bims-micesi 2024-09-08 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2024–09–08
ten papers selected by
Valentina Piano, Uniklinik Köln

The Mitotic Checkpoint Complex controls the association of Cdc20 regulatory protein with the ubiquitin ligase APC/C in mitosis.
Role of protein kinase PLK1 in the epigenetic maintenance of centromeres.
Distinct checkpoint and homolog biorientation pathways regulate meiosis I in Drosophila oocytes.
PLK1-mediated phosphorylation cascade activates Mis18 complex to ensure centromere inheritance.
The doublecortin-family kinase ZYG-8DCLK1 regulates microtubule dynamics and motor-driven forces to promote the stability of C. elegans acentrosomal spindles.
The three Plasmodium falciparum Aurora-related kinases display distinct temporal and spatial associations with mitotic structures in asexual blood stage parasites and gametocytes.
Spindle morphology changes between meiosis and mitosis driven by CK2 regulation of the Ran pathway.
CDK5-cyclin B1 regulates mitotic fidelity.
A phosphorylation-controlled switch confers cell cycle-dependent protein relocalization.
mInsc coordinates Par3 and NuMA condensates for assembly of the spindle orientation machinery in asymmetric cell division.

Proc Natl Acad Sci U S A. 2024 Sep 10. 121(37): e2413089121

The Mitotic Checkpoint Complex controls the association of Cdc20 regulatory protein with the ubiquitin ligase APC/C in mitosis.

Danielle Sitry-Shevah, Shirly Miniowitz-Shemtov, Tanya Liburkin Dan, Avram Hershko.

  The ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) and its regulatory protein Cdc20 play important roles in the control of different stages of mitosis. APC/C associated with Cdc20 is active and promotes metaphase-anaphase transition by targeting for degradation inhibitors of anaphase initiation. Earlier in mitosis, premature action of APC/C is prevented by the mitotic checkpoint (or spindle assembly checkpoint) system, which ensures that anaphase is not initiated until all chromosomes are properly attached to the mitotic spindle. The active mitotic checkpoint system promotes the assembly of a Mitotic Checkpoint Complex (MCC), which binds to APC/C and inhibits its activity. The interaction of MCC with APC/C is strongly enhanced by Cdc20 bound to APC/C. While the association of Cdc20 with APC/C was known to be essential for both these stages of mitosis, it was not known how Cdc20 remains bound in spite of ongoing processes, phosphorylation and ubiquitylation, that stimulate its release from APC/C. We find that MCC strongly inhibits the release of Cdc20 from APC/C by the action of mitotic protein kinase Cdk1-cyclin B. This is not due to protection from phosphorylation of specific sites in Cdc20 that affect its interaction with APC/C. Rather, MCC stabilizes the binding to APC/C of partially phosphorylated forms of Cdc20. MCC also inhibits the autoubiquitylation of APC/C-bound Cdc20 and its ubiquitylation-promoted release from APC/C. We propose that these actions of MCC to maintain Cdc20 bound to APC/C in mitosis are essential for the control of mitosis during active mitotic checkpoint and in subsequent anaphase initiation.

Keywords:  cell cycle; mitosis; ubiquitin

DOI:  https://doi.org/10.1073/pnas.2413089121
Science. 2024 Sep 06. 385(6713): 1091-1097

Role of protein kinase PLK1 in the epigenetic maintenance of centromeres.

Duccio Conti, Arianna Esposito Verza, Marion E Pesenti, Verena Cmentowski, Ingrid R Vetter, Dongqing Pan, Andrea Musacchio.

The centromere, a chromosome locus defined by the histone H3-like protein centromeric protein A (CENP-A), promotes assembly of the kinetochore to bind microtubules during cell division. Centromere maintenance requires CENP-A to be actively replenished by dedicated protein machinery in the early G1 phase of the cell cycle to compensate for its dilution after DNA replication. Cyclin-dependent kinases (CDKs) limit CENP-A deposition to once per cell cycle and function as negative regulators outside of early G1. Antithetically, Polo-like kinase 1 (PLK1) promotes CENP-A deposition in early G1, but the molecular details of this process are still unknown. We reveal here a phosphorylation network that recruits PLK1 to the deposition machinery to control a conformational switch required for licensing the CENP-A deposition reaction. Our findings clarify how PLK1 contributes to the epigenetic maintenance of centromeres.

