bims-micesi 2024-11-03 papers

Issue of 2024–11–03
eight papers selected by
Valentina Piano, Uniklinik Köln

Drug Discov Ther. 2024 Oct 26.

Mitotic abnormalities and spindle assembly checkpoint inactivation in a cell model of Shwachman-Diamond syndrome with mutations in the Shwachman-Bodian-Diamond syndrome gene, 258+2T > C.

Yukihiro Sera, Tsuneo Imanaka, Yusuke Iguchi, Masafumi Yamaguchi.

Hematologic abnormalities are the most common symptoms of Shwachman-Diamond syndrome (SDS). The causative gene for SDS is the Shwachman-Bodian-Diamond syndrome (SBDS) gene; however, the function of SBDS and pathogenesis of each condition in SDS are largely unknown. SBDS is known to be localized at mitotic spindles and stabilizes microtubules. Previously, we demonstrated that SBDS is ubiquitinated and subsequently degraded in the mitotic phase, thereby accelerating mitotic progression. In this study, we examined mitosis in a myeloid cell model of SDS (SDS cells). 4',6-Diamidino-2-phenylindole (DAPI)-stained chromosome observation and cell cycle analysis of nocodazole-synchronized cells revealed that the SDS cells have abnormally rapid mitosis. In addition, many lagging chromosomes and micronuclei were detected. Moreover, the phosphorylation of threonine tyrosine kinase, the crucial kinase of the spindle assembly checkpoint (SAC), was suppressed. Chromosomal instability caused by SAC dysfunction may cause a variety of clinical conditions, including hematologic tumors in patients with SDS.

Keywords: Shwachman-Diamond syndrome; chromosomal instability; mitosis; spindle assembly checkpoint

DOI: https://doi.org/10.5582/ddt.2024.01070

Nat Commun. 2024 Oct 29. 15(1): 9213

Regulation of minimal spindle midzone organization by mitotic kinases.

Wei Ming Lim, Wei-Xiang Chew, Arianna Esposito Verza, Marion Pesenti, Andrea Musacchio, Thomas Surrey.

During cell division, the microtubule cytoskeleton undergoes dramatic cell cycle-driven reorganizations of its architecture. Coordinated by changes in the phosphorylation patterns of a multitude of microtubule associated proteins, the mitotic spindle first self-assembles to capture the chromosomes and then reorganizes in anaphase as the chromosomes are segregated. A key protein for this reorganization is PRC1 which is differentially phosphorylated by the mitotic kinases CDK1 and PLK1. How the phosphorylation state of PRC1 orchestrates spindle reorganization is not understood mechanistically. Here, we reconstitute in vitro the transition between metaphase and anaphase-like microtubule architectures triggered by the changes in PRC1 phosphorylation. We find that whereas PLK1 regulates its own recruitment by PRC1, CDK1 controls the affinity of PRC1 for antiparallel microtubule binding. Dephosphorylation of CDK1-phosphorylated PRC1 is required and sufficient to trigger the reorganization of a minimal anaphase midzone in the presence of the midzone length controlling kinesin KIF4A. These results demonstrate how phosphorylation-controlled affinity changes regulate the architecture of active microtubule networks, providing new insight into the mechanistic underpinnings of the cell cycle-driven reorganization of the central spindle during mitosis.

DOI: https://doi.org/10.1038/s41467-024-53500-1

Proc Natl Acad Sci U S A. 2024 Nov 05. 121(45): e2413873121

A noncanonical GTPase signaling mechanism controls exit from mitosis in budding yeast.

Xiaoxue Zhou, Shannon Y Weng, Stephen P Bell, Angelika Amon.

In the budding yeast Saccharomyces cerevisiae, exit from mitosis is coupled to spindle position to ensure successful genome partitioning between mother and daughter cells. This coupling occurs through a GTPase signaling cascade known as the mitotic exit network (MEN). The MEN senses spindle position via a Ras-like GTPase Tem1 which localizes to the spindle pole bodies (SPBs, yeast equivalent of centrosomes) during anaphase and signals to its effector protein kinase Cdc15. How Tem1 couples the status of spindle position to MEN activation is not fully understood. Here, we show that Cdc15 has a relatively weak preference for Tem1GTP and Tem1's nucleotide state does not change upon MEN activation. Instead, we find that Tem1's nucleotide cycle establishes a localization-based concentration difference in the cell where only Tem1GTP is recruited to the SPB, and spindle position regulates the MEN by controlling Tem1 localization to the SPB. SPB localization of Tem1 primarily functions to promote Tem1-Cdc15 interaction for MEN activation by increasing the effective concentration of Tem1. Consistent with this model, we demonstrate that artificially tethering Tem1 to the SPB or concentrating Tem1 in the cytoplasm with genetically encoded multimeric nanoparticles could bypass the requirement of Tem1GTP and correct spindle position for MEN activation. This localization/concentration-based GTPase signaling mechanism for Tem1 differs from the canonical Ras-like GTPase signaling paradigm and is likely relevant to other localization-based signaling scenarios.

