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Partial inhibition of Cdk1 in G2 phase overrides the SAC and decouples mitotic events
Cell Cycle
Abstract
Entry and progression through mitosis has traditionally been linked directly to the activity of cyclin-dependent kinase 1 (Cdk1). In this study we utilized low doses of the Cdk1-specific inhibitor, RO3306 from early G 2 phase onwards. Addition of low doses of RO3306 in G 2 phase induced minor chromosome congression and segregation defects. In contrast, mild doses of RO3306 during G 2 phase resulted in cells entering an aberrant mitosis, with cells fragmenting centrosomes and failing to fully disassemble the nuclear envelope. Cells often underwent cytokinesis and metaphase simultaneously, despite the presence of an active spindle assembly checkpoint, which prevented degradation of cyclin B1 and securin, resulting in the random partitioning of whole chromosomes. This highly aberrant mitosis produced a significant increase in the proportion of viable polyploid cells present up to 3 days post-treatment. Furthermore, cells treated with medium doses of RO3306 were only able to reach the threshold of Cdk1 substrate phosphorylation required to initiate nuclear envelope breakdown, but failed to reach the levels of phosphorylation required to correctly complete pro-metaphase. Treatment with low doses of Okadaic acid, which primarily inhibits PP2A, rescued the mitotic defects and increased the number of cells that completed a normal mitosis. This supports the current model that PP2A is the primary phosphatase that counterbalances the activity of Cdk1 during mitosis. Taken together these results show that continuous and subtle disruption of Cdk1 activity from G 2 phase onwards has deleterious consequences on mitotic progression by disrupting the balance between Cdk1 and PP2A.
... The Corrected Total Cell Fluorescence (CTCF) signal for Nmb, Gpr4 and Task2 mRNAs was quantified as previously described (Cardani et al., 2022;McCloy et al., 2014). Briefly, a Leica TCS SP5 (B-120G) Laser Scanning Confocal microscope was used to acquire images of the tissue. ...
... Exposure time and acquisition parameters were set for the naive group and kept unchanged for the entire dataset acquisition. The collected images were then analysed by selecting a single cell at a time and measuring the area, integrated density and mean grey value (McCloy et al., 2014). For each image, three background areas were used to normalize against autofluorescence. ...
PHOX2B is a transcription factor essential for the development of different classes of neurons in the central and peripheral nervous system. Heterozygous mutations in the PHOX2B coding region are responsible for the occurrence of Congenital Central Hypoventilation Syndrome (CCHS), a rare neurological disorder characterised by inadequate chemosensitivity and life-threatening sleep-related hypoventilation. Animal studies suggest that chemoreflex defects are caused in part by the improper development or function of PHOX2B expressing neurons in the retrotrapezoid nucleus (RTN), a central hub for CO 2 chemosensitivity. Although the function of PHOX2B in rodents during development is well established, its role in the adult respiratory network remains unknown. In this study, we investigated whether reduction in PHOX2B expression in chemosensitive neuromedin-B (NMB) expressing neurons in the RTN altered respiratory function. Four weeks following local RTN injection of a lentiviral vector expressing the short hairpin RNA (shRNA) targeting Phox2b mRNA, a reduction of PHOX2B expression was observed in Nmb neurons compared to both naive rats and rats injected with the non-target shRNA. PHOX2B knockdown did not affect breathing in room air or under hypoxia, but ventilation was significantly impaired during hypercapnia. PHOX2B knockdown did not alter Nmb expression but it was associated with reduced expression of both Task2 and Gpr4 , two CO 2 /pH sensors in the RTN. We conclude that PHOX2B in the adult brain has an important role in CO 2 chemoreception and reduced PHOX2B expression in CCHS beyond the developmental period may contribute to the impaired central chemoreflex function.
... Then three selections of the background were taken and the average for each measurement was found. The corrected total cell fluorescence (CTCF) equation, a quantitative fluorescence imaging analysis, was used to calculate the final measurement: CTCF = Mean Integrated Density − (Mean area of the cell*Mean Gray Value of the background) [41]. CTCF can be used for any fluorescent material within a restricted area. ...
... Then three selections of the background were taken and the average for each measurement was found. The corrected total cell fluorescence (CTCF) equation, a quantitative fluorescence imaging analysis, was used to calculate the final measurement: CTCF = Mean Integrated Density − (Mean area of the cell * Mean Gray Value of the background) [41]. CTCF can be used for any fluorescent material within a restricted area. ...
