bims-micesi 2024-12-01 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2024–12–01
24 papers selected by
Valentina Piano, Uniklinik Köln

Generation of Cdc20 RNAi-Sensitive Cell Lines to Study Mitotic Exit.
Chromosome alignment and Kif18A action rely on spindle-localized control of Cdk1 activity.
The Use of APC/C Antagonists to Promote Mitotic Catastrophe in Cancer Cells.
Inducing Exit from Mitosis by Inactivating Cdk1/Cyclin B in Metaphase-Arrested Cells: A Tool to Study Mitotic Exit and Reestablishment of Interphase.
Determination of Anaphase Duration by Time-Lapse Microscopy in Budding Yeast.
FRET-FLIM for the Study of Protein-Protein Interactions Underpinning Mitosis Checkpoints.
Visualizing Cartwheel Disassembly Process During Mitosis in Fixed and Live Cells by Fluorescence Microscope.
A Transgenic Method to Measure Mitotic Exit in Drosophila Embryos.
Dynamic remodelling of the endoplasmic reticulum for mitosis.
Quantitative Live-Cell Imaging to Study Chromatin Segregation and Nuclear Reformation.
Overexpression of GFP Fusions of Regulators of the SAC from Arabidopsis thaliana.
The Role of Polo-Like Kinase 1 (PLK1) O-GlcNAcylation in Mitosis.
Targeting Mitotic Exit in Malignant Cells.
Luciferase-Based Reporter Assay for the Assessment of Aurora A-Kinase Activity in Mitotic Cycle.
Fibrous corona is reduced in cancer cell lines that attenuate microtubule nucleation from kinetochores.
Targeting APC/C Ubiquitin E3-Ligase Activation with Pyrimidinethylcarbamate Apcin Analogues for the Treatment of Breast Cancer.
Force transmission through the inner kinetochore is enhanced by centromeric DNA sequences.
Methods for Detecting Phosphorylated Proteins and Observing Intracellular Localization During Mitosis.
The kinetoplastid kinetochore protein KKT23 acetyltransferase is a structural homolog of GCN5 that acetylates the histone H2A C-terminal tail.
Time-Lapse Imaging to Analyze Cell Fate in Response to Antimitotics.
Studying Mitotic Phosphorylation in Drosophila.
Production of SAC Proteins in a Baculovirus-Based Heterologous Gene Expression System.
ChromEMT: visualizing and reconstructing chromatin ultrastructure and 3D organization in situ.
Delays in the final stages of fertilization are strongly associated with trichotomous cytokinesis and cleavage arrest.

Methods Mol Biol. 2025 ;2874 9-20

Generation of Cdc20 RNAi-Sensitive Cell Lines to Study Mitotic Exit.

Tingting Lei, Gang Zhang.

  Accurate mitotic progression ensures the fidelity of genome passage. Cdc20 is a key mitotic regulator. It promotes mitotic exit by activating the anaphase-promoting complex or cyclosome (APC/C) and monitors kinetochore-microtubule attachment through activating the spindle assembly checkpoint (SAC). Precise characterization of Cdc20 requires efficient depletion of endogenous Cdc20, which is extremely difficult to achieve by RNA interference (RNAi). This chapter describes the methodology to generate Cdc20 RNAi-sensitive cell lines with the help of CRISPR/Cas9 technology. These cell lines are highly sensitive to Cdc20 RNAi and provide a very useful tool for Cdc20 functionality investigation without the interference of endogenous Cdc20 protein. Similar strategy could be applied to other genes.

Keywords:  CRISPR/Cas9; Cdc20; RNAi

DOI:  https://doi.org/10.1007/978-1-0716-4236-8_2
Front Cell Dev Biol. 2024 ;12 1490781

Chromosome alignment and Kif18A action rely on spindle-localized control of Cdk1 activity.

Angela Flavia Serpico, Caterina Pisauro, Asia Trano, Domenico Grieco.

