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The mitotic spindle is the bipolar, microtubule-based structure that segregates chromosomes at each cell division. Aberrant spindles are frequently observed in cancer cells, but how oncogenic transformation affects spindle mechanics and function, particularly in the mechanical context of solid tumors, remains poorly understood. Here, we constitutively overexpress the oncogene cyclin D1 in human MCF10A cells to probe its effects on spindle architecture and response to compressive force. We find that cyclin D1 overexpression increases the incidence of spindles with extra poles, centrioles, and chromosomes. However, it also protects spindle poles from fracturing under compressive force, a deleterious outcome linked to multipolar cell divisions. Our findings suggest that cyclin D1 overexpression may adapt cells to increased compressive stress, possibly contributing to its prevalence in cancers such as breast cancer by allowing continued proliferation in mechanically challenging environments.

Video-microscopy is a technology widely used to follow, in a single cell manner, cell behavior. A number of new studies are searching a way to track these behaviors by artificial intelligence; unfortunately some real-time events still have to be track manually. For that reason, we developed a software that helps the experimenter to analyze collected data. Toto-cell is very simple to use and it can be adapted at different type of analyses or treatments. It allows a wide new range of parameters that were nearly impossible to calculate only by hand. We thus developed this new software using HEC-1-A endometrial cell line to track different cellular parameters such as: the number of normal/abnormal mitosis, the ratio per day of death, mitosis, cell fusions or finally the length between two mitosis cycles. We treated our cells with cisplatin, doxorubicin or AZD5363 (an Akt inhibitor) to obtain different cellular events. What emerged is a huge heterogeneity for these analyzed parameters between the cells in a single treatment which is clearly demonstrated by the results provided by Toto-Cell. In conclusion, our software is an important tool to facilitate the analysis of video-microscopy, in a quantifying and qualifying manner. It enables a higher accuracy when compared to manual calculations. [ABSTRACT FROM AUTHOR]

Mitosis is the process by which eukaryotic cells divide to produce two similar daughter cells with identical genetic material. Research into the process of mitosis is therefore of critical importance both for the basic understanding of cell biology and for the clinical approach to manifold pathologies resulting from its malfunctioning, including cancer. In this paper, we propose an approach to study mitotic progression automatically using deep learning. We used neural networks to predict different mitosis stages. We extracted video sequences of cells undergoing division and trained a Recurrent Neural Network (RNN) to extract image features. The use of RNN enabled better extraction of features. The RNN-based approach gave better performance compared to classifier based feature extraction methods which do not use time information. Evaluation of precision, recall, and F-score indicates the superiority of the proposed model compared to the baseline. To study the loss in performance due to confusion between adjacent classes, we plotted the confusion matrix as well. In addition, we visualized the feature space to understand why RNNs are better at classifying the mitosis stages than other classifier models, which indicated the formation of strong clusters for the different classes, clearly confirming the advantage of the proposed RNN-based approach. [ABSTRACT FROM AUTHOR]

The mitotic regulator, Aurora kinase B (AURKB), is frequently overexpressed in malignancy and is a target for therapeutic intervention. The compound, LXY18, is a potent, orally available small molecule that inhibits the proper localization of AURKB during late mitosis, without affecting its kinase activity. In this study, we demonstrate that LXY18 elicits apoptosis in cancer cells derived from various indications, but not in non-transformed cell lines. The apoptosis is p53-independent, triggered by a prolonged mitotic arrest and occurs predominantly in mitosis. Some additional cells succumb post-mitotic slippage. We also demonstrate that cancer cell lines refractory to AURKB kinase inhibitors are sensitive to LXY18. The mitotic proteins MKLP2, NEK6, NEK7 and NEK9 are known regulators of AURKB localization during the onset of anaphase. LXY18 fails to inhibit the catalytic activity of these AURKB localization factors. Overall, our findings suggest a novel activity for LXY18 that produces a prolonged mitotic arrest and lethality in cancer cells, leaving non-transformed cells healthy. This new activity suggests that the compound may be a promising drug candidate for cancer treatment and that it can also be used as a tool compound to further dissect the regulatory network controlling AURKB localization. [ABSTRACT FROM AUTHOR]

Understanding the mitotic DNA damage response (DDR) is critical to our comprehension of cancer, premature aging and developmental disorders which are marked by DNA repair deficiencies. In this study we use a micro-focused laser to induce DNA damage in selected mitotic chromosomes to study the subsequent repair response. Our findings demonstrate that (1) mitotic cells are capable of DNA repair as evidenced by DNA synthesis at damage sites, (2) Repair is attenuated when DNA-PKcs and ATM are simultaneously compromised, (3) Laser damage may permit the observation of previously undetected DDR proteins when damage is elicited by other methods in mitosis, and (4) Twenty five percent of mitotic DNA-damaged cells undergo a subsequent mitosis. Together these findings suggest that mitotic DDR is more complex than previously thought and may involve factors from multiple repair pathways that are better understood in interphase.

Understanding the mitotic DNA damage response (DDR) is critical to our comprehension of cancer, premature aging and developmental disorders which are marked by DNA repair deficiencies. In this study we use a micro-focused laser to induce DNA damage in selected mitotic chromosomes to study the subsequent repair response. Our findings demonstrate that (1) mitotic cells are capable of DNA repair as evidenced by DNA synthesis at damage sites, (2) Repair is attenuated when DNA-PKcs and ATM are simultaneously compromised, (3) Laser damage may permit the observation of previously undetected DDR proteins when damage is elicited by other methods in mitosis, and (4) Twenty five percent of mitotic DNA-damaged cells undergo a subsequent mitosis. Together these findings suggest that mitotic DDR is more complex than previously thought and may involve factors from multiple repair pathways that are better understood in interphase.

During mitosis, the structure of the Endoplasmic Reticulum (ER) displays a dramatic reorganization and remodeling, however, the mechanism driving these changes is poorly understood. Hairpin-containing ER transmembrane proteins that stabilize ER tubules have been identified as possible factors to promote these drastic changes in ER morphology. Recently, the Reticulon and REEP family of ER shaping proteins have been shown to heavily influence ER morphology by driving the formation of ER tubules, which are known for their close proximity with microtubules. Here, we examine the role of microtubules and other cytoskeletal factors in the dynamics of a Drosophila Reticulon, Reticulon-like 1 (Rtnl1), localization to spindle poles during mitosis in the early embryo. At prometaphase, Rtnl1 is enriched to spindle poles just prior to the ER retention motif KDEL, suggesting a possible recruitment role for Rtnl1 in the bulk localization of ER to spindle poles. Using image analysis-based methods and precise temporal injections of cytoskeletal inhibitors in the early syncytial Drosophila embryo, we show that microtubules are necessary for proper Rtnl1 localization to spindles during mitosis. Lastly, we show that astral microtubules, not microfilaments, are necessary for proper Rtnl1 localization to spindle poles, and is largely independent of the minus-end directed motor protein dynein. This work highlights the role of the microtubule cytoskeleton in Rtnl1 localization to spindles during mitosis and sheds light on a pathway towards inheritance of this major organelle.

Preterm birth (PTB) is leading contributor to infant death in the United States and globally, yet the underlying mechanistic causes are not well understood. Histopathological studies of preterm birth suggest advanced villous maturity may have a role in idiopathic spontaneous preterm birth (isPTB). To better understand pathological and molecular basis of isPTB, we compared placental villous transcriptomes from carefully phenotyped cohorts of PTB due to infection or isPTB between 28-36 weeks gestation and healthy term placentas. Transcriptomic analyses revealed a unique expression signature for isPTB distinct from the age-matched controls that were delivered prematurely due to infection. This signature included the upregulation of three IGF binding proteins (IGFBP1, IGFBP2, and IGFBP6), supporting a role for aberrant IGF signaling in isPTB. However, within the isPTB expression signature, we detected secondary signature of inflammatory markers including TNC, C3, CFH, and C1R, which have been associated with placental maturity. In contrast, the expression signature of the gestational age-matched infected samples included upregulation of proliferative genes along with cell cycling and mitosis pathways. Together, these data suggest an isPTB molecular signature of placental hypermaturity, likely contributing to the premature activation of inflammatory pathways associated with birth and providing a molecular basis for idiopathic spontaneous birth.