Simple Summary
Insects possess a very strong and flexible protein in their appendages and wing joints called resilin. In flying insects, resilin provides wing flexibility that improves flight and movement dynamics. In our study, we investigated how resilin changes with age in honey bees. We discovered that resilin genes are expressed at high levels in worker honey bee pupae, but then levels decline significantly with age. We also determined that the resilin protein is found at higher levels in some joints in the newly emerged honey bees compared to older age classes, further confirming the age-dependent nature of resilin presence in honey bee wings. Specifically, resilin is highly expressed in pupae, deposited at high amounts in some joints in newly emerged honey bees, and then expression and deposition both decline rapidly with age. Resilin must be kept hydrated to stay functional, so these results suggest that although resilin genes may not be expressed significantly after emerging, the resilin protein is likely maintained through fluid support to allow for strong flight dynamics throughout life. Resilin expression and maintenance should be explored for their potential to be used as an indicator of wing development and overall wing health in aging honey bees in the future.
Abstract
The presence of resilin, an elastomeric protein, in insect vein joints provides the flexible, passive deformations that are crucial to flapping flight. This study investigated the resilin gene expression and autofluorescence dynamics among Apis mellifera (honey bee) worker age classes and drone honey bees. Resilin gene expression was determined via ddPCR on whole honey bees and resilin autofluorescence was measured in the 1m-cu, 2m-cu, Cu-V, and Cu2-V joints on the forewing and the Cu-V joint of the hindwing. Resilin gene expression varied significantly with age, with resilin activity being highest in the pupae. Autofluorescence of the 1m-cu and the Cu-V joints on the ventral forewing and the Cu-V joint on the ventral hindwing varied significantly between age classes on the left and right sides of the wing, with the newly emerged honey bees having the highest level of resilin autofluorescence compared to all other groups. The results of this study suggest that resilin gene expression and deposition on the wing is age-dependent and may inform us more about the physiology of aging in honey bees.
... Using a selection tool, Spar-positive areas were demarcated, and measurements recorded. Corrected total cell fluorescence (CTCF), in arbitrary units, was measured for each third instar brain as follows: CTCF = integrated density -(area of selected cell × mean fluorescence of background readings) (Bora et al., 2021;McCloy et al., 2014). Calculated CTCFs were represented in the form of boxplots (n=12 each for w 1118 , Alk Y1255S , Alk RA . ...
Numerous roles for the Alk receptor tyrosine kinase have been described in Drosophila , including functions in the central nervous system (CNS), however the molecular details are poorly understood. To gain mechanistic insight, we employed Targeted DamID (TaDa) transcriptional profiling to identify targets of Alk signaling in the larval CNS. TaDa was employed in larval CNS tissues, while genetically manipulating Alk signaling output. The resulting TaDa data were analyzed together with larval CNS scRNA-seq datasets performed under similar conditions, identifying a role for Alk in the transcriptional regulation of neuroendocrine gene expression. Further integration with bulk and scRNA-seq datasets from larval brains in which Alk signaling was manipulated identified a previously uncharacterized Drosophila neuropeptide precursor encoded by CG4577 as an Alk signaling transcriptional target. CG4577 , which we named Sparkly (Spar) , is expressed in a subset of Alk-positive neuroendocrine cells in the developing larval CNS, including circadian clock neurons. In agreement with our TaDa analysis, overexpression of the Drosophila Alk ligand Jeb resulted in increased levels of Spar protein in the larval CNS. We show that Spar protein is expressed in circadian (clock) neurons, and flies lacking Spar exhibit defects in sleep and circadian activity control. In summary, we report a novel activity regulating neuropeptide precursor gene that is regulated by Alk signaling in the Drosophila CNS.
... Corrected fluorescence intensity was calculated from immunofluorescence images using Fiji/ImageJ. For fluorescence intensity (such as FN1, Phalloidin), images were transformed to 8-bit and the average of mean gray values of three circles on non-fluorescence regions was calculated as the background fluorescence using standard methods (McCloy et al., 2014;Shihan et al., 2021). The corrected fluorescence per ROI was calculated as integrated density -(area of ROI × average of background fluorescence). ...
