bims-micesi 2024-06-09 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2024–06–09
seventeen papers selected by
Valentina Piano, Uniklinik Köln

Chromosome segregation: Mps1 and Dam1 battle to bind a shared interaction site at the kinetochore.
ZW10: an emerging orchestrator of organelle dynamics during the cell division cycle.
CENP-E inhibition induces chromosomal instability and synergizes with diverse microtubule-targeting agents in breast cancer.
Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint.
The ubiquitin E3 ligase APC/CCdc20 mediates mitotic degradation of OGT.
Dissecting the Mechanical Control of Mitotic Entry Using a Cell Confinement Setup.
Haspin kinase binds to a nucleosomal DNA supergroove.
Native and tagged CENP-A histones are functionally inequivalent.
Helical Coiled Nucleosome Chromosome Architectures during Cell Cycle Progression.
Reconstitution of nuclear envelope subdomain formation on mitotic chromosomes in semi-intact cells.
Cell cycle machinery in oncology: A comprehensive review of therapeutic targets.
Linking phosphoinositide function to mitosis.
CEP41, a ciliopathy-linked centrosomal protein, regulates microtubule assembly and cell proliferation.
Spatiotemporal regulation of CENP-E-guided chromosomes using a fast-relaxing arylazopyrazole photoswitch.
Low-Cost Histopathological Mitosis Detection for Microscope-acquired Images.
Advanced environmental scanning electron microscopy reveals natural surface nano-morphology of condensed mitotic chromosomes in their native state.
Mutations in Tyrosyl-DNA Phosphodiesterase 2 suppress top-2 induced chromosome segregation defects during C. elegans spermatogenesis.

Curr Biol. 2024 Jun 03. pii: S0960-9822(24)00583-9. [Epub ahead of print]34(11): R530-R533

Chromosome segregation: Mps1 and Dam1 battle to bind a shared interaction site at the kinetochore.

Soni Lacefield.

The attachment of kinetochores to spindle microtubules is highly regulated to ensure proper chromosome segregation. Three new studies identify an interaction hub at the kinetochore that integrates kinetochore attachment state with spindle checkpoint activity and kinetochore assembly.

DOI: https://doi.org/10.1016/j.cub.2024.04.070
J Mol Cell Biol. 2024 Jun 03. pii: mjae026. [Epub ahead of print]

ZW10: an emerging orchestrator of organelle dynamics during the cell division cycle.

Sm Faysal Bellah, Fengrui Yang, Fangyuan Xiong, Zhen Dou, Xuebiao Yao, Xing Liu.

  Zeste white 10 (ZW10) was first identified as a centromere/kinetochore protein encoded by the ZW10 gene in Drosophila. ZW10 guides the spindle assembly checkpoint signaling during mitotic chromosome segregation in metazoans. Recent studies have shown that ZW10 is also involved in membranous organelle interactions during interphase and plays a vital role in membrane transport between the endoplasmic reticulum and Golgi apparatus. Despite these findings, the precise molecular mechanisms by which ZW10 regulates interactions between membranous organelles in interphase and the assembly of membraneless organelle kinetochore in mitosis remain elusive. Here, we highlight how ZW10 forms context-dependent protein complexes during the cell cycle. These complexes are essential for mediating membrane trafficking in interphase and ensuring the accurate segregation of chromosomes in mitosis.

Keywords:  ZW10; kinetochore; membraneless organelle; membranous organelle; mitosis

DOI:  https://doi.org/10.1093/jmcb/mjae026
Cancer Res. 2024 Jun 04.

CENP-E inhibition induces chromosomal instability and synergizes with diverse microtubule-targeting agents in breast cancer.

John B Tucker, Caleb L Carlsen, Christina M Scribano, Srishrika M Pattaswamy, Mark E Burkard, Beth A Weaver.

