bims-micesi 2022-05-15 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2022–05–15
twenty papers selected by
Valentina Piano, Max Planck Institute of Molecular Physiology

Recent Progress on the Localization of PLK1 to the Kinetochore and Its Role in Mitosis.
Polar Chromosomes-Challenges of a Risky Path.
Investigating the Transition of the Core Centromeric Nucleosome Complex from Interphase to Mitosis using Chemical Biology Tools.
Structure of the human inner kinetochore CCAN complex and its significance for human centromere organization.
Hydrogen-Deuterium Exchange Identifies the Structural Basis of Phase Separation of the Chromosome Passenger Complex.
SUMO Regulation of Bir1 in Mitotic Progression.
Considerations for studying phosphorylation of the mitotic checkpoint pseudokinase BUBR1.
The chirality of the mitotic spindle provides a mechanical response to forces and depends on microtubule motors and augmin.
SIN-Like Pathway Kinases Regulate the End of Mitosis in the Methylotrophic Yeast Ogataea polymorpha.
PLEKHA5 Regulates Mitotic Progression by Promoting APC/C Localization to Microtubules.
Too close for comfort? Endomembranes promote missegregation by enclosing lost chromosomes.
Quantitative Mass Spectrometry Reveals a Proteome-wide Role for Cyclin A and Cks1 in Multisite, Non-Proline Directed Phosphorylation by CDK1.
Single-cell monitoring of dry mass and dry mass density reveals exocytosis of cellular dry contents in mitosis.
A proposed unified mitotic chromosome architecture.
Epigenetic, genetic and maternal effects enable stable centromere inheritance.
Combination of CENP-B Box Positive and Negative Synthetic Alpha Satellite Repeats Improves De Novo Human Artificial Chromosome Formation.
Integrative Multi-Omics Analysis of Identified SKA3 as a Candidate Oncogene Correlates with Poor Prognosis and Immune Infiltration in Lung Adenocarcinoma.
Inhibition of PLK-1 by using a Small Molecule Inhibitor HMN-214 is a Novel Therapeutic Approach for High-risk Neuroblastoma.
SKA3 is a prognostic biomarker and associated with immune infiltration in bladder cancer.
Nuclear focal adhesion kinase induces APC/C activator protein CDH1-mediated cyclin-dependent kinase4/6 degradation and inhibits melanoma proliferation.

Int J Mol Sci. 2022 May 08. pii: 5252. [Epub ahead of print]23(9):

Recent Progress on the Localization of PLK1 to the Kinetochore and Its Role in Mitosis.

Taekyung Kim.

  The accurate distribution of the replicated genome during cell division is essential for cell survival and healthy organismal development. Errors in this process have catastrophic consequences, such as birth defects and aneuploidy, a hallmark of cancer cells. PLK1 is one of the master kinases in mitosis and has multiple functions, including mitotic entry, chromosome segregation, spindle assembly checkpoint, and cytokinesis. To dissect the role of PLK1 in mitosis, it is important to understand how PLK1 localizes in the specific region in cells. PLK1 localizes at the kinetochore and is essential in spindle assembly checkpoint and chromosome segregation. However, how PLK1 localizes at the kinetochore remains elusive. Here, we review the recent literature on the kinetochore recruitment mechanisms of PLK1 and its roles in spindle assembly checkpoint and attachment between kinetochores and spindle microtubules. Together, this review provides an overview of how the local distribution of PLK1 could regulate major pathways in mitosis.

Keywords:  PLK1 kinase; PP1; PP2A; cell cycle; chromosome segregation; kinetochore; spindle assembly checkpoint

DOI:  https://doi.org/10.3390/ijms23095252
Cells. 2022 May 03. pii: 1531. [Epub ahead of print]11(9):

Polar Chromosomes-Challenges of a Risky Path.

Kruno Vukušić, Iva M Tolić.

  The process of chromosome congression and alignment is at the core of mitotic fidelity. In this review, we discuss distinct spatial routes that the chromosomes take to align during prometaphase, which are characterized by distinct biomolecular requirements. Peripheral polar chromosomes are an intriguing case as their alignment depends on the activity of kinetochore motors, polar ejection forces, and a transition from lateral to end-on attachments to microtubules, all of which can result in the delayed alignment of these chromosomes. Due to their undesirable position close to and often behind the spindle pole, these chromosomes may be particularly prone to the formation of erroneous kinetochore-microtubule interactions, such as merotelic attachments. To prevent such errors, the cell employs intricate mechanisms to preposition the spindle poles with respect to chromosomes, ensure the formation of end-on attachments in restricted spindle regions, repair faulty attachments by error correction mechanisms, and delay segregation by the spindle assembly checkpoint. Despite this protective machinery, there are several ways in which polar chromosomes can fail in alignment, mis-segregate, and lead to aneuploidy. In agreement with this, polar chromosomes are present in certain tumors and may even be involved in the process of tumorigenesis.