The skull roof, or calvaria, is comprised of interlocking plates of bones that encase the brain. Separating these bones are fibrous sutures that permit growth. Currently, we do not understand the instructions for directional growth of the calvaria, a process which is error-prone and can lead to skeletal deficiencies or premature suture fusion (craniosynostosis, CS). Here, we identify graded expression of fibronectin (FN1) in the mouse embryonic cranial mesenchyme (CM) that precedes the apical expansion of calvaria. Conditional deletion of Fn1 or Wasl leads to diminished frontal bone expansion by altering cell shape and focal actin enrichment, respectively, suggesting defective migration of calvarial progenitors. Interestingly, Fn1 mutants have premature fusion of coronal sutures. Consistently, syndromic forms of CS in humans exhibit dysregulated FN1 expression, and we also find FN1 expression altered in a mouse CS model of Apert syndrome. These data support a model of FN1 as a directional substrate for calvarial osteoblast migration that may be a common mechanism underlying many cranial disorders of disparate genetic etiologies.
... 30 neurons were analyzed in their cytoplasm and nucleus areas. Fluorescence images of the perifocal area 0.3-0.7 mm from the infarct core perimeter were examined (McCloy et al., 2014). ...
This study focuses on understanding the role of c-Myc, a cancer-associated transcription factor, in the penumbra following ischemic stroke. While its involvement in cell death and survival is recognized, its post-translational modifications, particularly acetylation, remain understudied in ischemia models. Investigating these modifications could have significant clinical implications for controlling c-Myc activity in the central nervous system. Although previous studies on c-Myc acetylation have been limited to non-neuronal cells, our research examines its expression in perifocal cells during stroke recovery to explore regulatory mechanisms via acetylation. We found that in peri-infarct neurons, c-Myc is upregulated with acetylation at K148 but not K323 during the acute phase of stroke, with SIRT2 deacetylase primarily affecting K148 acetylation. Molecular dynamics simulations suggest that lysine 148 plays a crucial role in stabilizing c-Myc spatial structure. Increased acetylation at K148 reduces c-Myc compaction, potentially limiting its nuclear penetration, promoting calpain-mediated cleavage, and decreasing nuclear localization. Additionally, cytoplasmic acetylation at K148 may alter c-Myc's interaction with unidentified proteins, potentially influencing its pro-apoptotic effects and promoting cytoplasmic accumulation. Targeting SIRT2 with selective inhibitors could be a promising avenue for future stroke therapy strategies.
... converted to optical density (OD) by the formula OD = log (255mean intensity).For fluorescence stains (lectins, anti-Muc6, anti-H + /K + -ATPase), the corrected total cell fluorescence (CTCF) for each cell was computed(McCloy et al., 2014). Details for computing were given elsewhere(Carlucci et al., 2019). ...
Aquaporins (AQPs) are important for water transport in the gastrointestinal tract. Changes in their expression and/or localization could cause in disorders and be used as therapeutic targets. Aquaporin‐4 (AQP4) is expressed predominantly on the basolateral membrane of the parietal cells in the corpus of the murine gastric glands. Although the secretion of gastric juice is not affected in AQP4‐deficient knockout, we evaluated by light microscopy whether the lack of AQP4 affects the glycopatterns of secreting gastric cells. Wild type (WT) and AQP4‐deficient knockout mice (KO) were fed a standard diet ad libitum before sacrifice. Segments of stomach corpus were collected, fixed in buffered formalin, and embedded in paraffin wax. Sections, 5‐μm thick, were analyzed by histochemical methods (Periodic acid‐Schiff, Alcian Blue pH 2.5), and binding of lectins specific to GalNAc (SBA, DBA), Gal (PNA) GlcNAc (WGA, GSAII) mannose and/or glucose (ConA), and fucose (UEA‐I, AAA, LTA). Immunohistochemical methods such as anti‐Muc6 for neck cells and anti‐ β‐ H ⁺ /K ⁺ ‐ATPase for parietal cells were also performed. Compared to WT mice, in the mucous cells of KO lower amounts of glycans with galactosyl/galactosaminylated, glycosyl/glycosaminylated, and fucosylated residues were observed; lower fucosylation resulted also in the parietal cells. The observed differences of KO in respect to WT could lead to severer pathological conditions.
Research Highlights
Glycopatterns in gastric glands were compared between wild type (WT) and AQP4‐deficient knockout (KO) mice by histochemical and lectin‐binding methods.
In the mucous cells of KO lower amounts of glycans with galactosyl/galactosaminylated, glycosyl/glycosaminylated and fucosylated residues were observed.
In the parietal cells lower fucosylation also resulted.
AQP4‐deficiency affects glycosylation and could result in altered functionality and pathological conditions.
... The fluorescent images were then analyzed using ImageJ software (version 1.48) by selecting one cell at a time in an image and measuring the area, integrated density, and mean gray value. Using the calculation for corrected total cell fluorescence (CTCF) = integrated density-(area of selected cell × mean fluorescence of background readings), as described by McCloy et al. 48 , the fluorescence intensity of each cell was calculated using Excel (Microsoft Office 365) and GraphPad Prism (version 8.4.3; GraphPad Software). ...
