bims-micesi 2022-10-09 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2022–10–09
twelve papers selected by
Valentina Piano, Max Planck Institute of Molecular Physiology

Hyper-active RAS/MAPK introduces cancer-specific mitotic vulnerabilities.
Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes.
Mitotic Checkpoint Imbalances in Familial Cancer.
Bcl-xL activity influences outcome of the mitotic arrest.
Acetylation of Nup62 by TIP60 ensures accurate chromosome segregation in mitosis.
Synergy between inhibitors of two mitotic spindle assembly motors undermines an adaptive response.
Strain stiffening of Ndc80 complexes attached to microtubule plus ends.
Polo-like kinase and Aurora B kinase phosphorylate and cooperate with the CIF1-CIF2 complex to promote cytokinesis initiation in Trypanosoma brucei.
The multifaceted role of micronuclei in tumour progression: A whole organism perspective.
The chromatin remodeler RSC prevents ectopic CENP-A propagation into pericentromeric heterochromatin at the chromatin boundary.
Mitotic effects of copper oxide nanoparticle on root development and root tip cells of Phaseolus vulgaris L. seeds.
Membrane compartmentalization of Ect2/Cyk4/Mklp1 and NuMA/dynein regulates cleavage furrow formation.

Proc Natl Acad Sci U S A. 2022 Oct 11. 119(41): e2208255119

Hyper-active RAS/MAPK introduces cancer-specific mitotic vulnerabilities.

Jacob A Herman, Romario R Romain, Pia Hoellerbauer, Hazheen K Shirnekhi, David C King, Keith F DeLuca, Erin Osborne Nishimura, Patrick J Paddison, Jennifer G DeLuca.

  Aneuploidy, the incorrect number of whole chromosomes, is a common feature of tumors that contributes to their initiation and evolution. Preventing aneuploidy requires properly functioning kinetochores, which are large protein complexes assembled on centromeric DNA that link mitotic chromosomes to dynamic spindle microtubules and facilitate chromosome segregation. The kinetochore leverages at least two mechanisms to prevent aneuploidy: error correction and the spindle assembly checkpoint (SAC). BubR1, a factor involved in both processes, was identified as a cancer dependency and therapeutic target in multiple tumor types; however, it remains unclear what specific oncogenic pressures drive this enhanced dependency on BubR1 and whether it arises from BubR1's regulation of the SAC or error-correction pathways. Here, we use a genetically controlled transformation model and glioblastoma tumor isolates to show that constitutive signaling by RAS or MAPK is necessary for cancer-specific BubR1 vulnerability. The MAPK pathway enzymatically hyperstimulates a network of kinetochore kinases that compromises chromosome segregation, rendering cells more dependent on two BubR1 activities: counteracting excessive kinetochore-microtubule turnover for error correction and maintaining the SAC. This work expands our understanding of how chromosome segregation adapts to different cellular states and reveals an oncogenic trigger of a cancer-specific defect.

Keywords:  BubR1; MAPK; aneuploidy; kinetochore; mitosis

DOI:  https://doi.org/10.1073/pnas.2208255119
J Vis Exp. 2022 Sep 13.

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes.

Mansour Aboelenain, Karen Schindler, Cecilia S Blengini.

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.

DOI: https://doi.org/10.3791/64459
Cancer Res. 2022 10 04. 82(19): 3432-3434

Mitotic Checkpoint Imbalances in Familial Cancer.

Carolina Villarroya-Beltri, Marcos Malumbres.

Numerical chromosomal aberrations are highly frequent in cancer cells. However, tumor-associated mutations in regulators of the mitotic machinery that controls chromosome segregation are rather rare. By sequencing families with hereditary cancer, Chen and colleagues report two novel heterozygous mutations in CDC20, a coactivator of the anaphase-promoting complex (APC/C) and a target of the spindle assembly checkpoint (SAC) that prevents chromosome missegregation during mitosis. CDC20 mutations result in partial SAC functionality and segregate with tumor susceptibility in families with aneuploid ovarian cancers and other malignancies. The expression of these mutations in a knock-in mouse model accelerates the development of Myc-induced lymphomas and mortality, strongly supporting the notion that partial dysfunction of mitotic regulators may have profound implications in spontaneous and hereditary cancer. See related article by Chen et al., p. 3499.