   Introduction: During mitosis, chromosome alignment at the mitotic spindle equator grants correct chromosome segregation and proper nuclei formation in daughter cells. The kinesin 8 family member Kif18A plays a crucial role for chromosome alignment by localizing at the kinetochore-microtubule (K-MT) plus ends to dampen MT dynamics and stabilize K-MT attachments. Kif18A action is directly antagonized by the master mitotic kinase cyclin B-dependent kinase 1 (Cdk1) and is promoted by protein phosphatase 1 (PP1). Since chromosome alignment precedes Cdk1 inactivation by cyclin B proteolysis, it is unclear how Kif18A evades Cdk1 inhibition.
Methods: We analyzed chromosome alignment and Kif18A in mitotic cells upon genetic perturbation of the phosphorylation-dependent inhibitory control of Cdk1 activity by immunofluorescence and cell fractionation experiments.
Results: We show here that chromosome alignment in human cells relies on a recently identified fraction of Cdk1 that is inhibited by Wee1-dependent phosphorylation in mitosis (i-Cdk1, standing for inhibited/inactive-Cdk1) and that localized at spindle structures where it promotes proper spindle assembly. Indeed, the reduction of i-Cdk1 led to several spindle defects including spindles with misaligned, bipolarly attached chromosomes showing poor Kif18A localization at their K-MT plus ends. Restoring i-Cdk1 reversed both alignment defects and Kif18A localization. In cells with lowered i-Cdk1, expressing a phosphonull Kif18A mutant version at the sites that serve as Cdk1 substrate significantly rescued the alignment defects.
Discussion: Mechanistically, our evidence suggests that i-Cdk1 and active PP1 facilitated the dephosphorylation and reactivation of spindle-localized Kif18A. Considering the relevance of Kif18A for survival of aneuploid cancer cells and the potential therapeutic targeting of both Kif18A and Wee1, these findings could also be relevant for cancer therapy.

Keywords:  Wee1; aneuploidy; cancer vulnerability; chromosome alignment; i-Cdk1; micronuclei; spindle assembly

DOI:  https://doi.org/10.3389/fcell.2024.1490781
Methods Mol Biol. 2025 ;2874 207-213

The Use of APC/C Antagonists to Promote Mitotic Catastrophe in Cancer Cells.

Ammar Mayah, Raúl Benito Arenas, Agatha Bastida, Victor M Bolanos-Garcia.

  The multiprotein subunit E3 ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) plays a key role in the control of mitosis progression. APC/C is the ultimate effector of the Spindle Assembly Checkpoint (SAC), the signaling system of higher organisms including the human that monitors the proper attachment of chromosomes to microtubules during cell division. Defects in this process result in genome instability, aneuploidy, premature aging, and cancer. APC/C roles in the SAC require its activation by the protein Cdc20. Interfering with APC/C activation by Cdc20 impairs APC/C substrate recognition, resulting in a delayed mitotic exit and eventually inducing cell death. This may be advantageous for the treatment of cancer and malignancies associated with SAC dysregulation. Here we describe a protocol to interfere with mitotic exit through the use of commercially available (Apcin, proTAME) as well as innovative small molecules we have developed that function as antagonists of APC/C activation by Cdc20. We show that the use of these molecules alone and in combination is effective to promote mitotic catastrophe and suppress cell expansion in 2D and 3D (spheroids) cancer cells of different tissue origin, including breast, cervical, and ovarian cancer.

Keywords:  3D cancer cells (spheroids); Anaphase-Promoting Complex/Cyclosome (APC/C); Apcin analogues; Cdc20; Mitotic exit; Spindle Assembly Checkpoint (SAC)

DOI:  https://doi.org/10.1007/978-1-0716-4236-8_17
Methods Mol Biol. 2025 ;2874 183-198

Inducing Exit from Mitosis by Inactivating Cdk1/Cyclin B in Metaphase-Arrested Cells: A Tool to Study Mitotic Exit and Reestablishment of Interphase.

James R Paulson.

  Treatments which inhibit or inactivate Cdk1/cyclin B in metaphase-arrested mammalian cells and budding yeast are described. These treatments induce the cells to exit mitosis and return to interphase, though without chromosome segregation or cytokinesis, and they provide the basis for a method to identify enzymes or other proteins which act "downstream" from Cdk1 inactivation and to elucidate the roles of those proteins in mitotic exit. In this method, inactivation of Cdk1 is combined with inhibition or inactivation of a protein of interest and the effects are observed. This approach should be particularly useful for determining which protein phosphatases are involved in the transition from mitosis to G1-phase and for identifying their substrates.

Keywords:  1NM-PP1; Cdk1/Cyclin B; Cell cycle; FT210; HeLa; Mitotic exit; Protein kinase inhibitors; Saccharomyces cerevisiae

DOI:  https://doi.org/10.1007/978-1-0716-4236-8_15
Methods Mol Biol. 2025 ;2874 61-75

Determination of Anaphase Duration by Time-Lapse Microscopy in Budding Yeast.

Mariam Huda, Ayse Koca Caydasi.