Fluorescence nanosilica-based cell tracker has been explored and applied in cell biological research. However, the aggregation of these nanoparticles at physiological pH is still the main limitation. In this research, we introduced a novel fluorescence nano-based cell tracker suitable for application in live cells. The silica-coated fluorescein isothiocyanate isomer (FITC-SiO2) nanoparticles (NPs) were modified with carboxymethylsilanetriol disodium salt (FITC-SiO2-COOH), integrating the dianion form of FITC molecules. This nanosystem exhibited superior dispersion in aqueous solutions and effectively mitigated dye leakage. These labeled NPs displayed notable biocompatibility and minimal cytotoxicity in both in vitro and in vivo conditions. Significantly, the NPs did not have negative implications on cell migration or angiogenesis. They successfully penetrated primary fibroblasts, human umbilical vein endothelial cells and HeLa cells in both 2D and 3D cultures, with the fluorescence signal enduring for over 72 h. Furthermore, the NP signals were consistently observed in the developing gastrointestinal tract of live medaka fish larvae for extended periods during phases of subdued digestive activity, without manifesting any apparent acute toxicity. These results underscore the promising utility of FITC-SiO2-COOH NPs as advanced live cell trackers in biological research.
... committed cells) (8), and subsequently progressed to prometaphase with a fully active Cdk1 kinase (13,14) might lead to conflicting orders and mitotic disarray (Fig. 3), as Cdk1 is needed for spindle formation at this stage (20,21,56,57). Another consequence of interfering with Cdk1 activity in committed mitotic cells might be mitotic abortion and cell death (15). This is precisely what happened in HeLa cells treated with the Survivin peptide. ...
The Survivin protein has roles in repairing incorrect microtubule-kinetochore attachments at prometaphase, and the faithful execution of cytokinesis, both as part of the chromosomal passenger complex (CPC) (1). In this context, errors frequently lead to aneuploidy, polyploidy and cancer (1). Adding to these well-known roles of this protein, this paper now shows for the first time that Survivin is required for cancer cells to enter mitosis, and that, in its absence, HeLa cells accumulate at early prophase, or prior to reported before (2, 3). This early prophase blockage is demonstrated by the presence of an intact nuclear lamina and low Cdk1 activity (4). Importantly, escaping the arrest induced by Survivin abrogation leads to multiple mitotic defects, or mitotic catastrophe, and eventually cell death. Mechanistically, Cdk1 does not localize at the centrosome in the absence of Survivin. Also, the recombinant Survivin protein can induce the activation of the Cdk1 kinase via the Cdc25 phosphatase in vitro . Moreover, Survivin directly interacts with the Cdc25B isoform, both in vitro and in vivo , and in its absence, an inactive cytosolic Cdc25B-Cdk1-Cyclin B1 complex accumulates. Finally, in agreement with a role for Survivin in the early activation of Cdk1, the early prophase accumulation induced in HeLa cells by Survivin abrogation could be bypassed by a gain-of-function Cdc25B mutant, which drove cells back into mitosis.
... Fluorescence intensity analysis was then obtained by using ImageJ via the formula reported: CTCF = Integrated Density -(Area of selected cell × Mean fluorescence of background readings) as described by McCloy et al. [41]. The fluorescence intensity of each cell was calculated using Excel (Microsoft Office). ...
Human angiogenin (hANG) is the most studied stress-induced ribonuclease (RNase). In physiological conditions it performs its main functions in nucleoli, promoting cell proliferation by rDNA transcription, whereas it is strongly limited by its inhibitor (RNH1) throughout the rest of the cell. In stressed cells hANG dissociates from RNH1 and thickens in the cytoplasm where it manages the translational arrest and the recruitment of stress granules, thanks to its propensity to cleave tRNAs and to induce the release of active halves. Since it exists a clear connection between hANG roles and its intracellular routing, starting from our recent findings on heterologous ANG (ANG) properties in human keratinocytes (HaCaT cells), here we designed a variant unable to translocate into the nucleus with the aim of thoroughly verifying its potentialities under stress. This variant, widely characterized for its structural features and biological attitudes, shows more pronounced aid properties than unmodified protein. The collected evidence thus fully prove that ANG stress-induced skills in assisting cellular homeostasis are strictly due to its cytosolic localization. This study opens an interesting scenario for future studies regarding both the strengthening of skin defences and in understanding the mechanism of action of these special enzymes potentially suitable for any cell type.
