bims-micesi 2022-01-23 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2022–01–23
fourteen papers selected by
Valentina Piano, Max Planck Institute of Molecular Physiology

Biallelic BUB1 mutations cause microcephaly, developmental delay, and variable effects on cohesion and chromosome segregation.
Spatial and Temporal Scaling of Microtubules and Mitotic Spindles.
Kre28-Spc105 interaction is essential for Spc105 loading at the kinetochore.
PCTAIRE1 promotes mitotic progression and resistance against antimitotic and apoptotic signals.
The roles of transcription, chromatin organisation and chromosomal processes in holocentromere establishment and maintenance.
The phytochemical, corynoline, diminishes Aurora kinase B activity to induce mitotic defect and polyploidy.
Breaking malignant nuclei as a non-mitotic mechanism of taxol/paclitaxel.
Nuclear export restricts Gdown1 to a mitotic function.
Genetic interaction between PLK1 and downstream MCPH proteins in the control of centrosome asymmetry and cell fate during neural progenitor division.
A review of Plks: Thinking outside the (polo) box.
Keep Calm and Carry on with Extra Centrosomes.
Machine learning classification of trajectories from molecular dynamics simulations of chromosome segregation.
Single-molecule analysis of specificity and multivalency in binding of short linear substrate motifs to the APC/C.
Low-invasive 5D visualization of mitotic progression by two-photon excitation spinning-disk confocal microscopy.

Sci Adv. 2022 Jan 21. 8(3): eabk0114

Biallelic BUB1 mutations cause microcephaly, developmental delay, and variable effects on cohesion and chromosome segregation.

Sara Carvalhal, Ingrid Bader, Martin A Rooimans, Anneke B Oostra, Jesper A Balk, René G Feichtinger, Christine Beichler, Michael R Speicher, Johanna M van Hagen, Quinten Waisfisz, Mieke van Haelst, Martijn Bruijn, Alexandra Tavares, Johannes A Mayr, Rob M F Wolthuis, Raquel A Oliveira, Job de Lange.

Budding uninhibited by benzimidazoles (BUB1) contributes to multiple mitotic processes. Here, we describe the first two patients with biallelic BUB1 germline mutations, who both display microcephaly, intellectual disability, and several patient-specific features. The identified mutations cause variable degrees of reduced total protein level and kinase activity, leading to distinct mitotic defects. Both patients' cells show prolonged mitosis duration, chromosome segregation errors, and an overall functional spindle assembly checkpoint. However, while BUB1 levels mostly affect BUBR1 kinetochore recruitment, impaired kinase activity prohibits centromeric recruitment of Aurora B, SGO1, and TOP2A, correlating with anaphase bridges, aneuploidy, and defective sister chromatid cohesion. We do not observe accelerated cohesion fatigue. We hypothesize that unresolved DNA catenanes increase cohesion strength, with concomitant increase in anaphase bridges. In conclusion, BUB1 mutations cause a neurodevelopmental disorder, with clinical and cellular phenotypes that partially resemble previously described syndromes, including autosomal recessive primary microcephaly, mosaic variegated aneuploidy, and cohesinopathies.

DOI: https://doi.org/10.1126/sciadv.abk0114
Cells. 2022 Jan 12. pii: 248. [Epub ahead of print]11(2):

Spatial and Temporal Scaling of Microtubules and Mitotic Spindles.

Benjamin Lacroix, Julien Dumont.

  During cell division, the mitotic spindle, a macromolecular structure primarily comprised of microtubules, drives chromosome alignment and partitioning between daughter cells. Mitotic spindles can sense cellular dimensions in order to adapt their length and mass to cell size. This scaling capacity is particularly remarkable during early embryo cleavage when cells divide rapidly in the absence of cell growth, thus leading to a reduction of cell volume at each division. Although mitotic spindle size scaling can occur over an order of magnitude in early embryos, in many species the duration of mitosis is relatively short, constant throughout early development and independent of cell size. Therefore, a key challenge for cells during embryo cleavage is not only to assemble a spindle of proper size, but also to do it in an appropriate time window which is compatible with embryo development. How spatial and temporal scaling of the mitotic spindle is achieved and coordinated with the duration of mitosis remains elusive. In this review, we will focus on the mechanisms that support mitotic spindle spatial and temporal scaling over a wide range of cell sizes and cellular contexts. We will present current models and propose alternative mechanisms allowing cells to spatially and temporally coordinate microtubule and mitotic spindle assembly.