DOI: https://doi.org/10.1158/0008-5472.CAN-22-2400
Front Pharmacol. 2022 ;13 933112

Bcl-xL activity influences outcome of the mitotic arrest.

M Suleimenov, S Bekbayev, M Ten, N Suleimenova, M Tlegenova, A Nurmagambetova, S Kauanova, I Vorobjev.

  Microtubule-targeting (MT) drugs taxanes and vinca alkaloids are widely used as chemotherapeutic agents against different tumors for more than 30 years because of their ability to block mitotic progression by disrupting the mitotic spindle and activating the spindle assembly checkpoint (SAC) for a prolonged period of time. However, responses to mitotic arrest are different-some cells die during mitotic arrest, whereas others undergo mitotic slippage and survive becoming able for proliferation. Using normal fibroblasts and several cancer cell types we determined two critical doses, T1 and T2, of mitotic inhibitors (nocodazole, Taxol, and vinorelbine). T1 is the maximal dose cells can tolerate undergoing normal division, and T2 is the minimal mitostatic dose, wherein > 90% of mitotic cells are arrested in mitosis. In all studied cell lines after treatment with mitotic inhibitors in a dose above T2 cells had entered mitosis either die or undergo mitotic slippage. We show that for all three drugs used cell death during mitotic arrest and after slippage proceeded via mitochondria-dependent apoptosis. We determined two types of cancer cells: sensitive to mitotic arrest, that is, undergoing death in mitosis (DiM) frequently, and resistant to mitotic arrest, that is, undergoing mitotic slippage followed by prolonged survival. We then determined that inhibition of Bcl-xL, but not other anti-apoptotic proteins of the Bcl-2 group that regulate MOMP, make resistant cells susceptible to DiM induced by mitotic inhibitors. Combined treatment with MT drugs and highly specific Bcl-xL inhibitors A-1155643 or A-1331852 allows achieving 100% DiM in a time significantly shorter than maximal duration of mitotic arrest in all types of cultured cells tested. We further examined efficacy of sequential treatment of cultured cells using mitotic inhibitors followed by inhibitors of Bcl-xL anti-apoptotic protein and for the first time show that sensitivity to Bcl-xL inhibitors rapidly declines after mitotic slippage. Thus sequential use of mitotic inhibitors and inhibitors of Bcl-xL anti-apoptotic protein will be efficient only if the Bcl-xL inhibitor will be added before mitotic slippage occurs or soon afterward. The combined treatment proposed might be an efficient approach to anti-cancer therapy.

Keywords:  Bcl-2 proteins; anti-mitotic drugs; apoptosis; flow cytometry; live cell imaging; mitotic arrest

DOI:  https://doi.org/10.3389/fphar.2022.933112
J Mol Cell Biol. 2022 Oct 03. pii: mjac056. [Epub ahead of print]

Acetylation of Nup62 by TIP60 ensures accurate chromosome segregation in mitosis.

Hameed Akbar, Jun Cao, Dongmei Wang, Xiao Yuan, Manjuan Zhang, Saravanakumar Muthusamy, Xiaoyu Song, Xu Liu, Felix Aikhionbare, Xuebiao Yao, Xinjiao Gao, Xing Liu.

  Stable transmission of genetic information during cell division requires faithful mitotic spindle assembly and chromosome segregation. In eukaryotic cells, nuclear envelope breakdown (NEBD) is required for proper chromosome segregation. Although a list of mitotic kinases has been implicated in NEBD, how they coordinate their activity to dissolve nuclear envelope and protein machinery such as nuclear pore complexes was unclear. Here, we identified a regulatory mechanism in which Nup62 is acetylated by TIP60 in human cell division. Nup62 is a novel substrate of TIP60, and the acetylation of Lys432 by TIP60 dissolves nucleoporin Nup62-Nup58-Nup54 complex during entry into mitosis. Importantly, this acetylation-elicited remodeling of nucleoporin complex promotes the distribution of Nup62 to mitotic spindle, which is indispensable for orchestrating correct spindle orientation. Moreover, suppression of Nup62 perturbs accurate chromosome segregation during mitosis. These results establish a previously uncharacterized regulatory mechanism in which TIP60-elicited nucleoporin dynamics promotes chromosome segregation in mitosis.