Drugs that perturb microtubules are commonly used to treat breast cancers of all subtypes in both early stage and metastatic disease, but they are only effective in approximately 50% of patients. High concentrations of microtubule-targeting agents can elicit mitotic arrest in cell culture models; however, recent evidence from primary and metastatic breast cancers revealed that they only accumulate at intratumoral levels capable of inducing abnormal multipolar mitotic spindles, not mitotic arrest. While maintenance of multipolar spindles can generate cytotoxic rates of chromosomal instability (CIN), focusing of aberrant multipolar spindles into normal bipolar spindles dramatically reduces CIN and confers resistance to microtubule poisons. Here, we showed that inhibition of the mitotic kinesin CENP-E overcomes resistance caused by focusing multipolar spindles. Clinically relevant microtubule-targeting agents used a mechanistically conserved pathway to induce multipolar spindles without requiring centrosome amplification. Focusing could occur at any point in mitosis, with earlier focusing conferring greater resistance to anti-microtubule agents. CENP-E inhibition increased CIN on focused spindles by generating chromosomes that remained misaligned at spindle poles during anaphase, which substantially increased death in the resulting daughter cells. CENP-E inhibition synergized with diverse, clinically relevant microtubule poisons to potentiate cell death in cell lines and suppress tumor growth in orthotopic tumor models. These results suggest that primary resistance to microtubule-targeting drugs can be overcome by simultaneous inhibition of CENP-E.

DOI: https://doi.org/10.1158/0008-5472.CAN-23-3332
PLoS Genet. 2024 Jun 03. 20(6): e1011302

Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint.

Koly Aktar, Thomas Davies, Ioanna Leontiou, Ivan Clark, Christos Spanos, Edward Wallace, Laura Tuck, A Arockia Jeyaprakash, Kevin G Hardwick.

Cryptococcus neoformans is an opportunistic, human fungal pathogen which undergoes fascinating switches in cell cycle control and ploidy when it encounters stressful environments such as the human lung. Here we carry out a mechanistic analysis of the spindle checkpoint which regulates the metaphase to anaphase transition, focusing on Mps1 kinase and the downstream checkpoint components Mad1 and Mad2. We demonstrate that Cryptococcus mad1Δ or mad2Δ strains are unable to respond to microtubule perturbations, continuing to re-bud and divide, and die as a consequence. Fluorescent tagging of Chromosome 3, using a lacO array and mNeonGreen-lacI fusion protein, demonstrates that mad mutants are unable to maintain sister-chromatid cohesion in the absence of microtubule polymers. Thus, the classic checkpoint functions of the SAC are conserved in Cryptococcus. In interphase, GFP-Mad1 is enriched at the nuclear periphery, and it is recruited to unattached kinetochores in mitosis. Purification of GFP-Mad1 followed by mass spectrometric analysis of associated proteins show that it forms a complex with Mad2 and that it interacts with other checkpoint signalling components (Bub1) and effectors (Cdc20 and APC/C sub-units) in mitosis. We also demonstrate that overexpression of Mps1 kinase is sufficient to arrest Cryptococcus cells in mitosis, and show that this arrest is dependent on both Mad1 and Mad2. We find that a C-terminal fragment of Mad1 is an effective in vitro substrate for Mps1 kinase and map several Mad1 phosphorylation sites. Some sites are highly conserved within the C-terminal Mad1 structure and we demonstrate that mutation of threonine 667 (T667A) leads to loss of checkpoint signalling and abrogation of the GAL-MPS1 arrest. Thus Mps1-dependent phosphorylation of C-terminal Mad1 residues is a critical step in Cryptococcus spindle checkpoint signalling. We conclude that CnMps1 protein kinase, Mad1 and Mad2 proteins have all conserved their important, spindle checkpoint signalling roles helping ensure high fidelity chromosome segregation.

DOI: https://doi.org/10.1371/journal.pgen.1011302
J Biol Chem. 2024 Jun 04. pii: S0021-9258(24)01949-5. [Epub ahead of print] 107448

The ubiquitin E3 ligase APC/CCdc20 mediates mitotic degradation of OGT.