Keywords:  allometry; embryonic development; microtubule dynamics; mitotic spindle; spatial scaling; temporal scaling

DOI:  https://doi.org/10.3390/cells11020248
Open Biol. 2022 Jan;12(1): 210274

Kre28-Spc105 interaction is essential for Spc105 loading at the kinetochore.

Babhrubahan Roy, Janice Sim, Simon J Y Han, Ajit P Joglekar.

  Kinetochore (KTs) are macromolecular protein assemblies that attach sister chromatids to spindle microtubules (MTs) and mediate accurate chromosome segregation during mitosis. The outer KT consists of the KMN network, a protein super-complex comprising Knl1 (yeast Spc105), Mis12 (yeast Mtw1), and Ndc80 (yeast Ndc80), which harbours sites for MT binding. Within the KMN network, Spc105 acts as an interaction hub of components involved in spindle assembly checkpoint (SAC) signalling. It is known that Spc105 forms a complex with KT component Kre28. However, where Kre28 physically localizes in the budding yeast KT is not clear. The exact function of Kre28 at the KT is also unknown. Here, we investigate how Spc105 and Kre28 interact and how they are organized within bioriented yeast KTs using genetics and cell biological experiments. Our microscopy data show that Spc105 and Kre28 localize at the KT with a 1 : 1 stoichiometry. We also show that the Kre28-Spc105 interaction is important for Spc105 protein turn-over and essential for their mutual recruitment at the KTs. We created several truncation mutants of kre28 that affect Spc105 loading at the KTs. When over-expressed, these mutants sustain the cell viability, but SAC signalling and KT biorientation are impaired. Therefore, we conclude that Kre28 contributes to chromosome biorientation and high-fidelity segregation at least indirectly by regulating Spc105 localization at the KTs.

Keywords:  kinetochore; microtubule; spindle assembly checkpoint

DOI:  https://doi.org/10.1098/rsob.210274
J Cell Sci. 2022 Jan 19. pii: jcs.258831. [Epub ahead of print]

PCTAIRE1 promotes mitotic progression and resistance against antimitotic and apoptotic signals.

Syed Qaaifah Gillani, Irfana Reshi, Nusrat Nabi, Misbah Un Nisa, Zarka Sarwar, Sameer Bhat, Thomas M Roberts, Jonathan M G Higgins, Shaida Andrabi.

  PCTAIRE1 is a serine-threonine kinase implicated in physiological processes like neuronal development, vesicle trafficking, spermatogenesis, and cell proliferation. However, its exact role in cell division remains unclear. In this study, using a library screening approach, we identified PCTAIRE1 among several candidates that resisted mitotic arrest and mitotic cell death induced by polyoma virus small T (PolST) expression. Our study showed that PCTAIRE1 is a mitotic kinase that localizes at centrosomes during G2 and at spindle poles as the cells enter mitosis, and then at midbody during cytokinesis. We also report that PCTAIRE1 protein levels fluctuate through cell cycle, and reach their peak at mitosis, during which there is an increase in PCTAIRE1 phosphorylation as well. Interestingly, knockdown of PCTAIRE1 resulted in aberrant mitosis by interfering with spindle assembly and chromosome segregation. Further, we found that PCTAIRE1 promotes resistance of cancer cells to antimitotic drugs, thus underscoring the significance of PCTAIRE1 as a potential drug target for overcoming chemotherapeutic resistance. Taken together, these studies establish PCTAIRE1 as a critical mediator of mitotic progression and highlight its role in chemotherapeutic resistance.