DOI: https://doi.org/10.1126/science.ado5178
bioRxiv. 2024 Aug 21. pii: 2024.08.21.608908. [Epub ahead of print]

Distinct checkpoint and homolog biorientation pathways regulate meiosis I in Drosophila oocytes.

Joanatta G Shapiro, Neha Changela, Janet K Jang, Jay N Joshi, Kim S McKim.

Mitosis and meiosis have two mechanisms for regulating the accuracy of chromosome segregation: error correction and the spindle assembly checkpoint (SAC). We have investigated the function of several checkpoint proteins in meiosis I of Drosophila oocytes. Evidence of a SAC response by several of these proteins is found upon depolymerization of microtubules by colchicine. However, unattached kinetochores or errors in biorientation of homologous chromosomes does not induce a SAC response. Furthermore, the metaphase I arrest does not depend on SAC genes, suggesting the APC is inhibited even if the SAC is silenced. Two SAC proteins, ROD of the ROD-ZW10-Zwilch (RZZ) complex and MPS1, are also required for the biorientation of homologous chromosomes during meiosis I, suggesting an error correction function. Both proteins aid in preventing or correcting erroneous attachments and depend on SPC105R for localization to the kinetochore. We have defined a region of SPC105R, amino acids 123-473, that is required for ROD localization and biorientation of homologous chromosomes at meiosis I. Surprisingly, ROD removal, or "streaming", is independent of the dynein adaptor Spindly and is not linked to the stabilization of end-on attachments. Instead, meiotic RZZ streaming appears to depend on cell cycle stage and may be regulated independently of kinetochore attachment or biorientation status. We also show that dynein adaptor Spindly is also required for biorientation at meiosis I, and surprisingly, the direction of RZZ streaming.
Author Summary: The Spindle Assembly Checkpoint (SAC) is known to delay cell cycle progression until chromosomes are properly attached to microtubules. Meiotic cells often have modified cell cycle phases, and natural arrest points such as metaphase I in Drosophila . We show that in Drosophila oocytes, the SAC is sensitive to loss of microtubules, but not sensitive to a variety of kinetochore attachment errors. Thus, the function of the SAC appears to be limited to monitoring oocyte spindle assembly, and not required for accurate chromosome segregation. However, two of the SAC genes, rod and Mps1 , are required for the biorientation of homologous chromosomes during meiosis I, suggesting an error correction function. Rod is part of the RZZ complex and is notable for its property of streaming off the kinetochores. However, our results show that streaming off the kinetochore may not contribute to RZZ regulation of microtubule attachments, and only be associated with SAC function. Instead, the establishment of stable end-on attachments may occur while RZZ is still present at kinetochore. We suggest that RZZ interacts with multiple motors to promote bidirectional movement of kinetochores along microtubules, which allows chromosomes to find and attach to the correct pole.

DOI: https://doi.org/10.1101/2024.08.21.608908
Science. 2024 Sep 06. 385(6713): 1098-1104

PLK1-mediated phosphorylation cascade activates Mis18 complex to ensure centromere inheritance.

Pragya Parashara, Bethan Medina-Pritchard, Maria Alba Abad, Paula Sotelo-Parrilla, Reshma Thamkachy, David Grundei, Juan Zou, Christos Spanos, Chandni Natalia Kumar, Claire Basquin, Vimal Das, Zhaoyue Yan, Asma Abdullah Al-Murtadha, David A Kelly, Toni McHugh, Axel Imhof, Juri Rappsilber, A Arockia Jeyaprakash.