Li Meng, Rui Dong, Weixiao Mi, Ke Qin, Kunfu Ouyang, Jianwei Sun, Jing Li.

  O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole enzyme that catalyzes all O-GlcNAcylation reactions intracellularly. Previous investigations have found that OGT levels oscillate during the cell division process. Specifically, OGT abundance is downregulated during mitosis, but the underlying mechanism is lacking. Here we demonstrate that OGT is ubiquitinated by the ubiquitin E3 ligase, anaphase promoting complex/cyclosome (APC/C)-cell division cycle 20 (Cdc20). We show that APC/CCdc20 interacts with OGT through a conserved destruction box (D-box): Arg-351/Leu-354, the abrogation of which stabilizes OGT. As APC/CCdc20-substrate binding is often preceded by a priming ubiquitination event, we also used mass spectrometry and mapped OGT Lys-352 to be a ubiquitination site, which is a prerequisite for OGT association with APC/C subunits. Interestingly in The Cancer Genome Atlas, R351C is a uterine carcinoma mutant, suggesting that mutations of the D-box are linked with tumorigenesis. Paradoxically, we found that both R351C and the D-box mutants (R351A/L354A) inhibit uterine carcinoma in mouse xenograft models, probably due to impaired cell division and proliferation. In sum, we propose a model where OGT Lys-352 ubiquitination primes its binding with APC/C, and then APC/CCdc20 partners with OGT through the D-box for its mitotic destruction. Our work not only highlights the key mechanism that regulates OGT during the cell cycle, but also reveals the mutual coordination between glycosylation and the cell division machinery.

Keywords:  APC/C(Cdc20); OGT; cell cycle; ubiquitination; uterine carcinoma

DOI:  https://doi.org/10.1016/j.jbc.2024.107448
Bio Protoc. 2024 Mar 20. 14(6): e4959

Dissecting the Mechanical Control of Mitotic Entry Using a Cell Confinement Setup.

Margarida Dantas, Débora Vareiro, Jorge G Ferreira.

  Proliferating cells need to cope with extensive cytoskeletal and nuclear remodeling as they prepare to divide. These events are tightly regulated by the nuclear translocation of the cyclin B1-CDK1 complex, that is partly dependent on nuclear tension. Standard experimental approaches do not allow the manipulation of forces acting on cells in a time-resolved manner. Here, we describe a protocol that enables dynamic mechanical manipulation of single cells with high spatial and temporal resolution and its application in the context of cell division. In addition, we also outline a method for the manipulation of substrate stiffness using polyacrylamide hydrogels. Finally, we describe a static cell confinement setup, which can be used to study the impact of prolonged mechanical stimulation in populations of cells. Key features • Protocol for microfabrication of confinement devices. • Single-cell dynamic confinement coupled with high-resolution microscopy. • Static cell confinement protocol that can be combined with super-resolution STED microscopy. • Analysis of the mechanical control of mitotic entry in a time-resolved manner.

Keywords:  Cell confinement; Cyclin B1; G2-M transition; Hydrogels; Live-cell microscopy; Mechanical forces; Mitotic entry; Nucleus

DOI:  https://doi.org/10.21769/BioProtoc.4959
bioRxiv. 2024 May 23. pii: 2024.05.21.595243. [Epub ahead of print]

Haspin kinase binds to a nucleosomal DNA supergroove.

Chad W Hicks, Colin R Gliech, Xiangbin Zhang, Sanim Rahman, Stacy Vasquez, Andrew J Holland, Cynthia Wolberger.

Phosphorylation of histone H3 threonine 3 (H3T3) by Haspin recruits the chromosomal passenger complex to the inner centromere and ensures proper cell cycle progression through mitosis. The mechanism by which Haspin binds to nucleosomes to phosphorylate H3T3 is not known. We report here cryo-EM structures of the Haspin kinase domain bound to a nucleosome. In contrast with previous structures of histone-modifying enzymes, Haspin solely contacts the nucleosomal DNA, inserting into a supergroove formed by apposing major grooves of two DNA gyres. This unique binding mode provides a plausible mechanism by which Haspin can bind to nucleosomes in a condensed chromatin environment to phosphorylate H3T3. We identify key basic residues in the Haspin kinase domain that are essential for phosphorylation of nucleosomal histone H3 and binding to mitotic chromatin. Our structure is the first of a kinase domain bound to a nucleosome and is the first example of a histone-modifying enzyme that binds to nucleosomes solely through DNA contacts.

