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There is growing evidence that in the absence of pathology, serine-129 phosphorylation of α-synuclein (αS) serves the purpose of fine-tuning the synaptic function of αS. We posit that pS129 has a dual role in health and disease: a physiological role in regulating αS biology, and a pathological role related to the deposition of insoluble αS into Lewy bodies. We further propose that there is no direct conversion of physiological to pathological pS129. Instead, physiological pS129 is driven by a pathway that involves neuronal activity, calcium, calcineurin, polo-like kinase 2 (Plk2), and protein phosphatase 2A (PP2A), and is readily reversible. Pathological pS129 likely accumulates because aggregated αS is easier for Plk2 to phosphorylate and/or more difficult for PP2A to dephosphorylate than native αS. α-Synuclein (αS) is an abundant presynaptic protein that regulates neurotransmission. It is also a key protein implicated in a broad class of neurodegenerative disorders termed synucleinopathies, including Parkinson's disease (PD) and Lewy body dementia (LBD). Pathological αS deposits in these diseases, Lewy bodies (LBs)/neurites (LNs), contain about 90% of αS in its phospho-serine129 (pS129) form. Therefore, pS129 is widely used as a surrogate marker of pathology. However, recent findings demonstrate that pS129 is also physiologically triggered by neuronal activity to positively regulate synaptic transmission. In this opinion article, we contrast the literature on pathological and physiological pS129, with a special focus on the latter. We emphasize that pS129 is ambiguous and knowledge about the context is necessary to correctly interpret changes in pS129. [ABSTRACT FROM AUTHOR]

Abstract Background Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease with few effective therapeutic options. Recently, drug repositioning, which involves identifying novel therapeutic potentials for existing drugs, has been popularized as a new approach for the development of novel therapeutic reagents. However, this approach has not yet been fully utilized in the field of pulmonary fibrosis. Methods The present study identified novel therapeutic options for pulmonary fibrosis using a systematic computational approach for drug repositioning based on integration of public gene expression signatures of drug and diseases (in silico screening approach). Results Among the top compounds predicted to be therapeutic for IPF by the in silico approach, we selected BI2536, a polo-like kinase (PLK) 1/2 inhibitor, as a candidate for treating pulmonary fibrosis using an in silico analysis. However, BI2536 accelerated mortality and weight loss rate in an experimental mouse model of pulmonary fibrosis. Because immunofluorescence staining revealed that PLK1 expression was dominant in myofibroblasts while PLK2 expression was dominant in lung epithelial cells, we next focused on the anti-fibrotic effect of the selective PLK1 inhibitor GSK461364. Consequently, GSK461364 attenuated pulmonary fibrosis with acceptable mortality and weight loss in mice. Conclusions These findings suggest that targeting PLK1 may be a novel therapeutic approach for pulmonary fibrosis by inhibiting lung fibroblast proliferation without affecting lung epithelial cells. In addition, while in silico screening is useful, it is essential to fully determine the biological activities of candidates by wet-lab validation studies.

Genome haploidization at meiosis depends on two consecutive nuclear divisions, which are controlled by an oscillatory system consisting of Cdk1‐cyclin B and the APC/C bound to the Cdc20 activator. How the oscillator generates exactly two divisions has been unclear. We have studied this question in yeast where exit from meiosis involves accumulation of the APC/C activator Ama1 at meiosis II. We show that inactivation of the meiosis I‐specific protein Spo13/MEIKIN results in a single‐division meiosis due to premature activation of APC/CAma1. In the wild type, Spo13 bound to the polo‐like kinase Cdc5 prevents Ama1 synthesis at meiosis I by stabilizing the translational repressor Rim4. In addition, Cdc5‐Spo13 inhibits the activity of Ama1 by converting the B‐type cyclin Clb1 from a substrate to an inhibitor of Ama1. Cdc20‐dependent degradation of Spo13 at anaphase I unleashes a feedback loop that increases Ama1's synthesis and activity, leading to irreversible exit from meiosis at the second division. Thus, by repressing the exit machinery at meiosis I, Cdc5‐Spo13 ensures that cells undergo two divisions to produce haploid gametes. Synopsis: Deletion of SPO13 in yeast and Meikin in male mammals results in exit from meiosis after a single division. A complex of Spo13 and polo‐like kinase ensures a two‐division meiosis by preventing premature activation of the APC/CAma1 ubiquitin ligase at meiosis I. Yeast spo13Δ mutants undergo only one division because they prematurely synthesize and activate Ama1, an APC/C co‐activator dedicated to exit from meiosis.Spo13 prevents a kinase cascade consisting of Cdc5/Plk1, Ime2, and CK1δ/Hrr25 from mediating degradation of the translational repressor Rim4.By phosphorylating the B‐type cyclin Clb1, the Cdc5‐Spo13 kinase converts Cdk1‐Clb1 from a target into an inhibitor of Ama1‐dependent proteolysis. [ABSTRACT FROM AUTHOR]

Background: Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease with few effective therapeutic options. Recently, drug repositioning, which involves identifying novel therapeutic potentials for existing drugs, has been popularized as a new approach for the development of novel therapeutic reagents. However, this approach has not yet been fully utilized in the field of pulmonary fibrosis. Methods: The present study identified novel therapeutic options for pulmonary fibrosis using a systematic computational approach for drug repositioning based on integration of public gene expression signatures of drug and diseases (in silico screening approach). Results: Among the top compounds predicted to be therapeutic for IPF by the in silico approach, we selected BI2536, a polo-like kinase (PLK) 1/2 inhibitor, as a candidate for treating pulmonary fibrosis using an in silico analysis. However, BI2536 accelerated mortality and weight loss rate in an experimental mouse model of pulmonary fibrosis. Because immunofluorescence staining revealed that PLK1 expression was dominant in myofibroblasts while PLK2 expression was dominant in lung epithelial cells, we next focused on the anti-fibrotic effect of the selective PLK1 inhibitor GSK461364. Consequently, GSK461364 attenuated pulmonary fibrosis with acceptable mortality and weight loss in mice. Conclusions: These findings suggest that targeting PLK1 may be a novel therapeutic approach for pulmonary fibrosis by inhibiting lung fibroblast proliferation without affecting lung epithelial cells. In addition, while in silico screening is useful, it is essential to fully determine the biological activities of candidates by wet-lab validation studies. [ABSTRACT FROM AUTHOR]