  Time-lapse microscopy is a valuable, widely employed technique for investigating cell cycle dynamics. This chapter will guide you in using this tool to determine the duration of anaphase as a measure of mitotic exit kinetics in budding yeast cells. The methodology explained here is based on monitoring the mitotic spindle and provides a comprehensive guide covering all aspects, from sample preparation and microscopy to data analysis.

Keywords:  Anaphase; Anaphase onset; Budding yeast; Mitotic exit; Mitotic spindle; Saccharomyces cerevisiae; Spindle breakdown; Spindle elongation; Time-lapse microscopyTime-lapse microscopy

DOI:  https://doi.org/10.1007/978-1-0716-4236-8_6
Methods Mol Biol. 2025 ;2874 87-97

FRET-FLIM for the Study of Protein-Protein Interactions Underpinning Mitosis Checkpoints.

Charlotte Pain.

  Cell division is a key cellular process that ensures the continuation of life on Earth. In order to protect the genetic integrity of organisms, cell division must happen accurately, ensuring each daughter cell receives a complete copy of the original genome. The accuracy of this process is, in part, preserved by various cell cycle checkpoints. These checkpoints rely on the physical interactions of their components to ensure proper function. The spindle assembly checkpoint (SAC), for example, produces an inhibitory complex of BUBR1-BUB3 and MAD2 bound to CDC20. Many of these cell cycle checkpoint components have been identified in plants, but it has not yet been established whether plants have a mitotic checkpoint architecture that is similar to mammalian cells. To understand the function of plant cell cycle homologues, it is imperative to characterize their interactions in vivo. FRET-FLIM (Förster resonance energy transfer-fluorescence lifetime imaging microscopy), is a rapidly expanding technique that can be used to rapidly and simply characterize protein-protein interactions.

Keywords:  FRET-FLIM; Fluorescence imaging; Spindle assembly checkpoint

DOI:  https://doi.org/10.1007/978-1-0716-4236-8_8
Methods Mol Biol. 2025 ;2874 77-85

Visualizing Cartwheel Disassembly Process During Mitosis in Fixed and Live Cells by Fluorescence Microscope.

Fan Huang, Chuanmao Zhang.

  Centrosome is an evolutionarily conserved organelle that comprises two barrel-shaped centrioles surrounded by pericentriolar material (PCM). It functions as the major microtubule-organizing center (MTOC) to regulate cell polarity, motility, intracellular material transport during interphase, and bipolar spindle assembly during mitosis. Cartwheel assembly is considered the first step in the initiation of procentriole biogenesis at early S phase. In human cells, cartwheel is a transient scaffolding structure that is disassembled during procentriole maturation at mitotic exit. This chapter describes methods for synchronizing mammalian cells into mitosis by small molecule inhibitor treatment and visualizing cartwheel disassembly process during mitosis in fixed and live cells by fluorescence microscope.

Keywords:  Cartwheel disassembly; Centriole; Centrosome; Fluorescence microscope; MTOC; Mitosis; Mitotic exit

DOI:  https://doi.org/10.1007/978-1-0716-4236-8_7
Methods Mol Biol. 2025 ;2874 1-8

A Transgenic Method to Measure Mitotic Exit in Drosophila Embryos.

Shige Yamada, Aaron N Johnson, Shuo Yang.

  Mitotic exit is a necessary step for highly specialized cells to terminally differentiate and acquire unique functions. The FUCCI system can be used to visualize mitotic and post-mitotic cells during development and regeneration in both live organisms and fixed tissues. Here we describe a Fly-FUCCI protocol for assaying mitotic exit in Drosophila embryos.

Keywords:  Drosophila embryogenesis; Fly-FUCCI; Mef2; Mitotic exit; Myogenesis

DOI:  https://doi.org/10.1007/978-1-0716-4236-8_1
J Cell Sci. 2024 Nov 15. pii: jcs261444. [Epub ahead of print]137(22):

Dynamic remodelling of the endoplasmic reticulum for mitosis.

Suzan Kors, Anne-Lore Schlaitz.

  The endoplasmic reticulum (ER) is a dynamic and continuous membrane network with roles in many cellular processes. The importance and maintenance of ER structure and function have been extensively studied in interphase cells, yet recent findings also indicate crucial roles of the ER in mitosis. During mitosis, the ER is remodelled significantly with respect to composition and morphology but persists as a continuous network. The ER interacts with microtubules, actin and intermediate filaments, and concomitant with the mitotic restructuring of all cytoskeletal systems, ER dynamics and distribution change. The ER is a metabolic hub and several examples of altered ER functions during mitosis have been described. However, we lack an overall understanding of the ER metabolic pathways and functions that are active during mitosis. In this Review, we will discuss mitotic changes to the ER at different organizational levels to explore how the mitotic ER, with its distinct properties, might support cell division.