Poly (ADP-ribose) polymerase (PARP) is responsible for the synthesis of ADP-ribose polymers, which are involved in a wide range of cellular processes such as preservation of genome integrity, DNA damage signaling and repair, molecular switches between distinct cell death pathways, and cell cycle progression. Previously, we demonstrated that the only PARP present in T. cruzi migrates to the nucleus upon genotoxic stimulus. In this work, we identify the N-terminal domain as being sufficient for TcPARP nuclear localization and describe for the first time that TcPARP is enriched in the parasite's nucleolus. We also describe that TcPARP is present in a thread-like structure that connects two dividing nuclei and co-localizes with nucleolar material and microtubules. Furthermore, ADP-ribose polymers could also be detected in this thread during mitosis. These findings represent a first approach to new potential TcPARP functions inside the nucleus and will help understand its role well beyond the largely described DNA damage response protein in trypanosomatids. [ABSTRACT FROM AUTHOR]

Promastigote Leishmania mexicana have a complex cell division cycle characterised by the ordered replication of several single-copy organelles, a prolonged S phase and rapid G2 and cytokinesis phases, accompanied by cell cycle stage-associated morphological changes. Here we exploit these morphological changes to develop a high-throughput and semi-automated imaging flow cytometry (IFC) pipeline to analyse the cell cycle in live L. mexicana. Firstly, we demonstrate that, unlike several other DNA stains, Vybrant™ DyeCycle™ Orange (DCO) is non-toxic and enables quantitative DNA imaging in live promastigotes. Secondly, by tagging the orphan spindle kinesin, KINF, with mNeonGreen, we describe KINF's cell cycle-dependent expression and localisation. Then, by combining manual gating of DCO DNA intensity profiles with automated masking and morphological measurements of parasite images, visual determination of the number of flagella per cell, and automated masking and analysis of mNG:KINF fluorescence, we provide a newly detailed description of L. mexicana promastigote cell cycle events that, for the first time, includes the durations of individual G2, mitosis and post-mitosis phases, and identifies G1 cells within the first 12 minutes of the new cell cycle. Our custom-developed masking and gating scheme allowed us to identify elusive G2 cells and to demonstrate that the CDK-inhibitor, flavopiridol, arrests cells in G2 phase, rather than mitosis, providing proof-of-principle of the utility of IFC for drug mechanism-of-action studies. Further, the high-throughput nature of IFC allowed the close examination of promastigote cytokinesis, revealing considerable flexibility in both the timing of cytokinesis initiation and the direction of furrowing, in contrast to the related kinetoplastid parasite, Trypanosoma brucei and many other cell types. Our new pipeline offers many advantages over traditional methods of cell cycle analysis such as fluorescence microscopy and flow cytometry and paves the way for novel high-throughput analysis of Leishmania cell division., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Howell et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Live-cell imaging has become state of the art to accurately identify the nature of mitotic and cell cycle defects. Low- and high-throughput microscopy setups have yield huge data amounts of cells recorded in different experimental and pathological conditions. Tailored semi-automated and automated image analysis approaches allow the analysis of high-content screening data sets, saving time and avoiding bias. However, they were mostly designed for very specific experimental setups, which restricts their flexibility and usability. The general need for dedicated experiment-specific user-annotated training sets and experiment-specific user-defined segmentation parameters remains a major bottleneck for fully automating the analysis process. In this work we present LiveCellMiner, a highly flexible open-source software tool to automatically extract, analyze and visualize both aggregated and time-resolved image features with potential biological relevance. The software tool allows analysis across high-content data sets obtained in different platforms, in a quantitative and unbiased manner. As proof of principle application, we analyze here the dynamic chromatin and tubulin cytoskeleton features in human cells passing through mitosis highlighting the versatile and flexible potential of this tool set. [ABSTRACT FROM AUTHOR]

Factor quinolinone inhibitors (FQIs), a first-in-class set of small molecule inhibitors targeted to the transcription factor LSF (TFCP2), exhibit promising cancer chemotherapeutic properties. FQI1, the initial lead compound identified, unexpectedly induced a concentration-dependent delay in mitotic progression. Here, we show that FQI1 can rapidly and reversibly lead to mitotic arrest, even when added directly to mitotic cells, implying that FQI1-mediated mitotic defects are not transcriptionally based. Furthermore, treatment with FQIs resulted in a striking, concentration-dependent diminishment of spindle microtubules, accompanied by a concentration-dependent increase in multi-aster formation. Aberrant γ-tubulin localization was also observed. These phenotypes suggest that perturbation of spindle microtubules is the primary event leading to the mitotic delays upon FQI1 treatment. Previously, FQIs were shown to specifically inhibit not only LSF DNA-binding activity, which requires LSF oligomerization to tetramers, but also other specific LSF-protein interactions. Other transcription factors participate in mitosis through non-transcriptional means, and we recently reported that LSF directly binds α-tubulin and is present in purified cellular tubulin preparations. Consistent with a microtubule role for LSF, here we show that LSF enhanced the rate of tubulin polymerization in vitro, and FQI1 inhibited such polymerization. To probe whether the FQI1-mediated spindle abnormalities could result from inhibition of mitotic LSF-protein interactions, mass spectrometry was performed using as bait an inducible, tagged form of LSF that is biotinylated by endogenous enzymes. The global proteomics analysis yielded expected associations for a transcription factor, notably with RNA processing machinery, but also to nontranscriptional components. In particular, and consistent with spindle disruption due to FQI treatment, mitotic, FQI1-sensitive interactions were identified between the biotinylated LSF and microtubule-associated proteins that regulate spindle assembly, positioning, and dynamics, as well as centrosome-associated proteins. Probing the mitotic LSF interactome using small molecule inhibitors therefore supported a non-transcriptional role for LSF in mediating progression through mitosis. [ABSTRACT FROM AUTHOR]

The female gametophyte of flowering plants, called the embryo sac, develops from a haploid cell named the functional megaspore, which is specified after meiosis by the diploid sporophyte. In Arabidopsis, the functional megaspore undergoes three syncitial mitotic divisions followed by cellularization to form seven cells of four cell types including two female gametes. The plant hormone auxin is important for sporophytic developmental processes, and auxin levels are known to be regulated by biosynthesis and transport. Here, we investigated the role of auxin biosynthetic genes and auxin influx carriers in embryo sac development. We find that genes from the YUCCA/TAA pathway (YUC1, YUC2, YUC8, TAA1, TAR2) are expressed asymmetrically in the developing ovule and embryo sac from the two-nuclear syncitial stage until cellularization. Mutants for YUC1 and YUC2 exhibited defects in cell specification, whereas mutations in YUC8, as well as mutations in TAA1 and TAR2, caused defects in nuclear proliferation, vacuole formation and anisotropic growth of the embryo sac. Additionally, expression of the auxin influx carriers AUX1 and LAX1 were observed at the micropylar pole of the embryo sac and in the adjacent cells of the ovule, and the aux1 lax1 lax2 triple mutant shows multiple gametophyte defects. These results indicate that both localized auxin biosynthesis and auxin import, are required for mitotic divisions, cell expansion and patterning during embryo sac development.

We previously defined macrophages harvested from the peritoneal cavity of nude mice with subcutaneous human pancreatic tumors as "tumor-educated-macrophages" (Edu) and macrophages harvested from mice without tumors as "naïve-macrophages" (Naïve), and demonstrated that Edu-macrophages promoted tumor growth and metastasis. In this study, Edu- and Naïve-macrophages were compared for their ability to enhance pancreatic cancer malignancy at the cellular level in vitro and in vivo. The inhibitory efficacy of Zoledronic acid (ZA) on Edu-macrophage-enhanced metastasis was also determined. XPA1 human pancreatic cancer cells in Gelfoam co-cultured with Edu-macrophages proliferated to a greater extent compared to XPA1 cells cultured with Naïve-macrophages (P = 0.014). XPA1 cells exposed to conditioned medium harvested from Edu culture significantly increased proliferation (P = 0.016) and had more migration stimulation capability (P

The germinal zones of the embryonic macaque neocortex comprise the ventricular zone (VZ) and the subventricular zone (SVZ). The mammalian SVZ is subdivided into an inner SVZ and an outer SVZ, with the outer SVZ being particularly large in primates. The existence of distinct precursor cell types in the neocortical proliferative zones was inferred over 100 years ago and recent evidence supports this concept. Precursor cells exhibiting diverse morphologies, patterns of transcription factor expression, and fate potential have been identified in the neocortical proliferative zones. Neurogenic precursor cells are thought to exhibit characteristics of glial cells, but the existence of neurogenic precursor cells that do not share glial specific properties has also been proposed. Therefore, one question that remains is whether neural precursor cells in the prenatal neocortex belong within the astroglial cell class, as they do in neurogenic regions of the adult neocortex, or instead include a diverse collection of precursor cells belonging to distinct cell classes. We examined the expression of astroglial markers by mitotic precursor cells in the telencephalon of prenatal macaque and human. We show that in the dorsal neocortex all mitotic cells at the surface of the ventricle, and all Pax6+ and Tbr2+ mitotic cells in the proliferative zones, express the astroglial marker GFAP. The majority of mitotic cells undergoing division away from the ventricle express GFAP, and many of the GFAP-negative mitoses express markers of cells derived from the ventral telencephalon or extracortical sites. In contrast, a markedly lower proportion of precursor cells express GFAP in the ganglionic eminence. In conclusion, we propose that the heterogeneity of neural precursor cells in the dorsal cerebral cortex develops within the GFAP+ astroglial cell class.