Accurate chromosome segregation requires the attachment of microtubules to centromeres, epigenetically defined by the enrichment of CENP-A nucleosomes. During DNA replication, CENP-A nucleosomes undergo dilution. To preserve centromere identity, correct amounts of CENP-A must be restored in a cell cycle-controlled manner orchestrated by the Mis18 complex (Mis18α-Mis18β-Mis18BP1). We demonstrate here that PLK1 interacts with the Mis18 complex by recognizing self-primed phosphorylations of Mis18α (Ser54) and Mis18BP1 (Thr78 and Ser93) through its Polo-box domain. Disrupting these phosphorylations perturbed both centromere recruitment of the CENP-A chaperone HJURP and new CENP-A loading. Biochemical and functional analyses showed that phosphorylation of Mis18α and PLK1 binding were required to activate Mis18α-Mis18β and promote Mis18 complex-HJURP interaction. Thus, our study reveals key molecular events underpinning the licensing role of PLK1 in ensuring accurate centromere inheritance.

DOI: https://doi.org/10.1126/science.ado8270
PLoS Genet. 2024 Sep 03. 20(9): e1011373

The doublecortin-family kinase ZYG-8DCLK1 regulates microtubule dynamics and motor-driven forces to promote the stability of C. elegans acentrosomal spindles.

Emily R Czajkowski, Yuntong Zou, Nikita S Divekar, Sarah M Wignall.

Although centrosomes help organize spindles in most cell types, oocytes of most species lack these structures. During acentrosomal spindle assembly in C. elegans oocytes, microtubule minus ends are sorted outwards away from the chromosomes where they form poles, but then these outward forces must be balanced to form a stable bipolar structure. Simultaneously, microtubule dynamics must be precisely controlled to maintain spindle length and organization. How forces and dynamics are tuned to create a stable bipolar structure is poorly understood. Here, we have gained insight into this question through studies of ZYG-8, a conserved doublecortin-family kinase; the mammalian homolog of this microtubule-associated protein is upregulated in many cancers and has been implicated in cell division, but the mechanisms by which it functions are poorly understood. We found that ZYG-8 depletion from oocytes resulted in overelongated spindles with pole and midspindle defects. Importantly, experiments with monopolar spindles revealed that ZYG-8 depletion led to excess outward forces within the spindle and suggested a potential role for this protein in regulating the force-generating motor BMK-1/kinesin-5. Further, we found that ZYG-8 is also required for proper microtubule dynamics within the oocyte spindle and that kinase activity is required for its function during both meiosis and mitosis. Altogether, our findings reveal new roles for ZYG-8 in oocytes and provide insights into how acentrosomal spindles are stabilized to promote faithful meiosis.

DOI: https://doi.org/10.1371/journal.pgen.1011373
mSphere. 2024 Sep 05. e0046524

The three Plasmodium falciparum Aurora-related kinases display distinct temporal and spatial associations with mitotic structures in asexual blood stage parasites and gametocytes.

Matthias Wyss, Basil T Thommen, Jacob Kofler, Eilidh Carrington, Nicolas M B Brancucci, Till S Voss.