Keywords:  CDK16; Chemotherapeutic resistance; Mitotic arrest; PCTAIRE1; PLK1; PP2A

DOI:  https://doi.org/10.1242/jcs.258831
Semin Cell Dev Biol. 2022 Jan 15. pii: S1084-9521(22)00012-X. [Epub ahead of print]

The roles of transcription, chromatin organisation and chromosomal processes in holocentromere establishment and maintenance.

Yue Wang, Lillian Wu, Karen Wing Yee Yuen.

  The centromere is a unique functional region on each eukaryotic chromosome where the kinetochore assembles and orchestrates microtubule attachment and chromosome segregation. Unlike monocentromeres that occupy a specific region on the chromosome, holocentromeres are diffused along the length of the chromosome. Despite being less common, holocentromeres have been verified in almost 800 nematode, insect, and plant species. Understanding of the molecular and epigenetic regulation of holocentromeres is lagging that of monocentromeres. Here we review how permissive locations for holocentromeres are determined across the genome, potentially by chromatin organisation, transcription, and non-coding RNAs, specifically in the nematode C. elegans. In addition, we discuss how holocentric CENP-A or CENP-T-containing nucleosomes are recruited and deposited, through the help of histone chaperones, licensing factors, and condensin complexes, both during de novo holocentromere establishment, and in each mitotic cell cycle. The process of resolving sister centromeres after DNA replication in holocentric organisms is also mentioned. Conservation and diversity between holocentric and monocentric organisms are highlighted, and outstanding questions are proposed.

Keywords:  Cell cycle; Chromatin remodeling; Chromosome condensation; Holocentromere; Non-coding RNA; Transcription

DOI:  https://doi.org/10.1016/j.semcdb.2022.01.004
Biomed Pharmacother. 2022 Jan 17. pii: S0753-3322(22)00033-6. [Epub ahead of print]147 112645

The phytochemical, corynoline, diminishes Aurora kinase B activity to induce mitotic defect and polyploidy.

Ziqi Yan, Qiong Shi, Xumei Liu, Jinghua Li, Vidula Ahire, Shenqiu Zhang, Jing Zhang, Dun Yang, Thaddeus D Allen.

  Plants are a rich source for bioactive compounds. However, plant extracts can harbor a mixture of bioactive molecules that promote divergent phenotypes and potentially have confounding effects in bioassays. Even with further purification and identification, target deconvolution can be challenging. Corynoline and acetylcorynoline, are phytochemicals that were previously isolated through a screen for compounds able to induce mitotic arrest and polyploidy in oncogene expressing retinal pigment epithelial (RPE) cells. Here, we shed light on the mechanism by which these phytochemicals can attack human cancer cells. Mitotic arrest was coincident to the induction of centrosome amplification and declustering, causing multi-polar spindle formation. Corynoline was demonstrated to have true centrosome declustering activity in a model where A549 cells were chemically induced to have more than a regular complement of centrosomes. Corynoline could inhibit the centrosome clustering required for pseudo-bipolar spindle formation in these cells. The activity of AURKB, but not AURKA or polo-like kinase 4, was diminished by corynoline. It only partially inhibited AURKB, so it may be a partial antagonist or corynoline may work upstream on an unknown regulator of AURKB activity or localization. Nonetheless, corynoline and acetylcorynoline inhibited the viability of a variety of human cancer derived cell lines. These phytochemicals could serve as prototypes for a next-generation analog with improved potency, selectivity or in vivo bioavailability. Such an analog could be useful as a non-toxic component of combination therapies where inhibiting the chromosomal passenger protein complex is desired.

Keywords:  Aurora kinase; Centrosome; Corynoline; Mitosis; Phytochemical; Polyploidy

DOI:  https://doi.org/10.1016/j.biopha.2022.112645
J Cancer Biol. 2021 ;2(4): 86-93

Breaking malignant nuclei as a non-mitotic mechanism of taxol/paclitaxel.

Elizabeth R Smith, Xiang-Xi Xu.