A number of studies have indicated that the mitotic rate may be a predictive factor for poor prognosis in melanoma patients. The aim of this study was to investigate whether the mitotic rate is associated with other prognostic clinical and anatomopathological characteristics. After adjusting for other anatomopathological characteristics, we then verified the prognostic value of the number of mitoses, determining in which population subgroup this variable may have greater prognostic significance on 3-year mortality. The Veneto Cancer Registry (Registro Tumori del Veneto-RTV), a high-resolution population-based dataset covering the regional population of approximately 4.9 million residents, served as the clinical data source for the analysis. Inclusion criteria included all incident cases of invasive cutaneous malignant melanoma recorded in the RTV in 2015 (1,050 cases) and 2017 (1,205 cases) for which the number of mitoses was available. Mitotic classes were represented by Kaplan-Meier curves for short-term overall survival. Cox regression calculated hazard ratios in multivariable models to evaluate the independent prognostic role of different mitotic rate cut-offs. The results indicate that the mitotic rate is associated with other survival prognostic factors: the variables comprising the TNM stage (e.g., tumor thickness, ulceration, lymph node status and presence of metastasis) and the characteristics that are not included in the TNM stage (e.g., age, site of tumor, type of morphology, growth pattern and TIL). Moreover, this study demonstrated that, even after adjusting for these prognostic factors, mitoses per mm2 are associated with higher mortality, particularly in T2 patients. In conclusion, these findings revealed the need to include the mitotic rate in the histological diagnosis because it correlates with the prognosis as an independent factor. The mitotic rate can be used to develop a personalized medicine approach in the treatment and follow-up monitoring of melanoma patients., Competing Interests: The authors have no conflicts of interest to declare., (Copyright: © 2024 Buja et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cyclin-dependent kinase 1 (Cdk1) complexed with cyclin B phosphorylates multiple sites on hundreds of proteins during mitosis. However, it is not fully understood how multi-site mitotic phosphorylation by cyclin B-Cdk1 controls the structures and functions of individual substrates. Here we develop an easy-to-use protocol to express recombinant vertebrate cyclin B and Cdk1 in insect cells from a single baculovirus vector and to purify their complexes with excellent homogeneity. A series of in-vitro assays demonstrate that the recombinant cyclin B-Cdk1 can efficiently and specifically phosphorylate the SP and TP motifs in substrates. The addition of Suc1 (a Cks1 homolog in fission yeast) accelerates multi-site phosphorylation of an artificial substrate containing TP motifs. Importantly, we show that mitosis-specific multi-subunit and multi-site phosphorylation of the condensin I complex can be recapitulated in vitro using recombinant cyclin B-Cdk1-Suc1. The materials and protocols described here will pave the way for dissecting the biochemical basis of critical mitotic processes that accompany Cdk1-mediated large-scale phosphorylation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Shintomi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Recently, human asparagine synthetase has been found to be associated with the mitotic spindle. However, this event cannot be seen in yeast because yeast takes a different cell division process via closed mitosis (there is no nuclear envelope breakdown to allow the association between any cytosolic enzyme and mitotic spindle). To find out if yeast asparagine synthetase can also (but hiddenly) have this feature, the coding sequences of green fluorescent protein (GFP) and nuclear localization signal (NLS) were introduced downstream of ASN1 and ASN2, encoding asparagine synthetases Asn1p and Asn2p, respectively, in the yeast genome having mCherrry coding sequence downstream of TUB1 encoding alpha-tubulin, a building block of the mitotic spindle. The genomically engineered yeast strains showed co-localization of Asn1p-GFP-NLS (or Asn2p-GFP-NLS) and Tub1p-mCherry in dividing nuclei. In addition, an activity-disrupted mutation was introduced to ASN1 (or ASN2). The yeast mutants still exhibited co-localization between defective asparagine synthetase and mitotic spindle, indicating that the biochemical activity of asparagine synthetase is not required for its association with the mitotic spindle. Furthermore, nocodazole treatment was used to depolymerize the mitotic spindle, resulting in lack of association between the enzyme and the mitotic spindle. Although yeast cell division undergoes closed mitosis, preventing the association of its asparagine synthetase with the mitotic spindle, however, by using yeast constructs with re-localized Asn1/2p have suggested the moonlighting role of asparagine synthetase in cell division of higher eukaryotes. [ABSTRACT FROM AUTHOR]

During studies on the absorption and interactions between silver nanoparticles and mammalian cells grown in vitro it was observed that large extracellular rings of silver nanoparticles were deposited on the microscope slide, many located near post-mitotic cells. Silver nanoparticles (AgNP, 80nm), coated with citrate, were incubated at concentrations of 0.3 to 30 μg/ml with a human-derived culture of retinal pigment epithelial cells (ARPE-19) and observed using darkfield and fluorescent microscopy, 24 h after treatment. Approximately cell-sized extracellular rings of deposited AgNP were observed on the slides among a field of dispersed individual AgNP. The mean diameter of 45 nanoparticles circles was 62.5 +/-12 microns. Ring structures were frequently observed near what appeared to be post-mitotic daughter cells, giving rise to the possibility that cell membrane fragments were deposited on the slide during mitosis, and those fragments selectively attracted and retained silver nanoparticles from suspension in the cell culture medium. These circular structures were observable for the following technical reasons: 1) darkfield microscope could observe single nanoparticles below 100 nm in size, 2) a large concentration (108 and 109) of nanoparticles was used in these experiments 3) negatively charged nanoparticles were attracted to adhesion membrane proteins remaining on the slide from mitosis. The observation of silver nanoparticles attracted to apparent remnants of cellular mitosis could be a useful tool for the study of normal and abnormal mitosis. [ABSTRACT FROM AUTHOR]

The mammalian cerebral cortex arises from precursor cells that reside in a proliferative region surrounding the lateral ventricles of the developing brain. Recent work has shown that precursor cells in the subventricular zone (SVZ) provide a major contribution to prenatal cortical neurogenesis, and that the SVZ is significantly thicker in gyrencephalic mammals such as primates than it is in lissencephalic mammals including rodents. Identifying characteristics that are shared by or that distinguish cortical precursor cells across mammalian species will shed light on factors that regulate cortical neurogenesis and may point toward mechanisms that underlie the evolutionary expansion of the neocortex in gyrencephalic mammals. We immunostained sections of the developing cerebral cortex from lissencephalic rats, and from gyrencephalic ferrets and macaques to compare the distribution of precursor cell types in each species. We also performed time-lapse imaging of precursor cells in the developing rat neocortex. We show that the distribution of Pax6+ and Tbr2+ precursor cells is similar in lissencephalic rat and gyrencephalic ferret, and different in the gyrencephalic cortex of macaque. We show that mitotic Pax6+ translocating radial glial cells (tRG) are present in the cerebral cortex of each species during and after neurogenesis, demonstrating that the function of Pax6+ tRG cells is not restricted to neurogenesis. Furthermore, we show that Olig2 expression distinguishes two distinct subtypes of Pax6+ tRG cells. Finally we present a novel method for discriminating the inner and outer SVZ across mammalian species and show that the key cytoarchitectural features and cell types that define the outer SVZ in developing primates are present in the developing rat neocortex. Our data demonstrate that the developing rat cerebral cortex possesses an outer subventricular zone during late stages of cortical neurogenesis and that the developing rodent cortex shares important features with that of primates.