  Aurora kinases are crucial regulators of mitotic cell cycle progression in eukaryotes. The protozoan malaria parasite Plasmodium falciparum replicates via schizogony, a specialized mode of cell division characterized by consecutive asynchronous rounds of nuclear division by closed mitosis followed by a single cytokinesis event producing dozens of daughter cells. P. falciparum encodes three Aurora-related kinases (PfARKs) that have been reported essential for parasite proliferation, but their roles in regulating schizogony have not yet been explored in great detail. Here, we engineered transgenic parasite lines expressing GFP-tagged PfARK1-3 to provide a systematic analysis of their expression timing and subcellular localization throughout schizogony as well as in the non-dividing gametocyte stages, which are essential for malaria transmission. We demonstrate that all three PfARKs display distinct and highly specific and exclusive spatiotemporal associations with the mitotic machinery. In gametocytes, PfARK3 is undetectable, and PfARK1 and PfARK2 show male-specific expression in late-stage gametocytes, consistent with their requirement for endomitosis during male gametogenesis in the mosquito vector. Our combined data suggest that PfARK1 and PfARK2 have non-overlapping roles in centriolar plaque maturation, assembly of the mitotic spindle, kinetochore-spindle attachment and chromosome segregation, while PfARK3 seems to be exquisitely involved in daughter cell cytoskeleton assembly and cytokinesis. These important new insights provide a reliable foundation for future research aiming at the functional investigation of these divergent and possibly drug-targetable Aurora-related kinases in mitotic cell division of P. falciparum and related apicomplexan parasites.IMPORTANCEMalaria parasites replicate via non-conventional modes of mitotic cell division, such as schizogony, employed by the disease-causing stages in the human blood or endomitosis during male gametogenesis in the mosquito vector. Understanding the molecular mechanisms regulating cell division in these divergent unicellular eukaryotes is not only of scientific interest but also relevant to identify potential new antimalarial drug targets. Here, we carefully examined the subcellular localization of all three Plasmodium falciparum Aurora-related kinases (ARKs), distantly related homologs of Aurora kinases that coordinate mitosis in model eukaryotes. Detailed fluorescence microscopy-based analyses revealed distinct, specific, and exclusive spatial associations for each parasite ARK with different components of the mitotic machinery and at different phases of the cell cycle during schizogony and gametocytogenesis. This comprehensive set of results closes important gaps in our fragmentary knowledge on this important group of kinases and offers a valuable source of information for future functional studies.

Keywords:  Aurora-related kinase; Plasmodium falciparum; cell division; gametocytes; malaria; mitosis; schizogony

DOI:  https://doi.org/10.1128/msphere.00465-24
bioRxiv. 2024 Jul 25. pii: 2024.07.25.605073. [Epub ahead of print]

Spindle morphology changes between meiosis and mitosis driven by CK2 regulation of the Ran pathway.

Helena Cantwell, Hieu Nguyen, Arminja Kettenbach, Rebecca Heald.

The transition from meiotic divisions in the oocyte to embryonic mitoses is a critical step in animal development. Despite negligible changes to cell size and shape, following fertilization the small, barrel-shaped meiotic spindle is replaced by a large zygotic spindle that nucleates abundant astral microtubules at spindle poles. To probe underlying mechanisms, we applied a drug screening approach using Ciona eggs and found that inhibition of Casein Kinase 2 (CK2) caused a shift from meiotic to mitotic-like spindle morphology with nucleation of robust astral microtubules, an effect reproduced in cytoplasmic extracts prepared from Xenopus eggs. In both species, CK2 activity decreased at fertilization. Phosphoproteomic differences between Xenopus meiotic and mitotic extracts that also accompanied CK2 inhibition pointed to RanGTP-regulated factors as potential targets. Interfering with RanGTP-driven microtubule formation suppressed astral microtubule growth caused by CK2 inhibition. These data support a model in which CK2 activity attenuation at fertilization leads to activation of RanGTP-regulated microtubule effectors that induce mitotic spindle morphology.

DOI: https://doi.org/10.1101/2024.07.25.605073
Nature. 2024 Sep 04.

CDK5-cyclin B1 regulates mitotic fidelity.

Xiao-Feng Zheng, Aniruddha Sarkar, Humphrey Lotana, Aleem Syed, Huy Nguyen, Richard G Ivey, Jacob J Kennedy, Jeffrey R Whiteaker, Bartłomiej Tomasik, Kaimeng Huang, Feng Li, Alan D D'Andrea, Amanda G Paulovich, Kavita Shah, Alexander Spektor, Dipanjan Chowdhury.

CDK1 has been known to be the sole cyclin-dependent kinase (CDK) partner of cyclin B1 to drive mitotic progression1. Here we demonstrate that CDK5 is active during mitosis and is necessary for maintaining mitotic fidelity. CDK5 is an atypical CDK owing to its high expression in post-mitotic neurons and activation by non-cyclin proteins p35 and p392. Here, using independent chemical genetic approaches, we specifically abrogated CDK5 activity during mitosis, and observed mitotic defects, nuclear atypia and substantial alterations in the mitotic phosphoproteome. Notably, cyclin B1 is a mitotic co-factor of CDK5. Computational modelling, comparison with experimentally derived structures of CDK-cyclin complexes and validation with mutational analysis indicate that CDK5-cyclin B1 can form a functional complex. Disruption of the CDK5-cyclin B1 complex phenocopies CDK5 abrogation in mitosis. Together, our results demonstrate that cyclin B1 partners with both CDK5 and CDK1, and CDK5-cyclin B1 functions as a canonical CDK-cyclin complex to ensure mitotic fidelity.