PTEN, dual phosphatase tumor suppressor protein, is found to be frequently mutated in various cancers. Post-translational modification of PTEN is important for its sub-cellular localization and catalytic functions. But how these modifications affect cytological damage and aneuploidy is not studied in detail. We focus on the role of phosphatase activity along with C-terminal phosphorylation of PTEN in perspective of cytological damage like micronucleus, nuclear bud, and nuclear bridge formation. Our data suggest that wild-type PTEN, but not phospho-mutant PTEN significantly reduces cytological damage in PTEN null PC3 cells. In case of phosphatase-dead PTEN, cytological damage markers are increased during 24 h recovery after DNA damage. When we use phosphorylation and phosphatase-dead dual mutant PTEN, the extent of different cytological DNA damage parameters are similar to phosphatase-dead PTEN. We also find that both of those activities are essential for maintaining chromosome numbers. PTEN null cells exhibit significantly aberrant γ-tubulin pole formation during metaphase. Interestingly, we observed that p-PTEN localized to spindle poles along with PLK1 and Aurora Kinase A. Further depletion of phosphorylation and phosphatase activity of PTEN increases the expression of p-Aurora Kinase A (T288) and p-PLK1 (T210), compared to cells expressing wild-type PTEN. Again, wild-type PTEN but not phosphorylation-dead mutant is able to physically interact with PLK1 and Aurora Kinase A. Thus, our study suggests that the phosphorylation-dependent interaction of PTEN with PLK1 and Aurora Kinase A causes dephosphorylation of those mitotic kinases and by lowering their hyperphosphorylation status, PTEN prevents aberrant chromosome segregation in metaphase. [ABSTRACT FROM AUTHOR]

Meiotic chromosome segregation relies on synapsis and crossover (CO) recombination between homologous chromosomes. These processes require multiple steps that are coordinated by the meiotic cell cycle and monitored by surveillance mechanisms. In diverse species, failures in chromosome synapsis can trigger a cell cycle delay and/or lead to apoptosis. How this key step in ‘homolog engagement’ is sensed and transduced by meiotic cells is unknown. Here we report that in C. elegans, recruitment of the Polo-like kinase PLK-2 to the synaptonemal complex triggers phosphorylation and inactivation of CHK-2, an early meiotic kinase required for pairing, synapsis, and double-strand break (DSB) induction. Inactivation of CHK-2 terminates DSB formation and enables CO designation and cell cycle progression. These findings illuminate how meiotic cells ensure CO formation and accurate chromosome segregation.

Proper peripheral myelination depends upon the balance between Schwann cell proliferation and differentiation programs. The serine/threonine kinase mTOR integrates various environmental cues to serve as a central regulator of cell growth, metabolism, and function. We report here that tuberous sclerosis complex 1 (TSC1), a negative regulator of mTOR activity, establishes a stage-dependent program for Schwann cell lineage progression and myelination by controlling cell proliferation and myelin homeostasis. Tsc1 ablation in Schwann cell progenitors in mice resulted in activation of mTOR signaling, and caused over-proliferation of Schwann cells and blocked their differentiation, leading to hypomyelination. Transcriptome profiling analysis revealed that mTOR activation in Tsc1 mutants resulted in upregulation of a polo-like kinase (PLK)-dependent pathway and cell cycle regulators. Attenuation of mTOR or pharmacological inhibition of polo-like kinases partially rescued hypomyelination caused by Tsc1 loss in the developing peripheral nerves. In contrast, deletion of Tsc1 in mature Schwann cells led to redundant and overgrown myelin sheaths in adult mice. Together, our findings indicate stage-specific functions for the TSC1-mTOR-PLK signaling axis in controlling the transition from proliferation to differentiation and myelin homeostasis during Schwann cell development.

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

We propose that MuSC-derived myoblasts in PAD have transcriptomic differences that can highlight underlying causes of ischemia-induced myopathy. Differentiation capacity among perfused and ischemic human myoblasts was compared. Following next generation sequencing of mRNA, Ingenuity Pathway Analysis (IPA) was performed for canonical pathway enrichment. Live cell imaging and immunofluorescence were performed to determine myocyte fusion index and protein expression based on insights from IPA, specifically concerning cell cycle regulators including cell-division cycle protein 2 (CDC2) and polo-like kinase 1 (PLK1). Ischemic myoblasts formed attenuated myotubes indicative of reduced fusion. Additionally, myoblasts from ischemic segments showed significant differences in canonical pathways associated with PLK1 (upregulated) and G2/M DNA damage checkpoint regulation (downregulated). PLK1 inhibition with BI2536 did not affect cell viability in any group over 24 h but deterred fusion more significantly in PAD myoblasts. Furthermore, PLK1 inhibition reduced the expression of checkpoint protein CDC2 in perfused but not ischemic cells. Differentiating myoblasts derived from ischemic muscle have significant differences in gene expression including those essential to DNA-damage checkpoint regulation and cell cycle progress. DNA-damage checkpoint dysregulation may contribute to myopathy in PAD. • 1.Myoblasts from skeletal muscle in peripheral arterial disease (PAD) proliferate normally ex vivo. • 2.Despite viability and higher proliferative capacity, myoblast differentiation is significantly deterred in PAD with potential functional consequences. • Differentially expressed genetic analysis demonstrate marked upregulation of polo-like kinase (PLK1) associated mitotic pathways and concomitant downregulation in DNA checkpoint pathways in PAD. • Inhibiting PLK1 deterred differentiation significantly in PAD. [ABSTRACT FROM AUTHOR]

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor protein with dual phosphatase activity, is found to be frequently mutated in various cancers. PTEN is post-translationally modulated at various amino acid residues which are crucial for sub-cellular localization as well as its catalytic functions rendering genomic stability. Recent reports suggest that PTEN also acts as a DNA repair protein. But how post-translational modulation of PTEN affects cytological damage and aneuploidy is not studied in detail. Here, we focus on the role of sumoylation of PTEN in context with DNA damage induced cytological damage like micronucleus (MNi), nuclear bud (NB), and nuclear bridge formation. Our data suggest that wild type PTEN but not sumo-dead PTEN significantly reduces cytological damage in PTEN mutant PC3 cells. In case of sumo-dead PTEN, the cytological parameters are increased during 24 h recovery time point after DNA damage. Next, we measured the effectiveness of the sumo-dead (PTEN-K254R) mutant on aneuploidy, where we found that sumoylation is essential for maintaining chromosome number. As chromosome number variation in daughter cell is due to multiple spindle pole formation, we qualitatively and quantitatively evaluate the γ tubulin pole formation in PTEN-K254R clone transfected cells. We found aberrant pole formation is significantly increased in PTEN-K254R transfected cells compared to wild-type PTEN. Further depletion of sumoylation activity of PTEN increases the expression of phosphorylated form of Aurora kinase A (AURKA) (T288) and PLK1 (T210) proteins with or without nocodazole, a microtubule depolymerizing agent compared to cells expressing wild-type PTEN. Thus, sumoylation of PTEN is essential for maintaining genomic stability. [ABSTRACT FROM AUTHOR]