Keywords:  Cell division; ER; ER dynamics and morphology; ER–cytoskeleton contacts; Endoplasmic reticulum; Membrane contact sites; Mitosis

DOI:  https://doi.org/10.1242/jcs.261444
Methods Mol Biol. 2025 ;2874 47-60

Quantitative Live-Cell Imaging to Study Chromatin Segregation and Nuclear Reformation.

Anja Scheufen, Daniel Moreno-Andrés.

  Live-cell imaging is a powerful tool for the investigation of different steps of the life and fate of single cells and cell populations. In this chapter, we describe how to perform live-cell imaging in tissue culture cells and the subsequent image analysis to precisely characterize the cytological events occurring during mitotic exit and nuclear reformation.

Keywords:  Chromatin decondensation; Image analysis; Live-cell imaging; Mitotic exit; Nuclear reformation

DOI:  https://doi.org/10.1007/978-1-0716-4236-8_5
Methods Mol Biol. 2025 ;2874 21-32

Overexpression of GFP Fusions of Regulators of the SAC from Arabidopsis thaliana.

Charlotte Pain.

  Cell division is a fundamental biological process, essential for sustaining life on Earth. Accurate replication followed by uniform segregation of the genome is required to ensure cell division is sustainable and reduces the likelihood of aneuploidy. The cell cycle has various checkpoints to safeguard proper replication, for example, the spindle assembly checkpoint (SAC) which ensures that all chromosomes are correctly aligned and attached to the spindle, before the transition to anaphase. The precise function of the SAC and SAC components in plants is so far unclear. First, the high level of polyploidy in plants raises concerns about the efficacy of the SAC. Second, many plant SAC components are implicated in other cellular processes, such as MAD1, which has been implicated in the reproductive transition of Arabidopsis thaliana. Overexpression of GFP fusions of core SAC components provides a key route to establish the functions of the different SAC components in plants. Here we describe two methods for agrobacterium-mediated transformation of plants.

Keywords:  Agrobacterium; Arabidopsis thaliana; Floral dip; GFP; Spindle assembly checkpoint (SAC); Transient expression

DOI:  https://doi.org/10.1007/978-1-0716-4236-8_3
Methods Mol Biol. 2025 ;2874 127-137

The Role of Polo-Like Kinase 1 (PLK1) O-GlcNAcylation in Mitosis.

Jie Li, Guangcan Shao, Bin Peng, Xingzhi Xu, Meng-Qiu Dong, Jing Li.

  Polo-like kinase 1 (PLK1) is a crucial mitotic kinase that is implicated in various aspects of cell cycle. Many post-translational modifications have been identified on PLK1 to regulate its activation, stability, and localization. PLK1 has been shown previously to colocalize with the O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT), and OGT regulates PLK1 stability. In our recent work, we show that PLK1 is O-GlcNAcylated by click chemistry. Using stepped collisional energy/higher energy collision dissociation mass spectrometry, we mapped the PLK1 O-GlcNAc site to be T291. We further utilized fluorescent activated cell sorting and time-lapse microscopy to assess the mitotic defects of PLK1 O-GlcNAc mutants. In vivo studies in mouse xenograft demonstrated that it promoted uterine cancer tumorigenesis. In this chapter, we delineate the methodologies we used in studying PLK1 O-GlcNAcylation, including click chemistry, stepped collisional energy/higher energy collision dissociation mass spectrometry, fluorescent activated cell sorting, time-lapse microscopy, and mouse xenograft assays.

Keywords:  Mass spectrometry; Mitosis; O-GlcNAcylation; PLK1; Uterine carcinoma

DOI:  https://doi.org/10.1007/978-1-0716-4236-8_11
Methods Mol Biol. 2025 ;2874 199-205

Targeting Mitotic Exit in Malignant Cells.

Christine Greil, Monika Engelhardt, Julia Felthaus, Ralph Wäsch.