Keywords:  CENP-E; aneuploidy; chromosome congression; chromosome segregation; dynein; mitosis; mitotic spindle; motor proteins; polar chromosomes; polar ejection force; prometaphase; spindle assembly; tumors

DOI:  https://doi.org/10.3390/cells11091531
FASEB J. 2022 May;36 Suppl 1

Investigating the Transition of the Core Centromeric Nucleosome Complex from Interphase to Mitosis using Chemical Biology Tools.

Praveen Kumar Allu, Rachel M Lackner, David M Chenoweth, Ben E Black.

Centromeres are the location on each chromosome where kinetochores form in mitosis to connect to spindle microtubules that physically separate sister chromatids to the daughter cells. A 16-subunit complex termed the constitutive centromere associated network (CCAN) is bound to nucleosomes harboring the histone variant, CENP-A, and is essential for assembling the mitotic kinetochore. The core centromere nucleosome complex (CCNC) consists of CENP-A nucleosomes and CCAN subunits, CENP-C and CENP-N. In prior studies, we proposed that the CCNC undergoes an alteration at mitotic onset where one of two copies of CENP-N is lost. This model indicates that there would remain only one CENP-A nucleosome surface with both CENP-N and CENP-C that is competent to recruit the CCAN and, in turn, assemble the kinetochore. We hypothesize that the asymmetric surfaces on CENP-A nucleosomes provide a level of organization of centromeric chromatin that is important for chromosome segregation. We are developing a system to study these functional relationships in cells using chemical biology approaches. We have created a cell line with complementary SNAP and HALO tags on the CCNC components and employed the SNAP-HALO cross linker, HaXS8, to tether CCNC components (CENP-A with CENP-N) irreversibly on nucleosomes. Initial experiments indicate that this system is able to retain CENP-N at high levels on mitotic centromeres in an HaXS8 concentration-dependent fashion. This provides additional support for the model of a centromeric chromatin transition as cells enter mitosis, and our system holds promise to test predictions that arise from it.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.R4505
Mol Cell. 2022 May 06. pii: S1097-2765(22)00390-2. [Epub ahead of print]

Structure of the human inner kinetochore CCAN complex and its significance for human centromere organization.

Marion E Pesenti, Tobias Raisch, Duccio Conti, Kai Walstein, Ingrid Hoffmann, Dorothee Vogt, Daniel Prumbaum, Ingrid R Vetter, Stefan Raunser, Andrea Musacchio.

  Centromeres are specialized chromosome loci that seed the kinetochore, a large protein complex that effects chromosome segregation. A 16-subunit complex, the constitutive centromere associated network (CCAN), connects between the specialized centromeric chromatin, marked by the histone H3 variant CENP-A, and the spindle-binding moiety of the kinetochore. Here, we report a cryo-electron microscopy structure of human CCAN. We highlight unique features such as the pseudo GTPase CENP-M and report how a crucial CENP-C motif binds the CENP-LN complex. The CCAN structure has implications for the mechanism of specific recognition of the CENP-A nucleosome. A model consistent with our structure depicts the CENP-C-bound nucleosome as connected to the CCAN through extended, flexible regions of CENP-C. An alternative model identifies both CENP-C and CENP-N as specificity determinants but requires CENP-N to bind CENP-A in a mode distinct from the classical nucleosome octamer.

Keywords:  CCAN; CENP-A; CENP-C; CENP-L; CENP-N; CENP-TW; centromere; hemisome; kinetochore; nucleosome

DOI:  https://doi.org/10.1016/j.molcel.2022.04.027
FASEB J. 2022 May;36 Suppl 1

Hydrogen-Deuterium Exchange Identifies the Structural Basis of Phase Separation of the Chromosome Passenger Complex.

Nikaela W Bryan, Ewa Niedzialkowska, P T Stukenberg, Ben E Black.