Abstract Background Polo-like kinases (PLKs) are conserved serine/threonine kinases that regulate the cell cycle. To date, the role of Giardia lamblia PLK (GlPLK) in cells has not been studied. Here, we report our investigation on the function of GlPLK to provide insight into the role of this PKL in Giardia cell division, especially during cytokinesis and flagella formation. Methods To assess the function of GIPLK, Giardia trophozoites were treated with the PLK-specific inhibitor GW843286X (GW). Using a putative open reading frame for the PLK identified in the Giardia genomic database, we generated a transgenic Giardia expressing hemagglutinin (HA)-tagged GlPLK and used this transgenic for immunofluorescence assays (IFAs). GlPLK expression was knocked down using an anti-glplk morpholino to observe its effect on the number of nuclei number and length of flagella. Giardia cells ectopically expressing truncated GlPLKs, kinase domain + linker (GlPLK-KDL) or polo-box domains (GlPLK-PBD) were constructed for IFAs. Mutant GlPLKs at Lys51, Thr179 and Thr183 were generated by site-directed mutagenesis and then used for the kinase assay. To elucidate the role of phosphorylated GlPLK, the phosphorylation residues were mutated and expressed in Giardia trophozoites Results After incubating trophozoites with 5 μM GW, the percentage of cells with > 4 nuclei and longer caudal and anterior flagella increased. IFAs indicated that GlPLK was localized to basal bodies and flagella and was present at mitotic spindles in dividing cells. Morpholino-mediated GlPLK knockdown resulted in the same phenotypes as those observed in GW-treated cells. In contrast to Giardia expressing GlPLK-PBD, Giardia expressing GlPLK-KDL was defective in terms of GIPLK localization to mitotic spindles and had altered localization of the basal bodies in dividing cells. Kinase assays using mutant recombinant GlPLKs indicated that mutation at Lys51 or at both Thr179 and Thr183 resulted in loss of kinase activity. Giardia expressing these mutant GlPLKs also demonstrated defects in cell growth, cytokinesis and flagella formation. Conclusions These data indicate that GlPLK plays a role in Giardia cell division, especially during cytokinesis, and that it is also involved in flagella formation.

Centrosomes consist of two centrioles and the surrounding pericentriolar material (PCM). The PCM expands during mitosis in a process called centrosome maturation, in which PCM scaffold proteins play pivotal roles to recruit other centrosomal proteins. In Caenorhabditis elegans, the scaffold protein SPD-5 forms a PCM scaffold in a polo-like kinase 1 (PLK-1) phosphorylation-dependent manner. However, how phosphorylation of SPD-5 promotes PCM scaffold assembly is unclear. Here, we identified three functional domains of SPD-5 through in vivo domain analyses, and propose that sequential domain interactions of SPD-5 are required for mitotic PCMscaffold assembly. Firstly, SPD-5 is targeted to centrioles through a direct interaction between its centriole localization (CL) domain and the centriolar protein PCMD-1. Then, intramolecular and intermolecular interactions between the SPD-5 phospho-regulated multimerization (PReM) domain and the PReM association (PA) domain are enhanced by phosphorylation by PLK-1, which leads to PCM scaffold expansion. Our findings suggest that the sequential domain interactions of scaffold proteins mediated by PLK-1 phosphorylation is an evolutionarily conserved mechanism of PCM scaffold assembly. [ABSTRACT FROM AUTHOR]

Simple Summary: We determined the sensitivity of several conjunctival melanoma cell lines to kinase inhibitors as well as a few other inhibitors using an image-based high throughput drug screening assay with 542 compounds. All cell lines demonstrated sensitivity to cell cycle inhibition, especially with polo-like inhibitors. The response to MAPK pathway inhibition was better in the presence of BRAFV600E mutations, while the response to PI3k/mTOR inhibition was better in the presence of the NRASQ61L mutation. Our study uncovers a large panel of new vulnerabilities in conjunctival melanoma and establishes the background for the expansion of therapeutic options in the management of this tumor. Recent evidence suggests that numerous similarities exist between the genomic landscapes of both conjunctival and cutaneous melanoma. Since alterations of several components of the MAP kinases, PI3K/mTOR, and cell cycle pathways have been reported in conjunctival melanoma, we decided to assess the sensitivity of conjunctival melanoma to targeted inhibition mostly of kinase inhibitors. A high content drug screening assay based on automated fluorescence microscopy was performed in three conjunctival melanoma cell lines with different genomic backgrounds with 489 kinase inhibitors and 53 other inhibitors. IC50 and apoptosis induction were respectively assessed for 53 and 48 compounds. The genomic background influenced the response to MAK and PI3K/mTOR inhibition, more specifically cell lines with BRAF V600E mutations were more sensitive to BRAF/MEK inhibition, while CRMM2 bearing the NRASQ61L mutation was more sensitive to PI3k/mTOR inhibition. All cell lines demonstrated sensitivity to cell cycle inhibition, being more pronounced in CRMM2, especially with polo-like inhibitors. Our data also revealed new vulnerabilities to Hsp90 and Src inhibition. This study demonstrates that the genomic background partially influences the response to targeted therapy and uncovers a large panel of potential vulnerabilities in conjunctival melanoma that may expand available options for the management of this tumor. [ABSTRACT FROM AUTHOR]

Previously, we reported that the Polo-like kinase PLK-1 phosphorylates the single Caenorhabditis elegans lamin (LMN-1) to trigger lamina depolymerization during mitosis. We showed that this event is required to form a pronuclear envelope scission event that removes membranes on the juxtaposed oocyte and sperm pronuclear envelopes in the zygote, allowing the parental chromosomes to merge in a single nucleus after segregation (Velez-Aguilera et al., 2020). Here, we show that cortical microtubule pulling forces contribute to pronuclear envelopes scission by promoting mitotic spindle elongation, and conversely, nuclear envelopes remodeling facilitates spindle elongation. We also demonstrate that weakening the pronuclear envelopes via PLK-1-mediated lamina depolymerization, is a prerequisite for the astral microtubule pulling forces to trigger pronuclear membranes scission. Finally, we provide evidence that PLK-1 mainly acts via lamina depolymerization in this process. These observations thus indicate that temporal coordination between lamina depolymerization and mitotic spindle elongation facilitates pronuclear envelopes scission and parental genomes unification.

Cytokinesis in Trypanosoma brucei, an early branching protozoan, occurs along its longitudinal axis uni-directionally from the anterior tip of the new flagellum attachment zone filament toward the cell's posterior end. However, the underlying mechanisms remain elusive. Here we report that cytokinesis in T. brucei is regulated by a concerted action of Polo-like kinase, Aurora B kinase, and a trypanosome-specific protein CIF1. Phosphorylation of CIF1 by Polo-like kinase targets it to the anterior tip of the new flagellum attachment zone filament, where it subsequently recruits Aurora B kinase to initiate cytokinesis. Consistent with its role, CIF1 depletion inhibits cytokinesis initiation from the anterior end of the cell, but, surprisingly, triggers cytokinesis initiation from the posterior end of the cell, suggesting the activation of an alternative cytokinesis from the opposite cell end. Our results reveal the mechanistic roles of CIF1 and Polo-like kinase in cytokinesis initiation and elucidate the mechanism underlying the recruitment of Aurora B kinase to the cytokinesis initiation site at late anaphase. These findings also delineate a signaling cascade controlling cytokinesis initiation from the anterior end of the cell and uncover a backup cytokinesis that is initiated from the posterior end of the cell when the typical anterior-to-posterior cytokinesis is compromised.