As the microenvironment of a cell changes, associated mechanical cues may lead to changes in biochemical signaling and inherently mechanical processes such as mitosis. Here we explore the effects of confined mechanical environments on cellular responses during mitosis. Previously, effects of mechanical confinement have been difficult to optically observe in three-dimensional and in vivo systems. To address this challenge, we present a novel microfluidic perfusion culture system that allows controllable variation in the level of confinement in a single axis allowing observation of cell growth and division at the single-cell level. The device is capable of creating precise confinement conditions in the vertical direction varying from high (3 µm) to low (7 µm) confinement while also varying the substrate stiffness (E = 130 kPa and 1 MPa). The Human cervical carcinoma (HeLa) model with a known 3N+ karyotype was used for this study. For this cell line, we observe that mechanically confined cell cycles resulted in stressed cell divisions: (i) delayed mitosis, (ii) multi- daughter mitosis events (from 3 up to 5 daughter cells), (iii) unevenly sized daughter cells, and (iv) induction of cell death. In the highest confined conditions, the frequency of divisions producing more than two progeny was increased an astounding 50-fold from unconfined environments, representing about one half of all successful mitotic events. Notably, the majority of daughter cells resulting from multipolar divisions were viable after cytokinesis and, perhaps suggesting another regulatory checkpoint in the cell cycle, were in some cases observed to re-fuse with neighboring cells post-cytokinesis. The higher instances of abnormal mitosis that we report in confined mechanically stiff spaces, may lead to increased rates of abnormal, viable, cells in the population. This work provides support to a hypothesis that environmental mechanical cues influences structural mechanisms of mitosis such as geometric orientation of the mitotic plane or planes.

The relationships between cellular, structural and dynamical properties of tumors have traditionally been studied separately. Here, we construct a quantitative, predictive theory of solid tumor growth, metabolic rate, vascularization and necrosis that integrates the relationships between these properties. To accomplish this, we develop a comprehensive theory that describes the interface and integration of the tumor vascular network and resource supply with the cardiovascular system of the host. Our theory enables a quantitative understanding of how cells, tissues, and vascular networks act together across multiple scales by building on recent theoretical advances in modeling both healthy vasculature and the detailed processes of angiogenesis and tumor growth. The theory explicitly relates tumor vascularization and growth to metabolic rate, and yields extensive predictions for tumor properties, including growth rates, metabolic rates, degree of necrosis, blood flow rates and vessel sizes. Besides these quantitative predictions, we explain how growth rates depend on capillary density and metabolic rate, and why similar tumors grow slower and occur less frequently in larger animals, shedding light on Peto's paradox. Various implications for potential therapeutic strategies and further research are discussed.

Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow

Genome maintenance is ensured by a variety of biochemical sensors and pathways that repair accumulated damage. During mitosis, the mechanisms that sense and resolve DNA damage remain elusive. Studies have demonstrated that damage accumulated on lagging chromosomes can activate the spindle assembly checkpoint. However, there is little known regarding damage to DNA after anaphase onset. In this study, we demonstrate that laser-induced damage to chromosome tips (presumptive telomeres) in anaphase of Potorous tridactylis cells (PtK2) inhibits cytokinesis. In contrast, equivalent irradiation of non-telomeric chromosome regions or control irradiations in either the adjacent cytoplasm or adjacent to chromosome tips near the spindle midzone during anaphase caused no change in the eventual completion of cytokinesis. Damage to only one chromosome tip caused either complete absence of furrow formation, a prolonged delay in furrow formation, or furrow regression. When multiple chromosome tips were irradiated in the same cell, the cytokinesis defects increased, suggesting a potential dose-dependent mechanism. These results suggest a mechanism in which dysfunctional telomeres inhibit mitotic exit.

BMI1 is a core protein of the polycomb repressive complex 1 (PRC1) that is overexpressed in several cancer types, making it a promising target for cancer therapies. However, the underlying mechanisms and interactions associated with BMI1-induced tumorigenesis are often context-dependent and complex. Here, we performed a drug resistance screen on mutagenized human haploid HAP1 cells treated with BMI1 inhibitor PTC-318 to find new genetic and mechanistic features associated with BMI1-dependent cancer cell proliferation. Our screen identified NUMA1-mutations as the most significant inducer of PTC-318 cell death resistance. Independent validations on NUMA1-proficient HAP1 and non-small cell lung cancer cell lines exposed to BMI1 inhibition by PTC-318 or BMI1 knockdown resulted in cell death following mitotic arrest. Interestingly, cells with CRISPR-Cas9 derived NUMA1 knockout also showed a mitotic arrest phenotype following BMI1 inhibition but, contrary to cells with wildtype NUMA1, these cells were resistant to BMI1-dependent cell death. The current study brings new insights to BMI1 inhibition-induced mitotic lethality in cancer cells and presents a previously unknown role of NUMA1 in this process. [ABSTRACT FROM AUTHOR]

Background: Many genomic analyses of cortisol-producing adrenocortical carcinoma (ACC) have been reported, but very few have come from East Asia. The first objective of this study is to verify the genetic difference with the previous reports by analyzing targeted deep sequencing of 7 Japanese ACC cases using next-generation sequencing (NGS). The second objective is to compare the somatic variant findings identified by NGS analysis with clinical and pathological findings, aiming to acquire new knowledge about the factors that contribute to the poor prognosis of ACC and to find new targets for the treatment of ACC. Method: DNA was extracted from ACC tissue of seven patients and two reference blood samples. Targeted deep sequencing was performed using the MiSeq system for 12 genes, and the obtained results were analyzed using MuTect2. The hypothesis was obtained by integrating the somatic variant findings with clinical and pathological data, and it was further verified using The Cancer Genome Atlas (TCGA) dataset for ACC. Results: Six possible pathogenic and one uncertain significance somatic variants including a novel PRKAR1A (NM_002734.4):c.545C>A (p.T182K) variant were found in five of seven cases. By integrating these data with pathological findings, we hypothesized that cases with TP53 variants were more likely to show atypical mitotic figures. Using TCGA dataset, we found that atypical mitotic figures were associated with TP53 somatic variant, and mRNA expression of CCNB2 and AURKA was significantly high in TP53 mutated cases and atypical mitotic figure cases. Conclusion: We believe this is the first report that discusses the relationship between atypical mitotic figures and TP53 somatic variant in ACC. We presumed that overexpression of CCNB2 and AURKA mRNA may cause atypical mitosis in TP53 somatic mutated cases. Because AURKA is highly expressed in atypical mitotic cases, it may be an appropriate indicator for AURKA inhibitors. [ABSTRACT FROM AUTHOR]

Background: Poorly differentiated thyroid cancer (PDTC) is a rare but aggressive type of thyroid cancer (TC) and the main cause of death from non-anaplastic follicular cell-derived TC. Although the Turin criteria are well defined, the pathological features that could serve as diagnostic and prognostic factors remain controversial. Materials and methods: Forty-nine consecutive PDTC cases were identified in a single cancer center between 2000 and 2018. We analyzed the impact of routine histopathological and immunohistochemical features and several parameters that are not routinely included in pathology reports such as the presence of atypical mitoses, the amount of necrosis, or insulin-like growth factor-II mRNA-binding protein 3 immunostaining on the survival of patients with PDTC. Overall survival (OS) and disease-specific survival (DSS) were calculated using the Kaplan-Meier method. Results: Of the 49 PDTC 34 (69.4%) showed the insular pattern of growth. The median of poorly differentiated area was 95% (range, 1–100), and 30 (61.2%) patients had a predominant (>50%) insular area. The 5-year OS and DSS rates at a median follow-up of 57 months were 60.6% and 64.3%, respectively. Univariate analysis showed that tumor size >4 cm, presence of atypical mitoses, Ki-67 >5%, and thyroglobulin (Tg)-negative immunostaining were associated with a higher risk of PDTC-related death. Atypical mitoses and Tg negativity were independent factors of worse DSS in multivariate analysis. Patients with insular and predominant insular areas showed a 3- and 6-fold higher risk of PDTC death when they displayed atypical mitoses. Conclusions: In PDTC, the presence of atypical mitoses may be helpful in identifying patients with poorer outcome and worth including in pathology reports, particularly in tumors with a dominant insular pattern of growth. Additionally, the inclusion of Tg immunostaining may be considered in a prognostic context, and not only as a diagnostic feature. [ABSTRACT FROM AUTHOR]