DOI: https://doi.org/10.1101/2024.05.21.595243
Epigenetics Chromatin. 2024 Jun 02. 17(1): 19

Native and tagged CENP-A histones are functionally inequivalent.

Minh Bui, Songjoon Baek, Reda S Bentahar, Daniël P Melters, Yamini Dalal.

   BACKGROUND: Over the past several decades, the use of biochemical and fluorescent tags has elucidated mechanistic and cytological processes that would otherwise be impossible. The challenging nature of certain nuclear proteins includes low abundancy, poor antibody recognition, and transient dynamics. One approach to get around those issues is the addition of a peptide or larger protein tag to the target protein to improve enrichment, purification, and visualization. However, many of these studies were done under the assumption that tagged proteins can fully recapitulate native protein function.
RESULTS: We report that when C-terminally TAP-tagged CENP-A histone variant is introduced, it undergoes altered kinetochore protein binding, differs in post-translational modifications (PTMs), utilizes histone chaperones that differ from that of native CENP-A, and can partially displace native CENP-A in human cells. Additionally, these tagged CENP-A-containing nucleosomes have reduced centromeric incorporation at early G1 phase and poorly associates with linker histone H1.5 compared to native CENP-A nucleosomes.
CONCLUSIONS: These data suggest expressing tagged versions of histone variant CENP-A may result in unexpected utilization of non-native pathways, thereby altering the biological function of the histone variant.

Keywords:  Atomic force microscopy; CENP-A; Centromere; Chromatin; Chromatin immuno-precipitation; Deep sequencing; Epitope tags; Immuno-fluorescence; Post-translational modifications

DOI:  https://doi.org/10.1186/s13072-024-00543-9
bioRxiv. 2024 May 25. pii: 2024.05.25.595892. [Epub ahead of print]

Helical Coiled Nucleosome Chromosome Architectures during Cell Cycle Progression.

Angus McDonald, Cornelis Murre, John Sedat.

Recent studies showed an interphase chromosome architecture, --- a specific coiled nucleosome structure, --- derived from cryo-preserved EM tomograms, and dispersed throughout the nucleus. The images were computationally processed to fill in the missing wedges of data caused by incomplete tomographic tilts. The resulting structures increased z-resolution enabling an extension of the proposed architecture to that of mitotic chromosomes. Here we provide additional insights and details into the coiled nucleosome chromosome architectures. We build on the defined chromosomes time-dependent structures in an effort to probe their dynamics. Variants of the coiled chromosome structures, possibly further defining specific regions, are discussed. We propose, based on generalized specific uncoiling of mitotic chromosomes in telophase, large-scale re-organization of interphase chromosomes. Chromosome territories, organized as micron-sized small patches, are constructed, satisfying complex volume considerations. Finally, we unveiled the structures of replicated coiled chromosomes, still attached to centromeres, as part of chromosome architecture.
Significance Statement: This study places all 46 sequenced human chromosomes, --- correctly filled with nucleosomes and in micron sized chromosome territories - into 10micron (average sized) nuclei. The chromosome architecture used a helical nucleosome coiled structure discerned from cryo-EM tomography, as was recently published ( https://doi.org/10.1073/pnas.2119101119 ). This chromosome architecture was further modeled to dynamic structures, structure variations and chromosome replication centromere complications. Finally, this chromosome architecture was modified to allow seamless transition through the cell cycle.

DOI: https://doi.org/10.1101/2024.05.25.595892
Cell Struct Funct. 2024 Jun 04.