Background: Silybum marianum (SM) seeds contain a family of flavonolignans. Many studies have revealed the role of SM in regulating cancer cell proliferation and apoptosis, but research has rarely focused on the correlation between cancer cell regulation and SM seed (SMS). Breast cancer is the most commonly diagnosed cancer in women worldwide. In addition to clinical treatments, natural products reportedly act as auxiliary agents or treatments for breast cancer. Purpose: This study investigated the effect of SMS water extract (SMSWE) on breast cancer and explored the potential molecular mechanism of its effect. Methods: We used SMSWE to treat breast cancer cells and animals orthotopically injected with breast cancer cells. Results: The results indicated that SMSWE decreased breast cancer cell viability by disrupting mitosis. The mechanism was modulated by the downregulation of proteins involved in spindle assembly, including polo-like kinase 1 (PLK1) and the EXTAH complex (Eg5, XMAP215, Tpx2, Aurora A, and HURP). In mice orthotopically injected with breast cancer cells and treated with SMSWE, tumor growth significantly decreased and PLK1 expression decreased compared with controls. Conclusions: These current evidences provide that SMSWE treatment can decelerate breast cancer cell growth, and the possible mechanism involves mitosis disruption through decreased PLK1 expression.

The serine/threonine kinase polo-like kinase 1 (PLK1) is a master regulator of cell proliferation and contraction, but its physiological role in the lower urinary tract is unknown. We utilized transcriptomic programs of human bladder smooth muscle cells (hBSMCs), 3D bladder spheroid viability assays, and human ureterovesical junction contractility measurements to elucidate the impacts of PLK1 inhibition. This work reveals PLK1 reduction with the selective inhibitor TAK-960 (500 nM) suppresses high K+-evoked contractions of human urinary smooth muscle ex vivo while decreasing urothelial cell viability. Transcriptomic analysis of hBSMCs treated with TAK-960 shows modulation of cell cycle and contraction pathways, specifically through altered expression of Cys2/His2-type zinc finger transcription factors. In bladder spheroids, PLK1 inhibition also suppresses smooth muscle contraction protein filamin. Taken together, these findings establish PLK1 is a critical governor of urinary smooth muscle contraction and urothelial proliferation with implications for lower urinary tract disorders. Targeting PLK1 pharmacologically may therefore offer therapeutic potential to ameliorate hypercontractility and aberrant growth. Further elucidation of PLK1 signaling networks promises new insights into pathogenesis and much needed treatment advances for debilitating urinary symptoms., (© 2024 Wiley Periodicals LLC.)

Background: Polo-like kinases (PLKs) are conserved serine/threonine kinases that regulate the cell cycle. To date, the role of Giardia lamblia PLK (GlPLK) in cells has not been studied. Here, we report our investigation on the function of GlPLK to provide insight into the role of this PKL in Giardia cell division, especially during cytokinesis and flagella formation. Methods: To assess the function of GIPLK, Giardia trophozoites were treated with the PLK-specific inhibitor GW843286X (GW). Using a putative open reading frame for the PLK identified in the Giardia genomic database, we generated a transgenic Giardia expressing hemagglutinin (HA)-tagged GlPLK and used this transgenic for immunofluorescence assays (IFAs). GlPLK expression was knocked down using an anti-glplk morpholino to observe its effect on the number of nuclei number and length of flagella. Giardia cells ectopically expressing truncated GlPLKs, kinase domain + linker (GlPLK-KDL) or polo-box domains (GlPLK-PBD) were constructed for IFAs. Mutant GlPLKs at Lys51, Thr179 and Thr183 were generated by site-directed mutagenesis and then used for the kinase assay. To elucidate the role of phosphorylated GlPLK, the phosphorylation residues were mutated and expressed in Giardia trophozoites Results: After incubating trophozoites with 5 μM GW, the percentage of cells with > 4 nuclei and longer caudal and anterior flagella increased. IFAs indicated that GlPLK was localized to basal bodies and flagella and was present at mitotic spindles in dividing cells. Morpholino-mediated GlPLK knockdown resulted in the same phenotypes as those observed in GW-treated cells. In contrast to Giardia expressing GlPLK-PBD, Giardia expressing GlPLK-KDL was defective in terms of GIPLK localization to mitotic spindles and had altered localization of the basal bodies in dividing cells. Kinase assays using mutant recombinant GlPLKs indicated that mutation at Lys51 or at both Thr179 and Thr183 resulted in loss of kinase activity. Giardia expressing these mutant GlPLKs also demonstrated defects in cell growth, cytokinesis and flagella formation. Conclusions: These data indicate that GlPLK plays a role in Giardia cell division, especially during cytokinesis, and that it is also involved in flagella formation. [ABSTRACT FROM AUTHOR]

The anaphase‐promoting complex/cyclosome (APC/C), a multi‐subunit ubiquitin ligase essential for cell cycle control, is regulated by reversible phosphorylation. APC/C phosphorylation by cyclin‐dependent kinase 1 (Cdk1) promotes Cdc20 co‐activator loading in mitosis to form active APC/C‐Cdc20. However, detailed phospho‐regulation of APC/C dynamics through other kinases and phosphatases is still poorly understood. Here, we show that an interplay between polo‐like kinase (Plx1) and PP2A‐B56 phosphatase on a flexible loop domain of the subunit Apc1 (Apc1‐loop500) controls APC/C activity and mitotic progression. Plx1 directly binds to the Apc1‐loop500 in a phosphorylation‐dependent manner and promotes the formation of APC/C‐Cdc20 via Apc3 phosphorylation. Upon phosphorylation of loop residue T532, PP2A‐B56 is recruited to the Apc1‐loop500 and differentially promotes dissociation of Plx1 and PP2A‐B56 through dephosphorylation of Plx1‐binding sites. Stable Plx1 binding, which prevents PP2A‐B56 recruitment, prematurely activates the APC/C and delays APC/C dephosphorylation during mitotic exit. Furthermore, the phosphorylation status of the Apc1‐loop500 is controlled by distant Apc3‐loop phosphorylation. Our study suggests that phosphorylation‐dependent feedback regulation through flexible loop domains within a macromolecular complex coordinates the activity and dynamics of the APC/C during the cell cycle. SYNOPSIS: The APC/C, a mitotic E3 ubiquitin ligase, is regulated by reversible phosphorylation. This study shows how dynamic APC/C regulation in mitosis is coordinated by Polo‐like kinase Plx1 and phosphatase PP2A‐B56 through a flexible loop domain on the Apc1 subunit. Plx1 directly binds to the Apc1‐loop500, and promotes the formation of an active APC/C‐Cdc20 complex.PP2A‐B56 is recruited to the Apc1‐loop500 and promotes dissociation of Plx1.Recruitment of Plx1 and PP2A‐B56 is determined by phosphorylation of CDK sites (T532, T539, S558) on the Apc1‐loop500.Phosphorylation status of the Apc1‐loop500 is controlled by phosphorylation of a flexible loop domain of Apc3.Crosstalk between remote flexible APC/C loop domains is important for fine‐tuning APC/C‐Cdc20 activity with CDK activity. [ABSTRACT FROM AUTHOR]

A central player in meiotic chromosome dynamics is the conserved Polo-like kinase (PLK) family. PLKs are dynamically localized to distinct structures during meiotic prophase and phosphorylate a diverse group of substrates to control homolog pairing, synapsis, and meiotic recombination. In a recent study, we uncovered the mechanisms that control the targeting of a meiosis-specific PLK-2 in C. elegans. In early meiotic prophase, PLK-2 localizes to special chromosome regions known as pairing centers and drives homolog pairing and synapsis. PLK-2 then relocates to the synaptonemal complex (SC) after crossover designation and mediates chromosome remodeling required for homolog separation. What controls this intricate targeting of PLK-2 in space and time? We discuss recent findings and remaining questions for the future. [ABSTRACT FROM AUTHOR]