  Discovered in a large-scale screening of natural plant chemicals, Taxol/paclitaxel and the taxane family of compounds are surprisingly successful anti-cancer drugs, used in treatment of the majority of solid tumors, and especially suitable for metastatic and recurrent cancer. Paclitaxel is often used in combination with platinum agents and is administrated in a dose dense regimen to treat recurrent cancer. The enthusiasm and clinical development were prompted by the discovery that Taxol binds beta-tubulins specifically found within microtubules and stabilizes the filaments, and consequently inhibits mitosis. However, questions on how paclitaxel suppresses cancer persist, as other specific mitotic inhibitors are impressive in pre-clinical studies but fail to achieve significant clinical activity. Thus, additional mechanisms, such as promoting mitotic catastrophe and impacting non-mitotic targets, have been proposed and studied. A good understanding of how paclitaxel, and additional new microtubule stabilizing agents, kill cancer cells will advance the clinical application of these common chemotherapeutic agents. A recent study provides a potential non-mitotic mechanism of paclitaxel action, that paclitaxel-induced rigid microtubules act to break malleable cancer nuclei into multiple micronuclei. Previous studies have established that cancer cells have a less sturdy, more pliable nuclear envelope due to the loss or reduction of lamin A/C proteins. Such changes in nuclear structure provide a selectivity for paclitaxel to break the nuclear membrane and kill cancer cells over non-neoplastic cells that have a sturdier nuclear envelope. The formation of multiple micronuclei appears to be an important aspect of paclitaxel in the killing of cancer cells, either by a mitotic or non-mitotic mechanism. Additionally, by binding to microtubule, paclitaxel is readily sequestered and concentrated within cells. This unique pharmacokinetic property allows the impact of paclitaxel on cells to persist for several days, even though the circulating drug level is much reduced following drug administration/infusion. The retention of paclitaxel within cells likely is another factor contributing to the efficacy of the drugs. Overall, the new understanding of Taxol/paclitaxel killing mechanism-rigid microtubule-induced multiple micronucleation-will likely provide new strategies to overcome drug resistance and for rational drug combination.

Keywords:  Cell cycle; Drug mechanism; Drug resistance; Microtubules; Mitosis; Nuclear envelope; Paclitaxel; Taxanes; Taxol

DOI:  https://doi.org/10.46439/cancerbiology.2.031
Nucleic Acids Res. 2022 Jan 20. pii: gkac015. [Epub ahead of print]

Nuclear export restricts Gdown1 to a mitotic function.

Christopher B Ball, Mrutyunjaya Parida, Juan F Santana, Benjamin M Spector, Gustavo A Suarez, David H Price.

Approximately half of purified mammalian RNA polymerase II (Pol II) is associated with a tightly interacting sub-stoichiometric subunit, Gdown1. Previous studies have established that Gdown1 inhibits transcription initiation through competitive interactions with general transcription factors and blocks the Pol II termination activity of transcription termination factor 2 (TTF2). However, the biological functions of Gdown1 remain poorly understood. Here, we utilized genetic, microscopic, and multi-omics approaches to functionally characterize Gdown1 in three human cell lines. Acute depletion of Gdown1 caused minimal direct effects on transcription. We show that Gdown1 resides predominantly in the cytoplasm of interphase cells, shuttles between the cytoplasm and nucleus, and is regulated by nuclear export. Gdown1 enters the nucleus at the onset of mitosis. Consistently, genetic ablation of Gdown1 is associated with partial de-repression of mitotic transcription, and Gdown1 KO cells present with evidence of aberrant mitoses coupled to p53 pathway activation. Evidence is presented demonstrating that Gdown1 modulates the combined functions of purified productive elongation factors PAF1C, RTF1, SPT6, DSIF and P-TEFb in vitro. Collectively, our findings support a model wherein the Pol II-regulatory function of Gdown1 occurs during mitosis and is required for genome integrity.

DOI: https://doi.org/10.1093/nar/gkac015
Cell Death Differ. 2022 Jan 20.

Genetic interaction between PLK1 and downstream MCPH proteins in the control of centrosome asymmetry and cell fate during neural progenitor division.