Reconstitution of nuclear envelope subdomain formation on mitotic chromosomes in semi-intact cells.

Tomoko Funakoshi, Naoko Imamoto.

  In metazoans, the nuclear envelope (NE) disassembles during the prophase and reassembles around segregated chromatids during the telophase. The process of NE formation has been extensively studied using live-cell imaging. At the early step of NE reassembly in human cells, specific pattern-like localization of inner nuclear membrane (INM) proteins, connected to the nuclear pore complex (NPC), was observed in the so-called "core" region and "noncore" region on telophase chromosomes, which corresponded to the "pore-free" region and the "pore-rich" region, respectively, in the early G1 interphase nucleus. We refer to these phenomena as NE subdomain formation. To biochemically investigate this process, we aimed to develop an in vitro NE reconstitution system using digitonin-permeabilized semi-intact mitotic human cells coexpressing two INM proteins, emerin and lamin B receptor, which were labeled with fluorescent proteins. The targeting and accumulation of INM proteins to chromosomes before and after anaphase onset in semi-intact cells were observed using time-lapse imaging. Our in vitro NE reconstitution system recapitulated the formation of the NE subdomain, as in living cells, although chromosome segregation and cytokinesis were not observed. This in vitro NE reconstitution required the addition of a mitotic cytosolic fraction supplemented with a cyclin-dependent kinase inhibitor and energy sources. The cytoplasmic soluble factor(s) dependency of INM protein targeting differed among the segregation states of chromosomes. Furthermore, the NE reconstituted on segregated chromosomes exhibited active nucleocytoplasmic transport competency. These results indicate that the chromosome status changes after anaphase onset for recruiting NPC components.Key words: nuclear envelope reassembly, inner nuclear membrane protein, nuclear pore complex, semi-intact cell, in vitro reconstitution.

Keywords:  in vitro reconstitution; inner nuclear membrane protein; nuclear envelope reassembly; nuclear pore complex; semi-intact cell

DOI:  https://doi.org/10.1247/csf.24003
FASEB J. 2024 Jun 15. 38(11): e23734

Cell cycle machinery in oncology: A comprehensive review of therapeutic targets.

Simona Cavalu, Amir Mohamed Abdelhamid, Sameh Saber, Elsayed A Elmorsy, Rabab S Hamad, Mustafa Ahmed Abdel-Reheim, Galal Yahya, Mohamed M Salama.

  The cell cycle is tightly regulated to ensure controlled cell proliferation. Dysregulation of the cell cycle machinery is a hallmark of cancer that leads to unchecked growth. This review comprehensively analyzes key molecular regulators of the cell cycle and how they contribute to carcinogenesis when mutated or overexpressed. It focuses on cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, checkpoint kinases, and mitotic regulators as therapeutic targets. Promising strategies include CDK4/6 inhibitors like palbociclib, ribociclib, and abemaciclib for breast cancer treatment. Other possible targets include the anaphase-promoting complex/cyclosome (APC/C), Skp2, p21, and aurora kinase inhibitors. However, challenges with resistance have limited clinical successes so far. Future efforts should focus on combinatorial therapies, next-generation inhibitors, and biomarkers for patient selection. Targeting the cell cycle holds promise but further optimization is necessary to fully exploit it as an anti-cancer strategy across diverse malignancies.

Keywords:  CDK; cell cycle; cyclins; mitosis; therapeutic target

DOI:  https://doi.org/10.1096/fj.202400769R
Cell Rep. 2024 Jun 04. pii: S2211-1247(24)00601-6. [Epub ahead of print]43(6): 114273

Linking phosphoinositide function to mitosis.

Lorenzo Prever, Gabriele Squillero, Emilio Hirsch, Federico Gulluni.