Jan A Stratmann, Martin SebastianDepartment of Internal Medicine II, University Clinic of Frankfurt, 60596 Frankfurt, GermanyAbstract: Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. Due to often unspecific disease symptoms, locally advanced or metastatic disease is diagnosed in the majority of all cases. Palliative treatment options comprise of conventional cytotoxic agents, immunotherapy with checkpoint inhibitors and the use of specific small-molecule tyrosine kinase inhibitors (TKI). However, these TKIs are mainly restricted to a small proportion of patients with lung cancer that harbor activating driver mutations. Still, the effectiveness and favorable safety profile of these compounds have prompted a systematic search for specific driver mechanisms of tumorigenesis and moreover the development of corresponding kinase inhibitors. In recent years, the Polo-like kinase (PLK) family has emerged as a key regulator in mitotic regulation. Its role in cell proliferation and the frequently observed overexpression in various tumor entities have raised much interest in basic and clinical oncology aiming to attenuate tumor growth by targeting the PLK. In this review, we give a comprehensive summary on the (pre-) clinical development of the different types of PLK inhibitors in lung cancer and summarize their mechanisms of action, safety and efficacy data and give an overview on translational research aiming to identify predictive biomarkers for a rational use of PLK inhibitors.Keywords: non-small cell lung cancer, Polo-like kinase, targeted therapy, therapy resistance

The establishment of bipolar spindles during meiotic divisions ensures faithful chromosome segregation to prevent gamete aneuploidy. We analyzed centriole duplication, as well as centrosome maturation and separation during meiosis I and II using mouse spermatocytes. The first round of centriole duplication occurs during early prophase I, and then, centrosomes mature and begin to separate by the end of prophase I to prime formation of bipolar metaphase I spindles. The second round of centriole duplication occurs at late anaphase I, and subsequently, centrosome separation coordinates bipolar segregation of sister chromatids during meiosis II. Using a germ cell‐specific conditional knockout strategy, we show that Polo‐like kinase 1 and Aurora A kinase are required for centrosome maturation and separation prior to metaphase I, leading to the formation of bipolar metaphase I spindles. Furthermore, we show that PLK1 is required to block the second round of centriole duplication and maturation until anaphase I. Our findings emphasize the importance of maintaining strict spatiotemporal control of cell cycle kinases during meiosis to ensure proficient centrosome biogenesis and, thus, accurate chromosome segregation during spermatogenesis. Synopsis: Polo‐like kinase 1 and Aurora A kinase are required for the maturation and separation of centrosomes prior to the first meiotic division in mouse spermatocytes, which ensure accurate segregation of chromosomes during spermatogenesis. Unlike in somatic cells, centrioles duplicate independently of the G to S phase transition in mouse spermatocytes.Centrioles duplicate twice during spermatogenesis; once when spermatocytes enter meiotic prophase, and again following the first meiotic division.Depletion of PLK1 and AURKA in mice impairs centrosome separation and maturation and spindle morphology during meiosis I.Plk1 haploinsufficiency leads to centriole overduplication, formation of multipolar spindles and chromosome missegregation. [ABSTRACT FROM AUTHOR]

The kinetochore is a mega-dalton protein assembly that forms within centromeric regions of chromosomes and directs their segregation during cell division. Here we review cell cycle-mediated phosphorylation events at the kinetochore, with a focus on the budding yeast Saccharomyces cerevisiae and the insight gained from forced associations of kinases and phosphatases. The point centromeres found in the budding yeast S. cerevisiae are one of the simplest such structures found in eukaryotes. The S. cerevisiae kinetochore comprises a single nucleosome, containing a centromere-specific H3 variant Cse4CENP-A, bound to a set of kinetochore proteins that connect to a single microtubule. Despite the simplicity of the budding yeast kinetochore, the proteins are mostly homologous with their mammalian counterparts. In some cases, human proteins can complement their yeast orthologs. Like its mammalian equivalent, the regulation of the budding yeast kinetochore is complex: integrating signals from the cell cycle, checkpoints, error correction, and stress pathways. The regulatory signals from these diverse pathways are integrated at the kinetochore by post-translational modifications, notably phosphorylation and dephosphorylation, to control chromosome segregation. Here we highlight the complex interplay between the activity of the different cell-cycle kinases and phosphatases at the kinetochore, emphasizing how much more we have to understand this essential structure. [ABSTRACT FROM AUTHOR]

Polo‐like kinases (PLKs) are potent regulators of cell proliferation and cell survival. Polo‐like kinases are potential targets in the treatment of anaplastic thyroid cancer (ATC), a rare but deadly disease. The therapeutic effects of volasertib, a PLK inhibitor, was evaluated for the treatment of ATC either alone or in combination with sorafenib. Volasertib decreased cell viability in three ATC cell lines (8505C, 8305C, and KAT18) in a dose‐dependent manner. Volasertib caused ATC cells to accumulate in G2/M phase, activated caspase‐3 activity, and induced apoptosis. Combination therapy using volasertib and sorafenib in ATC cells showed mostly synergistic effects. In vivo studies revealed that combination therapy of volasertib and sorafenib was effective in the treatment of 8505C xenografts. Single‐agent volasertib treatment was sufficient to retard 8305C tumor growth. No substantial morbidity was observed in animals that received either single‐agent or combination treatment. These preclinical findings suggest that volasertib could be an effective drug in treating ATC. [ABSTRACT FROM AUTHOR]

Polo-like kinases (Plks) are evolutionarily conserved serine/threonine protein kinases playing crucial roles during multiple stages of mitosis and cytokinesis in yeast and animals. Plks are characterized by a unique Polo-box domain, which plays regulatory roles in controlling Plk activation, interacting with substrates and targeting Plk to specific subcellular locations. Plk activity and protein abundance are subject to temporal and spatial control through transcription, phosphorylation and proteolysis. In the early branching protists, Plk orthologues are present in some taxa, such as kinetoplastids and Giardia, but are lost in apicomplexans, such as Plasmodium. Works from characterizing a Plk orthologue in Trypanosoma brucei, a kinetoplastid protozoan, discover its essential roles in regulating the inheritance of flagellum-associated cytoskeleton and the initiation of cytokinesis, but not any stage of mitosis. These studies reveal evolutionarily conserved and species-specific features in the control of Plk activation, substrate recognition and protein abundance, and suggest the divergence of Plk function and regulation for specialized needs in this flagellated unicellular eukaryote.

