the nuclear pore complexThe importance of Mrc1 (hClaspin) implicates fork uncoupling as the initial checkpoint signalPhosphorylation of the Cep3 kinetochore protein by Dun1 kinase modulates centromere release, which is critical for collapsed fork repositioningDamage-inducible nuclear microtubules colocalize with the CAG repeat locus and are required for relocalizationEstablishes a new role for the DNA replication and DNA damage checkpoint response to trigger repositioning of collapsed forks within the nucleus.

DOI: https://doi.org/10.1101/2024.06.17.599319