  Phosphoinositides (PtdIns) are a family of differentially phosphorylated lipid second messengers localized to the cytoplasmic leaflet of both plasma and intracellular membranes. Kinases and phosphatases can selectively modify the PtdIns composition of different cellular compartments, leading to the recruitment of specific binding proteins, which control cellular homeostasis and proliferation. Thus, while PtdIns affect cell growth and survival during interphase, they are also emerging as key drivers in multiple temporally defined membrane remodeling events of mitosis, like cell rounding, spindle orientation, cytokinesis, and abscission. In this review, we summarize and discuss what is known about PtdIns function during mitosis and how alterations in the production and removal of PtdIns can interfere with proper cell division.

Keywords:  CP: Cell biology

DOI:  https://doi.org/10.1016/j.celrep.2024.114273
J Cell Sci. 2024 Jun 06. pii: jcs.261927. [Epub ahead of print]

CEP41, a ciliopathy-linked centrosomal protein, regulates microtubule assembly and cell proliferation.

Shweta Shyam Prassanawar, Tuhin Sarkar, Dulal Panda.

  Centrosomal proteins play pivotal roles in orchestrating microtubule dynamics, and their dysregulation leads to disorders, including cancer and ciliopathies. Understanding the multifaceted roles of centrosomal proteins is vital to comprehend their involvement in disease development. Here, we report novel cellular functions of CEP41, a centrosomal and ciliary protein implicated in Joubert syndrome. We show that CEP41 is an essential microtubule-associated protein with microtubule-stabilizing activity. Purified CEP41 binds to preformed microtubules, promotes microtubule nucleation, and suppresses microtubule disassembly. When overexpressed in cultured cells, CEP41 localizes to microtubules and promotes microtubule bundling. Conversely, shRNA-mediated knockdown of CEP41 disrupts the interphase microtubule network and delays microtubule reassembly, emphasizing its role in microtubule organization. Further, we demonstrate that CEP41's association with microtubules relies on its conserved rhodanese homology domain (RHOD) and the N-terminal region. Interestingly, a disease-causing mutation in the RHOD domain impairs CEP41-microtubule interaction. Moreover, depletion of CEP41 inhibits cell proliferation and disrupts cell cycle progression, suggesting its potential involvement in cell cycle regulation. These insights into the cellular functions of CEP41 hold promise for unraveling the impact of its mutations in ciliopathies.

Keywords:  CEP41; Centrosomal proteins; Ciliopathy; Microtubule stabilization; Microtubules; Rhodanese-like domain.

DOI:  https://doi.org/10.1242/jcs.261927
Chem Commun (Camb). 2024 Jun 07.

Spatiotemporal regulation of CENP-E-guided chromosomes using a fast-relaxing arylazopyrazole photoswitch.

Kazuya Matsuo, Ryota Uehara, Takashi Kikukawa, Tomonori Waku, Akio Kobori, Nobuyuki Tamaoki.

We developed a centromere-associated protein E (CENP-E) inhibitor employing trans to cis photoisomerization with 405 nm visible light illumination and fast thermal relaxation. This photoswitching characteristic of the inhibitor enabled selective blockage or release of the motion of particular chromosomes within a single mitotic cell. Using this technique, we successfully demonstrated targeted chromosome gain and loss in daughter cells by introducing asymmetric chromosome segregation.

DOI: https://doi.org/10.1039/d4cc01922a
AMIA Jt Summits Transl Sci Proc. 2024 ;2024 409-418

Low-Cost Histopathological Mitosis Detection for Microscope-acquired Images.

Bilal Shabbir, Saira Saleem, Iffat Aleem, Nida Babar, Hammad Farooq, Asif Loya, Hammad Naveed.

Cancer outcomes are poor in resource-limited countries owing to high costs and insufficient pathologist-population ratio. The advent of digital pathology has assisted in improving cancer outcomes, however, Whole Slide Image scanners are expensive and not affordable in low-income countries. Microscope-acquired images on the other hand are cheap to collect and can be more viable for automation of cancer detection. In this study, we propose LCH-Network, a novel method to identify the cancer mitotic count from microscope-acquired images. We introduced Label Mix, and also synthesized images using GANs to handle data imbalance. Moreover, we applied progressive resolution to handle different image scales for mitotic localization. We achieved F1-Score of 0.71 and outperformed other existing techniques. Our findings enable mitotic count estimation from microscopic images with a low-cost setup. Clinically, our method could help avoid presumptive treatment without a confirmed cancer diagnosis.