Keywords:  Nup62; TIP60; acetylation; mitosis; spindle

DOI:  https://doi.org/10.1093/jmcb/mjac056
Mol Biol Cell. 2022 Oct 06. mbcE22060225

Synergy between inhibitors of two mitotic spindle assembly motors undermines an adaptive response.

April L Solon, Taylor M Zaniewski, Patrick O'Brien, Martin Clasby, William O Hancock, Ryoma Ohi.

Mitosis is the cellular process that ensures accurate segregation of the cell's genetic material into two daughter cells. Mitosis is often deregulated in cancer, thus drugs that target mitosis-specific proteins represent attractive targets for anticancer therapy. Numerous inhibitors have been developed against kinesin-5 Eg5, a kinesin essential for bipolar spindle assembly. Unfortunately, Eg5 inhibitors (K5Is) have been largely ineffective in the clinic, possibly due to the activity of a second kinesin, KIF15, that can suppress the cytotoxic effect of K5Is by driving spindle assembly through an Eg5-independent pathway. We hypothesized that pairing of K5Is with small molecule inhibitors of KIF15 will be more cytotoxic than either inhibitor alone. Here, we present the results of a high-throughput screen from which we identified two inhibitors that inhibit the motor activity of KIF15 both in vitro and in cells. These inhibitors selectively inhibit KIF15 over other molecular motors and differentially affect the ability of KIF15 to bind microtubules. Finally, we find that chemical inhibition of KIF15 reduces the ability of cells to acquire resistance to K5Is, highlighting the centrality of KIF15 to K5I resistance and the value of these inhibitors as tools with which to study KIF15 in a physiological context.

DOI: https://doi.org/10.1091/mbc.E22-06-0225
Biophys J. 2022 Oct 04. pii: S0006-3495(22)00816-5. [Epub ahead of print]

Strain stiffening of Ndc80 complexes attached to microtubule plus ends.

Felix Schwietert, Vladimir A Volkov, Pim J Huis In 't Veld, Marileen Dogterom, Andrea Musacchio, Jan Kierfeld.

In the mitotic spindle, microtubules attach to chromosomes via kinetochores. The microtubule-binding Ndc80 complex is an integral part of kinetochores, and is essential for kinetochores to attach to microtubules and to transmit forces from dynamic microtubule ends to the chromosomes. The Ndc80 complex has a rod-like appearance with globular domains at its ends that are separated by a long coiled coil. Its mechanical properties are considered important for the dynamic interaction between kinetochores and microtubules. Here, we present a novel method that allows us to time-trace the effective stiffness of Ndc80 complexes following shortening microtubule ends against applied force in optical trap experiments. Applying this method to wild type Ndc80 and three variants (CH-domains mutated or Hec1-tail unphosphorylated, phosphorylated, or truncated), we reveal that each variant exhibits strain stiffening, i.e., the effective stiffness increases under tension that is built up by a depolymerizing microtubule. The strain stiffening relation is roughly linear and independent of the state of the microtubule. We introduce structure-based models, which show that the strain stiffening can be traced back to the specific architecture of the Ndc80 complex with a characteristic flexible kink, to thermal fluctuations of the microtubule, and to the bending elasticity of flaring protofilaments, which exert force to move the Ndc80 complexes. Our model accounts for changes in the amount of load-bearing attachments at various force levels and reproduces the roughly linear strain stiffening behavior, highlighting the importance of force-dependent binding affinity.

DOI: https://doi.org/10.1016/j.bpj.2022.09.039
Open Biol. 2022 Oct;12(10): 220197

Polo-like kinase and Aurora B kinase phosphorylate and cooperate with the CIF1-CIF2 complex to promote cytokinesis initiation in Trypanosoma brucei.

Yasuhiro Kurasawa, Kyu Joon Lee, Huiqing Hu, Kieu T M Pham, Ziyin Li.