  In order to sustain genomic stability by correct DNA replication and mitosis and thus avoid malignant transformation of cells, the cell cycle is a strictly regulated process. Aberrant cell cycle regulation and defects in mitosis in malignant cells are targets of various cancer therapies. Cancer cells may survive antimitotic treatment due to mitotic slippage with a residual activity of the ubiquitin ligase anaphase-promoting complex (APC/C) and a continuous slow ubiquitin-proteasome-dependent cyclin B-degradation leading to mitotic exit. The combination of antimitotic chemotherapeutics with proteasome inhibitors to block cyclin B-proteolysis or with targeted inhibitors of the APC/C and the antiapoptotic protein Mcl-1 seems a promising approach to improve treatment response in different malignancies by enhancing mitotic arrest and apoptosis.The influence of conventional spindle poisons and new targeted substances and of their combinations on mitosis and apoptosis has not yet been conclusively clarified. Most models have been verified on cell lines whose biology may differ from that of tumors growing in vivo. To study the impact of various antimitotic substances on cell proliferation, especially detect onset of apoptosis depending on different cell cycle phases and thus to identify a possibly entity-dependent mechanism of those agents and their combinations, a combined approach with live-cell imaging and soft-agar colony assays in cultured patient-derived xenografts (PDX) was established.

Keywords:  Anaphase-promoting complex/cyclosome (APC/C); Antimitotic therapy; Cyclin B; Live-cell imaging; Mitotic exit; Mitotic slippage; Patient-derived xenograft (PDX); Soft-agar colony assay

DOI:  https://doi.org/10.1007/978-1-0716-4236-8_16
Methods Mol Biol. 2025 ;2874 139-152

Luciferase-Based Reporter Assay for the Assessment of Aurora A-Kinase Activity in Mitotic Cycle.

Homa Fatma, Hifzur R Siddique.

  Luciferase-based reporter assay is an important tool that employs bioluminescence to quickly and precisely investigate the gene of interest's promoter activity by reporter gene expression at the transcriptional level. The promoter of the gene of interest is fused with the reporter gene (a gene that produces luciferase enzymes) and then transfected into the cells. Luciferase is an enzyme that catalyzes a chemical reaction to produce light. The bioluminescence activity of the luciferase gene in the transfected cells is directly proportional to the expression of the gene of interest, which is measured by using a luminometer. In this chapter, we outline the use of a dual-reporter luciferase assay to measure Aurora A kinase activity during the mitotic cycle.

Keywords:  Aurora A Kinase; Bioluminescence; Dual-reporter Luciferase Assay; Luminometer; Mitotic cycle; Promoter

DOI:  https://doi.org/10.1007/978-1-0716-4236-8_12
Cancer Sci. 2024 Nov 27.

Fibrous corona is reduced in cancer cell lines that attenuate microtubule nucleation from kinetochores.

Yudai Ishikawa, Hirotaka Fukue, Runa Iwakami, Masanori Ikeda, Kenji Iemura, Kozo Tanaka.

  Most cancer cells show increased chromosome missegregation, known as chromosomal instability (CIN), which promotes cancer progression and drug resistance. The underlying causes of CIN in cancer cells are not fully understood. Here we found that breast cancer cell lines show a reduced kinetochore localization of ROD, ZW10, and Zwilch, components of the fibrous corona, compared with non-transformed breast epithelial cell lines. The fibrous corona is a structure formed on kinetochores before their end-on attachment to microtubules and plays a role in efficient kinetochore capture and the spindle assembly checkpoint. The reduction in the fibrous corona was not due to reduced expression levels of the fibrous corona components or to a reduction in outer kinetochore components. Kinetochore localization of Bub1 and CENP-E, which play a role in the recruitment of the fibrous corona to kinetochores, was reduced in cancer cell lines, presumably due to reduced activity of Mps1, which is required for their recruitment to kinetochores through phosphorylating KNL1. Increasing kinetochore localization of Bub1 and CENP-E in cancer cells restored the level of the fibrous corona. Cancer cell lines showed a reduced capacity to nucleate microtubules from kinetochores, which was recently shown to be dependent on the fibrous corona, and increasing kinetochore localization of Bub1 and CENP-E restored the microtubule nucleation capacity on kinetochores. Our study revealed a distinct feature of cancer cell lines that may be related to CIN.

Keywords:  Bub1; CENP‐E; RZZ complex; breast cancer; chromosomal instability; fibrous corona

DOI:  https://doi.org/10.1111/cas.16406
Biomolecules. 2024 Nov 12. pii: 1439. [Epub ahead of print]14(11):

Targeting APC/C Ubiquitin E3-Ligase Activation with Pyrimidinethylcarbamate Apcin Analogues for the Treatment of Breast Cancer.

Maria Kapanidou, Natalie L Curtis, Sandra S Diaz-Minguez, Sandra Agudo-Alvarez, Alfredo Rus Sanchez, Ammar Mayah, Rosette Agena, Paul Brennan, Paula Morales, Raul Benito-Arenas, Agatha Bastida, Victor M Bolanos-Garcia.