In a process linked to DNA replication, duplicated chromosomes are entrapped in large, circular cohesin complexes and functional sister chromatid cohesion (SCC) is established by acetylation of the SMC3 cohesin subunit. Roberts Syndrome (RBS) and Warsaw Breakage Syndrome (WABS) are rare human developmental syndromes that are characterized by defective SCC. RBS is caused by mutations in the SMC3 acetyltransferase ESCO2, whereas mutations in the DNA helicase DDX11 lead to WABS. We found that WABS-derived cells predominantly rely on ESCO2, not ESCO1, for residual SCC, growth and survival. Reciprocally, RBS-derived cells depend on DDX11 to maintain low levels of SCC. Synthetic lethality between DDX11 and ESCO2 correlated with a prolonged delay in mitosis, and was rescued by knockdown of the cohesin remover WAPL. Rescue experiments using human or mouse cDNAs revealed that DDX11, ESCO1 and ESCO2 act on different but related aspects of SCC establishment. Furthermore, a DNA binding DDX11 mutant failed to correct SCC in WABS cells and DDX11 deficiency reduced replication fork speed. We propose that DDX11, ESCO1 and ESCO2 control different fractions of cohesin that are spatially and mechanistically separated. [ABSTRACT FROM AUTHOR]

Condensins I and II in vertebrates are essential ATP-dependent complexes necessary for chromosome condensation in mitosis. Condensins depletion is known to perturb structure and function of centromeres, however the mechanism of this functional link remains elusive. Depletion of condensin activity is now shown to result in a significant loss of loading of CENP-A, the histone H3 variant found at active centromeres and the proposed epigenetic mark of centromere identity. Absence of condensins and/or CENP-A insufficiency produced a specific kinetochore defect, such that a functional mitotic checkpoint cannot prevent chromosome missegregation resulting from improper attachment of sister kinetochores to spindle microtubules. Spindle microtubule-dependent deformation of both inner kinetochores and the HEC1/Ndc80 microtubule-capturing module, then results in kinetochore separation from the Aurora B pool and ensuing reduced kinase activity at centromeres. Moreover, recovery from mitosis-inhibition by monastrol revealed a high incidence of merotelic attachment that was nearly identical with condensin depletion, Aurora B inactivation, or both, indicating that the Aurora B dysfunction is the key defect leading to chromosome missegregation in condensin-depleted cells. Thus, beyond a requirement for global chromosome condensation, condensins play a pivotal role in centromere assembly, proper spatial positioning of microtubule-capturing modules and positioning complexes of the inner centromere versus kinetochore plates.

SUMO is a protein modifier that is vital for multicellular development. Here we present the first system-wide analysis, combining multiple approaches, to correlate the sumoylated proteome (SUMO-ome) in a multicellular organism with the developmental roles of SUMO. Using mass-spectrometry-based protein identification, we found over 140 largely novel SUMO conjugates in the early Drosophila embryo. Enriched functional groups include proteins involved in Ras signaling, cell cycle, and pattern formation. In support of the functional significance of these findings, sumo germline clone embryos exhibited phenotypes indicative of defects in these same three processes. Our cell culture and immunolocalization studies further substantiate roles for SUMO in Ras signaling and cell cycle regulation. For example, we found that SUMO is required for efficient Ras-mediated MAP kinase activation upstream or at the level of Ras activation. We further found that SUMO is dynamically localized during mitosis to the condensed chromosomes, and later also to the midbody. Polo kinase, a SUMO substrate found in our screen, partially colocalizes with SUMO at both sites. These studies show that SUMO coordinates multiple regulatory processes during oogenesis and early embryogenesis. In addition, our database of sumoylated proteins provides a valuable resource for those studying the roles of SUMO in development.

BackgroundChromosomal instability (CIN), a feature widely shared by cells from solid tumors, is caused by occasional chromosome missegregations during cell division. Two of the causes of CIN are weakened mitotic checkpoint signaling and persistent merotelic attachments that result in lagging chromosomes during anaphase.Principal findingsHere we identify an autophosphorylation event on Mps1 that is required to prevent these two causes of CIN. Mps1 is phosphorylated in mitotic cells on at least 7 residues, 4 of which by autophosphorylation. One of these, T676, resides in the activation loop of the kinase domain and a mutant that cannot be phosphorylated on T676 is less active than wild-type Mps1 but is not kinase-dead. Strikingly, cells in which endogenous Mps1 was replaced with this mutant are viable but missegregate chromosomes frequently. Anaphase is initiated in the presence of misaligned and lagging chromosomes, indicative of a weakened checkpoint and persistent merotelic attachments, respectively.Conclusions/significanceWe propose that full activity of Mps1 is essential for maintaining chromosomal stability by allowing resolution of merotelic attachments and to ensure that single kinetochores achieve the strength of checkpoint signaling sufficient to prevent premature anaphase onset and chromosomal instability. To our knowledge, phosphorylation of T676 on Mps1 is the first post-translational modification in human cells of which the absence causes checkpoint weakening and CIN without affecting cell viability.

Aurora B kinase is a key component of the chromosomal passenger complex (CPC), which regulates chromosome segregation and cytokinesis. An ortholog of Aurora B was characterized in Trypanosoma brucei (TbAUK1), but other conserved components of the complex have not been found. Here we identified four novel TbAUK1 associated proteins by tandem affinity purification and mass spectrometry. Among these four proteins, TbKIN-A and TbKIN-B are novel kinesin homologs, whereas TbCPC1 and TbCPC2 are hypothetical proteins without any sequence similarity to those known CPC components from yeasts and metazoans. RNAi-mediated silencing of each of the four genes led to loss of spindle assembly, chromosome segregation and cytokinesis. TbKIN-A localizes to the mitotic spindle and TbKIN-B to the spindle midzone during mitosis, whereas TbCPC1, TbCPC2 and TbAUK1 display the dynamic localization pattern of a CPC. After mitosis, the CPC disappears from the central spindle and re-localizes at a dorsal mid-point of the mother cell, where the anterior tip of the daughter cell is tethered, to start cell division toward the posterior end, indicating a most unusual CPC-initiated cytokinesis in a eukaryote.

Aurora B kinase plays essential roles in mitosis and cytokinesis in eukaryotes. In the procyclic form of Trypanosoma brucei, the Aurora B homolog TbAUK1 regulates mitosis and cytokinesis, phosphorylates the Tousled-like kinase TbTLK1, interacts with two mitotic kinesins TbKIN-A and TbKIN-B and forms a novel chromosomal passenger complex (CPC) with two novel proteins TbCPC1 and TbCPC2. Here we show with time-lapse video microscopy the time course of CPC trans-localization from the spindle midzone in late anaphase to the dorsal side of the cell where the anterior end of daughter cell is tethered, and followed by a glide toward the posterior end to divide the cell, representing a novel mode of cytokinesis in eukaryotes. The three subunits of CPC, TbKIN-B and TbTLK1 interact with one another suggesting a close association among the five proteins. An ablation of TbTLK1 inhibited the subsequent trans-localization of CPC and TbKIN-B, whereas a knockdown of CPC or TbKIN-B disrupted the spindle pole localization of TbTLK1 during mitosis. In the bloodstream form of T. brucei, the five proteins also play essential roles in chromosome segregation and cytokinesis and display subcellular localization patterns similar to that in the procyclic form. The CPC in bloodstream form also undergoes a trans-localization during cytokinesis similar to that in the procyclic form. All together, our results indicate that the five-protein complex CPC-TbTLK1-TbKIN-B plays key roles in regulating chromosome segregation in the early phase of mitosis and that the highly unusual mode of cytokinesis mediated by CPC occurs in both forms of trypanosomes.

The human NAD+-dependent protein deacetylase SIRT2 resides predominantly in the cytoplasm where it functions as a tubulin deacetylase. Here we report that SIRT2 maintains a largely cytoplasmic localization during interphase by active nuclear export in a Crm1-dependent manner. We identified a functional, leptomycin B-sensitive, nuclear export signal sequence within SIRT2. During the cell cycle, SIRT2 becomes enriched in the nucleus and is associated with mitotic structures, beginning with the centrosome during prophase, the mitotic spindle during metaphase, and the midbody during cytokinesis. Cells overexpressing wild-type or a catalytically inactive SIRT2 exhibit an increase in multinucleated cells. The findings suggest a novel mechanism of regulating SIRT2 function by nucleo-cytoplasmic shuttling, as well as a role for SIRT2 in the nucleus during interphase and throughout mitosis.