José González-Martínez, Andrzej W Cwetsch, Javier Gilabert-Juan, Jesús Gómez, Guillermo Garaulet, Paulina Schneider, Guillermo de Cárcer, Francisca Mulero, Eduardo Caleiras, Diego Megías, Eva Porlan, Marcos Malumbres.

Alteration of centrosome function and dynamics results in major defects during chromosome segregation and is associated with primary autosomal microcephaly (MCPH). Despite the knowledge accumulated in the last few years, why some centrosomal defects specifically affect neural progenitors is not clear. We describe here that the centrosomal kinase PLK1 controls centrosome asymmetry and cell fate in neural progenitors during development. Gain- or loss-of-function mutations in Plk1, as well as deficiencies in the MCPH genes Cdk5rap2 (MCPH3) and Cep135 (MCPH8), lead to abnormal asymmetry in the centrosomes carrying the mother and daughter centriole in neural progenitors. However, whereas loss of MCPH proteins leads to increased centrosome asymmetry and microcephaly, deficient PLK1 activity results in reduced asymmetry and increased expansion of neural progenitors and cortical growth during mid-gestation. The combination of PLK1 and MCPH mutations results in increased microcephaly accompanied by more aggressive centrosomal and mitotic abnormalities. In addition to highlighting the delicate balance in the level and activity of centrosomal regulators, these data suggest that human PLK1, which maps to 16p12.1, may contribute to the neurodevelopmental defects associated with 16p11.2-p12.2 microdeletions and microduplications in children with developmental delay and dysmorphic features.

DOI: https://doi.org/10.1038/s41418-022-00937-w
Mol Carcinog. 2022 Jan 20.

A review of Plks: Thinking outside the (polo) box.

Julianna Korns, Xiaoqi Liu, Vinita Takiar.

  The polo-like kinase (Plk) family is comprised of five different members (Plk1-5), each with their own distinct functions. Plk family members participate in pivotal cell division processes as well as in non-mitotic roles. Importantly, Plk expression has been correlated with various disease states, including cancer. Multiples therapies, which primarily target Plk1, are currently being investigated alone or in combination with other agents for clinical use in different cancers. As the role of Plks in disease progression becomes more prominent, it is important to outline their functions as cell cycle regulators and more. This review summarizes the structure and both mitotic and non-mitotic functions of each of the five Plk family members, sequentially. Additionally, the proposed mechanisms for how Plks contribute to tumorigenesis and the therapeutics currently under investigation are outlined.

Keywords:  Plk1; Polo-like kinase; cancer; radiation; therapeutics

DOI:  https://doi.org/10.1002/mc.23388
Cancers (Basel). 2022 Jan 17. pii: 442. [Epub ahead of print]14(2):

Keep Calm and Carry on with Extra Centrosomes.

Batuhan Mert Kalkan, Selahattin Can Ozcan, Nicholas J Quintyne, Samantha L Reed, Ceyda Acilan.

  Aberrations in the centrosome number and structure can readily be detected at all stages of tumor progression and are considered hallmarks of cancer. Centrosome anomalies are closely linked to chromosome instability and, therefore, are proposed to be one of the driving events of tumor formation and progression. This concept, first posited by Boveri over 100 years ago, has been an area of interest to cancer researchers. We have now begun to understand the processes by which these numerical and structural anomalies may lead to cancer, and vice-versa: how key events that occur during carcinogenesis could lead to amplification of centrosomes. Despite the proliferative advantages that having extra centrosomes may confer, their presence can also lead to loss of essential genetic material as a result of segregational errors and cancer cells must deal with these deadly consequences. Here, we review recent advances in the current literature describing the mechanisms by which cancer cells amplify their centrosomes and the methods they employ to tolerate the presence of these anomalies, focusing particularly on centrosomal clustering.

Keywords:  cancer; centrosome; chromosomal instability; clustering; multipolar spindles

DOI:  https://doi.org/10.3390/cancers14020442
PLoS One. 2022 ;17(1): e0262177

Machine learning classification of trajectories from molecular dynamics simulations of chromosome segregation.