  Activation of the ubiquitin ligase APC/C by the protein Cdc20 is an essential requirement for proper cell division in higher organisms, including humans. APC/C is the ultimate effector of the Spindle Assembly Checkpoint (SAC), the signalling system that monitors the proper attachment of chromosomes to microtubules during cell division. Defects in this process result in genome instability and cancer. Interfering with APC/C substrate ubiquitylation in cancer cells delays mitotic exit, which induces cell death. Therefore, impairing APC/C function represents an opportunity for the treatment of cancer and malignancies associated with SAC dysregulation. In this study, we report a new class of pyrimidinethylcarbamate apcin analogues that interfere with APC/C activity in 2D and 3D breast cancer cells. The new pyrimidinethylcarbamate apcin analogues exhibited higher cytotoxicity than apcin in all breast cancer cell subtypes investigated, with much lower cytotoxicity observed in fibroblasts and RPE-1 cells. Further molecular rationalisation of apcin and its derivatives was conducted using molecular docking studies. These structural modifications selected from the in silico studies provide a rational basis for the development of more potent chemotypes to treat highly aggressive breast cancer and possibly other aggressive tumour types of diverse tissue origins.

Keywords:  APC/C (Anaphase Promoting Complex/Cyclosome); apcin analogues; breast cancer; cell cycle regulation; genome instability; spindle assembly checkpoint (SAC)

DOI:  https://doi.org/10.3390/biom14111439
bioRxiv. 2024 Nov 14. pii: 2024.11.13.623448. [Epub ahead of print]

Force transmission through the inner kinetochore is enhanced by centromeric DNA sequences.

Elise Miedlar, Grace E Hamilton, Samuel R Witus, Sara Gonske, Michael Riffle, Alex Zelter, Rachel E Klevit, Charles L Asbury, Yoana N Dimitrova, Trisha N Davis.

Previously, we reconstituted a minimal functional kinetochore from recombinant S. cerevisiae proteins that was capable of transmitting force from dynamic microtubules to nucleosomes containing the centromere-specific histone variant Cse4 (Hamilton et al. 2020). This work revealed two paths of force transmission through the inner kinetochore: through Mif2 and through the Okp1/Ame1 complex (OA). Here, using a chimeric DNA sequence that contains crucial centromere-determining elements of the budding yeast point centromere, we demonstrate that the presence of centromeric DNA sequences in Cse4-containing nucleosomes significantly strengthens OA-mediated linkages. Our findings indicate that centromeric sequences are important for the transmission of microtubule-based forces to the chromosome.

DOI: https://doi.org/10.1101/2024.11.13.623448
Methods Mol Biol. 2025 ;2874 153-166

Methods for Detecting Phosphorylated Proteins and Observing Intracellular Localization During Mitosis.

Miharu Maeda, Kota Saito.

  This protocol describes the detection of phosphorylated proteins within cells and the identification of their intracellular localization, with a particular focus on mitotic cells. While the detection of phosphorylated proteins can be achieved using radioactive labeling with 32Pi, this method presents experimental challenges due to the requirement for radioisotopes. Alternatively, detection using phosphorylation-specific antibodies is a potent method; however, it necessitates the identification of phosphorylation sites and further requires the generation of antibodies targeting these sites, making it effective only for thoroughly analyzed phosphorylations. Here, we outline a convenient method for detecting intracellular phosphorylation using the Phos-tag technique. Additionally, we discuss the establishment of inducible stable cell lines using lentivirus, synchronization methods for cells in the mitotic phase using thymidine and nocodazole, as well as some tips for immunoprecipitation, Western blotting, and immunostaining from mitotic cells.

Keywords:  COPII; Immunoprecipitation; Mitosis; Phos-tag; Phosphorylation; Stable cell lines; Western blotting

DOI:  https://doi.org/10.1007/978-1-0716-4236-8_13
Structure. 2024 Nov 19. pii: S0969-2126(24)00486-6. [Epub ahead of print]

The kinetoplastid kinetochore protein KKT23 acetyltransferase is a structural homolog of GCN5 that acetylates the histone H2A C-terminal tail.

Patryk Ludzia, Midori Ishii, Gauri Deák, Christos Spanos, Marcus D Wilson, Christina Redfield, Bungo Akiyoshi.