Chronic myeloid leukemia (CML) is characterized by the constitutive tyrosine kinase activity of the oncoprotein BCR-ABL1 in myeloid progenitor cells that activates multiple signal transduction pathways leading to the leukemic phenotype. The tyrosine-kinase inhibitor (TKI) nilotinib inhibits the tyrosine kinase activity of BCR-ABL1 in CML patients. Despite the success of nilotinib treatment in patients with chronic-phase (CP) CML, a population of Philadelphia-positive (Ph+) quiescent stem cells escapes the drug activity and can lead to drug resistance. The molecular mechanism by which these quiescent cells remain insensitive is poorly understood. The aim of this study was to compare the gene expression profiling (GEP) of bone marrow (BM) CD34+/lin- cells from CP-CML patients at diagnosis and after 12 months of nilotinib treatment by microarray, in order to identify gene expression changes and the dysregulation of pathways due to nilotinib action. We selected BM CD34+/lin- cells from 78 CP-CML patients at diagnosis and after 12 months of first-line nilotinib therapy and microarray analysis was performed. GEP bioinformatic analyses identified 2,959 differently expressed probes and functional clustering determined some significantly enriched pathways between diagnosis and 12 months of nilotinib treatment. Among these pathways, we observed the under expression of 26 genes encoding proteins belonging to the cell cycle after 12 months of nilotinib treatment which led to the up-regulation of chromosome replication, cell proliferation, DNA replication, and DNA damage checkpoint at diagnosis. We demonstrated the under expression of the ATP-binding cassette (ABC) transporters ABCC4, ABCC5, and ABCD3 encoding proteins which pumped drugs out of the cells after 12 months of nilotinib. Moreover, GEP data demonstrated the deregulation of genes involved in the JAK-STAT signaling pathway. The down-regulation of JAK2, IL7, STAM, PIK3CA, PTPN11, RAF1, and SOS1 key genes after 12 months of nilotinib could demonstrate the up-regulation of cell cycle, proliferation and differentiation via MAPK and PI3K-AKT signaling pathways at diagnosis. [ABSTRACT FROM AUTHOR]

The cAMP-dependent protein kinase Pka1 is known as a regulator of glycogenesis, transition into meiosis, chronological aging, and stress responses in the fission yeast, Schizosaccharomyces pombe. We demonstrated here that Pka1 is responsible for normal growth in the presence of the microtubule-destabilization drug TBZ and proper chromosome segregation. The deletion of the pka1 gene resulted in the TBZ-sensitive phenotype and chromosome mis-segregation. We isolated the mal3 gene as a multi-copy suppressor of the TBZ-sensitive phenotype in the pka1Δ strains. Overexpression of the CH domain (1–143) or the high-affinity microtubule binding mutant (1–143 Q89R) of Mal3 rescued the TBZ-sensitive phenotype in the pka1Δ and mal3Δ strains, while the EB1 domain (135–308) and the mutants defective in microtubule binding (1–143 Q89E) failed to do so in the same strains. Chromosome mis-segregation caused by TBZ in the pka1Δ or mal3Δ strains was suppressed by the overexpression of the Mal3 CH domain (1–143), Mal3 CH domain with the coiled-coil domain (1–197), or full-length Mal3. Overexpression of EB1 orthologs from Saccharomyces cerevisiae, Arabidopsis thaliana, Mus musculus, or Homo sapiens suppressed the TBZ-sensitive phenotype in the pka1Δ strains, indicating their conserved functions. These findings suggest that Pka1 and the microtubule binding of the Mal3 CH domain play a role in the maintenance of proper chromosome segregation. [ABSTRACT FROM AUTHOR]

Cell therapy for diabetes could benefit from the identification of small-molecule compounds that increase the number of functional pancreatic beta cells. Using a newly developed screening assay, we previously identified glucocorticoids as potent stimulators of human and rat beta cell proliferation. We now compare the stimulatory action of these steroid hormones to a selection of checkpoint tyrosine kinase inhibitors that were also found to activate the cell cycle-in beta cells and analyzed their respective effects on DNA-synthesis, beta cell numbers and expression of cell cycle regulators. Our data using glucocorticoids in combination with a receptor antagonist, mifepristone, show that 48h exposure is sufficient to allow beta cells to pass the cell cycle restriction point and to become committed to cell division regardless of sustained glucocorticoid-signaling. To reach the end-point of mitosis another 40h is required. Within 14 days glucocorticoids stimulate up to 75% of the cells to undergo mitosis, which indicates that these steroid hormones act as proliferation competence-inducing factors. In contrast, by correlating thymidine-analogue incorporation to changes in absolute cell numbers, we show that the checkpoint kinase inhibitors, as compared to glucocorticoids, stimulate DNA-synthesis only during a short time-window in a minority of cells, insufficient to give a measurable increase of beta cell numbers. Glucocorticoids, but not the kinase inhibitors, were also found to induce changes in the expression of checkpoint regulators. Our data, using checkpoint kinase-specific inhibitors further point to a role for Chk1 and Cdk1 in G1/S transition and progression of beta cells through the cell cycle upon stimulation with glucocorticoids. [ABSTRACT FROM AUTHOR]

Chromosome organization during cell division is achieved through the timely association of proteins with chromatin and is regulated by protein phosphorylation. Kinesin family member 4A (KIF4A) plays an important role in the chromosome organization through the formation of the chromosome scaffold structure. However, the relationship between the function of KIF4A and its phosphorylation remains unclear. Here, we demonstrate that Cdk1-dependent phosphorylation of KIF4A at S1186 is required for chromosome binding and chromosome scaffold formation. The KIF4A mutant, which is not phosphorylated at S1186, was found to localize to the nucleus during interphase but did not accumulate in the chromosome scaffold after nuclear envelope breakdown. In addition, defects in KIF4A phosphorylation were found to disrupt the interaction of KIF4A with the condensin I complex. As a result, the morphology of the chromosomes was observed to be laterally decondensed, without condensin I in the chromosome scaffold. Additionally, a defect in chromosome segregation, chromosome bridge formation, was often observed. Although both KIF4A and condensin I disappeared from the chromosomes, the chromosomal localization of condensin II was not affected. Collectively, our novel results revealed that Cdk1-dependent KIF4A phosphorylation at S1186 is a trigger for chromosomal organization during early mitosis. [ABSTRACT FROM AUTHOR]

In Drosophila it has recently been demonstrated that a spindle matrix in the form of a membrane-less macromolecular assembly embeds the microtubule-based spindle apparatus. In addition, two of its constituents, Megator and Chromator, were shown to function as spatial regulators of spindle checkpoint proteins. However, whether the spindle matrix plays a wider functional role in spatially regulating cell cycle progression factors was unknown. Here using a live imaging approach we provide evidence that a number of key cell cycle proteins such as Cyclin B, Polo, and Ran co-localize with the spindle matrix during mitosis. Furthermore, prevention of spindle matrix formation by injection of a function blocking antibody against the spindle matrix protein Chromator results in cell cycle arrest prior to nuclear envelope breakdown. In such embryos the spatial dynamics of Polo and Cyclin B enrichment at the nuclear rim and kinetochores is abrogated and Polo is not imported into the nucleus. This is in contrast to colchicine-arrested embryos where the wild-type dynamics of these proteins are maintained. Taken together these results suggest that spindle matrix formation may be a general requirement for the localization and proper dynamics of cell cycle factors promoting signaling events leading to cell cycle progression. [ABSTRACT FROM AUTHOR]

