bims-micesi 2023-11-12 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2023–11–12
ten papers selected by
Valentina Piano, Uniklinik Köln

A farnesyl-dependent structural role for CENP-E in expansion of the fibrous corona.
Distinct Mitotic Functions of Nucleolar and Spindle-Associated Protein 1 (NuSAP1) Are Controlled by Two Consensus SUMOylation Sites.
Dynamic interplay between human alpha-satellite DNA structure and centromere functions.
Pericentric major satellite transcription is essential for meiotic chromosome stability and spindle pole organization.
Release of Histone H3K4-reading transcription factors from chromosomes in mitosis is independent of adjacent H3 phosphorylation.
Bioinformatics insights into CENP-T and CENP-W protein-protein interaction disruptive amino acid substitution in the CENP-T-W complex.
Δ3-tubulin impairs mitotic spindle morphology and increases nuclear size in pancreatic cancer cells.
Aurora A Kinase Plays a Key Role in Mitosis Skip during Senescence Induced by Ionizing Radiation.
PP2A inhibition causes synthetic lethality in BRCA2-mutated prostate cancer models via reactivating spindle assembly checkpoint.
Roles of ESCRT-III polymers in cell division across the tree of life.

J Cell Biol. 2024 Jan 01. pii: e202303007. [Epub ahead of print]223(1):

A farnesyl-dependent structural role for CENP-E in expansion of the fibrous corona.

Jingchao Wu, Maximilian W D Raas, Paula Sobrevals Alcaraz, Harmjan R Vos, Eelco C Tromer, Berend Snel, Geert J P L Kops.

Correct chromosome segregation during cell division depends on proper connections between spindle microtubules and kinetochores. During prometaphase, kinetochores are temporarily covered with a dense protein meshwork known as the fibrous corona. Formed by oligomerization of ROD/ZW10/ZWILCH-SPINDLY (RZZ-S) complexes, the fibrous corona promotes spindle assembly, chromosome orientation, and spindle checkpoint signaling. The molecular requirements for formation of the fibrous corona are not fully understood. Here, we show that the fibrous corona depends on the mitotic kinesin CENP-E and that poorly expanded fibrous coronas after CENP-E depletion are functionally compromised. This previously unrecognized role for CENP-E does not require its motor activity but instead is driven by farnesyl modification of its C-terminal kinetochore- and microtubule-binding domain. We show that in cells, CENP-E binds Spindly and recruits RZZ-S complexes to ectopic locations in a farnesyl-dependent manner. CENP-E is recruited to kinetochores following RZZ-S, and-while not required for RZZ-S oligomerization per se-promotes subsequent fibrous corona expansion. Our comparative genomics analyses suggest that the farnesylation motif in CENP-E orthologs emerged alongside the full RZZ-S module in an ancestral lineage close to the fungi-animal split (Obazoa), revealing potential conservation of the mechanisms for fibrous corona formation. Our results show that proper spindle assembly has a potentially conserved non-motor contribution from the kinesin CENP-E through stabilization of the fibrous corona meshwork during its formation.

DOI: https://doi.org/10.1083/jcb.202303007
Cells. 2023 Oct 30. pii: 2545. [Epub ahead of print]12(21):

Distinct Mitotic Functions of Nucleolar and Spindle-Associated Protein 1 (NuSAP1) Are Controlled by Two Consensus SUMOylation Sites.

Michela Damizia, Ludovica Altieri, Vincenzo Costanzo, Patrizia Lavia.

  Nucleolar and Spindle-Associated Protein 1 (NuSAP1) is an important mitotic regulator, implicated in control of mitotic microtubule stability and chromosome segregation. NuSAP1 regulates these processes by interacting with several protein partners. Its abundance, activity and interactions are therefore tightly regulated during mitosis. Protein conjugation with SUMO (Small Ubiquitin-like MOdifier peptide) is a reversible post-translational modification that modulates rapid changes in the structure, interaction(s) and localization of proteins. NuSAP1 was previously found to interact with RANBP2, a nucleoporin with SUMO ligase and SUMO-stabilizing activity, but how this interaction affects NuSAP1 activity has remained elusive. Here, we show that NuSAP1 interacts with RANBP2 and forms proximity ligation products with SUMO2/3 peptides in a RANBP2-dependent manner at key mitotic sites. A bioinformatic search identified two putative SUMO consensus sites in NuSAP1, within the DNA-binding and the microtubule-binding domains, respectively. Site-specific mutagenesis, and mitotic phenotyping in cell lines expressing each NuSAP1 mutant version, revealed selective roles of each individual site in control of NuSAP1 localization and in generation of specific mitotic defects and distinct fates in daughter cells. These results identify therefore two new regulatory sites for NuSAP1 functions and implicate RANBP2 in control of NuSAP1 activity.