DOI: https://doi.org/10.1038/s41586-024-07888-x
Nat Cell Biol. 2024 Aug 29.

A phosphorylation-controlled switch confers cell cycle-dependent protein relocalization.

Xiaofu Cao, Shiying Huang, Mateusz M Wagner, Yuan-Ting Cho, Din-Chi Chiu, Krista M Wartchow, Artur Lazarian, Laura Beth McIntire, Marcus B Smolka, Jeremy M Baskin.

Tools for acute manipulation of protein localization enable elucidation of spatiotemporally defined functions, but their reliance on exogenous triggers can interfere with cell physiology. This limitation is particularly apparent for studying mitosis, whose highly choreographed events are sensitive to perturbations. Here we exploit the serendipitous discovery of a phosphorylation-controlled, cell cycle-dependent localization change of the adaptor protein PLEKHA5 to develop a system for mitosis-specific protein recruitment to the plasma membrane that requires no exogenous stimulus. Mitosis-enabled anchor-away/recruiter system comprises an engineered, 15 kDa module derived from PLEKHA5 capable of recruiting functional protein cargoes to the plasma membrane during mitosis, either through direct fusion or via GFP-GFP nanobody interaction. Applications of the mitosis-enabled anchor-away/recruiter system include both knock sideways to rapidly extract proteins from their native localizations during mitosis and conditional recruitment of lipid-metabolizing enzymes for mitosis-selective editing of plasma membrane lipid content, without the need for exogenous triggers or perturbative synchronization methods.

DOI: https://doi.org/10.1038/s41556-024-01495-8
Int J Biol Macromol. 2024 Aug 30. pii: S0141-8130(24)05932-4. [Epub ahead of print]279(Pt 2): 135126

mInsc coordinates Par3 and NuMA condensates for assembly of the spindle orientation machinery in asymmetric cell division.

Shijing Huang, Minjie Fu, Aihong Gu, Ruiqian Zhao, Ziheng Liu, Wei Hua, Ying Mao, Wenyu Wen.

  As a fundamental process governing the self-renewal and differentiation of stem cells, asymmetric cell division is controlled by several conserved regulators, including the polarity protein Par3 and the microtubule-associated protein NuMA, which orchestrate the assembly and interplay of the Par3/Par6/mInsc/LGN complex at the apical cortex and the LGN/Gαi/NuMA/Dynein complex at the mitotic spindle to ensure asymmetric segregation of cell fate determinants. However, this model, which is well-supported by genetic studies, has been challenged by evidence of competitive interaction between NuMA and mInsc for LGN. Here, the solved crystal structure of the Par3/mInsc complex reveals that mInsc competes with Par6β for Par3, raising questions about how proteins assemble overlapping targets into functional macromolecular complexes. Unanticipatedly, we discover that Par3 can recruit both Par6β and mInsc by forming a dynamic condensate through phase separation. Similarly, the phase-separated NuMA condensate enables the coexistence of competitive NuMA and mInsc with LGN in the same compartment. Bridge by mInsc, Par3/Par6β and LGN/NuMA condensates coacervate, robustly enriching all five proteins both in vitro and within cells. These findings highlight the pivotal role of protein condensates in assembling multi-component signalosomes that incorporate competitive protein-protein interaction pairs, effectively overcoming stoichiometric constraints encountered in conventional protein complexes.

Keywords:  Asymmetric cell division; Competitive binding; NuMA condensate; Par3 condensate

DOI:  https://doi.org/10.1016/j.ijbiomac.2024.135126