bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2024–11–24
eleven papers selected by
Valentina Piano, Uniklinik Köln
  1. NuSAP4 regulates chromosome segregation in Trypanosoma brucei by promoting bipolar spindle assembly.
  2. A coordinated kinase and phosphatase network regulates Stu2 recruitment to yeast kinetochores.
  3. Nanoscale 3D DNA tracing reveals the mechanism of self-organization of mitotic chromosomes.
  4. Securin regulates the spatiotemporal dynamics of separase.
  5. Acentric chromosome congression and alignment on the metaphase plate via kinetochore-independent forces in Drosophila.
  6. Structure of the microtubule anchoring factor NEDD1 bound to the γ-tubulin ring complex.
  7. Cryo-EM structures of apo-APC/C and APC/CCDH1:EMI1 complexes provide insights into APC/C regulation.
  8. DSN1 Interaction With Centromere-Associated Proteins Promotes Chromosomal Instability in Hepatocellular Carcinoma.
  9. Transcription of a centromere-enriched retroelement and local retention of its RNA are significant features of the CENP-A chromatin landscape.
  10. RAD52 and ERCC6L/PICH have a compensatory relationship for genome stability in mitosis.
  11. TSG101 depletion dysregulates mitochondria and PML NBs, triggering MAD2-overexpressing interphase cell death (MOID) through AIFM1-PML-DAXX pathway.
  1. Commun Biol. 2024 Nov 16. 7(1): 1524
    NuSAP4 regulates chromosome segregation in Trypanosoma brucei by promoting bipolar spindle assembly.
    Qing Zhou, Ziyin Li.
      Faithful chromosome segregation in eukaryotes requires the assembly of a bipolar spindle and the faithful attachment of kinetochores to spindle microtubules, which are regulated by various spindle-associated proteins (SAPs) that play distinct functions in regulating spindle dynamics and microtubule-kinetochore attachment. The protozoan parasite Trypanosoma brucei employs evolutionarily conserved and kinetoplastid-specific proteins, including some kinetoplastid-specific nucleus- and spindle-associated proteins (NuSAPs), to regulate chromosome segregation. Here, we characterized NuSAP4 and its functional interplay with diverse SAPs in promoting chromosome segregation in T. brucei. NuSAP4 associates with the spindle during mitosis and concentrates at spindle poles where it interacts with SPB1 and MAP103. Knockdown of NuSAP4 impairs chromosome segregation by disrupting bipolar spindle assembly and spindle pole protein localization. These results uncover the mechanistic role of NuSAP4 in regulating chromosome segregation by promoting bipolar spindle assembly, and highlight the unusual features of mitotic regulation by spindle-associated proteins in this early divergent microbial eukaryote.
    DOI:  https://doi.org/10.1038/s42003-024-07248-5
  2. bioRxiv. 2024 Nov 03. pii: 2024.11.01.621564. [Epub ahead of print]
    A coordinated kinase and phosphatase network regulates Stu2 recruitment to yeast kinetochores.
    Michael G Stewart, Joseph S Carrier, Jacob A Zahm, Stephen C Harrison, Matthew P Miller.
      Cells coordinate diverse events at anaphase onset, including separase activation, cohesin cleavage, chromosome separation, and spindle reorganization. Regulation of the XMAP215 family member and microtubule polymerase, Stu2, at the metaphase-anaphase transition determines a specific redistribution from kinetochores to spindle microtubules. We show that cells modulate Stu2 kinetochore-microtubule localization by Polo-like kinase1/Cdc5-mediated phosphorylation of T866, near the Stu2 C-terminus, thereby promoting dissociation from the kinetochore Ndc80 complex. Cdk/Cdc28 likely primes Cdc5:Stu2 interaction. Cdc28 activity is also required for Stu2 nuclear import. PP2A Cdc55 actively opposes Cdc5 activity on Stu2 T866 during metaphase. This counter-regulation allows for switchlike redistribution of Stu2 pT866 at anaphase onset when separase inhibits PP2A Cdc55 . Blocking Stu2 T866 phosphorylation disrupts anaphase spindle progression, and we infer that PP2A Cdc55 regulates the mitotic spindle by dephosphorylating Stu2 and other MAPs. These data support a model in which increased phosphorylation at anaphase onset results from phosphatase inhibition and point to a larger regulatory network that facilitates rapid cytoskeletal modulation required for anaphase spindle maintenance.