... ; https://doi.org/10.1101/2023.12.21.572935 doi: bioRxiv preprint with a fully active Cdk1 kinase (13,14) might lead to conflicting orders and mitotic disarray (Fig. 3), as Cdk1 is needed for spindle formation at this stage (20,21,54,55). Another consequence of interfering with Cdk1 activity in committed mitotic cells might be mitotic abortion and cell death (15). This is precisely what happened in HeLa cells treated with the Regarding the cell death observed when HeLa cells were treated with the Survivin peptide, it has been shown that Cdk1-Cyclin B1 activity is required to phosphorylate procaspase 8 (57) and procaspase 9 (58), and subsequently block the extrinsic (57) and intrinsic (58) apoptotic pathways in cancer cell lines. ...
Survivin has roles in repairing incorrect microtubule-kinetochore attachments at prometaphase, and the execution of cytokinesis, both as part of the chromosomal passenger complex. Here, errors lead to aneuploidy, polyploidy and cancer. Adding to these roles of Survivin, this work shows that Survivin is also required for cancer cells to enter mitosis, and that, in its absence, HeLa cells accumulate at early prophase. This blockage is demonstrated by the presence of an intact nuclear lamina and low Cdk1 activity. Interestingly, Survivin and Cdk1 form a complex at mitosis, and its molecular targeting indicates that Survivin is needed for Cdk1 to activate. Subsequently, escaping the blockage induced by Survivin abrogation leads to mitotic defects and apoptosis. Mechanistically, recombinant Survivin induces the activation of Cdk1 via Cdc25 in vitro. Coincidentally, Cdk1 mislocalizes at the centrosome when Survivin is not expressed. Moreover, Survivin interacts with Cdc25B in vitro and in vivo, and when absent, an inactive cytosolic Cdc25B-Cdk1-Cyclin B1 complex accumulates. Finally, the increase in prophase cells when Survivin was absent, could be bypassed by a gain-of-function Cdc25B mutant.
... The Gray values were measured simultaneously for the immediate background of each cell. The calculated mean intensity values were then converted into Corrected Total Cell Fluorescent (CTCF) values by applying the equation described elsewhere [36]. The average CTCF values of all biological replicates were tabulated corresponding to the calibration graphs. ...
Background
In this study, we designed a novel genetic circuit sensitive to Cd ²⁺ , Zn ²⁺ and Pb ²⁺ by mimicking the CadA/CadR operon system mediated heavy metal homeostasis mechanism of Pseudomonas aeruginosa . The regular DNA motifs on natural operon were reconfigured and coupled with the enhanced Green Fluorescent Protein (eGFP) reporter to develop a novel basic NOT type logic gate CadA/CadR- eGFP to respond metal ions mentioned above. A Genetically Engineered Microbial (GEM)-based biosensor ( E.coli -BL21:pJET1.2- CadA/CadR- eGFP) was developed by cloning the chemically synthesised CadA/CadR- eGFP gene circuit into pJET1.2-plasmid and transforming into Escherichia coli ( E. coli )-BL21 bacterial cells.
Results
The GEM-based biosensor cells indicated the reporter gene expression in the presence of Cd ²⁺ , Zn ²⁺ and Pb ²⁺ either singly or in combination. Further, the same biosensor cells calibrated for fluorescent intensity against heavy metal concentration generated linear graphs for Cd ²⁺ , Zn ²⁺ and Pb ²⁺ with the R ² values of 0.9809, 0.9761 and 0.9758, respectively as compared to non-specific metals, Fe ³⁺ (0.0373), AsO 4 ³⁻ (0.3825) and Ni ²⁺ (0.8498) making our biosensor suitable for the detection of low concentration of the former metal ions in the range of 1–6 ppb. Furthermore, the GEM based biosensor cells were growing naturally within the concentration range of heavy metals, at 37 °C and optimum pH = 7.0 in the medium, resembling the characteristics of wildtype E.coli .
Conclusion
Finally, the novel GEM based biosensor cells developed in this study can be applied for detection of targeted heavy metals in low concentration ranges (1–6 ppb) at normal bacterial physiological conditions.
... Fluorescence intensity was analyzed using FIJI by measuring the area, integrated density, and mean gray value from three nonoverlapping images of each male worm. Fluorescence intensity was determined using corrected total cell fluorescence (CTCF) = integrated density -(area of selected cell x mean fluorescence of background readings, (31)). For each image, a background area was used to normalize against autofluorescence. ...
