Molecular basis promoting centriole triplet microtubule assembly.

The triplet microtubule, a core structure of centrioles crucial for the organization of centrosomes, cilia, and flagella, consists of unclosed incomplete microtubules. The mechanisms of its assembly represent a fundamental open question in biology. Here, we discover that the ciliopathy protein HYLS1 and the ß-tubulin isotype TUBB promote centriole triplet microtubule assembly. HYLS1 or a C-terminal tail truncated version of TUBB generates tubulin-based superstructures composed of centriole-like incomplete microtubule chains when overexpressed in human cells. AlphaFold-based structural models and mutagenesis analyses further suggest that the ciliopathy-related residue D211 of HYLS1 physically traps the wobbling C-terminal tail of TUBB, thereby suppressing its inhibitory role in the initiation of the incomplete microtubule assembly. Overall, our findings provide molecular insights into the biogenesis of atypical microtubule architectures conserved for over a billion years.

Predicting potential target genes in molecular biology experiments using machine learning and multifaceted data sources.

Experimental analysis of functionally related genes is key to understanding biological phenomena. The selection of genes to study is a crucial and challenging step, as it requires extensive knowledge of the literature and diverse biomedical data resources. Although software tools that predict relationships between genes are available to accelerate this process, they do not directly incorporate experiment information derived from the literature. Here, we develop LEXAS, a target gene suggestion system for molecular biology experiments. LEXAS is based on machine learning models trained with diverse information sources, including 24 million experiment descriptions extracted from full-text articles in PubMed Central by using a deep-learning-based natural language processing model. By integrating the extracted experiment contexts with biomedical data sources, LEXAS suggests potential target genes for upcoming experiments, complementing existing tools like STRING, FunCoup, and GOSemSim. A simple web interface enables biologists to consider newly derived gene information while planning experiments.

An antioxidant screen identifies ascorbic acid for prevention of light-induced mitotic prolongation in live cell imaging.

Phototoxicity is an important issue in fluorescence live imaging of light-sensitive cellular processes such as mitosis. Among several approaches to reduce phototoxicity, the addition of antioxidants to the media has been used as a simple method. Here, we analyzed the impact of phototoxicity on the mitotic progression in fluorescence live imaging of human cells and performed a screen to identify the most efficient antioxidative agents that reduce it. Quantitative analysis shows that high amounts of light illumination cause various mitotic defects such as prolonged mitosis and delays of chromosome alignment and centrosome separation. Among several antioxidants, our screen reveals that ascorbic acid significantly alleviates these phototoxic effects in mitosis. Furthermore, we demonstrate that adding ascorbic acid to the media enables fluorescence imaging of mitotic events at very high temporal resolution without obvious photodamage. Thus, this study provides an optimal method to effectively reduce the phototoxic effects in fluorescence live cell imaging.

Mitotic perturbation is a key mechanism of action of decitabine in myeloid tumor treatment.

Decitabine (DAC) is clinically used to treat myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our genome-wide CRISPR-dCas9 activation screen using MDS-derived AML cells indicates that mitotic regulation is critical for DAC resistance. DAC strongly induces abnormal mitosis (abscission failure or tripolar mitosis) in human myeloid tumors at clinical concentrations, especially in those with TP53 mutations or antecedent hematological disorders. This DAC-induced mitotic disruption and apoptosis are significantly attenuated in DNMT1-depleted cells. In contrast, overexpression of Dnmt1, but not the catalytically inactive mutant, enhances DAC-induced mitotic defects in myeloid tumors. We also demonstrate that DAC-induced mitotic disruption is enhanced by pharmacological inhibition of the ATR-CLSPN-CHK1 pathway. These data challenge the current assumption that DAC inhibits leukemogenesis through DNMT1 inhibition and subsequent DNA hypomethylation and highlight the potent activity of DAC to disrupt mitosis through aberrant DNMT1-DNA covalent bonds.

ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid RPE1 and HCT116 cells.

BACKGROUND: Recent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, partly due to the complexity of long ssDNA preparation, it remains unclear whether ssDNA is the optimal type of HDR donors for insertion of long transgenes such as fluorescent reporters in human cells. RESULTS: In this study, we established a nuclease-based simple method for the preparation of long ssDNA with high yield and purity, and comprehensively compared the performance of ssDNA and dsDNA donors with 90 bases of homology arms for endogenous gene tagging with long transgenes in human diploid RPE1 and HCT116 cells. Quantification using flow cytometry revealed lower efficiency of endogenous fluorescent tagging with ssDNA donors than with dsDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was lower with ssDNA donors than with dsDNA. Moreover, in off-target integration analyses using donors without homology arms, ssDNA and dsDNA were comparably prone to non-homologous integration. CONCLUSIONS: These results indicate that ssDNA is not superior to dsDNA as long HDR donors with relatively short homology arms for gene knock-in in human RPE1 and HCT116 cells.