The centromere is the locus that directs accurate chromosome segregation, and a master regulatory complex, the chromosome passenger complex (CPC) is enriched in a region of the mitotic centromere termed the inner centromere. In mitosis, the CPC monitors the connections of the centromere to the mitotic spindle, acting to destabilize incorrect attachments but permitting correct attachments to persist. The CPC contains the Aurora-B kinase and three regulatory subunits: INCENP, Survivin and Borealin. Recent findings support a role for CPC phase separation in its localization and function in mitotic error correction (Trivedi et al., 2019, Nat. Cell Biol. 21:1127-1137). Liquid-liquid phase separation is emerging as a central regulatory principle of cell biology. This form of phase separation is often driven by multivalency of adhesive domains and/or linear motifs. The sequences of these domains are often found in unstructured regions where they are enriched in uncharged polar side chains, charged amino acids, or aromatic residues; additionally, structural plasticity allows them to generate multiple, weakly-adhesive, inter- and intramolecular interactions. Currently there are no techniques to globally map the motifs on proteins that drive the multivalent interactions. In this context, we now employ hydrogen/deuterium exchange coupled to mass spectrometry (HXMS) to study dynamics of CPC phase separation. We have identified four regions within the regulatory CPC subunits that upon phase separation exhibit differential HX (three slower and one faster). Importantly, three of these regions map to structured regions of proteins while one maps to the expected intrinsically disordered regions. Each of the four regions is enriched with charged amino acids. Mutation of key residues in one of the differential HX regions within the INCENP subunit diminish droplet size and quantity, suggesting that we have successfully mapped a key surface for the phase separation properties of the CPC. In broader context we have shown for the first time how HXMS coupled with structural data and bioinformatic analysis can be applied to study molecular basics of protein liquid-liquid phase separation.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.R5784
FASEB J. 2022 May;36 Suppl 1

SUMO Regulation of Bir1 in Mitotic Progression.

Haley Stabile, Julie Cepec, Tori Valachovic, Yee Mon Thu.

Organisms utilize many mechanisms in order to preserve their genomic integrity when faced with DNA damage or chromosomal instability. One such mechanism is protein sumoylation, in which E1, E2, and E3 ligases covalently link small ubiquitin-like modifier (SUMO) peptides or chains to target proteins. The goal of this research is to examine the contribution of Bir1 sumoylation to genome stability in Saccharomyces cerevisiae. Bir1 is the target of one of the three S. cerevisiae E3 SUMO ligases, Mms21, and is a subunit of the chromosomal passenger complex (CPC). The CPC mediates the spindle assembly checkpoint (SAC) to establish chromosome bi-orientation during mitosis. Therefore, it has previously been hypothesized that the sumoylation of Bir1 influences mitotic progression. In order to explore this hypothesis, BIR1 was genetically modified to mimic conditions in which Bir1 is constantly sumoylated (BIR1-SuON) or cannot be sumoylated (BIR1-SuOFF). The BIR1-SuOFF mutants appeared to be more resistant to DNA damage caused by methyl methanesulfonate (MMS) and ultraviolet radiation than the BIR1-SuON mutant, as well as the control strain (BIR1-SuC). BIR1-SuOff mutants phenocopied the temperature sensitive mutant of Ipl1, ipl1-321, suggesting that mitotic checkpoint regulation may be affected in BIR1-SuOff mutants. However, cell cycle analyses of BIR1-SuOff mutants revealed that mitotic checkpoint activation was comparable to the control strain, at least in response to treatment with nocodazole, an agent that depolymerizes microtubules. We will further characterize the status of mitotic checkpoint activation in BIR1 mutants by examining Pds1 degradation and cell cycle analyses in the presence of other genotoxic agents.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.R3816
Methods Enzymol. 2022 ;pii: S0076-6879(22)00136-7. [Epub ahead of print]667 507-534

Considerations for studying phosphorylation of the mitotic checkpoint pseudokinase BUBR1.

Luciano Gama Braga, Chantal Garand, Sabine Elowe.

  Budding uninhibited by benzimidazole 1-related protein 1 (BUBR1) is a mitotic checkpoint (better known as the spindle assembly checkpoint) protein that forms part of an inhibitory complex required to delay mitosis when errors occur in the attachment between chromosomes and the mitotic spindle. If these errors remain uncorrected, it could result in unequal distribution of genetic material to each of the nascent daughter cells, leading to potentially disastrous consequences at both the cellular and organismal level. In some higher eukaryotes including vertebrates, BUBR1 has a C-terminal kinase fold that is largely thought to be inactive, whereas in many species this domain has been lost through evolution and the truncated protein is known as mitotic arrest deficient 3 (MAD3). Here we present advice and practical considerations for the design of experiments, their analysis and interpretation to study the functions of the vertebrate BUBR1 during mitosis with emphasis on analysis implicating the pseudokinase domain.

Keywords:  BUBR1; Kinetochores; Mitosis; PP2A; Pseudokinase

DOI:  https://doi.org/10.1016/bs.mie.2022.03.045
Curr Biol. 2022 May 04. pii: S0960-9822(22)00604-2. [Epub ahead of print]

The chirality of the mitotic spindle provides a mechanical response to forces and depends on microtubule motors and augmin.

Monika Trupinić, Barbara Kokanović, Ivana Ponjavić, Ivan Barišić, Siniša Šegvić, Arian Ivec, Iva M Tolić.