Keywords:  NuSAP1; RANBP2; SUMOylation; chromosome segregation; kinetochores; microtubules; mitosis

DOI:  https://doi.org/10.3390/cells12212545
Semin Cell Dev Biol. 2023 Nov 03. pii: S1084-9521(23)00171-4. [Epub ahead of print]

Dynamic interplay between human alpha-satellite DNA structure and centromere functions.

Elena Di Tommaso, Simona Giunta.

  Maintenance of genome stability relies on functional centromeres for correct chromosome segregation and faithful inheritance of the genetic information. The human centromere is the primary constriction within mitotic chromosomes made up of repetitive alpha-satellite DNA hierarchically organized in megabase-long arrays of near-identical higher order repeats (HORs). Centromeres are epigenetically specified by the presence of the centromere-specific histone H3 variant, CENP-A, which enables the assembly of the kinetochore for microtubule attachment. Notably, centromeric DNA is faithfully inherited as intact haplotypes from the parents to the offspring without intervening recombination, yet, outside of meiosis, centromeres are akin to common fragile sites (CFSs), manifesting crossing-overs and ongoing sequence instability. Consequences of DNA changes within the centromere are just starting to emerge, with unclear effects on intra- and inter-generational inheritance driven by centromere's essential role in kinetochore assembly. Here, we review evidence of meiotic selection operating to mitigate centromere drive, as well as recent reports on centromere damage, recombination and repair during the mitotic cell division. We propose an antagonistic pleiotropy interpretation to reconcile centromere DNA instability as both driver of aneuploidy that underlies degenerative diseases, while also potentially necessary for the maintenance of homogenized HORs for centromere function. We attempt to provide a framework for this conceptual leap taking into consideration the structural interface of centromere-kinetochore interaction and present case scenarios for its malfunctioning. Finally, we offer an integrated working model to connect DNA instability, chromatin, and structural changes with functional consequences on chromosome integrity.

Keywords:  Alpha-satellite; Aneuploidy; CENP-A; Cancer; Centromere; Instability

DOI:  https://doi.org/10.1016/j.semcdb.2023.10.002
Open Biol. 2023 Nov;13(11): 230133

Pericentric major satellite transcription is essential for meiotic chromosome stability and spindle pole organization.

Claudia Baumann, Xiangyu Zhang, Maria M Viveiros, Rabindranath De La Fuente.

  In somatic cells, mitotic transcription of major satellite non-coding RNAs is tightly regulated and essential for heterochromatin formation and the maintenance of genome integrity. We recently demonstrated that major satellite transcripts are expressed, and chromatin-bound during mouse oocyte meiosis. Pericentric satellite RNAs are also expressed in human oocytes. However, the specific biological function(s) during oocyte meiosis remain to be established. Here, we use validated locked nucleic acid gapmers for major satellite RNA depletion followed by live cell imaging, and superresolution analysis to determine the role of pericentric non-coding RNAs during female meiosis. Depletion of satellite RNA induces mesoscale changes in pericentric heterochromatin structure leading to chromosome instability, kinetochore attachment errors and abnormal chromosome alignment. Chromosome misalignment is associated with spindle defects, microtubule instability and, unexpectedly, loss of acentriolar microtubule organizing centre (aMTOC) tethering to spindle poles. Pericentrin fragmentation and failure to assemble ring-like aMTOCs with loss of associated polo-like kinase 1 provide critical insight into the mechanisms leading to impaired spindle pole integrity. Inhibition of transcription or RNA splicing phenocopies the chromosome alignment errors and spindle defects, suggesting that pericentric transcription during oocyte meiosis is required to regulate heterochromatin structure, chromosome segregation and maintenance of spindle organization.

Keywords:  aMTOC; major satellite transcripts; meiosis; non-coding RNA; pericentric heterochromatin; spindle

DOI:  https://doi.org/10.1098/rsob.230133
Nat Commun. 2023 Nov 09. 14(1): 7243

Release of Histone H3K4-reading transcription factors from chromosomes in mitosis is independent of adjacent H3 phosphorylation.

Rebecca J Harris, Maninder Heer, Mark D Levasseur, Tyrell N Cartwright, Bethany Weston, Jennifer L Mitchell, Jonathan M Coxhead, Luke Gaughan, Lisa Prendergast, Daniel Rico, Jonathan M G Higgins.

Histone modifications influence the recruitment of reader proteins to chromosomes to regulate events including transcription and cell division. The idea of a histone code, where combinations of modifications specify unique downstream functions, is widely accepted and can be demonstrated in vitro. For example, on synthetic peptides, phosphorylation of Histone H3 at threonine-3 (H3T3ph) prevents the binding of reader proteins that recognize trimethylation of the adjacent lysine-4 (H3K4me3), including the TAF3 component of TFIID. To study these combinatorial effects in cells, we analyzed the genome-wide distribution of H3T3ph and H3K4me2/3 during mitosis. We find that H3T3ph anti-correlates with adjacent H3K4me2/3 in cells, and that the PHD domain of TAF3 can bind H3K4me2/3 in isolated mitotic chromatin despite the presence of H3T3ph. Unlike in vitro, H3K4 readers are still displaced from chromosomes in mitosis in Haspin-depleted cells lacking H3T3ph. H3T3ph is therefore unlikely to be responsible for transcriptional downregulation during cell division.