    SUMMARY: Stu2 displays dynamic localization patterns in the cell cycle, with different kinetochore and microtubule distribution during distinct phases. Phosphorylation near Stu2's C-terminus reduces its attachment to kinetochores to promote its microtubule activity in anaphase. Cdc5 and PP2A Cdc55 play counteracting roles in this pathway to promote proper timing of Stu2 phosphorylation.
    DOI:  https://doi.org/10.1101/2024.11.01.621564
  3. bioRxiv. 2024 Oct 29. pii: 2024.10.28.620625. [Epub ahead of print]
    Nanoscale 3D DNA tracing reveals the mechanism of self-organization of mitotic chromosomes.
    Kai Sandvold Beckwith, Andreas Brunner, Natalia Rosalia Morero, Ralf Jungmann, Jan Ellenberg.
      How genomic DNA is folded during cell division to form the characteristic rod-shaped mitotic chromosomes essential for faithful genome inheritance is a long-standing open question in biology. Here, we use nanoscale DNA-tracing in single dividing cells to directly visualize how the 3D fold of genomic DNA changes during mitosis, at scales from single loops to entire chromosomes. Our structural analysis reveals a characteristic genome scaling minimum at 6-8 Mbp in mitosis. Combined with data-driven modeling and molecular perturbations, we can show that very large and strongly overlapping loops formed by Condensins are the fundamental structuring principle of mitotic chromosomes. These loops compact chromosomes locally and globally to the limit set by chromatin self-repulsion. The characteristic length, density and increasingly overlapping structure of mitotic loops we observe in 3D, fully explain how the rod-shaped mitotic chromosome structure emerges by self-organization during cell division.
    DOI:  https://doi.org/10.1101/2024.10.28.620625
  4. J Cell Biol. 2025 Feb 03. pii: e202312099. [Epub ahead of print]224(2):
    Securin regulates the spatiotemporal dynamics of separase.
    Christopher G Sorensen Turpin, Dillon Sloan, Marian LaForest, Lindsey Klebanow, Diana Mitchell, Aaron F Severson, Joshua N Bembenek.
      Separase regulates multiple aspects of the metaphase-to-anaphase transition. Separase cleaves cohesin to allow chromosome segregation and localizes to vesicles to promote exocytosis. The anaphase-promoting complex/cyclosome (APC/C) activates separase by ubiquitinating its inhibitory chaperone, securin, triggering its degradation. How this pathway controls the exocytic function of separase is unknown. During meiosis I, securin is degraded over several minutes, while separase rapidly relocalizes from kinetochore structures at the spindle and cortex to sites of action on chromosomes and vesicles at anaphase onset. The loss of cohesin coincides with the relocalization of separase to the chromosome midbivalent at anaphase onset. APC/C depletion prevents separase relocalization, while securin depletion causes precocious separase relocalization. Expression of non-degradable securin inhibits chromosome segregation, exocytosis, and separase localization to vesicles but not to the anaphase spindle. We conclude that APC/C-mediated securin degradation controls separase localization. This spatiotemporal regulation will impact the effective local concentration of separase for more precise targeting of substrates in anaphase.
    DOI:  https://doi.org/10.1083/jcb.202312099
  5. Genetics. 2024 Nov 18. pii: iyae188. [Epub ahead of print]
    Acentric chromosome congression and alignment on the metaphase plate via kinetochore-independent forces in Drosophila.
    Hannah Vicars, Alison Mills, Travis Karg, William Sullivan.