Keywords: GTPase signaling; cell cycle control; mitotic exit network; spindle position checkpoint

DOI: https://doi.org/10.1073/pnas.2413873121

J Cell Biol. 2025 Jan 06. pii: e202405176. [Epub ahead of print]224(1):

Kinetochores grip microtubules with directionally asymmetric strength.

Joshua D Larson, Natalie A Heitkamp, Lucas E Murray, Andrew R Popchock, Sue Biggins, Charles L Asbury.

For accurate mitosis, all chromosomes must achieve "biorientation," with replicated sister chromatids coupled via kinetochores to the plus ends of opposing microtubules. However, kinetochores first bind the sides of microtubules and subsequently find plus ends through a trial-and-error process; accurate biorientation depends on the selective release of erroneous attachments. Proposed mechanisms for error-correction have focused mainly on plus-end attachments. Whether erroneous side attachments are distinguished from correct side attachments is unknown. Here, we show that side-attached kinetochores are very sensitive to microtubule polarity, gripping sixfold more strongly when pulled toward plus versus minus ends. This directionally asymmetric grip is conserved in human and yeast subcomplexes, and it correlates with changes in the axial arrangement of subcomplexes within the kinetochore, suggesting that internal architecture dictates attachment strength. We propose that the kinetochore's directional grip promotes accuracy during early mitosis by stabilizing correct attachments even before both sisters have found plus ends.

DOI: https://doi.org/10.1083/jcb.202405176

Turk J Biol. 2024 ;48(5): 308-318

Disappearance of Cdc14 from the daughter spindle pole body requires Glc7-Bud14.

İdil Kirdök, Ayşe Koca Çaydaşi.

Background/aim: The conserved phosphatase Cdc14 facilitates mitotic exit in budding yeast by counteracting mitotic cyclin-dependent kinase activity. Cdc14 is kept in the nucleolus until anaphase onset, when it is released transiently into the nucleoplasm. In late anaphase, Cdc14 is fully released into the cytoplasm upon activation of the mitotic exit network (MEN) to trigger mitotic exit. Cdc14 also localizes to yeast spindle pole bodies (SPBs) in anaphase and dephosphorylates key targets residing on SPBs to allow SPB duplication and prime the MEN. Protein phosphatase 1 (Glc7) with regulatory subunit Bud14 is another phosphatase that plays a key role in the spatiotemporal control of mitotic exit. In this study, we investigated the regulation of Cdc14 localization by Bud14-Glc7.
Materials and methods: We used fluorescence microscopy to analyze Cdc14 localization in BUD14 wildtype and BUD14 knockout cells (bud14Δ) as well as in cells expressing a mutant allele of BUD14 (bud14-F379A) that cannot bind Glc7. We also utilized a yeast two-hybrid (Y2H) system to examine the interaction of Bud14 with Cdc14.
Results: We found that Cdc14 remains at the SPBs longer in bud14Δ and bud14-F379A compared to wildtype cells. This effect is limited to the SPB that has migrated to the daughter cell (dSPB). Cdc14 localizes to both SPBs shortly after anaphase onset. In mid-to-late anaphase, levels of Cdc14 increase at the dSPB in both wildtype and bud14Δ cells. With mitotic exit, Cdc14 disappears from the dSPB in wildtype cells but not in bud14Δ cells. Accordingly, 50% of bud14Δ cells in G1 have Cdc14 at their SPBs. We also found that Cdc14 localization at the dSPB was largely, but not entirely, dependent on Bfa1 in bud14Δ cells. Furthermore, Bud14 interacted with Cdc14 in the Y2H system.
Conclusion: Our results suggest that Glc7-Bud14 is part of a mechanism that promotes Cdc14 disappearance from the dSPB.

Keywords: Bud14; Cdc14; mitotic exit network; protein phosphatase 1; spindle pole body

DOI: https://doi.org/10.55730/1300-0152.2707

bioRxiv. 2024 Oct 25. pii: 2024.10.25.620315. [Epub ahead of print]

Palmitoylated Importin α Regulates Mitotic Spindle Orientation Through Interaction with NuMA.

Patrick James Sutton, Christopher W Brownlee.

Regulation of cell division orientation is a fundamental process critical to differentiation and tissue homeostasis. Microtubules emanating from the mitotic spindle pole bind a conserved complex of proteins at the cell cortex which orients the spindle and ultimately the cell division plane. Control of spindle orientation is of particular importance in developing tissues, such as the developing brain. Misorientation of the mitotic spindle and thus subsequent division plane misalignment can contribute to improper segregation of cell fate determinants in developing neuroblasts, leading to a rare neurological disorder known as microcephaly. We demonstrate that the nuclear transport protein importin α, when palmitoylated, plays a critical role in mitotic spindle orientation through localizing factors, such as NuMA, to the cell cortex. We also observe craniofacial developmental defects in Xenopus laevis when importin α palmitoylation is abrogated, including smaller head and brains, a hallmark of spindle misorientation and microcephaly. These findings characterize not only a role for importin α in spindle orientation, but also a broader role for importin α palmitoylation which has significance for many cellular processes.