David Geisel, Peter Lenz.

In contrast to the well characterized mitotic machinery in eukaryotes it seems as if there is no universal mechanism organizing chromosome segregation in all bacteria. Apparently, some bacteria even use combinations of different segregation mechanisms such as protein machines or rely on physical forces. The identification of the relevant mechanisms is a difficult task. Here, we introduce a new machine learning approach to this problem. It is based on the analysis of trajectories of individual loci in the course of chromosomal segregation obtained by fluorescence microscopy. While machine learning approaches have already been applied successfully to trajectory classification in other areas, so far it has not been possible to use them to discriminate segregation mechanisms in bacteria. A main obstacle for this is the large number of trajectories required to train machine learning algorithms that we overcome here by using trajectories obtained from molecular dynamics simulations. We used these trajectories to train four different machine learning algorithms, two linear models and two tree-based classifiers, to discriminate segregation mechanisms and possible combinations of them. The classification was performed once using the complete trajectories as high-dimensional input vectors as well as on a set of features which were used to transform the trajectories into low-dimensional input vectors for the classifiers. Finally, we tested our classifiers on shorter trajectories with duration times comparable (or even shorter) than typical experimental trajectories and on trajectories measured with varying temporal resolutions. Our results demonstrate that machine learning algorithms are indeed capable of discriminating different segregation mechanisms in bacteria and to even resolve combinations of the mechanisms on rather short time scales.

DOI: https://doi.org/10.1371/journal.pone.0262177
Nat Commun. 2022 Jan 17. 13(1): 341

Single-molecule analysis of specificity and multivalency in binding of short linear substrate motifs to the APC/C.

Nairi Hartooni, Jongmin Sung, Ankur Jain, David O Morgan.

Robust regulatory signals in the cell often depend on interactions between short linear motifs (SLiMs) and globular proteins. Many of these interactions are poorly characterized because the binding proteins cannot be produced in the amounts needed for traditional methods. To address this problem, we developed a single-molecule off-rate (SMOR) assay based on microscopy of fluorescent ligand binding to immobilized protein partners. We used it to characterize substrate binding to the Anaphase-Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that triggers chromosome segregation. We find that SLiMs in APC/C substrates (the D box and KEN box) display distinct affinities and specificities for the substrate-binding subunits of the APC/C, and we show that multiple SLiMs in a substrate generate a high-affinity multivalent interaction. The remarkably adaptable substrate-binding mechanisms of the APC/C have the potential to govern the order of substrate destruction in mitosis.

DOI: https://doi.org/10.1038/s41467-022-28031-2
Sci Rep. 2022 Jan 17. 12(1): 809

Low-invasive 5D visualization of mitotic progression by two-photon excitation spinning-disk confocal microscopy.

Takafumi Kamada, Kohei Otomo, Takashi Murata, Kaito Nakata, Shota Hiruma, Ryota Uehara, Mitsuyasu Hasebe, Tomomi Nemoto.

Non-linear microscopy, such as multi-photon excitation microscopy, offers spatial localities of excitations, thereby achieving 3D cross-sectional imaging with low phototoxicity even in thick biological specimens. We had developed a multi-point scanning two-photon excitation microscopy system using a spinning-disk confocal scanning unit. However, its severe color cross-talk has precluded multi-color simultaneous imaging. Therefore, in this study, we introduced a mechanical switching system to select either of two NIR laser light pulses and an image-splitting detection system for 3- or 4-color imaging. As a proof of concept, we performed multi-color fluorescent imaging of actively dividing human HeLa cells and tobacco BY-2 cells. We found that the proposed microscopy system enabled time-lapse multi-color 3D imaging of cell divisions while avoiding photodamage. Moreover, the application of a linear unmixing method to the 5D dataset enabled the precise separation of individual intracellular components in multi-color images. We thus demonstrated the versatility of our new microscopy system in capturing the dynamic processes of cellular components that could have multitudes of application.

DOI: https://doi.org/10.1038/s41598-021-04543-7