  Forces produced by motor proteins and microtubule dynamics within the mitotic spindle are crucial for proper chromosome segregation. In addition to linear forces, rotational forces or torques are present in the spindle, which are reflected in the left-handed twisted shapes of microtubule bundles that make the spindle chiral. However, the biological role and molecular origins of spindle chirality are unknown. By developing methods for measuring the spindle twist, we show that spindles are most chiral near the metaphase-to-anaphase transition. To assess the role of chirality in spindle mechanics, we compressed the spindles along their axis. This resulted in a stronger left-handed twist, suggesting that the twisted shape allows for a mechanical response to forces. Inhibition or depletion of motor proteins that perform chiral stepping, Eg5/kinesin-5, Kif18A/kinesin-8, MKLP1/kinesin-6, and dynein, decreased the left-handed twist or led to right-handed twist, implying that these motors regulate the twist by rotating microtubules within their antiparallel overlaps or at the spindle pole. A right-handed twist was also observed after the depletion of the microtubule nucleator augmin, indicating its contribution to the twist through the nucleation of antiparallel bridging microtubules. The uncovered switch from left-handed to right-handed twist reveals the existence of competing mechanisms that promote twisting in opposite directions. As round spindles are more twisted than the elongated ones are, we infer that bending and twisting moments are generated by similar molecular mechanisms and propose a physiological role for spindle chirality in allowing the spindle to absorb mechanical load.

Keywords:  augmin; chirality; kinesins; mitosis; mitotic spindle; motor proteins; rotation; spindle compression; torques; twist

DOI:  https://doi.org/10.1016/j.cub.2022.04.035
Cells. 2022 Apr 30. pii: 1519. [Epub ahead of print]11(9):

SIN-Like Pathway Kinases Regulate the End of Mitosis in the Methylotrophic Yeast Ogataea polymorpha.

Hiromi Maekawa, Shen Jiangyan, Kaoru Takegawa, Gislene Pereira.

  The mitotic exit network (MEN) is a conserved signalling pathway essential for the termination of mitosis in the budding yeast Saccharomyces cerevisiae. All MEN components are highly conserved in the methylotrophic budding yeast Ogataea polymorpha, except for Cdc15 kinase. Instead, we identified two essential kinases OpHcd1 and OpHcd2 (homologue candidate of ScCdc15) that are homologous to SpSid1 and SpCdc7, respectively, components of the septation initiation network (SIN) of the fission yeast Schizosaccharomyces pombe. Conditional mutants for OpHCD1 and OpHCD2 exhibited significant delay in late anaphase and defective cell separation, suggesting that both genes have roles in mitotic exit and cytokinesis. Unlike Cdc15 in S. cerevisiae, the association of OpHcd1 and OpHcd2 with the yeast centrosomes (named spindle pole bodies, SPBs) is restricted to the SPB in the mother cell body. SPB localisation of OpHcd2 is regulated by the status of OpTem1 GTPase, while OpHcd1 requires the polo-like kinase OpCdc5 as well as active Tem1 to ensure the coordination of mitotic exit (ME) signalling and cell cycle progression. Our study suggests that the divergence of molecular mechanisms to control the ME-signalling pathway as well as the loss of Sid1/Hcd1 kinase in the MEN occurred relatively recently during the evolution of budding yeast.

Keywords:  Cdc15 kinase; Cdc5 kinase; Ogataea polymorpha; SPB; mitotic exit network; septation initiation network

DOI:  https://doi.org/10.3390/cells11091519
FASEB J. 2022 May;36 Suppl 1

PLEKHA5 Regulates Mitotic Progression by Promoting APC/C Localization to Microtubules.

Xiaofu Cao, Adnan Shami Shah, Ethan J Sanford, Marcus B Smolka, Jeremy M Baskin.