Glutamate overactivity in basal ganglia critically contributes to the exacerbation of dopaminergic neuron degeneration in Parkinson’s disease (PD). Activation of group II metabotropic glutamate receptors (mGlu2/3 receptors), which can decrease excitatory glutamate neurotransmission, provides an opportunity to slow down the degeneration of the dopaminergic system. However, the roles of mGlu2/3 receptors in relation to PD pathology were partially recognized. By using mGlu2/3 receptors agonist (LY354740) and mGlu2/3 receptors antagonist (LY341495) in mice challenged with different cumulative doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we demonstrated that systemic injection of LY354740 reduced the level of extracellular glutamate and the extent of nigro-striatal degeneration in both acute and sub-acute MPTP mice, while LY341495 amplified the lesions in sub-acute MPTP mice only. LY354740 treatment improved behavioral dysfunctions mainly in acute MPTP mice and LY341495 treatment seemed to aggravate motor deficits in sub-acute MPTP mice. In addition, ligands of mGlu2/3 receptors also influenced the total amount of glutamate and dopamine in brain tissue. Interestingly, compared with normal mice, MPTP-treated mice abnormally up-regulated the expression of polo-like kinase 2 (PLK2)/pS129 α-synuclein and phosphorylation of Fyn/N-methyl-D-aspartate receptor subunit 2A/2B (GluN2A/2B). Both acute and sub-acute MPTP mice treated with LY354740 dose-dependently reduced all the above abnormal expression. Compared with MPTP mice treated with vehicle, mice pretreated with LY341495 exhibited much higher expression of p-Fyn Tyr416/p-GluN2B Tyr1472 and PLK2/pS129 α-synuclein in sub-acute MPTP mice models. Thus, our current data indicated that mGlu2/3 receptors ligands could influence MPTP-induced toxicity, which supported a role for mGlu2/3 receptors in PD pathogenesis.

The phenotypic spectrum of colorectal cancer (CRC) is remarkably diverse, with seemingly endless variations in cell shape, mitotic figures and multicellular configurations. Despite this morphological complexity, histological grading of collective phenotype patterns provides robust prognostic stratification in CRC. Although mechanistic understanding is incomplete, previous studies have shown that the cortical protein ezrin controls diversification of cell shape, mitotic figure geometry and multicellular architecture, in 3D organotypic CRC cultures. Because ezrin is a substrate of Src tyrosine kinase that is frequently overexpressed in CRC, we investigated Src regulation of ezrin and morphogenic growth in 3D CRC cultures. Here we show that Src perturbations disrupt CRC epithelial spatial organisation. Aberrant Src activity suppresses formation of the cortical ezrin cap that anchors interphase centrosomes. In CRC cells with a normal centrosome number, these events lead to mitotic spindle misorientation, perturbation of cell cleavage, abnormal epithelial stratification, apical membrane misalignment, multilumen formation and evolution of cribriform multicellular morphology, a feature of low‐grade cancer. In isogenic CRC cells with centrosome amplification, aberrant Src signalling promotes multipolar mitotic spindle formation, pleomorphism and morphological features of high‐grade cancer. Translational studies in archival human CRC revealed associations between Src intensity, multipolar mitotic spindle frequency and high‐grade cancer morphology. Collectively, our study reveals Src regulation of CRC morphogenic growth via ezrin–centrosome engagement and uncovers combined perturbations underlying transition to high‐grade CRC morphology. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Ultra Quenchbody (UQ‐body) is a biosensor that utilizes the quenching behavior of the fluorescent dye linked to the antibody V region. When the corresponding antigen is bound to the UQ‐body, the fluorescence is restored and allows the detection of target molecules easily and sensitively. In this paper, we constructed UQ‐bodies to sensitively detect the human epidermal growth factor receptor 2 (HER2) cancer marker in solution or on cancer cells, which was further used to kill the cancer cells. A synthetic Fab fragment of anti‐HER2 antibody Fab37 with many Trp residues at hypervariable region was prepared and labeled with fluorescent dyes to obtain the UQ‐bodies. The UQ‐body could detect HER2 in solution at concentrations as low as 20 pM with an EC50 of 0.3 nM with a fourfold response. Fluorescence imaging of HER2‐positive cells was successfully performed without any washing steps. To deliver small interfering RNA (siRNA) to cancer cells, a modified UQ‐body with C‐terminal 9R sequence was also prepared. HER2‐positive cancer cells were effectively killed by polo‐like kinase 1 siRNA intracellularly delivered by the UQ‐body‐9R. The novel approach employing siRNA‐empowered UQ‐body could detect and image the HER2 antigen easily and sensitively, and effectively kill the HER2‐positive cancer cells. [ABSTRACT FROM AUTHOR]

Radiation sensitizers that can selectively act on cancer cells hold great promise to patients who receive radiation therapy. We developed a novel targeted therapy and radiation sensitizer for non-small cell lung cancer (NSCLC) based on cetuximab conjugated nanoparticle that targets epidermal growth factor receptor (EGFR) and delivers small interfering RNA (siRNA) against polo-like kinase 1 (PLK1). EGFR is overexpressed in 50% of lung cancer patients and a mediator of DNA repair, while PLK1 is a key mitotic regulator whose inhibition enhances radiation sensitivity. The nanoparticle construct (C-siPLK1-NP) effectively targets EGFR + NSCLC cells and reduces PLK1 expression, leading to G2/M arrest and cell death. Furthermore, we show a synergistic combination between C-siPLK1-NP and radiation, which was confirmed in vivo in A549 flank tumors. We also demonstrate the translational potential of C-siPLK1-NP as a systemic therapeutic in an orthotopic lung tumor model, where administration of C-siPLK1-NP reduced tumor growth and led to prolonged survival. Our findings demonstrate that C-siPLK1-NP is effective as a targeted therapy and as a potent radiation sensitizer for NSCLC. Potential application to other EGFR + cancer types such as colorectal and breast cancer is also demonstrated. [ABSTRACT FROM AUTHOR]

The past few decades have witnessed significant progress in anticancer drug discovery. Small molecules containing heterocyclic moieties have attracted considerable interest for designing new antitumor agents. Of these, the pyrimidine ring system is found in multitude of drug structures, and being the building unit of DNA and RNA makes it an attractive scaffold for the design and development of anticancer drugs. Currently, 22 pyrimidine-containing entities are approved for clinical use as anticancer drugs by the FDA. An exhaustive literature search indicates several publications and more than 59 patents from the year 2009 onwards on pyrimidine derivatives exhibiting potent antiproliferative activity. These pyrimidine derivatives exert their activity via diverse mechanisms, one of them being inhibition of protein kinases. Aurora kinase (AURK) and polo-like kinase (PLK) are protein kinases involved in the regulation of the cell cycle. Within the numerous pyrimidine-based small molecules developed as anticancer agents, this review focuses on the pyrimidine fused heterocyclic compounds modulating the AURK and PLK proteins in different phases of clinical trials as anticancer agents. This article aims to provide a comprehensive overview of synthetic strategies for the preparation of pyrimidine derivatives and their associated biological activity on AURK/PLK. It will also present an overview of the synthesis of the heterocyclic-2-aminopyrimidine, 4-aminopyrimidine and 2,4-diaminopyrimidine scaffolds, and one of the pharmacophores in AURK/PLK inhibitors is described systematically.