  Cytokinesis in eukaryotes is regulated by a Polo-like kinase-mediated and Aurora B kinase-mediated signalling pathway that promotes the assembly of the actomyosin contractile ring, a cytokinesis machinery conserved across evolution from yeast to humans. Trypanosoma brucei, an early divergent parasitic protozoan, employs an actomyosin-independent mechanism for its unusual cytokinesis that is controlled by a regulatory pathway comprising the Polo-like kinase TbPLK, the Aurora B kinase TbAUK1 and multiple trypanosomatid-specific regulators. However, whether any of these trypanosomatid-specific regulators function as substrates of TbPLK and/or TbAUK1 and how they cooperate with TbPLK and TbAUK1 to promote cytokinesis remain unknown. Here, we demonstrate that TbPLK and TbAUK1 phosphorylate the cytokinesis regulators CIF1 and CIF2 on multiple sites within their intrinsically disordered regions. We further show that TbPLK localization depends on its interaction with CIF1 from S/G2 phases, that TbPLK maintains CIF1 and CIF2 localization from G2 phase until early mitosis, and that TbAUK1 maintains CIF1 and CIF2 localization from late mitosis. Finally, we demonstrate that the cytokinesis regulators CIF4 and FPRC are not substrates of TbPLK and TbAUK1, and that they function upstream of TbPLK and TbAUK1 in the cytokinesis regulatory pathway. Together, these results provide insights into the functional interplay and the order of actions between the two protein kinases and the trypanosomatid-specific cytokinesis regulators in T. brucei.

Keywords:  Aurora B kinase; CIF1; CIF2; Polo-like kinase; Trypanosoma brucei; cytokinesis

DOI:  https://doi.org/10.1098/rsob.220197
Int J Biochem Cell Biol. 2022 Sep 20. pii: S1357-2725(22)00145-5. [Epub ahead of print]152 106300

The multifaceted role of micronuclei in tumour progression: A whole organism perspective.

Molly Guscott, Akash Saha, Jovanna Maharaj, Sarah E McClelland.

  Within most tumour types, cancerous cells exist in a state of aneuploidy, an incorrect chromosome number or structure. Additionally, tumour cells frequently exhibit chromosomal instability; the ongoing loss or gain of whole or parts of chromosomes during cell division. Chromosomal instability results in a high rate of chromosome segregation defects, and a constantly changing genomic landscape. A second consequence of recurrent chromosome segregation defects is the exclusion of mis-segregated chromatin from the newly reforming nucleus. Chromosomes, or chromosome fragments that are not incorporated into the main nucleus are often packaged into extranuclear structures called micronuclei. While the initial impact of micronucleus formation is an imbalance or loss of genetic material in the resulting daughter cells, several other downstream consequences are now known to result from this process. In this review, we discuss the further consequences of micronucleus formation, including how structural changes to the micronuclear envelope, and the rupturing of micronuclear membranes can contribute to metastasis, immune cell activation and overall, tumour progression.

Keywords:  CGAS; Cancer; Chromosomal instability; Micronuclei; Nuclear envelope; STING

DOI:  https://doi.org/10.1016/j.biocel.2022.106300
Nucleic Acids Res. 2022 Oct 06. pii: gkac827. [Epub ahead of print]

The chromatin remodeler RSC prevents ectopic CENP-A propagation into pericentromeric heterochromatin at the chromatin boundary.

Satoru Tsunemine, Hiromi Nakagawa, Yutaka Suzuki, Yota Murakami.

Centromeres of most eukaryotes consist of two distinct chromatin domains: a kinetochore domain, identified by the histone H3 variant, CENP-A, and a heterochromatic domain. How these two domains are separated is unclear. Here, we show that, in Schizosaccharomyces pombe, mutation of the chromatin remodeler RSC induced CENP-ACnp1 misloading at pericentromeric heterochromatin, resulting in the mis-assembly of kinetochore proteins and a defect in chromosome segregation. We find that RSC functions at the kinetochore boundary to prevent CENP-ACnp1 from spreading into neighbouring heterochromatin, where deacetylated histones provide an ideal environment for the spread of CENP-ACnp1. In addition, we show that RSC decompacts the chromatin structure at this boundary, and propose that this RSC-directed chromatin decompaction prevents mis-propagation of CENP-ACnp1 into pericentromeric heterochromatin. Our study provides an insight into how the distribution of distinct chromatin domains is established and maintained.