Progression through major cell cycle checkpoints involves spatiotemporal coordination of cytoskeletal dynamics with changes in protein activities, chromosomal dynamics, and signaling events. A major role of the ubiquitin-proteasome system is to orchestrate cell cycle progression through temporally controlled protein degradation programs. The anaphase-promoting complex/cyclosome (APC/C) is the primary E3 ligase controlling all aspects of mitosis, including G2/M transition, early mitosis, the metaphase-anaphase transition, and mitotic exit, via polyubiquitination of important mitotic effector proteins. The temporal control of APC/C-mediated ubiquitination in these processes is well established, including identities of ubiquitination substrates and accessory factors that modulate E3 ligase activity. Much less is known, however, about the spatial organization of APC/C function, including its potential interplay with the microtubule cytoskeleton, which serves as a signaling platform and whose dynamic behavior is central to advancement through mitosis. Here, we investigate and describe pleckstrin homology domain-containing family A, member 5 (PLEKHA5) as a new regulator of APC/C function and the ubiquitination pathway in mitosis. We found that PLEKHA5 localized to the microtubule network and interacted with APC/C. PLEKHA5 knockdown antagonized mitotic entry and progression, causing a buildup of APC/C substrates implicated in both the G2/M and metaphase-anaphase transitions, in a manner dependent upon the PLEKHA5 interaction with APC/C. In vitro ubiquitination assays showed that APC/C isolated from mitotic cells lacking PLEKHA5 had lower catalytic activity and a decreased association with its key mitotic co-activator CDC20. We investigated the dynamic localizations of the APC/C and the potential role of PLEKHA5 in its regulation by developing a TurboID proximity biotinylation tool to assess microtubule localization of endogenous proteins. Microtubule-targeted TurboIDs revealed that PLEKHA5, APC/C subunits, and the co-activator CDC20 all localized to microtubules both in interphase and in M phase. Importantly, upon PLEKHA5 knockdown, a pool of APC/C, but not CDC20, lost its microtubule localization, which could explain the decreased APC/C-CDC20 association. We propose that PLEKHA5 functions as a novel APC/C adaptor to promote its subcellular localization to microtubules and enable APC/C to search for its co-activator CDC20 more efficiently in one dimension along the microtubule network. In this model PLEKHA5 acts to coordinate the spatial regulation of APC/CCDC20 and facilitate its efficient polyubiquitination of key mitotic effector proteins to ensure proper progression of mitosis.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.R3131
J Cell Biol. 2022 Jun 06. pii: e202204114. [Epub ahead of print]221(6):

Too close for comfort? Endomembranes promote missegregation by enclosing lost chromosomes.

Lisa Donker, Susana A Godinho.

Correct segregation of chromosomes during mitosis is essential to prevent aneuploidy. In this issue, Ferrandiz et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202203021) show that endomembranes can promote chromosome missegregation by "ensheathing" misaligned chromosomes, preventing their integration into the metaphase plate. Their findings point toward endomembranes as a potential risk factor for aneuploidy.

DOI: https://doi.org/10.1083/jcb.202204114
FASEB J. 2022 May;36 Suppl 1

Quantitative Mass Spectrometry Reveals a Proteome-wide Role for Cyclin A and Cks1 in Multisite, Non-Proline Directed Phosphorylation by CDK1.

Tony Ly, Aymen Al-Rawi, Jane Endicott, Svitlana Korolchuk.

Cdk1 is a master kinase of mitotic cell division, phosphorylating thousands of substrates in a highly temporally regulated manner. How Cdk1 phosphorylates the right substrate at the right time remains an open question. Cdk1 activity requires complex formation with a cyclin subunit: cyclin A or cyclin B. Additionally, active Cdk1 is found in complex with a small adaptor protein called Cks1. Cyclin subunits activate Cdk1 and tune Cdk1 substrate choice through interactions with short linear motifs on substrates. While Cks1 is essential in all organisms tested to date, a general function for Cks1 in Cdk1 substrate choice has not been demonstrated. In this study, we investigated the roles of the non-catalytic subunits of Cdk1 in substrate phosphorylation in human cells. We designed an in vitro assay in which we induced protein phosphorylation in fixed and permeabilized human lymphoblasts using active recombinant Cdk1 either in complex with cyclin A, cyclin B, or cyclin B+Cks1. Global phosphorylation levels were then measured using quantitative mass spectrometry. Our data demonstrate that, contrary to the textbook model of Cdk1 as a proline-directed kinase, both cyclin A and Cks1 facilitate widespread Cdk1 phosphorylation of sites lacking a +1 proline motif. Our results also suggest a role for Cks1 in multisite phosphorylation. We propose a model whereby cyclin A and Cks1 have indispensable functions in promoting non-proline directed, multisite phosphorylation of Cdk1 substrates to mediate dynamic, switch-like changes in protein function required for mitotic entry, proper chromosome segregation and cell division.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.0R351
Elife. 2022 May 10. pii: e76664. [Epub ahead of print]11

Single-cell monitoring of dry mass and dry mass density reveals exocytosis of cellular dry contents in mitosis.

Teemu P Miettinen, Kevin S Ly, Alice Lam, Scott R Manalis.