Parasitic flatworms cause various clinical and veterinary infections that impart a huge burden worldwide. The most clinically impactful infection is schistosomiasis, a neglected tropical disease caused by parasitic blood flukes. Schistosomiasis is treated with praziquantel (PZQ), an old drug introduced over 40 years ago. New drugs are urgently needed, as while PZQ is broadly effective it suffers from several limitations including poor efficacy against juvenile worms, which may prevent it from being completely curative. An old compound that retains efficacy against juvenile worms is the benzodiazepine meclonazepam (MCLZ). However, host side effects caused by benzodiazepines preclude development of MCLZ as a drug and MCLZ lacks an identified parasite target to catalyze rational drug design for engineering out human host activity. Here, we identify a transient receptor potential ion channel of the melastatin subfamily, named TRPMMCLZ, as a parasite target of MCLZ. MCLZ potently activates Schistosoma mansoni TRPMMCLZ through engagement of a binding pocket within the voltage-sensor-like domain of the ion channel to cause worm paralysis, tissue depolarization, and surface damage. TRPMMCLZ reproduces all known features of MCLZ action on schistosomes, including a lower activity versus Schistosoma japonicum, which is explained by a polymorphism within this voltage-sensor-like domain–binding pocket. TRPMMCLZ is distinct from the TRP channel targeted by PZQ (TRPMPZQ), with both anthelmintic chemotypes targeting unique parasite TRPM paralogs. This advances TRPMMCLZ as a novel druggable target that could circumvent any target-based resistance emerging in response to current mass drug administration campaigns centered on PZQ.
... The cells were analyzed using a Leica DMi8 microscope. Subsequently, the images were analyzed with ImageJ software version 1.8 according to the protocol used by McCloy et al. [62]. Briefly, a line was drawn around each individual cell to calculate the area and intensity of the emi ed fluorescence (integrated density). ...
Chlorogenic acid (CGA), a polyphenol found mainly in coffee and tea, exerts antioxidant, anti-inflammatory and anti-apoptotic effects at the gastrointestinal level. However, although CGA is known to cross the blood–brain barrier (BBB), its effects on the CNS are still unknown. Oligodendrocytes (OLs), the myelin-forming cells in the CNS, are the main target in demyelinating neuroinflammatory diseases such as multiple sclerosis (MS). We evaluated the antioxidant, anti-inflammatory and anti-apoptotic roles of CGA in M03-13, an immortalized human OL cell line. We found that CGA reduces intracellular superoxide ions, mitochondrial reactive oxygen species (ROS) and NADPH oxidases (NOXs) /dual oxidase 2 (DUOX2) protein levels. The stimulation of M03-13 cells with TNFα activates the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-kB) pathway, leading to an increase in superoxide ion, NOXs/DUOX2 and phosphorylated extracellular regulated protein kinase (pERK) levels. In addition, tumor necrosis factor alpha (TNF-α) stimulation induces caspase 8 activation and the cleavage of poly-ADP-ribose polymerase (PARP). All these TNFα-induced effects are reversed by CGA. Furthermore, CGA induces a blockade of proliferation, driving cells to differentiation, resulting in increased mRNA levels of myelin basic protein (MBP) and proteolipid protein (PLP), which are major markers of mature OLs. Overall, these data suggest that dietary supplementation with this polyphenol could play an important beneficial role in autoimmune neuroinflammatory diseases such as MS.
... html. The corrected total cell fluorescence (CTCF) was calculated using the formula: CTCF = Integrated Density − (Area of selected cell x Mean fluorescence of background readings) [42]. For manual counting, 10 images were captured at random locations using the confocal microscope at 20× magnitude and the mean is determined. ...
Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease, characterized by the loss of midbrain dopaminergic neurons which leads to impaired motor and cognitive functions. PD is predominantly an idiopathic disease; however, about 5% of cases are linked to hereditary mutations. The most common mutation in both familial and sporadic PD is the G2019S mutation of leucine-rich repeat kinase 2 (LRRK2). Currently, it is not fully understood how this mutation leads to PD pathology. In this study, we isolated self-renewable, multipotent neural stem cells (NSCs) from induced pluripotent stem cells (iPSCs) harboring the G2019S LRRK2 mutation and compared them with their isogenic gene corrected counterparts using single-cell RNA-sequencing. Unbiased single-cell transcriptomic analysis revealed perturbations in many canonical pathways, specifically NRF2-mediated oxidative stress response, and glutathione redox reactions. Through various functional assays, we observed that G2019S iPSCs and NSCs exhibit increased basal levels of reactive oxygen species (ROS). We demonstrated that mutant cells show significant increase in the expression for KEAP1 and decrease in NRF2 associated with a reduced antioxidant response. The decreased viability of mutant NSCs in the H2O2-induced oxidative stress assay was rescued by two potent antioxidant drugs, PrC-210 at concentrations of 500 µM and 1 mM and Edaravone at concentrations 50 µM and 100 µM. Our data suggest that the hyperactive LRRK2 G2019S kinase activity leads to increase in KEAP1, which binds NRF2 and leads to its degradation, reduction in the antioxidant response, increased ROS, mitochondria dysfunction and cell death observed in the PD phenotype.