  The kinetochore is the macromolecular protein machine that drives chromosome segregation in eukaryotes. In an evolutionarily divergent group of organisms called kinetoplastids, kinetochores are built using a unique set of proteins (KKT1-25 and KKIP1-12). KKT23 is a constitutively localized kinetochore protein containing a C-terminal acetyltransferase domain of unknown function. Here, using X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, we have determined the structure and dynamics of the KKT23 acetyltransferase domain from Trypanosoma brucei and found that it is structurally similar to the GCN5 histone acetyltransferase domain. We find that KKT23 can acetylate the C-terminal tail of histone H2A and that knockdown of KKT23 results in decreased H2A acetylation levels in T. brucei. Finally, we have determined the crystal structure of the N-terminal region of KKT23 and shown that it interacts with KKT22. Our study provides important insights into the structure and function of the unique kinetochore acetyltransferase in trypanosomes.

Keywords:  GCN5; KKT23; NMR spectroscopy; Trypanosoma brucei; X-ray crystallography; acetylation; acetyltransferase; histones; kinetochore; kinetoplastid

DOI:  https://doi.org/10.1016/j.str.2024.10.031
Methods Mol Biol. 2025 ;2874 115-126

Time-Lapse Imaging to Analyze Cell Fate in Response to Antimitotics.

Patrícia M A Silva, João P N Silva, Bárbara Pinto, Hassan Bousbaa.

  Time-lapse imaging is a powerful technique widely used in Cellular and Molecular Biology to capture and analyze the dynamic processes of living cells over specific time periods. Particularly, in the study of cell division, under normal conditions or after drug treatments, this methodology can provide essential data that is impossible to obtain from conventional cell-fixed-based assays. In the context of evaluating cell fate after antimitotic drug treatment, time-lapse imaging provides valuable insights into the mechanisms of action of these compounds. Antimitotic drugs belong to a class of anticancer compounds that interfere with mitosis by targeting microtubules or specific kinases and molecular motors involved in the mitotic apparatus. This chapter aims to highlight the significant advantage of using time-lapse microscopy to assess the effects of antimitotic drugs on cell behavior and fates. We describe the full protocol for time-lapse imaging, using the human lung adenocarcinoma A549 cell line as a model, after exposure to the antimitotic BI2536, a potent inhibitor of polo-like kinase 1 (PLK1). This description includes software for imaging acquisition and data analysis.

Keywords:  Antimitotic agents; BI2536; Cell dynamics; Cell fate; Cell tracking; Polo-like kinase 1; Time-lapse imaging

DOI:  https://doi.org/10.1007/978-1-0716-4236-8_10
Methods Mol Biol. 2025 ;2874 167-182

Studying Mitotic Phosphorylation in Drosophila.

Éric Bonneil, Myreille Larouche, Virginie Emond-Fraser, Peter Kubiniok, Cristina Mirela Pascariu, Pierre Thibault, Vincent Archambault.

  Mitosis is largely controlled by the reversible phosphorylation of effector proteins. The addition or removal of phosphate groups alters the activities of these proteins, resulting in changes in chromosome structure, cytoskeletal dynamics, nuclear envelope integrity, and other transformations that must occur as a cell progresses through mitosis. Drosophila has been instrumental in the elucidation of the molecular mechanisms of mitosis, which are mostly conserved among animals. In this model system, sophisticated genetic tools can be used to study mitosis in different tissues during development in vivo. Drosophila cell culture affords complementary possibilities. In this chapter, we present a phosphoproteomic protocol using Drosophila cell culture to identify phosphorylation sites that depend on mitotic kinases and phosphatases. We also provide protocols to dissect the roles of the identified sites in the regulation of protein interactions and localization during mitosis, using Drosophila embryos. We emphasize the advantages of the selected methods compared to possible alternatives in Drosophila or in other systems.

Keywords:  Biochemistry; Cell biology; Drosophila; Microscopy; Mitosis; Phosphorylation; Proteomics

DOI:  https://doi.org/10.1007/978-1-0716-4236-8_14
Methods Mol Biol. 2025 ;2874 33-45

Production of SAC Proteins in a Baculovirus-Based Heterologous Gene Expression System.

Victor M Bolanos-Garcia.

  The overproduction of certain protein components of the Spindle Assembly Checkpoint (SAC) such as human BUB3, CDH1, and CDC20 either in isolation or associated with other proteins in prokaryotic heterologue gene expression systems remains a technical challenge. Recent advances in virus engineering methods and the development of genetically modified insect cells have been exploited to overproduce these SAC proteins as natively folded, functional molecules. One widely used baculovirus-based expression system is flashBac, which exhibits several advantages over other insect cells-based expression systems such as the straightforward production of recombinant baculoviruses through homologous recombination and the positive selection of recombinant clones.