A CRISPR-del-based pipeline for complete gene knockout in human diploid cells.

The advance of CRISPR/Cas9 technology has enabled us easily to generate gene knockout cell lines by introducing insertion-deletion mutations (indels) at the target site via the error-prone non-homologous end joining repair system. Frameshift-promoting indels can disrupt gene functions by generation of a premature stop codon. However, there is growing evidence that targeted genes are not always knocked out by the indel-based gene disruption. Here, we established a pipeline of CRISPR-del, which induces a large chromosomal deletion by cutting two different target sites, to perform 'complete' gene knockout efficiently in human diploid cells. Quantitative analyses show that the frequency of gene deletion with this approach is much higher than that of conventional CRISPR-del methods. The lengths of the deleted genomic regions demonstrated in this study are longer than those of 95% of the human protein-coding genes. Furthermore, the pipeline enabled the generation of a model cell line having a bi-allelic cancer-associated chromosomal deletion. Overall, these data lead us to propose that the CRISPR-del pipeline is an efficient and practical approach for producing 'complete' gene knockout cell lines in human diploid cells.

Stress-dependent cell stiffening by tardigrade tolerance proteins that reversibly form a filamentous network and gel.

Tardigrades are able to tolerate almost complete dehydration by entering a reversible ametabolic state called anhydrobiosis and resume their animation upon rehydration. Dehydrated tardigrades are exceptionally stable and withstand various physical extremes. Although trehalose and late embryogenesis abundant (LEA) proteins have been extensively studied as potent protectants against dehydration in other anhydrobiotic organisms, tardigrades produce high amounts of tardigrade-unique protective proteins. Cytoplasmic-abundant heat-soluble (CAHS) proteins are uniquely invented in the lineage of eutardigrades, a major class of the phylum Tardigrada and are essential for their anhydrobiotic survival. However, the precise mechanisms of their action in this protective role are not fully understood. In the present study, we first postulated the presence of tolerance proteins that form protective condensates via phase separation in a stress-dependent manner and searched for tardigrade proteins that reversibly form condensates upon dehydration-like stress. Through a comprehensive search using a desolvating agent, trifluoroethanol (TFE), we identified 336 proteins, collectively dubbed "TFE-Dependent ReversiblY condensing Proteins (T-DRYPs)." Unexpectedly, we rediscovered CAHS proteins as highly enriched in T-DRYPs, 3 of which were major components of T-DRYPs. We revealed that these CAHS proteins reversibly polymerize into many cytoskeleton-like filaments depending on hyperosmotic stress in cultured cells and undergo reversible gel-transition in vitro. Furthermore, CAHS proteins increased cell stiffness in a hyperosmotic stress-dependent manner and counteract the cell shrinkage caused by osmotic pressure, and even improved the survival against hyperosmotic stress. The conserved putative helical C-terminal region is necessary and sufficient for filament formation by CAHS proteins, and mutations disrupting the secondary structure of this region impaired both the filament formation and the gel transition. On the basis of these results, we propose that CAHS proteins are novel cytoskeleton-like proteins that form filamentous networks and undergo gel-transition in a stress-dependent manner to provide on-demand physical stabilization of cell integrity against deformative forces during dehydration and could contribute to the exceptional physical stability in a dehydrated state.

Experimental and Natural Induction of de novo Centriole Formation.

In cycling cells, new centrioles are assembled in the vicinity of pre-existing centrioles. Although this canonical centriole duplication is a tightly regulated process in animal cells, centrioles can also form in the absence of pre-existing centrioles; this process is termed de novo centriole formation. De novo centriole formation is triggered by the removal of all pre-existing centrioles in the cell in various manners. Moreover, overexpression of polo-like kinase 4 (Plk4), a master regulatory kinase for centriole biogenesis, can induce de novo centriole formation in some cell types. Under these conditions, structurally and functionally normal centrioles can be formed de novo. While de novo centriole formation is normally suppressed in cells with intact centrioles, depletion of certain suppressor proteins leads to the ectopic formation of centriole-related protein aggregates in the cytoplasm. It has been shown that de novo centriole formation also occurs naturally in some species. For instance, during the multiciliogenesis of vertebrate epithelial cells, massive de novo centriole amplification occurs to form numerous motile cilia. In this review, we summarize the previous findings on de novo centriole formation, particularly under experimental conditions, and discuss its regulatory mechanisms.

Biophysical and biochemical properties of Deup1 self-assemblies: a potential driver for deuterosome formation during multiciliogenesis.