  Cell mass and composition change with cell cycle progression. Our previous work characterized buoyant mass dynamics in mitosis (Miettinen et al., 2019), but how dry mass and cell composition change in mitosis has remained unclear. To better understand mitotic cell growth and compositional changes, we develop a single-cell approach for monitoring dry mass and the density of that dry mass every ~75 s with 1.3% and 0.3% measurement precision, respectively. We find that suspension grown mammalian cells lose dry mass and increase dry mass density following mitotic entry. These changes display large, non-genetic cell-to-cell variability, and the changes are reversed at metaphase-anaphase transition, after which dry mass continues accumulating. The change in dry mass density causes buoyant and dry mass to differ specifically in early mitosis, thus reconciling existing literature on mitotic cell growth. Mechanistically, cells in early mitosis increase lysosomal exocytosis, and inhibition of lysosomal exocytosis decreases the dry mass loss and dry mass density increase in mitosis. Overall, our work provides a new approach for monitoring single-cell dry mass and dry mass density, and reveals that mitosis is coupled to extensive exocytosis-mediated secretion of cellular contents.

Keywords:  cell biology; cell growth; density; dry mass; exocytosis; human; mitosis; mouse; physics of living systems; quantitative biology

DOI:  https://doi.org/10.7554/eLife.76664
Proc Natl Acad Sci U S A. 2022 May 17. 119(20): e2119107119

A proposed unified mitotic chromosome architecture.

John Sedat, Angus McDonald, Herbert Kasler, Eric Verdin, Hu Cang, Muthuvel Arigovindan, Cornelis Murre, Michael Elbaum.

  SignificanceThe significance of this proposed mitotic chromosome architecture is that a specific, sequenced chromosome, human chromosome 10, can be built into a specific architecture that accounts for the dimensional values and cytological descriptions. Since this molecular architecture is an extension of the interphase chromosome structure, a coiling of the 11-nm nucleosome fiber with further coiling, a unifying molecular structure motif is present throughout the entire mitotic cycle, interphase through mitosis.

Keywords:  biophysics and computational structure analysis; cell biology; chromosome structure; computational structure analysis

DOI:  https://doi.org/10.1073/pnas.2119107119
Nat Cell Biol. 2022 May;24(5): 748-756

Epigenetic, genetic and maternal effects enable stable centromere inheritance.

Arunika Das, Aiko Iwata-Otsubo, Aspasia Destouni, Jennine M Dawicki-McKenna, Katelyn G Boese, Ben E Black, Michael A Lampson.

Centromeres are defined epigenetically by the histone H3 variant CENP-A. The propagation cycle by which pre-existing CENP-A nucleosomes serve as templates for nascent assembly predicts the epigenetic memory of weakened centromeres. Using a mouse model with reduced levels of CENP-A nucleosomes, we find that an embryonic plastic phase precedes epigenetic memory through development. During this phase, nascent CENP-A nucleosome assembly depends on the maternal Cenpa genotype rather than the pre-existing template. Weakened centromeres are thus limited to a single generation, and parental epigenetic differences are eliminated by equal assembly on maternal and paternal centromeres. These differences persist, however, when the underlying DNA of parental centromeres differs in repeat abundance, as assembly during the plastic phase also depends on sufficient repetitive centromere DNA. With contributions of centromere DNA and the Cenpa maternal effect, we propose that centromere inheritance naturally minimizes fitness costs associated with weakened centromeres or epigenetic differences between parents.

DOI: https://doi.org/10.1038/s41556-022-00897-w
Cells. 2022 Apr 19. pii: 1378. [Epub ahead of print]11(9):

Combination of CENP-B Box Positive and Negative Synthetic Alpha Satellite Repeats Improves De Novo Human Artificial Chromosome Formation.

Koei Okazaki, Megumi Nakano, Jun-Ichirou Ohzeki, Koichiro Otake, Kazuto Kugou, Vladimir Larionov, William C Earnshaw, Hiroshi Masumoto.

  Human artificial chromosomes (HACs) can be formed de novo by introducing large (>30 kb) centromeric sequences consisting of highly repeated 171-bp alpha satellite (alphoid) DNA into HT1080 cells. However, only a subset of transformed cells successfully establishes HACs. CENP-A chromatin and heterochromatin assemble on the HACs and play crucial roles in chromosome segregation. The CENP-B protein, which binds a 17-bp motif (CENP-B box) in the alphoid DNA, functions in the formation of alternative CENP-A chromatin or heterochromatin states. A balance in the coordinated assembly of these chromatin states on the introduced alphoid DNA is important for HAC formation. To obtain information about the relationship between chromatin architecture and de novo HAC formation efficiency, we tested combinations of two 60-kb synthetic alphoid sequences containing either tetO or lacO plus a functional or mutated CENP-B box combined with a multiple fusion protein tethering system. The combination of mutated and wild-type CENP-B box alphoid repeats significantly enhanced HAC formation. Both CENP-A and HP1α were enriched in the wild-type alphoid DNA, whereas H3K27me3 was enriched on the mutant alphoid array. The presence or absence of CENP-B binding resulted in differences in the assembly of CENP-A chromatin on alphoid arrays and the formation of H3K9me3 or H3K27me3 heterochromatin.