      Chromosome congression and alignment on the metaphase plate involves lateral and microtubule plus-end interactions with the kinetochore. Here we take advantage of our ability to efficiently generate a GFP-marked acentric X chromosome fragment in Drosophila neuroblasts to identify forces acting on chromosome arms that drive congression and alignment. We find acentrics efficiently congress and align on the metaphase plate, often more rapidly than kinetochore-bearing chromosomes. Unlike intact chromosomes, the paired sister acentrics oscillate as they move to and reside on the metaphase plate in a plane distinct and significantly further from the main mass of intact chromosomes. Consequently, at anaphase onset acentrics are oriented either parallel or perpendicular to the spindle. Parallel-oriented sisters separate by sliding while those oriented perpendicularly separate via unzipping. This oscillation, together with the fact that in the presence of spindles with disrupted interpolar microtubules acentrics are rapidly shunted away from the poles, indicates that distributed plus-end directed forces are primarily responsible for acentric migration. This conclusion is supported by the observation that reduction of EB1 preferentially disrupts acentric alignment. Taken together these studies suggest that plus-end forces mediated by the outer interpolar microtubules contribute significantly to acentric congression and alignment. Surprisingly, we observe disrupted telomere pairing and alignment of sister acentrics indicating that the kinetochore is required to ensure proper gene-to-gene alignment of sister chromatids. Finally, we demonstrate that like mammalian cells, the Drosophila congressed chromosomes on occasion exhibit a toroid configuration.
    Keywords:  Drosophila melanogaster; chromosome biology; kinetochore; mitosis
    DOI:  https://doi.org/10.1093/genetics/iyae188
  6. bioRxiv. 2024 Nov 05. pii: 2024.11.05.622067. [Epub ahead of print]
    Structure of the microtubule anchoring factor NEDD1 bound to the γ-tubulin ring complex.
    Hugo Muñoz-Hernández, Yixin Xu, Daniel Zhang, Allen Xue, Amol Aher, Aitor Pellicer Camardiel, Ellie Walker, Florina Marxer, Tarun M Kapoor, Michal Wieczorek.
      The γ-tubulin ring complex (γ-TuRC) is an essential multiprotein assembly, in which γ-tubulin, GCP2-6, actin, MZT1 and MZT2 form an asymmetric cone-shaped structure that provides a template for microtubule nucleation. The γ-TuRC is recruited to microtubule organizing centers (MTOCs), such as centrosomes and pre-existing mitotic spindle microtubules, via the evolutionarily-conserved attachment factor NEDD1. NEDD1 contains an N-terminal WD40 domain that binds to microtubules, and a C-terminal domain that associates with the γ-TuRC. However, the structural basis of the NEDD1-γ-TuRC interaction is not known. Here, we report cryo-electron microscopy (cryo-EM) structures of NEDD1 bound to the human γ-TuRC in the absence or presence of the activating factor CDK5RAP2, which interacts with GCP2 to induce conformational changes in the γ-TuRC and promote its microtubule nucleating function. We found that the C-terminus of NEDD1 forms a tetrameric α-helical assembly that contacts the lumen of the γ-TuRC cone, is anchored to GCP4, 5 and 6 via protein modules consisting of MZT1 & GCP3 subcomplexes, and orients its microtubule-binding WD40 domains away from the complex. We biochemically tested our structural models by identifying NEDD1 mutants unable to pull-down γ -tubulin from cultured cells. The structure of the γ-TuRC simultaneously bound to NEDD1 and CDK5RAP2 reveals that both factors can associate with the "open" conformation of the complex. Our results show that NEDD1 does not induce conformational changes in the γ-TuRC, but suggest that anchoring of γ-TuRC-capped microtubules by NEDD1 would be structurally compatible with the significant conformational changes experienced by the γ-TuRC during microtubule nucleation.
    DOI:  https://doi.org/10.1101/2024.11.05.622067
  7. Nat Commun. 2024 Nov 21. 15(1): 10074
    Cryo-EM structures of apo-APC/C and APC/CCDH1:EMI1 complexes provide insights into APC/C regulation.
    Anna Höfler, Jun Yu, Jing Yang, Ziguo Zhang, Leifu Chang, Stephen H McLaughlin, Geoffrey W Grime, Elspeth F Garman, Andreas Boland, David Barford.