Following a certain type-specific number of mitotic divisions, terminally differentiated cells undergo proliferative senescence, thwarting efforts to expand different cell populations in vitro for the needs of scientific research or medical therapies. The primary cause of this phenomenon is the progressive shortening of the telomeres and the subsequent activation of cell cycle control pathways leading to a block of cell proliferation. Restoration of telomere length by transgenic expression of telomerase reverse transcriptase (TERT) usually results in bypassing of the replicative senescence and ultimately in cell immortalization. To date, there have not been any reports regarding immortalization of cells from common marmoset (Callithrix jacchus), an important non-human primate model for various human diseases, with the use of exogenous human TERT (hTERT). In this study, marmoset fibroblasts were successfully immortalized with transposon-integrated transgenic hTERT and expanded in vitro for over 500 population doublings. Calculation of population doubling levels (PDL) showed that the derived hTERT-transgenic lines had significantly higher proliferation potential than the wild-type fibroblasts, which reached only a maximum of 46 doublings. However, the immortalized cells exhibited differences in the morphology compared with the control fibroblasts and transcriptome analysis also revealed changes in the gene expression patterns. Finally, the karyotypes of all hTERT-transgenic cell lines showed various aberrations such as presence of extra Chromosome 17, isochromosome 21q, or tetraploidy. By single-cell expansion of the least affected monoclonal immortalized line, one sub-clonal line with normal karyotype was established, suggesting the possibility to derive immortal marmoset cells with normal karyotypes. The results of this study are an important step towards the development and optimization of methods for the production of immortalized cells from common marmoset monkeys. [ABSTRACT FROM AUTHOR]

Histone H1 is an evolutionarily conserved linker histone protein that functions in arranging and stabilizing chromatin structure and is frequently fused to a fluorescent protein to track nuclei in live cells. In time-lapse analyses, we observed stochastic exchange of photoactivated Dendra2-histone H1 protein between nuclei within the same cellular compartment. We also observed exchange of histones between genetically distinct nuclei in a heterokaryon derived from fusion of strains carrying histone H1-RFP or H1-GFP. Subsequent analysis of the resulting uninucleate conidia containing both RFP- and GFP-labeled histone H1 proteins showed only parental genotypes, ruling out genetic recombination and diploidization. These data together suggest that the linker histone H1 protein can diffuse between non-daughter nuclei in the filamentous fungus Aspergillus nidulans. [ABSTRACT FROM AUTHOR]

Background: The grading of oral epithelial dysplasia is not possible in the atrophic epithelium of oral submucous fibrosis (OSMF). Recently, we found that features such as increased basal cell layer hyperplasia, abnormal superficial mitosis, increased nuclear-cytoplasmic ratio, increased nuclear size, and hyperchromasia represent transformation-relevant dysplastic features in the atrophic epithelium of OSMF. The presence of these features can be considered a high-risk feature for patients. However, these findings have not been tested and authenticated using markers relevant to oral carcinogenesis. Method: Paraffin-embedded tissues from 30 normal oral mucosa (NOM) and 50 OSMF were retrieved from 2008 to 2016 and subjected to immunohistochemical expression using Ki67, CD105 and α-SMA antibodies. Results: Ki67 LI showed significant increases from NOM (12.47±2.34) to LRED (23.47±3.75) to HRED (34.31±7.31) (<0.0001). Similarly, MVD was increased significantly from NOM (3.53±5.17) to LRED (27.57±12.25) to HRED (46.18±12.55) (p<0.0001). The expression of α-SMA was significantly increased from LRED (0.21±0.41) to HRED (1.13±0.56) (<0.0001). The Ki67 LI and α-SMA; MVD and α-SMA; and Ki67Ki67 LI and MVD in NOM, LRED and HRED showed a statistically significant positive correlation (P<0.0001). The increase in Ki67 LI was directly proportional to MVD and α-SMA expression from NOM to LRED to HRED (P<0.0001). The connective tissue stroma of NOM lacks α-SMA expression. Mild myofibroblast expression was noted in 4 cases of LRED (14.28%) and in 18 cases of HRED (81.81%). Moderate expression was noted only in 4 cases of HRED (22.22%). Conclusion: Ki67 LI, CD105, and α-SMA expression showed significant differences between normal, LRED and HRED. These findings further support that features such as increased basal cell layer hyperplasia, abnormal superficial mitosis, increased nuclear-cytoplasmic ratio, and hyperchromasia could be transformation-relevant dysplastic features. [ABSTRACT FROM AUTHOR]

DBP5, also known as DDX19, GLE1 and inositol hexakisphosphate (IP6) function in messenger RNA (mRNA) export at the cytoplasmic surface of the nuclear pore complex in eukaryotic cells. DBP5 is a DEAD-box RNA helicase, and its activity is stimulated by interactions with GLE1 and IP6. In addition, these three factors also have unique role(s). To investigate how these factors influenced the cytoplasmic mRNA expression and cell phenotype change, we performed RNA microarray analysis to detect the effect and function of DBP5, GLE1 and IP6 on the cytoplasmic mRNA expression. The expression of some cytoplasmic mRNA subsets (e.g. cell cycle, DNA replication) was commonly suppressed by the knock-down of DBP5, GLE1 and IPPK (IP6 synthetic enzyme). The GLE1 knock-down selectively reduced the cytoplasmic mRNA expression required for mitotic progression, results in an abnormal spindle phenotype and caused the delay of mitotic process. Meanwhile, G1/S cell cycle arrest was observed in DBP5 and IPPK knock-down cells. Several factors that function in immune response were also down-regulated in DBP5 or IPPK knock-down cells. Thereby, IFNβ-1 mRNA transcription evoked by poly(I:C) treatment was suppressed. These results imply that DBP5, GLE1 and IP6 have a conserved and individual function in the cytoplasmic mRNA expression. Variations in phenotype are due to the difference in each function of DBP5, GLE1 and IPPK in intracellular mRNA metabolism. [ABSTRACT FROM AUTHOR]

Revertant mosaicism, or “natural gene therapy”, is the phenomenon in which germline mutations are corrected by somatic events. In recent years, revertant mosaicism has been identified in all major types of epidermolysis bullosa, the group of heritable blistering disorders caused by mutations in the genes encoding epidermal adhesion proteins. Moreover, revertant mosaicism appears to be present in all patients with a specific subtype of recessive epidermolysis bullosa. We therefore hypothesized that revertant mosaicism should be expected at least in all patients with recessive forms of epidermolysis bullosa. Naturally corrected, patient-own cells are of extreme interest for their promising therapeutic potential, and their presence in all patients would open exciting, new treatment perspectives to those patients. To test our hypothesis, we determined the probability that single nucleotide reversions occur in patients’ skin using a mathematical developmental model. According to our model, reverse mutations are expected to occur frequently (estimated 216x) in each patient’s skin. Reverse mutations should, however, occur early in embryogenesis to be able to drive the emergence of recognizable revertant patches, which is expected to occur in only one per ~10,000 patients. This underestimate, compared to our clinical observations, can be explained by the “late-but-fitter revertant cell” hypothesis: reverse mutations arise at later stages of development, but provide revertant cells with a selective growth advantage in vivo that drives the development of recognizable healthy skin patches. Our results can be extrapolated to any other organ with stem cell division numbers comparable to skin, which may offer novel future therapeutic options for other genetic conditions if these revertant cells can be identified and isolated. [ABSTRACT FROM AUTHOR]

Argonaute family proteins are well conserved among all organisms. Its role in mitotic cell cycle progression and apoptotic cell elimination is poorly understood. Earlier we have established the contribution of Ago-1 in cell cycle control related to G2/M cyclin in Drosophila. Here we have extended our study in understanding the relationship of Ago-1 in regulating apoptosis during Drosophila development. Apoptosis play a critical role in controlling organ shape and size during development of multi cellular organism. Multifarious regulatory pathways control apoptosis during development among which highly conserved JNK (c-Jun N-terminal kinase) pathway play a crucial role. Here we have over expressed Ago-1 in Drosophila eye and brain by employing UAS (upstream activation sequence)-GAL4 system under the expression of eye and brain specific driver. Over expression of Ago-1 resulted in reduced number of ommatidia in the eye and produced smaller size brain in adult and larval Drosophila. A drastic reversal of the phenotype towards normal was observed upon introduction of a single copy of the dominant negative mutation of basket (bsk, Drosophila homolog of JNK) indicating an active and physical involvement of the bsk with Ago-1 in inducing developmental apoptotic process. Further study showed that Ago-1 stimulates phosphorylation of JNK through transforming growth factor-β activated kinase 1- hemipterous (Tak1-hep) axis of JNK pathway. JNK phosphorylation results in up regulation of pro-apoptotic genes head involution defective (hid), grim & reaper (rpr) and induces activation of Drosophila caspases (cysteinyl aspartate proteinases);DRONC (Death regulator Nedd2-like caspase), ICE (alternatively Drice, Death related ICE-like caspase) and DCP1 (Death caspase-1) by inhibiting apoptotic inhibitor protein DIAP1 (Death-associated inhibitor of apoptosis 1). Further, Ago-1 also inhibits miR-14 expression to trigger apoptosis. Our findings propose that Ago-1 acts as a key regulator in controlling cell death, tumor regression and stress response in metazoan providing a constructive bridge between RNAi machinery and cell death. [ABSTRACT FROM AUTHOR]