The CD37 targeting radioimmunoconjugate 177Lu-lilotomab satetraxetan (Betalutin) is currently being evaluated in a clinical phase 2b trial for patients with follicular lymphoma (FL) and in a phase 1 trial for patients with diffuse large B-cell lymphoma (DLBCL). Herein we have investigated the effect of 177Lu-lilotomab satetraxetan in seven activated B-cell like (ABC) DLBCL cell lines. Although the radioimmunoconjugate showed anti-tumor activity, primary resistance was observed in a subset of cell lines. Thus, we set out to identify drugs able to overcome the resistance to 177Lu-lilotomab satetraxetan in two resistant ABC-DLBCL cell lines. We performed a viability-based screen combining 177Lu-lilotomab satetraxetan with the 384-compound Cambridge Cancer Compound Library. Drug combinations were scored using Bliss and Chou-Talalay algorithms. We identified and characterized the dual-specific CDK1/2 and AURA/B kinase inhibitor JNJ-7706621 as compound able to revert the resistance to RIT, alongside topoisomerase and histone deacetylases (HDAC) inhibitors. [ABSTRACT FROM AUTHOR]

Proper partitioning of homologous chromosomes during meiosis relies on the coordinated execution of multiple interconnected events: Homologs must locate, recognize, and align with their correct pairing partners. Further, homolog pairing must be coupled to assembly of the synaptonemal complex (SC), a meiosis-specific tripartite structure that maintains stable associations between the axes of aligned homologs and regulates formation of crossovers between their DNA molecules to create linkages that enable their segregation. Here, we identify HAL-3 (Homolog Alignment 3) as an important player in coordinating these key events during Caenorhabditis elegans meiosis. HAL-3, and the previously identified HAL-2, are interacting and interdependent components of a protein complex that localizes to the nucleoplasm of germ cells. hal-3 (or hal-2) mutants exhibit multiple meiotic prophase defects including failure to establish homolog pairing, inappropriate loading of SC subunits onto unpaired chromosome axes, and premature loss of synapsis checkpoint protein PCH-2. Further, loss of hal function results in misregulation of the subcellular localization and activity of Polo-like kinases (PLK-1 and PLK-2), which dynamically localize to different defined subnuclear sites during wild-type prophase progression to regulate distinct cellular events. Moreover, loss of PLK-2 activity partially restores tripartite SC structure in a hal mutant background, suggesting that the defect in pairwise SC assembly in hal mutants reflects inappropriate PLK activity. Together, our data support a model in which the nucleoplasmic HAL-2/HAL-3 protein complex constrains both localization and activity of meiotic Polo-like kinases, thereby preventing premature interaction with stage-inappropriate targets.. [ABSTRACT FROM AUTHOR]

Polo-like kinase (PLK1) has been identified as a potential target for cancer treatment. Although a number of small molecules have been investigated as PLK1 inhibitors, many of which showed limited selectivity. PLK1 harbors a regulatory domain, the Polo box domain (PBD), which has a key regulatory function for kinase activity and substrate recognition. We report on 3-bromomethyl-benzofuran-2-carboxylic acid ethyl ester (designated: MCC1019) as selective PLK1 inhibitor targeting PLK1 PBD. Cytotoxicity and fluorescence polarization-based screening were applied to a library of 1162 drug-like compounds to identify potential inhibitors of PLK1 PBD. The activity of compound MC1019 against the PLK1 PBD was confirmed using fluorescence polarization and microscale thermophoresis. This compound exerted specificity towards PLK1 over PLK2 and PLK3. MCC1019 showed cytotoxic activity in a panel of different cancer cell lines. Mechanistic investigations in A549 lung adenocarcinoma cells revealed that MCC1019 induced cell growth inhibition through inactivation of AKT signaling pathway, it also induced prolonged mitotic arrest—a phenomenon known as mitotic catastrophe, which is followed by immediate cell death via apoptosis and necroptosis. MCC1019 significantly inhibited tumor growth in vivo in a murine lung cancer model without affecting body weight or vital organ size, and reduced the growth of metastatic lesions in the lung. We propose MCC1019 as promising anti-cancer drug candidate. MCC1019 is a novel anticancer candidate that selectively targets PLK1. It mediates G2/M cell cycle arrest and cell death through induction of apoptosis and necroptosis. Inhibition of PLK1 downstream effectors like spindle assembly check points and cell growth pathway was well characterized. In vivo models revealed inhibition of tumor growth and metastasis. fx1 [ABSTRACT FROM AUTHOR]

Trypanosoma brucei possesses a motile flagellum that determines cell morphology and the cell division plane. Inheritance of the newly assembled flagellum during the cell cycle is controlled by the Polo-like kinase homolog TbPLK, which also regulates cytokinesis initiation. How TbPLK is targeted to its subcellular locations remains poorly understood. Here we report the trypanosome-specific protein BOH1 that cooperates with TbPLK to regulate flagellum inheritance and cytokinesis initiation in the procyclic formof T. brucei. BOH1 localizes to an unusual sub-domain in the flagellum-associated hook complex, bridging the hook complex, the centrin arm and the flagellum attachment zone. Depletion of BOH1 disrupts hook-complex morphology, inhibits centrin-arm elongation and abolishes flagellum attachment zone assembly, leading to flagellum mis-positioning and detachment. Further, BOH1 deficiency impairs the localization of TbPLK and the cytokinesis regulator CIF1 to the cytokinesis initiation site, providing a molecular mechanism for its role in cytokinesis initiation. These findings reveal the roles of BOH1 in maintaining hookcomplex morphology and regulating flagellum inheritance, and establish BOH1 as an upstream regulator of the TbPLK-mediated cytokinesis regulatory pathway. [ABSTRACT FROM AUTHOR]

Centrosomes are major microtubule-organizing centers composed of centrioles surrounded by an extensive proteinacious layer called the pericentriolar material (PCM). In Caenorhabditis elegans embryos, the mitotic PCM expands by Polo-like kinase 1 (PLK-1) phosphorylation-accelerated assembly of SPD-5 molecules into supramolecular scaffolds. However, how PLK-1 phosphorylation regulates SPD-5 assembly is not known. We found that a mutant version of SPD-5 that is insensitive to PLK-1 phosphorylation (SPD-54A) could localize to PCM but was unable to rescue the reduction in PCM size and density when wild-type SPD-5 levels were decreased. In vitro, purified SPD-54A self-assembled into functional supramolecular scaffolds over long time scales, suggesting that phosphorylation only controls the rate of SPD-5 scaffold assembly. Furthermore, the SPD-5 scaffold, once assembled, remained intact and supported microtubule nucleation in the absence of PLK-1 activity in vivo. We conclude that PLK-1 is required for rapid assembly of the PCM scaffold but not for scaffold maintenance or function. Based on this idea, we developed a theoretical model that adequately predicted PCM growth rates in different mutant conditions in vivo. We propose that PLK-1 phosphorylation-dependent conversion of SPD-5 into an assembly-competent form underlies PCM formation in vivo and that the rate of this conversion determines final PCM size and density.

Adenomatous polyposis coli (APC) mutations cause aneuploidy and are responsible for familial adenomatous polyposis characterized by chromosomal instability. PLK1 contributes to sustain an intact spindle assembly checkpoint ensuring genomic stability. In our work using independent ApcMin/+ mouse models we revealed that PLK1 functions as tumor suppressor in APC-mutated colorectal cancers.