Keywords:  CENP-A; CENP-B; HAC; alphoid; centromere; chromatin; heterochromatin; histone

DOI:  https://doi.org/10.3390/cells11091378
Int J Gen Med. 2022 ;15 4635-4647

Integrative Multi-Omics Analysis of Identified SKA3 as a Candidate Oncogene Correlates with Poor Prognosis and Immune Infiltration in Lung Adenocarcinoma.

Yuansheng Lin, Jianzhong An, Xingli Zhuo, Yingzhuo Qiu, Wenjing Xie, Wei Yao, Dan Yin, Linpeng Wu, Dian Lei, Chenghui Li, Yuanguang Xie, Ahu Hu, Shengjun Li.

   Background: Spindle and kinetochore-associated complex subunit 3 (SKA3) plays important roles in promoting the migration and the invasion of various human cancer cells. There are a few studies on SKA3 in lung adenocarcinoma (LUAD), but the in-depth analysis of the expression of SKA3 and the correlated possible immune mechanism of SKA3 in LUAD are not clear.
Methods: In our study, the expression and survival data of SKA3 were analyzed in LUAD using TIMER, Oncomine, UALCAN, cBioPortal, LinkedOmics, Human Protein Atlas, and Kaplan-Meier plotter. Then, quantitative PCR was used to verify the expression differences of SKA3 between LUAD tissues of mice and the normal tissues.
Results: We established that the expression of SKA3 in the LUAD group was remarkably higher than that in the normal group. Additionally, high SKA3 expression was linked to poorer survival in LUAD. Moreover, SKA3 expression had a remarkable negative correlation with the immune infiltration of B cells, macrophages, and CD4+ T cells. SKA3 was markedly negatively related to the immune type biomarkers of T cells and B cells in LUAD. The elevated expression of SKA3 with LUAD in enriched B cells, CD4+ T cells, CD8+ T cells, macrophages and Treg cells had worse prognosis, respectively. Functional network analysis showed that SKA3 regulated the mitotic cell cycle, mitosis, chromosome segregation and cell division via pathways.
Conclusion: In summary, our study suggested that SKA3 was highly expressed in LUAD and SKA3 might function as a prognostic biomarker in LUAD. Besides, SKA3 may be a candidate oncogene, which correlates with poor prognosis and immune infiltration in lung adenocarcinoma.

Keywords:  LUAD; SKA3; immune infiltration; prognosis

DOI:  https://doi.org/10.2147/IJGM.S359987
FASEB J. 2022 May;36 Suppl 1

Inhibition of PLK-1 by using a Small Molecule Inhibitor HMN-214 is a Novel Therapeutic Approach for High-risk Neuroblastoma.

Rameswari Chilamakuri, Saurabh Agarwal.

Polo-like kinase1 (PLK-1), a member of the polo-like kinase family of serine/threonine protein kinases, plays an essential role in regulating the cell cycle. PLK-1 has a strong relationship with numerous regulatory events progressing during G2/M transition, chromosomal segregation, spindle assembly maturation, and mitotic exit. In addition, PLK-1 also regulates DNA damage response, DNA replication, transcription, translation, chromosomes dynamics, and checkpoint adoption. Elevated levels of PLK-1 have been observed in a plethora of cancers, including neuroblastoma (NB). NB is the most prevalent solid tumor that develops during the early embryonic stage and accounts for 15% of pediatric cancer-related deaths. Current cytotoxic treatment strategies for NB are limiting and failed to prevent relapse and metastasis. In the present study, we hypothesized that inhibition of cell cycle regulator PLK-1 by a small molecule inhibitor HMN-214 will inhibit NB growth. To determine the role of PLK-1 in NB, we analyzed multiple NB patient datasets and observed that PLK-1 expression is inversely correlated with overall patient survival. PLK-1 also strongly correlates with MYCN amplification and overall NB disease progression in our patient dataset analysis. Further, we used six NB cell lines, including MYCN- amplified and -non-amplified cell lines, to evaluate the anti-proliferative effects of HMN-214 in NB. Results showed that HMN-214 significantly and in a dose-dependent manner inhibits NB proliferation and colony formation capacity. Additionally, we found that HMN-214 significantly induces apoptosis and blocks cell cycle progression at the G2-M phase in treatment groups compared to controls. Our molecular analysis further revealed that HMN-214 significantly inhibits the mRNA and protein levels of PLK-1 and CDK1 in contrast to control treatments. Further, we developed a 3D spheroid tumor model for NB to mimic in vivo tumor growth and utilized it to determine the effects of HMN-214 on NB tumor growth. Results showed that HMN-214 significantly and in a dose-dependent manner inhibits NB 3D spheroidal tumor growth by specifically inducing tumor cell death. Overall, our data highlights the role of PLK-1 in the oncogenic progression of NB and showed the efficacy of HMN-214 in inhibiting NB growth. Our future efforts will be directed towards elucidating the role of PLK-1 in NB and developing effective therapeutic strategies incorporating PLK-1 inhibitor.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.R6149
Hereditas. 2022 May 11. 159(1): 20

SKA3 is a prognostic biomarker and associated with immune infiltration in bladder cancer.