DOI: https://doi.org/10.1038/s41467-023-43115-3
J Cell Biochem. 2023 Nov 09.

Bioinformatics insights into CENP-T and CENP-W protein-protein interaction disruptive amino acid substitution in the CENP-T-W complex.

Suryakanta Mohanty, Rajendra Bhadane, Shashank Kumar.

  Kinetochores are multi-protein assemblies present at the centromere of the human chromosome and play a crucial role in cellular mitosis. The CENP-T and CENP-W chains form a heterodimer, which is an integral part of the inner kinetochore, interacting with the linker DNA on one side and the outer kinetochore on the other. Additionally, the CENP-T-W dimer interacts with other regulatory proteins involved in forming inner kinetochores. The specific roles of different amino acids in the CENP-W at the protein-protein interaction (PPI) interface during the CENP-T-W dimer formation remain incompletely understood. Since cell division goes awry in diseases like cancer, this CENP-T-W partnership is a potential target for new drugs that could restore healthy cell division. We employed molecular docking, binding free energy calculations, and molecular dynamics (MD) simulations to investigate the disruptive effects of amino acids substitutions in the CENP-W chain on CENP-T-W dimer formation. By conducting a molecular docking study and analysing hydrogen bonding interactions, we identified key residues in CENP-W (ASN-46, ARG-53, LEU-83, SER-86, ARG-87, and GLY-88) for further investigation. Through site-directed mutagenesis and subsequent binding free energy calculations, we refined the selection of mutant. We chose four mutants (N46K, R53K, L83K, and R87E) of CENP-W to assess their comparative potential in forming CENP-T-W dimer. Our analysis from 250 ns long revealed that the substitution of LEU83 and ARG53 residues in CENP-W with the LYS significantly disrupts the formation of CENP-T-W dimer. In conclusion, LEU83 and ARG53 play a critical role in CENP-T and CENP-W dimerization which is ultimately required for cellular mitosis. Our findings not only deepen our understanding of cell division but also hint at exciting drug-target possibilities.

Keywords:  CENP-T; CENP-W; amino acid substitution; binding free energy; dimer formation; molecular dynamics (MD) simulation; protein-protein interaction

DOI:  https://doi.org/10.1002/jcb.30495
Med Mol Morphol. 2023 Nov 06.

Δ3-tubulin impairs mitotic spindle morphology and increases nuclear size in pancreatic cancer cells.

Kenta Baba, Kenichiro Uemura, Ryota Nakazato, Faryal Ijaz, Shinya Takahashi, Koji Ikegami.

  Cancer cell proliferation is affected by post-translational modifications of tubulin. Especially, overexpression or depletion of enzymes for modifications on the tubulin C-terminal region perturbs dynamic instability of the spindle body. Those modifications include processing of C-terminal amino acids of α-tubulin; detyrosination, and a removal of penultimate glutamic acid (Δ2). We previously found a further removal of the third last glutamic acid, which generates so-called Δ3-tubulin. The effects of Δ3-tubulin on spindle integrities and cell proliferation remain to be elucidated. In this study, we investigated the impacts of forced expression of Δ3-tubulin on the structure of spindle bodies and cell division in a pancreatic cancer cell line, PANC-1. Overexpression of HA-tagged Δ3-tubulin impaired the morphology and orientation of spindle bodies during cell division in PANC-1 cells. In particular, spindle bending was most significantly increased. Expression of EGFP-tagged Δ3-tubulin driven by the endogenous promoter of human TUBA1B also deformed and misoriented spindle bodies. Spindle bending and condensation defects were significantly observed by EGFP-Δ3-tubulin expression. Furthermore, EGFP-Δ3-tubulin expression increased the nuclear size in a dose-dependent manner of EGFP-Δ3-tubulin expression. The expression of EGFP-Δ3-tubulin tended to slow down cell proliferation. Taken together, our results demonstrate that Δ3-tubulin affects the spindle integrity and cell division.

Keywords:  Microtubule; Mitosis; Pancreatic cancer; Post-translational modification; Spindle morphology; Δ3-tubulin

DOI:  https://doi.org/10.1007/s00795-023-00373-w
Biomed Environ Sci. 2023 Oct 20. 36(10): 903-916

Aurora A Kinase Plays a Key Role in Mitosis Skip during Senescence Induced by Ionizing Radiation.