The deuterosome is a non-membranous organelle involved in large-scale centriole amplification during multiciliogenesis. Deuterosomes are specifically assembled during the process of multiciliogenesis. However, the molecular mechanisms underlying deuterosome formation are poorly understood. In this study, we investigated the molecular properties of deuterosome protein 1 (Deup1), an essential protein involved in deuterosome assembly. We found that Deup1 has the ability to self-assemble into macromolecular condensates both in vitro and in cells. The Deup1-containing structures formed in multiciliogenesis and the Deup1 condensates self-assembled in vitro showed low turnover of Deup1, suggesting that Deup1 forms highly stable structures. Our biochemical analyses revealed that an increase of the concentration of Deup1 and a crowded molecular environment both facilitate Deup1 self-assembly. The self-assembly of Deup1 relies on its N-terminal region, which contains multiple coiled coil domains. Using an optogenetic approach, we demonstrated that self-assembly and the C-terminal half of Deup1 were sufficient to spatially compartmentalize centrosomal protein 152 (Cep152) and polo like kinase 4 (Plk4), master components for centriole biogenesis, in the cytoplasm. Collectively, the present data suggest that Deup1 forms the structural core of the deuterosome through self-assembly into stable macromolecular condensates.This article has an associated First Person interview with the first author of the paper.

Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly.

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. Here, we show the cooperative roles of the centriole and PCM scaffold proteins, pericentrin and CDK5RAP2, in the recruitment of CEP192 to spindle poles during mitosis. Systematic depletion of PCM proteins revealed that CEP192, but not pericentrin and/or CDK5RAP2, was crucial for bipolar spindle assembly in HeLa, RPE1, and A549 cells with centrioles. Upon double depletion of pericentrin and CDK5RAP2, CEP192 that remained at centriole walls was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the perturbation of PLK1, a critical kinase for PCM assembly, efficiently suppressed bipolar spindle formation in mitotic cells with one centrosome. Overall, these data suggest that the centriole and PCM scaffold proteins cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation.

Cep57 and Cep57L1 maintain centriole engagement in interphase to ensure centriole duplication cycle.

Centrioles duplicate in interphase only once per cell cycle. Newly formed centrioles remain associated with their mother centrioles. The two centrioles disengage at the end of mitosis, which licenses centriole duplication in the next cell cycle. Therefore, timely centriole disengagement is critical for the proper centriole duplication cycle. However, the mechanisms underlying centriole engagement during interphase are poorly understood. Here, we show that Cep57 and Cep57L1 cooperatively maintain centriole engagement during interphase. Codepletion of Cep57 and Cep57L1 induces precocious centriole disengagement in interphase without compromising cell cycle progression. The disengaged daughter centrioles convert into centrosomes during interphase in a Plk1-dependent manner. Furthermore, the centrioles reduplicate and the centriole number increases, which results in chromosome segregation errors. Overall, these findings demonstrate that the maintenance of centriole engagement by Cep57 and Cep57L1 during interphase is crucial for the tight control of centriole copy number and thus for proper chromosome segregation.

Emerging insights into symmetry breaking in centriole duplication: updated view on centriole duplication theory.

Centriole duplication occurs once per cell cycle. Since only a single daughter centriole is assembled adjacent to each mother centriole, symmetry around the mother centriole must be broken in the process of centriole duplication. Recent studies have established that Plk4, a master kinase for centriole duplication, can self-assemble into condensates, and have suggested that this Plk4 self-assembly is the key to symmetry breaking. Here, we present the current hypotheses for how Plk4 could break symmetry around the mother centriole via autonomous regulation. After this initial symmetry-breaking process, the ring-to-dot conversion of Plk4 around the mother centriole completes the selection of the site for procentriole formation. We also discuss how this dynamic transition contributes to the strict regulation of centriole duplication.

The centriole protein CEP76 negatively regulates PLK1 activity in the cytoplasm for proper mitotic progression.

Polo-like kinase 1 (PLK1) dynamically changes its localization and plays important roles in proper mitotic progression. In particular, strict control of cytoplasmic PLK1 is needed to prevent mitotic defects. However, the regulation of cytoplasmic PLK1 is not fully understood. In this study, we show that CEP76, a centriolar protein, physically interacts with PLK1 and tightly controls the activation of cytoplasmic PLK1 during mitosis in human cells. We found that removal of centrosomes induced ectopic aggregation of PLK1, which is highly phosphorylated, in the cytoplasm during mitosis. Importantly, a targeted RNAi screen revealed that depletion of CEP76 resulted in a similar phenotype. In addition, depletion of CEP76 caused defective spindle orientation and mitotic delay. Moreover, the formation of ectopic PLK1 aggregates and defective spindle orientation were significantly suppressed by the inhibition of PLK1 kinase activity. Overall, these results demonstrate that CEP76 suppresses the aberrant activation of cytoplasmic PLK1 for proper mitotic progression.This article has an associated First Person interview with the first author of the paper.

Mechanisms of spindle bipolarity establishment in acentrosomal human cells.