DOI: https://doi.org/10.1093/nar/gkac827
Microsc Res Tech. 2022 Oct 07.

Mitotic effects of copper oxide nanoparticle on root development and root tip cells of Phaseolus vulgaris L. seeds.

Neslihan Tasar.

  Copper oxide nanoparticle (CuO NP) is used widely in many fields in nanotechnology. For this reason, both production, use, and release to the environment are increasing with each passing day. With the increased use of products that contain nanoparticles (NP) (<100 nm), plants and organisms that constitute the food chain are at risk. In the present study, Phaseolus vulgaris L., a very common food plant, was exposed to metal-based CuO NPs. The anomalies that were caused by CuO NP in germination and mitosis of P. vulgaris were investigated. In the trials, a total of 4 groups (Control, 50, 150, and 300 ppm) were formed and examined in three replications. The determination of the accumulation and elimination rate because of NPs in P. vulgaris that was used in the study was made through X-ray diffraction (XRD), scanning electron microscope (SEM), mapping image, and EDX characteristic spectrum analysis. Also, the mitotic effects on germination, root development, and root tip cells of seeds that were grown by treatment with control, 50, 150, and 300 ppm concentrations were investigated. The study was conducted in three replications in a laboratory setting. All concentrations of CuO NPs caused significant decreases in the mitotic index in the root tip cells of P. vulgaris when compared to the control. The mitotic index reached the lowest level, especially at the highest concentration. Multiple analyzes in the study showed that CuO NPs cause abnormalities in cell division such as C-metaphase, distorted metaphase, distorted anaphase and telophase, chromosome breakage, asynchronous division, advanced chromosomes, micronucleus, and loss of genetic material. These findings also support that the Cytogenetic Test of P. vulgaris can be used to evaluate the genotoxicity of new nanomaterials that are used in many consumer products. In this respect, NPs that are taken up by the organisms in the food chain may pose a danger to higher consumer organisms when they accumulate in the tissue. A control mechanism must be established for the use and contamination of these particles and wider studies must be conducted regarding their effects. HIGHLIGHTS: The effects of CuO nanoparticle, which has a very wide usage area, on root development and mitosis of Phaseolus vulgaris L. plant were investigated in the study. The abnormalities of mitotic division on interphase, prophase, metaphase, anaphase, and telophase were visualized. Evaluation was made considering scanning electron microscopy (SEM) and X-ray diffraction (XRD) results as well.

Keywords:  CuO; genotoxicity; mitotic anomaly; nanoparticle; nanotoxicity

DOI:  https://doi.org/10.1002/jemt.24239
J Cell Biol. 2022 Dec 05. pii: e202203127. [Epub ahead of print]221(12):

Membrane compartmentalization of Ect2/Cyk4/Mklp1 and NuMA/dynein regulates cleavage furrow formation.

Shrividya Sana, Ashwathi Rajeevan, Sachin Kotak.

In animal cells, spindle elongation during anaphase is temporally coupled with cleavage furrow formation. Spindle elongation during anaphase is regulated by NuMA/dynein/dynactin complexes that occupy the polar region of the cell membrane and are excluded from the equatorial membrane. How NuMA/dynein/dynactin are excluded from the equatorial membrane and the biological significance of this exclusion remains unknown. Here, we show that the centralspindlin (Cyk4/Mklp1) and its interacting partner RhoGEF Ect2 are required for NuMA/dynein/dynactin exclusion from the equatorial cell membrane. The Ect2-based (Ect2/Cyk4/Mklp1) and NuMA-based (NuMA/dynein/dynactin) complexes occupy mutually exclusive membrane surfaces during anaphase. The equatorial membrane enrichment of Ect2-based complexes is essential for NuMA/dynein/dynactin exclusion and proper spindle elongation. Conversely, NuMA-based complexes at the polar region of the cell membrane ensure spatially confined localization of Ect2-based complexes and thus RhoA. Overall, our work establishes that membrane compartmentalization of NuMA-based and Ect2-based complexes at the two distinct cell surfaces restricts dynein/dynactin and RhoA for coordinating spindle elongation with cleavage furrow formation.

DOI: https://doi.org/10.1083/jcb.202203127