Xu Rui Zhang, Tong Shan Zhang, Ya Nan Zhang, Jun Rui Hua, Ju Fang Wang, Jin Peng He.

   Objective: To investigate the fate and underlying mechanisms of G2 phase arrest in cancer cells elicited by ionizing radiation (IR).
Methods: Human melanoma A375 and 92-1 cells were treated with X-rays radiation or Aurora A inhibitor MLN8237 (MLN) and/or p21 depletion by small interfering RNA (siRNA). Cell cycle distribution was determined using flow cytometry and a fluorescent ubiquitin-based cell cycle indicator (FUCCI) system combined with histone H3 phosphorylation at Ser10 (pS10 H3) detection. Senescence was assessed using senescence-associated-β-galactosidase (SA-β-Gal), Ki67, and γH2AX staining. Protein expression levels were determined using western blotting.
Results: Tumor cells suffered severe DNA damage and underwent G2 arrest after IR treatment. The damaged cells did not successfully enter M phase nor were they stably blocked at G2 phase but underwent mitotic skipping and entered G1 phase as tetraploid cells, ultimately leading to senescence in G1. During this process, the p53/p21 pathway is hyperactivated. Accompanying p21 accumulation, Aurora A kinase levels declined sharply. MLN treatment confirmed that Aurora A kinase activity is essential for mitosis skipping and senescence induction.
Conclusion: Persistent p21 activation during IR-induced G2 phase blockade drives Aurora A kinase degradation, leading to senescence via mitotic skipping.

Keywords:  G2 arrest; Ionizing radiation; Mitosis skipping; Senescence; Tetraploid

DOI:  https://doi.org/10.3967/bes2023.119
J Clin Invest. 2023 Nov 07. pii: e172137. [Epub ahead of print]

PP2A inhibition causes synthetic lethality in BRCA2-mutated prostate cancer models via reactivating spindle assembly checkpoint.

Jian Wang, Yuke Chen, Shiwei Li, Wanchang Liu, Xiao Albert Zhou, Yefei Luo, Zhanzhan Xu, Yundong Xiong, Kaiqi Cheng, Mingjian Ruan, Wei Yu, Xiaoman Li, Weibin Wang, Jiadong Wang.

  Mutations in the BRCA2 tumor suppressor gene have been associated with an increased risk of developing prostate cancer. One of the paradoxes concerning BRCA2 is the fact that its inactivation affects genetic stability and is deleterious for cellular and organismal survival, while BRCA2-mutated cancer cells adapt to this detriment and malignantly proliferate. Therapeutic strategies for tumors arising from BRCA2 mutations may be discovered by understanding these adaptive mechanisms. In this study, we conducted forward genetic synthetic viability screenings in C. elegans brc-2 (Cebrc-2) mutants and found that Ceubxn-2 inactivation rescued the viability of Cebrc-2 mutants. Moreover, loss of NSFL1C, the mammalian ortholog of CeUBXN-2, suppressed the spindle assembly checkpoint (SAC) activation and promoted the survival of BRCA2-deficient cells. Mechanistically, NSFL1C recruited USP9X to inhibit the polyubiquitination of AURKB and reduce the removal of AURKB from the centromeres by VCP, which is essential for SAC activation. SAC inactivation is common in BRCA2-deficient prostate cancer patients, but PP2A inhibitors could reactivate the SAC and achieve BRCA2-deficient prostate tumor synthetic lethality. Our research reveals the survival adaptation mechanism of BRCA2-deficient prostate tumor cells and provides different angles for exploring synthetic lethal inhibitors in addition to targeting DNA damage repair pathways.

Keywords:  Cell Biology; DNA repair; Oncology; Prostate cancer; Tumor suppressors

DOI:  https://doi.org/10.1172/JCI172137
Curr Opin Cell Biol. 2023 Nov 07. pii: S0955-0674(23)00123-0. [Epub ahead of print]85 102274

Roles of ESCRT-III polymers in cell division across the tree of life.

Jeremy Graham Carlton, Buzz Baum.

Every cell becomes two through a carefully orchestrated process of division. Prior to division, contractile machinery must first be assembled at the cell midzone to ensure that the cut, when it is made, bisects the two separated copies of the genetic material. Second, this contractile machinery must be dynamically tethered to the limiting plasma membrane so as to bring the membrane with it as it constricts. Finally, the connecting membrane must be severed to generate two physically separate daughter cells. In several organisms across the tree of life, Endosomal Sorting Complex Required for Transport (ESCRT)-III family proteins aid cell division by forming composite polymers that function together with the Vps4 AAA-ATPase to constrict and cut the membrane tube connecting nascent daughter cells from the inside. In this review, we discuss unique features of ESCRT-III that enable it to play this role in division in many archaea and eukaryotes.

DOI: https://doi.org/10.1016/j.ceb.2023.102274