The centrosome serves as the main microtubule-organizing center in metazoan cells, yet despite its functional importance, little is known mechanistically about the structure and organizational principles that dictate protein organization in the centrosome. In particular, the protein-protein interactions that allow for the massive structural transition between the tightly organized interphase centrosome and the highly expanded matrix-like arrangement of the mitotic centrosome have been largely uncharacterized. Among the proteins that undergo a major transition is the Drosophila melanogaster protein centrosomin that contains a conserved carboxyl terminus motif, CM2. Recent crystal structures have shown this motif to be dimeric and capable of forming an intramolecular interaction with a central region of centrosomin. Here we use a combination of in-cell microscopy and in vitro oligomer assessment to show that dimerization is not necessary for CM2 recruitment to the centrosome and that CM2 alone undergoes significant cell cycle dependent rearrangement. We use NMR binding assays to confirm this intramolecular interaction and show that residues involved in solution are consistent with the published crystal structure and identify L1137 as critical for binding. Additionally, we show for the first time an in vitro interaction of CM2 with the Drosophila pericentrin-like-protein that exploits the same set of residues as the intramolecular interaction. Furthermore, NMR experiments reveal a calcium sensitive interaction between CM2 and calmodulin. Although unexpected because of sequence divergence, this suggests that centrosomin-mediated assemblies, like the mammalian pericentrin, may be calcium regulated. From these results, we suggest an expanded model where during interphase CM2 interacts with pericentrin-like-protein to form a layer of centrosomin around the centriole wall and that at the onset of mitosis this population acts as a nucleation site of intramolecular centrosomin interactions that support the expansion into the metaphase matrix. [ABSTRACT FROM AUTHOR]

Ring chromosomes are circular chromosomal abnormalities that have been reported in association with some genetic disorders and cancers. In Schizosaccharomyces pombe, lack of function of protection of telomere 1 (Pot1) or telomerase catalytic subunit (Trt1) results in survivors with circular chromosomes. Hitherto, it is poorly understood how cells with circular chromosomes survive and how circular chromosomes are maintained. Fission yeast Cut17/Bir1, Ark1, Pic1, and Nbl1 is a conserved chromosome passenger complex (CPC) functioning mainly throughout mitosis. Here, using a temperature-sensitive mutant of CPC subunits, we determined that CPC is synthetically lethal in combination with either Pot1 or Trt1. The pot1Δ pic1-T269 double mutant, which has circular chromosomes, showed a high percentage of chromosome mis-segregation and DNA damage foci at 33°C. We furthermore found that neither Shugoshin Sgo2 nor heterochromatin protein Swi6, which contribute to the centromeric localization of CPC, were required for the survival in the absence of Pot1. Both the pot1Δ sgo2Δ and pot1Δ swi6Δ double mutants displayed a high percentage of DNA damage foci, but a low percentage of chromosome mis-segregation, suggesting the link between the high percentage of chromosome mis-segregation and the lethality of the CPC pot1Δ double mutant. Our results suggest that CPC is required for the survival of cells with circular chromosomes and sheds light on the possible roles of CPC in the maintenance of circular chromosomes. [ABSTRACT FROM AUTHOR]

Correct segregation of the mitotic chromosomes into daughter cells is a highly regulated process critical to safeguard genome stability. During M phase the spindle assembly checkpoint (SAC) ensures that all kinetochores are correctly attached before its inactivation allows progression into anaphase. Upon SAC inactivation, the anaphase promoting complex/cyclosome (APC/C) E3 ligase ubiquitinates and targets cyclin B and securin for proteasomal degradation. Here, we describe the identification of Ribonucleic Acid Export protein 1 (RAE1), a protein previously shown to be involved in SAC regulation and bipolar spindle formation, as a novel substrate of the deubiquitinating enzyme (DUB) Ubiquitin Specific Protease 11 (USP11). Lentiviral knock-down of USP11 or RAE1 in U2OS cells drastically reduces cell proliferation and increases multipolar spindle formation. We show that USP11 is associated with the mitotic spindle, does not regulate SAC inactivation, but controls ubiquitination of RAE1 at the mitotic spindle, hereby functionally modulating its interaction with Nuclear Mitotic Apparatus protein (NuMA). [ABSTRACT FROM AUTHOR]

The repressive role of p53 on the human mitotic centromere-associated kinesin (MCAK) core promoter from ‒266 to +54, relative to the transcription start site, has been determined. The MCAK mRNA and protein levels were 2.1- and 3.0-fold higher, respectively, in HCT116 (p53‒/‒) than in HCT116 (p53+/+) cells. Enforced down-regulation of p53 levels either in HCT116 (p53+/+) cells by p53 RNAi treatment or in MCF-7 cells using shRNA for p53 (shp53) resulted in a remarkable increase in the MCAK protein level. Site-directed mutagenesis and ChIP analyses showed that p53-mediated repression of the MCAK core promoter activity was not directly exerted by p53-binding to putative p53-response elements (p53-RE1 at −173/−166 and p53-RE2 at −245/−238), but indirectly by attenuating Sp1 binding to GC-motifs (GC1 at −93/−84 and GC2 at −119/−110). Treatment of HEK-293 cells bearing the MCAK core promoter-reporter (pGL2-320-Luc) with mithramycin A, which down-regulates Sp1 gene expression, reduced the promoter activity as well as endogenous MCAK levels. Exposure of HCT116 (p53+/+) cells to nutlin-3a, a validated activator of p53, caused a simultaneous reduction in Sp1 and MCAK protein levels, but not in HCT116 (p53−/−) cells. In contrast to wild-type (wt)-p53, tumor-derived p53 mutants (p53V143A, p53R248W, and p53R273H) failed to repress the Sp1-dependent activation of the MCAK promoter and to down-regulate endogenous levels of Sp1 and MCAK proteins. Collectively, these findings demonstrate that p53 can repress MCAK promoter activity indirectly via down-regulation of Sp1 expression level, and suggest that MCAK elevation in human tumor cells might be due to p53 mutation. [ABSTRACT FROM AUTHOR]

Endogenously tagging proteins with green fluorescent protein (GFP) enables the visualization of the tagged protein using live cell microscopy. GFP-tagging is widely utilized to study biological processes in model experimental organisms including filamentous fungi such as Aspergillus nidulans. Many strains of A. nidulans have therefore been generated with different proteins endogenously tagged with GFP. To further enhance experimental approaches based upon GFP-tagging, we have adapted the FP inding Protein (GBP) system for A. nidulans. GBP is a genetically encoded Llama single chain antibody against GFP which binds GFP with high affinity. Using gene replacement approaches, it is therefore possible to link GBP to anchor proteins, which will then retarget GFP-tagged proteins away from their normal location to the location of the anchor-GBP protein. To facilitate this approach in A. nidulans, we made four base plasmid cassettes that can be used to generate gene replacement GBP-tagging constructs by utilizing fusion PCR. Using these base cassettes, fusion PCR, and gene targeting approaches, we generated strains with SPA10-GBP and Tom20-GBP gene replacements. These strains enabled test targeting of GFP-tagged proteins to septa or to the surface of mitochondria respectively. SPA10-GBP is shown to effectively target GFP-tagged proteins to both forming and mature septa. Tom20-GBP has a higher capacity to retarget GFP-tagged proteins being able to relocate all Nup49-GFP from its location within nuclear pore complexes (NPCs) to the cytoplasm in association with mitochondria. Notably, removal of Nup49-GFP from NPCs causes cold sensitivity as does deletion of the nup49 gene. The cassette constructs described facilitate experimental approaches to generate precise protein-protein linkages in fungi. The A. nidulans SPA10-GBP and Tom20-GBP strains can be utilized to modulate other GFP-tagged proteins of interest. [ABSTRACT FROM AUTHOR]