Keywords:  BUB3BUB3; BaculovirusBaculovirus; CDC20Cdc20; CDH1CDH1; Flashback system; Insect cells heterologous gene expression; Protein overproductionProtein overproduction; Spindle Assembly Checkpoint (SAC)Spindle Assembly Checkpoint (SAC)

DOI:  https://doi.org/10.1007/978-1-0716-4236-8_4
Nat Protoc. 2024 Nov 29.

ChromEMT: visualizing and reconstructing chromatin ultrastructure and 3D organization in situ.

Horng D Ou, Sebastien Phan, Thomas J Deerinck, Akiko Inagaki, Mark H Ellisman, Clodagh C O'Shea.

Structure determines function. The discovery of the DNA double-helix structure revealed how genetic information is stored and copied. In the mammalian cell nucleus, up to two meters of DNA is compacted by histones to form nucleosome/DNA particle chains that form euchromatin and heterochromatin domains, chromosome territories and mitotic chromosomes upon cell division. A critical question is what are the structures, interactions and 3D organization of DNA as chromatin in the nucleus and how do they determine DNA replication timing, gene expression and ultimately cell fate. To visualize genomic DNA across these different length scales in the nucleus, we developed ChromEMT, a method that selectively enhances the electron density and contrast of DNA and interacting nucleosome particles, which enables nucleosome chains, chromatin domains, chromatin ultrastructure and 3D organization to be imaged and reconstructed by using multi-tilt electron microscopy tomography (EMT). ChromEMT exploits a membrane-permeable, fluorescent DNA-binding dye, DRAQ5, which upon excitation drives the photo-oxidation and precipitation of diaminobenzidine polymers on the surface of DNA/nucleosome particles that are visible in the electron microscope when stained with osmium. Here, we describe a detailed protocol for ChromEMT, including DRAQ5 staining, photo-oxidation, sample preparation and multi-tilt EMT that can be applied broadly to reconstruct genomic DNA structure and 3D interactions in cells and tissues and different kingdoms of life. The entire procedure takes ~9 days and requires expertise in electron microscopy sample sectioning and acquisition of multi-tilt EMT data sets.

DOI: https://doi.org/10.1038/s41596-024-01071-2
J Assist Reprod Genet. 2024 Nov 28.

Delays in the final stages of fertilization are strongly associated with trichotomous cytokinesis and cleavage arrest.

Giovanni Coticchio, Lorena Marchio, Alessandro Bartolacci, Danilo Cimadomo, Carlotta Zacà, Cristina Lagalla, Nicoletta Tarozzi, Andrea Borini, Laura Rienzi.

   PURPOSE: Recent evidence showed that the phase between pronuclear fading and the first cleavage is a perilous bridge connecting the zygote and the embryo. Indeed, delay in the short interval between pronuclear breakdown (PNBD) and the first cytokinesis may result in chromosome segregation errors. We tested the hypothesis that delays in this final phase of fertilization are associated with a detrimental impact on embryo development.
METHODS: This is a retrospective study of 1315 zygotes cultured using time lapse technologies generated in 205 first ICSI-cycles.
RESULTS: We observed an association between increasing times of the pronuclear fading-first cleavage interval (t2-tPNf) and the rates of trichotomous/direct unequal cleavage at the first (DUC-1) and second (DUC-2) mitotic cycle. Moreover, we observed a reduced blastulation rate. No significant associations were observed between rates of direct unequal cleavage at the third mitotic cycle (DUC-3) and top-quality blastocysts, euploidy, and live births. To evaluate whether the interval t2-tPNf could have a predictive value for the onset of DUC-1 and DUC-2, ROC curve analyses were performed. The area under the curve values obtained for DUC-1 showed a significant prediction accuracy. The best cut-offs to identify zygotes with a high risk of DUC-1 and DUC-2 occurrence were t2-tPNf > 2.78 (hours) and t2-tPNf > 2.50 (hours), respectively.
CONCLUSION: Delay in the short interval between PNBD and the first cytokinesis result in trichotomous cleavage and early developmental arrest. However, if the embryos reach the blastocyst stage, rates of euploidy and live birth do not appear to be compromised.

Keywords:  Cell division; Direct unequal cleavage; Embryo; Fertilization; Pronuclei; Time-lapse microscopy

DOI:  https://doi.org/10.1007/s10815-024-03330-3