... For each cell, the corrected total cell fluorescence (CTCF) was calculated as described by McCloy et. al, with the following formula: CTCF = Integrated Density-(Area of selected cell X Mean fluorescence of background readings) [55]. The production on the bar graphs and statistical analysis (One-way ANOVA and Tukey's multiple comparisons test) were performed using GraphPad Prism v.8.4.2. ...
Chemotherapy using temozolomide is the standard treatment for patients with glioblastoma. Despite treatment, prognosis is still poor largely due to the emergence of temozolomide resistance. This resistance is closely linked to the widely recognized inter- and intra-tumoral heterogeneity in glioblastoma, although the underlying mechanisms are not yet fully understood. To induce temozolomide resistance, we subjected 21 patient-derived glioblastoma cell cultures to Temozolomide treatment for a period of up to 90 days. Prior to treatment, the cells’ molecular characteristics were analyzed using bulk RNA sequencing. Additionally, we performed single-cell RNA sequencing on four of the cell cultures to track the evolution of temozolomide resistance. The induced temozolomide resistance was associated with two distinct phenotypic behaviors, classified as “adaptive” (ADA) or “non-adaptive” (N-ADA) to temozolomide. The ADA phenotype displayed neurodevelopmental and metabolic gene signatures, whereas the N-ADA phenotype expressed genes related to cell cycle regulation, DNA repair, and protein synthesis. Single-cell RNA sequencing revealed that in ADA cell cultures, one or more subpopulations emerged as dominant in the resistant samples, whereas N-ADA cell cultures remained relatively stable. The adaptability and heterogeneity of glioblastoma cells play pivotal roles in temozolomide treatment and contribute to the tumor’s ability to survive. Depending on the tumor’s adaptability potential, subpopulations with acquired resistance mechanisms may arise.
... The pixel intensity was measured using ImageJ (https://celldivisionlab. com/2015/08/12/using-imagej-to-measure-cell-fluorescenc/) [24]. The respective H&E slides (cytology) were interpreted by two oral pathologists blinded to histology diagnosis. ...
The high prevalence of oral potentially-malignant disorders exhibits diverse severity and risk of malignant transformation, which mandates a Point-of-Care diagnostic tool. Low patient compliance for biopsies underscores the need for minimally-invasive diagnosis. Oral cytol-ogy, an apt method, is not clinically applicable due to a lack of definitive diagnostic criteria and subjective interpretation. The primary objective of this study was to identify and evaluate the efficacy of biomarkers for cytology-based delineation of high-risk oral lesions. A comprehensive systematic review and meta-analysis of biomarkers recognized a panel of markers (n: 10) delineating dysplastic oral lesions. In this observational cross sectional study, immu-nohistochemical validation (n: 131) identified a four-marker panel, CD44, Cyclin D1, SNA-1, and MAA, with the best sensitivity (>75%; AUC>0.75) in delineating benign, hyperplasia, and mild-dysplasia (Low Risk Lesions; LRL) from moderate-severe dysplasia (High Grade Dysplasia: HGD) along with cancer. Independent validation by cytology (n: 133) showed that expression of SNA-1 and CD44 significantly delineate HGD and cancer with high sensitivity (>83%). Multiplex validation in another cohort (n: 138), integrated with a machine learning model incorporating clinical parameters, further improved the sensitivity and specificity (>88%). Additionally, image automation with SNA-1 profiled data set also provided a high sensitivity (sensitivity: 86%). In the present study, cytology with a two-marker panel, detecting aberrant glycosylation and a glycoprotein, provided efficient risk stratification of oral lesions. Our study indicated that use of a two-biomarker panel (CD44/SNA-1) integrated with clinical parameters or SNA-1 with automated image analysis (Sensitivity >85%) or mul-tiplexed two-marker panel analysis (Sensitivity: >90%) provided efficient risk stratification of PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.