Centrosomes are not absolutely essential for cell division; acentrosomal bipolar spindles can be established in oocytes and centrosome-eliminated somatic cells. However, the detailed mechanisms describing how spindle bipolarity is established without centrosomes are not completely understood. We have recently demonstrated that in acentrosomal human cells, nuclear mitotic apparatus protein (NuMA) assemblies-mediated microtubule asters and EG5 promote spindle bipolarization in early mitosis.

Centrosomal and Non-centrosomal Functions Emerged through Eliminating Centrosomes.

Centrosomes are highly conserved organelles that act as the major microtubule-organizing center (MTOC) in animal somatic cells. Through their MTOC activity, centrosomes play various roles throughout the cell cycle, such as supporting cell migration in interphase and spindle organization and positioning in mitosis. Various approaches for removing centrosomes from somatic cells have been developed and applied over the past few decades to understand the precise roles of centrosomes. Centrinone, a reversible and selective PLK4 (polo-like kinase 4) inhibitor, has recently emerged as an efficient approach to eliminate centrosomes. In this review, we describe the latest findings on centrosome function that have been revealed using various centrosome-eliminating approaches. In addition, we discuss our recent findings on the mechanism of centrosome-independent spindle bipolarization, discovered through the use of centrinone.Key words: centrosome, centrinone, mitotic spindle, bipolarity, NuMA.

The Emerging Role of ncRNAs and RNA-Binding Proteins in Mitotic Apparatus Formation.

Mounting experimental evidence shows that non-coding RNAs (ncRNAs) serve a wide variety of biological functions. Recent studies suggest that a part of ncRNAs are critically important for supporting the structure of subcellular architectures. Here, we summarize the current literature demonstrating the role of ncRNAs and RNA-binding proteins in regulating the assembly of mitotic apparatus, especially focusing on centrosomes, kinetochores, and mitotic spindles.

NuMA assemblies organize microtubule asters to establish spindle bipolarity in acentrosomal human cells.

In most animal cells, mitotic spindle formation is mediated by coordination of centrosomal and acentrosomal pathways. At the onset of mitosis, centrosomes promote spindle bipolarization. However, the mechanism through which the acentrosomal pathways facilitate the establishment of spindle bipolarity in early mitosis is not completely understood. In this study, we show the critical roles of nuclear mitotic apparatus protein (NuMA) in the generation of spindle bipolarity in acentrosomal human cells. In acentrosomal human cells, we found that small microtubule asters containing NuMA formed at the time of nuclear envelope breakdown. In addition, these asters were assembled by dynein and the clustering activity of NuMA. Subsequently, NuMA organized the radial array of microtubules, which incorporates Eg5, and thus facilitated spindle bipolarization. Importantly, in cells with centrosomes, we also found that NuMA promoted the initial step of spindle bipolarization in early mitosis. Overall, these data suggest that canonical centrosomal and NuMA-mediated acentrosomal pathways redundantly promote spindle bipolarity in human cells.

Feedback loops in the Plk4-STIL-HsSAS6 network coordinate site selection for procentriole formation.

Centrioles are duplicated once in every cell cycle, ensuring the bipolarity of the mitotic spindle. How the core components cooperate to achieve high fidelity in centriole duplication remains poorly understood. By live-cell imaging of endogenously tagged proteins in human cells throughout the entire cell cycle, we quantitatively tracked the dynamics of the critical duplication factors: Plk4, STIL and HsSAS6. Centriolar Plk4 peaks and then starts decreasing during the late G1 phase, which coincides with the accumulation of STIL at centrioles. Shortly thereafter, the HsSAS6 level increases steeply at the procentriole assembly site. We also show that both STIL and HsSAS6 are necessary for attenuating Plk4 levels. Furthermore, our mathematical modeling and simulation suggest that the STIL-HsSAS6 complex in the cartwheel has a negative feedback effect on centriolar Plk4. Combined, these findings illustrate how the dynamic behavior of and interactions between critical duplication factors coordinate the centriole-duplication process.This article has an associated First Person interview with the first author of the paper.

A theory of centriole duplication based on self-organized spatial pattern formation.

In each cell cycle, centrioles are duplicated to produce a single copy of each preexisting centriole. At the onset of centriole duplication, the master regulator Polo-like kinase 4 (Plk4) undergoes a dynamic change in its spatial pattern around the preexisting centriole, forming a single duplication site. However, the significance and mechanisms of this pattern transition remain unknown. Using super-resolution imaging, we found that centriolar Plk4 exhibits periodic discrete patterns resembling pearl necklaces, frequently with single prominent foci. Mathematical modeling and simulations incorporating the self-organization properties of Plk4 successfully generated the experimentally observed patterns. We therefore propose that the self-patterning of Plk4 is crucial for the regulation of centriole duplication. These results, defining the mechanisms of self-organized regulation, provide a fundamental principle for understanding centriole duplication.