      APC/C is a multi-subunit complex that functions as a master regulator of cell division. It controls progression through the cell cycle by timely marking mitotic cyclins and other cell cycle regulatory proteins for degradation. The APC/C itself is regulated by the sequential action of its coactivator subunits CDC20 and CDH1, post-translational modifications, and its inhibitory binding partners EMI1 and the mitotic checkpoint complex. In this study, we took advantage of developments in cryo-electron microscopy to determine the structures of human APC/CCDH1:EMI1 and apo-APC/C at 2.9 Å and 3.2 Å resolution, respectively, providing insights into the regulation of APC/C activity. The high-resolution maps allow the unambiguous assignment of an α-helix to the N-terminus of CDH1 (CDH1α1) in the APC/CCDH1:EMI1 ternary complex. We also identify a zinc-binding module in APC2 that confers structural stability to the complex, and we confirm the presence of zinc ions experimentally. Finally, due to the higher resolution and well defined density of these maps, we are able to build, aided by AlphaFold predictions, several intrinsically disordered regions in different APC/C subunits that likely play a role in proper APC/C assembly and regulation of its activity.
    DOI:  https://doi.org/10.1038/s41467-024-54398-5
  8. Mol Carcinog. 2024 Nov 19.
    DSN1 Interaction With Centromere-Associated Proteins Promotes Chromosomal Instability in Hepatocellular Carcinoma.
    Hongrui Zhou, Mengxue Zhang, Jiabing Lian, Ruichang Wang, Zhe Yang, Jin Wang, Xiuli Bi.
      Hepatocellular carcinoma (HCC) is the most prevalent type of liver cancer. Dosage suppressor of NNF1 (DSN1), a component of the MIS12 kinetochore complex, encodes a kinetochore protein crucial for proper mitotic assembly. The role of DSN1 in HCC remains to be elucidated. In this study, we utilized The Cancer Genome Atlas, the Hepatocellular carcinoma Cell Database, and other databases to analyze DSN1 expression and prognosis in samples from patients with HCC. We investigated the signaling pathways regulated by DSN1 and their implications in HCC. Additionally, we engineered siRNAsiRNA/shRNA/shRNA and overexpression vectors for DSN1 and assessed the specific mechanisms of regulatory pathways of DSN1 in hepatoma cell lines and subcutaneous tumor xenograft model. Our findings revealed that DSN1 expression was significantly upregulated in patients with HCC, correlating with decreased survival rates. Elevated DSN1 expression led to the overproduction of cell cycle-related proteins through direct interaction with Centromere Protein T. This interaction contributes to chromosomal instability in patients with HCC, resulting in an aberrant cell cycle and fostering the development and progression of HCC. Increased DSN1 expression is pivotal in HCC initiation and progression. Investigating DSN1 offers valuable insights into the pathogenesis, treatment, and prevention of HCC.
    Keywords:  DSN1; cell cycle regulation; chromosomal instability; hepatocellular carcinoma
    DOI:  https://doi.org/10.1002/mc.23845
  9. Genome Biol. 2024 Nov 18. 25(1): 295
    Transcription of a centromere-enriched retroelement and local retention of its RNA are significant features of the CENP-A chromatin landscape.
    B J Chabot, R Sun, A Amjad, S J Hoyt, L Ouyang, C Courret, R Drennan, L Leo, A M Larracuente, L J Core, R J O'Neill, B G Mellone.
       BACKGROUND: Centromeres depend on chromatin containing the conserved histone H3 variant CENP-A for function and inheritance, while the role of centromeric DNA repeats remains unclear. Retroelements are prevalent at centromeres across taxa and represent a potential mechanism for promoting transcription to aid in CENP-A incorporation or for generating RNA transcripts to maintain centromere integrity.
    RESULTS: In this study, we probe into the transcription and RNA localization of the centromere-enriched retroelement G2/Jockey-3 (hereafter referred to as Jockey-3) in Drosophila melanogaster, currently the only in vivo model with assembled centromeres. We find that Jockey-3 is a major component of the centromeric transcriptome and produces RNAs that localize to centromeres in metaphase. Leveraging the polymorphism of Jockey-3 and a de novo centromere system, we show that these RNAs remain associated with their cognate DNA sequences in cis, suggesting they are unlikely to perform a sequence-specific function at all centromeres. We show that Jockey-3 transcription is positively correlated with the presence of CENP-A and that recent Jockey-3 transposition events have occurred preferentially at CENP-A-containing chromatin.