The polo-box domain of polo-like kinase 1 (PLK1-PBD) is proved to have crucial roles in cell proliferation. Designing PLK1-PBD inhibitors is challenging due to their poor cellular penetration. In this study, we applied a virtual screening workflow based on a combination of structure-based pharmacophore modeling with molecular docking screening techniques, so as to discover potent PLK1-PBD peptide inhibitors. The resulting 9 virtual screening peptides showed affinities for PLK1-PBD in a competitive binding assay. In particular, peptide 5 exhibited an approximately 100-fold increase in inhibitory activity (IC50 = 70 nM), as compared with the control poloboxtide. Moreover, cell cycle experiments indicated that peptide 5 effectively inhibited the expression of p-Cdc25C and cell cycle regulatory proteins by affecting the function of PLK1-PBD, thereby inducing mitotic arrest at the G2/M phase. Overall, peptide 5 can serve as a potent lead for further investigation as PLK1-PBD inhibitors.

Phosphorylation of the α-synuclein (α-syn) protein at Ser129 [P(S129)-α-syn] was found to be the most abundant form in intracellular inclusions in brains from Parkinson's disease (PD) patients. This finding suggests that P(S129)-α-syn plays a central role in the pathogenesis of PD. However, it is at present unclear whether P(S129)-α-syn is pathogenic driving the neurodegenerative process. Rodent studies using neither the phosphomimics of human α-syn nor co-expression of human wild-type α-syn and kinases phosphorylating α-syn at Ser129 gave consistent results. One major concern in interpreting these findings is that human α-syn was expressed above physiological levels inducing neurodegeneration in rat nigral neurons. In order to exclude this confounding factor, we took a different approach and increased the phosphorylation level of endogenous α-syn. For this purpose, we took advantage of recombinant adeno-associated viral (rAAV) vectors to deliver polo-like kinase (PLK) 2 or PLK3 in the substantia nigra and investigated whether increased levels of P(S129)-α-syn compromised the function and survival of nigral dopaminergic neurons. Interestingly, we observed that hyperphosphorylated α-syn did not induce nigral dopaminergic cell death, as assessed at 1 and 4 months. Furthermore, histological analysis did not show any accumulation of α-syn protein or formation of inclusions. Using in vivo microdialysis, we found that the only measurable functional alteration was the depolarisation-induced release of dopamine, while the in vivo synthesis rate of DOPA and dopamine baseline release remained unaltered. Taken together, our results suggest that phosphorylation of α-syn at Ser129 does not confer a toxic gain of function per se.

Genome haploidization at meiosis depends on two consecutive nuclear divisions, which are controlled by an oscillatory system consisting of Cdk1-cyclin B and the APC/C bound to the Cdc20 activator. How the oscillator generates exactly two divisions has been unclear. We have studied this question in yeast where exit from meiosis involves accumulation of the APC/C activator Ama1 at meiosis II. We show that inactivation of the meiosis I-specific protein Spo13/MEIKIN results in a single-division meiosis due to premature activation of APC/C Ama1 . In the wild type, Spo13 bound to the polo-like kinase Cdc5 prevents Ama1 synthesis at meiosis I by stabilizing the translational repressor Rim4. In addition, Cdc5-Spo13 inhibits the activity of Ama1 by converting the B-type cyclin Clb1 from a substrate to an inhibitor of Ama1. Cdc20-dependent degradation of Spo13 at anaphase I unleashes a feedback loop that increases Ama1's synthesis and activity, leading to irreversible exit from meiosis at the second division. Thus, by repressing the exit machinery at meiosis I, Cdc5-Spo13 ensures that cells undergo two divisions to produce haploid gametes., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Cyclin-dependent kinases (CDKs) and Polo-like kinases (PLKs) play key role in the regulation of the cell cycle. The aim of our study was originally the further development of our recently discovered polo-like kinase 1 (PLK1) inhibitors. A series of new 2,4-disubstituted pyrimidine derivatives were synthesized around the original hit, but their PLK1 inhibitory activity was very poor. However the novel compounds showed nanomolar CDK9 inhibitory activity and very good antiproliferative effect on multiple myeloma cell lines (RPMI-8226). [ABSTRACT FROM AUTHOR]

ABSTRACT Giardia intestinalis is a protozoan parasite that causes giardiasis, a form of severe and infectious diarrhea. Despite the importance of the cell cycle in the control of proliferation and differentiation during a giardia infection, it has been difficult to study this process due to the absence of a synchronization procedure that would not induce cellular damage resulting in artifacts. We utilized counterflow centrifugal elutriation (CCE), a size-based separation technique, to successfully obtain fractions of giardia cultures enriched in G1, S, and G2. Unlike drug-induced synchronization of giardia cultures, CCE did not induce double-stranded DNA damage or endoreplication. We observed increases in the appearance and size of the median body in the cells from elutriation fractions corresponding to the progression of the cell cycle from early G1 to late G2. Consequently, CCE could be used to examine the dynamics of the median body and other structures and organelles in the giardia cell cycle. For the cell cycle gene expression studies, the actin-related gene was identified by the program geNorm as the most suitable normalizer for reverse transcription-quantitative PCR (RT-qPCR) analysis of the CCE samples. Ten of 11 suspected cell cycle-regulated genes in the CCE fractions have expression profiles in giardia that resemble those of higher eukaryotes. However, the RNA levels of these genes during the cell cycle differ less than 4-fold to 5-fold, which might indicate that large changes in gene expression are not required by giardia to regulate the cell cycle. IMPORTANCE Giardias are among the most commonly reported intestinal protozoa in the world, with infections seen in humans and over 40 species of animals. The life cycle of giardia alternates between the motile trophozoite and the infectious cyst. The regulation of the cell cycle controls the proliferation of giardia trophozoites during an active infection and contains the restriction point for the differentiation of trophozoite to cyst. Here, we developed counterflow centrifugal elutriation as a drug-free method to obtain fractions of giardia cultures enriched in cells from the G1, S, and G2 stages of the cell cycle. Analysis of these fractions showed that the cells do not show side effects associated with the drugs used for synchronization of giardia cultures. Therefore, counterflow centrifugal elutriation would advance studies on key regulatory events during the giardia cell cycle and identify potential drug targets to block giardia proliferation and transmission.

Cytoplasmic microtubules (cMT) control mitotic spindle positioning in many organisms, and are therefore pivotal for successful cell division. Despite its importance, the temporal control of cMT formation remains poorly understood. Here we show that unlike the best-studied yeast Saccharomyces cerevisiae, position of pre-anaphase nucleus is not strongly biased toward bud neck in Ogataea polymorpha and the regulation of spindle positioning becomes active only shortly before anaphase. This is likely due to the unstable property of cMTs compared to those in S. cerevisiae. Furthermore, we show that cMT nucleation/anchoring is restricted at the level of recruitment of the γ-tubulin complex receptor, Spc72, to spindle pole body (SPB), which is regulated by the polo-like kinase Cdc5. Additionally, electron microscopy revealed that the cytoplasmic side of SPB is structurally different between G1 and anaphase. Thus, polo-like kinase dependent recruitment of γ-tubulin receptor to SPBs determines the timing of spindle orientation in O. polymorpha.