Chenyang Wang, Shasha Liu, Xinhong Zhang, Yan Wang, Peng Guan, Fanyou Bu, Hao Wang, Dawen Wang, Yi Fan, Sichuan Hou, Zhilei Qiu.

   BACKGROUND: Spindle and kinetochore‑associated complex subunit 3 (SKA3) has recently been considered a key regulator of carcinogenesis. However, the connection between SKA3 and immune cell infiltration remains unknown.
METHODS: The current study investigated the expression mode, prognostic effect, and functional role of SKA3 in different tumors, particularly bladder cancer using numerous databases, comprising TIMER, GEPIA, HPA, UALCAN, PrognoScan, and Kaplan-Meier Plotter. Differentially expressed gene and enrichment analyses were implemented on SKA3 using R packages "edgR" and "clusterProfiler". Immunohistochemistry was further used to validate the expression of SKA3 gene in bladder cancer. Following that, the relevance of SKA3 expression to immune infiltration level in bladder cancer was evaluated using TIMER.
RESULTS: Overall, the level of SKA3 expression in tumor tissue significantly increased than in normal tissue. In bladder cancer and other tumors, patients with high SKA3 expression levels had worse overall survival (OS) (p = 0.016), disease-specific survival (DSS) (p = 0.00004), and disease-free survival (DFS) (p = 0.032). Additionally, the major molecular functions for SKA3 included nuclear division, mitotic nuclear division, mitotic sister chromatid segregation, humoral immune response, and cell chemotaxis. Additionally, SKA3 expression was found to be positively associated with enhanced M2 macrophage and T helper (Th) 2 cell infiltration in bladder cancer.
CONCLUSIONS: Our study implies that SKA3 contributes to M2 macrophage and Th2 cell polarization by acting as an oncogene in bladder cancer. SKA3 might be a novel biomarker for evaluating prognosis and immune infiltration in bladder cancer.

Keywords:  Biomarker; Bladder cancer; Immune infiltration; Prognosis; Spindle and kinetochore-associated complex subunit 3

DOI:  https://doi.org/10.1186/s41065-022-00234-z
J Biol Chem. 2022 May 04. pii: S0021-9258(22)00453-7. [Epub ahead of print] 102013

Nuclear focal adhesion kinase induces APC/C activator protein CDH1-mediated cyclin-dependent kinase4/6 degradation and inhibits melanoma proliferation.

James M Murphy, Kyuho Jeong, Eun-Young Erin Ahn, Ssang-Taek Steve Lim.

  Dysregulation of cyclin-dependent kinases (CDKs) can promote unchecked cell proliferation and cancer progression. Although focal adhesion kinase (FAK) contributes to regulating cell cycle progression, the exact molecular mechanism remains unclear. Here, we found that FAK plays a key role in cell cycle progression potentially through regulation of CDK4/6 protein expression. We show that FAK inhibition increased its nuclear localization and induced G1 arrest in B16F10 melanoma cells. Mechanistically, we demonstrate nuclear FAK associated with CDK4/6 and promoted their ubiquitination and proteasomal degradation through recruitment of CDH1, an activator and substrate recognition subunit of the anaphase promoting complex/cyclosome (APC/C) E3 ligase complex. We found the FAK N-terminal FERM domain acts as a scaffold to bring CDK4/6 and CDH1 within close proximity. However, overexpression of nonnuclear-localizing mutant (NLM) FAK FERM failed to function as a scaffold for CDK4/6 and CDH1. Further, short hairpin RNA (shRNA) knockdown of CDH1 increased CDK4/6 protein expression and blocked FAK inhibitor-induced reduction of CDK4/6 in B16F10 cells. In vivo, we show pharmacological FAK inhibition reduced B16F10 tumor size, correlating with increased FAK nuclear localization and decreased CDK4/6 expression compared to vehicle controls. In patient-matched healthy skin and melanoma biopsies, we found FAK was mostly inactive and nuclear-localized in healthy skin, while melanoma lesions showed increased active cytoplasmic FAK and elevated CDK4 expression. Taken together, our data demonstrate that FAK inhibition blocks tumor proliferation by inducing G1 arrest, in part through decreased CDK4/6 protein stability by nuclear FAK.

Keywords:  CDH1; CDK4/6; FAK; melanoma

DOI:  https://doi.org/10.1016/j.jbc.2022.102013