DOI: https://doi.org/10.1101/2024.10.25.620315

Front Plant Sci. 2024 ;15 1467236

Plant kinetochore complex: composition, function, and regulation.

Yuqian Xie, Mingliang Wang, Beixin Mo, Chao Liang.

The kinetochore complex, an important protein assembly situated on the centromere, plays a pivotal role in chromosome segregation during cell division. Like in animals and fungi, the plant kinetochore complex is important for maintaining chromosome stability, regulating microtubule attachment, executing error correction mechanisms, and participating in signaling pathways to ensure accurate chromosome segregation. This review summarizes the composition, function, and regulation of the plant kinetochore complex, emphasizing the interactions of kinetochore proteins with centromeric DNAs (cenDNAs) and RNAs (cenRNAs). Additionally, the applications of the centromeric histone H3 variant (the core kinetochore protein CENH3, first identified as CENP-A in mammals) in the generation of ploidy-variable plants and synthesis of plant artificial chromosomes (PACs) are discussed. The review serves as a comprehensive roadmap for researchers delving into plant kinetochore exploration, highlighting the potential of kinetochore proteins in driving technological innovations in synthetic genomics and plant biotechnology.

Keywords: CENH3; CENP-C; cenDNAs; cenRNAs; centromere; kinetochore complex; plant artificial chromosomes

DOI: https://doi.org/10.3389/fpls.2024.1467236

mBio. 2024 Oct 30. e0293624

On the possibility of yet a third kinetochore system in the protist phylum Euglenozoa.

Corinna Benz, Maximilian W D Raas, Pragya Tripathi, Drahomíra Faktorová, Eelco C Tromer, Bungo Akiyoshi, Julius Lukeš.

Transmission of genetic material from one generation to the next is a fundamental feature of all living cells. In eukaryotes, a macromolecular complex called the kinetochore plays crucial roles during chromosome segregation by linking chromosomes to spindle microtubules. Little is known about this process in evolutionarily diverse protists. Within the supergroup Discoba, Euglenozoa forms a speciose group of unicellular flagellates-kinetoplastids, euglenids, and diplonemids. Kinetoplastids have an unconventional kinetochore system, while euglenids have subunits that are conserved among most eukaryotes. For diplonemids, a group of extremely diverse and abundant marine flagellates, it remains unclear what kind of kinetochores are present. Here, we employed deep homology detection protocols using profile-versus-profile Hidden Markov Model searches and AlphaFold-based structural comparisons to detect homologies that might have been previously missed. Interestingly, we still could not detect orthologs for most of the kinetoplastid or canonical kinetochore subunits with few exceptions including a putative centromere-specific histone H3 variant (cenH3/CENP-A), the spindle checkpoint protein Mad2, the chromosomal passenger complex members Aurora and INCENP, and broadly conserved proteins like CLK kinase and the meiotic synaptonemal complex proteins SYCP2/3 that also function at kinetoplastid kinetochores. We examined the localization of five candidate kinetochore-associated proteins in the model diplonemid, Paradiplonema papillatum. PpCENP-A shows discrete dots in the nucleus, implying that it is likely a kinetochore component. PpMad2, PpCLKKKT10/19, PpSYCP2L1KKT17/18, and PpINCENP reside in the nucleus, but no clear kinetochore localization was observed. Altogether, these results point to the possibility that diplonemids evolved a hitherto unknown type of kinetochore system.
IMPORTANCE: A macromolecular assembly called the kinetochore is essential for the segregation of genetic material during eukaryotic cell division. Therefore, characterization of kinetochores across species is essential for understanding the mechanisms involved in this key process across the eukaryotic tree of life. In particular, little is known about kinetochores in divergent protists such as Euglenozoa, a group of unicellular flagellates that includes kinetoplastids, euglenids, and diplonemids, the latter being a highly diverse and abundant component of marine plankton. While kinetoplastids have an unconventional kinetochore system and euglenids have a canonical one similar to traditional model eukaryotes, preliminary searches detected neither unconventional nor canonical kinetochore components in diplonemids. Here, we employed state-of-the-art deep homology detection protocols but still could not detect orthologs for the bulk of kinetoplastid-specific nor canonical kinetochore proteins in diplonemids except for a putative centromere-specific histone H3 variant. Our results suggest that diplonemids evolved kinetochores that do not resemble previously known ones.

Keywords: Diplonemea; Kinetoplastea; Paradiplonema; cell division; cenH3/CENP-A; kinetochore

DOI: https://doi.org/10.1128/mbio.02936-24