Keywords: Generative AI; Low-Cost Histopathology; Machine Learning; Medical Imaging
Sci Rep. 2024 06 06. 14(1): 12998

Advanced environmental scanning electron microscopy reveals natural surface nano-morphology of condensed mitotic chromosomes in their native state.

Vilém Neděla, Eva Tihlaříková, Petr Cápal, Jaroslav Doležel.

The challenge of in-situ handling and high-resolution low-dose imaging of intact, sensitive and wet samples in their native state at nanometer scale, including live samples is met by Advanced Environmental Scanning Electron Microscopy (A-ESEM). This new generation of ESEM utilises machine learning-based optimization of thermodynamic conditions with respect to sample specifics to employ a low temperature method and an ionization secondary electron detector with an electrostatic separator. A modified electron microscope was used, equipped with temperature, humidity and gas pressure sensors for in-situ and real-time monitoring of the sample. A transparent ultra-thin film of ionic liquid is used to increase thermal and electrical conductivity of the samples and to minimize sample damage by free radicals. To validate the power of the new method, we analyze condensed mitotic metaphase chromosomes to reveal new structural features of their perichromosomal layer, and the organization of chromatin fibers, not observed before by any microscopic technique. The ability to resolve nano-structural details of chromosomes using A-ESEM is validated by measuring gold nanoparticles with achievable resolution in the lower nanometre units.

DOI: https://doi.org/10.1038/s41598-024-63515-9
J Biol Chem. 2024 Jun 04. pii: S0021-9258(24)01947-1. [Epub ahead of print] 107446

Mutations in Tyrosyl-DNA Phosphodiesterase 2 suppress top-2 induced chromosome segregation defects during C. elegans spermatogenesis.

Ji Kent Kwah, Nirajan Bhandari, Christine Rourke, Gabriella Gassaway, Aimee Jaramillo-Lambert.

  Meiosis reduces ploidy through two rounds of chromosome segregation preceded by one round of DNA replication. In meiosis I, homologous chromosomes segregate while in meiosis II, sister chromatids separate from each other. Topoisomerase II (Topo II) is a conserved enzyme that alters DNA structure by introducing transient double strand breaks. During mitosis, Topo II relieves topological stress associated with unwinding DNA during replication, recombination, and sister chromatid segregation. Topo II also plays a role in maintaining mitotic chromosome structure. However, the role and regulation of Topo II during meiosis is not well defined. Previously, we found an allele of Topo II, top-2(it7), disrupts homologous chromosome segregation during meiosis I of C. elegans spermatogenesis. In a genetic screen, we identified different point mutations in 5'-tyrosyl-DNA phosphodiesterase two (Tdp2, C. elegans tdpt-1) that suppress top-2(it7) embryonic lethality. Tdp2 removes trapped Top-2-DNA complexes. The tdpt-1 suppressing mutations rescue embryonic lethality, ameliorate chromosome segregation defects, and restore TOP-2 protein levels of top-2(it7). Here, we show that both TOP-2 and TDPT-1 are expressed in germ line nuclei but occupy different compartments until late meiotic prophase. We also demonstrate that tdpt-1 suppression is due to loss of function of the protein and that the tdpt-1 mutations do not have a phenotype independent of top-2(it7) in meiosis. Lastly, we found that the tdpt-1 suppressing mutations either impair the phosphodiesterase activity, affect the stability of TDPT-1, or disrupt protein interactions. This suggests that the wild-type TDPT-1 protein is inhibiting chromosome biological functions of an impaired TOP-2 during meiosis.

Keywords:  C. elegans; TDPT-1; TOP-2; Tdp2; chromosome structure; meiosis; topoisomerase II

DOI:  https://doi.org/10.1016/j.jbc.2024.107446