    CONCLUSIONS: We propose that Jockey-3 preferentially inserts at the centromere to ensure its own selfish propagation, while contributing to transcription across these regions. Given the conservation of retroelements as centromere components through evolution, our findings may offer a basis for understanding similar associations in other species.
    DOI:  https://doi.org/10.1186/s13059-024-03433-1
  10. PLoS Genet. 2024 Nov 19. 20(11): e1011479
    RAD52 and ERCC6L/PICH have a compensatory relationship for genome stability in mitosis.
    Beth Osia, Arianna Merkell, Felicia Wednesday Lopezcolorado, Xiaoli Ping, Jeremy M Stark.
      Mammalian RAD52 is a DNA repair factor with strand annealing and recombination mediator activities that appear important in both interphase and mitotic cells. Nonetheless, RAD52 is dispensable for cell viability. To query RAD52 synthetic lethal relationships, we performed genome-wide CRISPR knock-out screens and identified hundreds of candidate synthetic lethal interactions. We then performed secondary screening and identified genes for which depletion causes reduced viability and elevated genome instability (increased 53BP1 nuclear foci) in RAD52-deficient cells. One such factor was ERCC6L, which marks DNA bridges during anaphase, and hence is important for genome stability in mitosis. Thus, we investigated the functional interrelationship between RAD52 and ERCC6L. We found that RAD52 deficiency increases ERCC6L-coated anaphase ultrafine bridges, and that ERCC6L depletion causes elevated RAD52 foci in prometaphase and interphase cells. These effects were enhanced with replication stress (i.e. hydroxyurea) and topoisomerase IIα inhibition (ICRF-193), where post-treatment effect timings were consistent with defects in addressing stress in mitosis. Altogether, we suggest that RAD52 and ERCC6L co-compensate to protect genome stability in mitosis.
    DOI:  https://doi.org/10.1371/journal.pgen.1011479
  11. Cell Death Dis. 2024 Nov 17. 15(11): 838
    TSG101 depletion dysregulates mitochondria and PML NBs, triggering MAD2-overexpressing interphase cell death (MOID) through AIFM1-PML-DAXX pathway.
    Yao Xi, Rui Xu, Shengnan Chen, Jiezhu Fang, Xiang Duan, Yidan Zhang, Guoli Zhong, Zhifei He, Yan Guo, Xinyu Li, Wenzhi Tao, Yang Li, Yan Li, Lei Fang, Yohei Niikura.
      Overexpression of mitotic arrest deficiency 2 (MAD2/MAD2L1), a pivotal component of the spindle assembly checkpoint (SAC), resulted in many types of cancer. Here we show that the depletion of tumor susceptibility gene 101 (TSG101), causes synthetic dosage lethality (SDL) in MAD2-overexpressing cells, and we term this cell death MAD2-overexpressing interphase cell death (MOID). The induction of MOID depends on PML and DAXX mediating mitochondrial AIFM1-release. MAD2, TSG101, and AIF-PML-DAXX axis regulate mitochondria, PML nuclear bodies (NBs), and autophagy with close inter-dependent protein stability in survival cells. Loss of C-terminal phosphorylation(s) of TSG101 and closed (C-)MAD2-overexpression contribute to induce MOID. In survival cells, both MAD2 and TSG101 localize at PML NBs in interphase, and TSG101 Y390 phosphorylation is required for localization of TSG101 to PML NBs. PML release from PML NBs through PML deSUMOylation contributes to induce MOID. The post-transcriptional/translational cell death machinery and the non-canonical transcriptional regulation are intricately linked to MOID, and ER-MAM, may serve as a crucial intersection for MOID signaling.
    DOI:  https://doi.org/10.1038/s41419-024-07229-w
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