... Quantification of the Cy5 fluorescence was performed as previously described. 59,60 In brief, five separate images (unless stated otherwise) containing approximately 10 cells per image were analyzed using ImageJ (version 1.53f51). A square mask of 184.52 μm 2 was used to quantify the raw integrated density (sum of all pixels in a region of interest). ...
The formation of noncovalent complexes by mixing of positively charged polymers with negatively charged oligonucleotides (ONs) is a widely explored concept in nanomedicine to achieve cellular delivery of ONs. Uptake of ON complexes occurs through endocytosis, which then requires release of ON from endosomes. As one type of polymer, cell-penetrating peptides (CPPs) are being used which are peptides of about 8−30 amino acids in length. However, only a few CPPs yield effective cytosolic ON delivery and activity. Several strategies have been devised to increase cellular uptake and enhance endosomal release, among which an increase of osmotic pressure through the so-called proton sponge effect, disruption of membrane integrity through membrane activity, and disulfide-mediated polymerization. Here, we address the relevance of these concepts for mRNA delivery by incorporating structural features into the human lactoferrin-derived CPP, which shows uptake but not delivery. The incorporation of histidines was explored to address osmotic pressure and structural motifs of the delivery-active CPP PepFect14 (PF14) to address membrane disturbance, and finally, the impact of polymerization was explored. Whereas oligomerization increased the stability of polyplexes against heparin-induced decomplexation, neither this approach nor the incorporation of histidine residues to promote a proton-sponge effect yielded activity. Also, the replacement of arginine residues with lysine or ornithine residues, as in PF14, was without effect, even though all polyplexes showed cellular uptake. Ultimately, sufficient activity could only be achieved by transferring amphipathic sequence motifs from PF14 into the hLF context with some benefit of oligomerization demonstrating overarching principles of delivery for CPPs, lipid nanoparticles, and other types of delivery polymers.
... Secondary antibod ies were applied for 1 h at room temperature with an appropriate Alexa Fluor-conjuga ted antibody. Quantification of immunofluorescence was performed by calculating the corrected total cell fluorescence = integrated density -(area of selected cell × mean fluorescence of background readings) (62). Co-localization analysis was performed using the JACoP plugin from ImageJ using Pearson's correlation coefficient (63). ...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the Coronavirus disease-19 (COVID-19) pandemic, utilizes angiotensin-converting enzyme 2 (ACE2) as a receptor for virus infection. However, the expression pattern of ACE2 does not coincide with the tissue tropism of SARS-CoV-2, hinting that other host proteins might be involved in facilitating SARS-CoV-2 entry. To explore potential host factors for SARS-CoV-2 entry, we performed an arrayed shRNA screen in H1650 and HEK293T cells. Here, we identified a disintegrin and a metalloproteinase domain 9 (ADAM9) protein as an important host factor for SARS-CoV-2 entry. Our data showed that silencing ADAM9 reduced virus entry, while its overexpression promoted infection. The knockdown of ADAM9 decreased the infectivity of the variants of concern tested—B.1.1.7 (alpha), B.1.617.2 (delta), and B.1.1.529 (omicron). Furthermore, mechanistic studies indicated that ADAM9 is involved in the binding and endocytosis stages of SARS-CoV-2 entry. Through immunoprecipitation experiments, we demonstrated that ADAM9 binds to the S1 subunit of the SARS-CoV-2 Spike. Additionally, ADAM9 can interact with ACE2, and co-expression of both proteins markedly enhances virus infection. Moreover, the enzymatic activity of ADAM9 facilitates virus entry. Our study reveals an insight into the mechanism of SARS-CoV-2 virus entry and elucidates the role of ADAM9 in virus infection.
IMPORTANCE
COVID-19, an infectious respiratory disease caused by SARS-CoV-2, has greatly impacted global public health and the economy. Extensive vaccination efforts have been launched worldwide over the last couple of years. However, several variants of concern that reduce the efficacy of vaccines have kept emerging. Thereby, further understanding of the mechanism of SARS-CoV-2 entry is indispensable, which will allow the development of an effective antiviral strategy. Here, we identify a disintegrin and metalloproteinase domain 9 (ADAM9) protein as a co-factor of ACE2 important for SARS-CoV-2 entry, even for the variants of concern, and show that ADAM9 interacts with Spike to aid virus entry. This virus-host interaction could be exploited to develop novel therapeutics against COVID-19.
... ). Corrected nuclear fluorescence was calculated accordingMcCloy et al. (2014). For each sample, three technical replications were performed and 100 nuclear areas per replication were measured. ...
Protoplast technology uses protoplasts (e.g., cells whose cell wall has been removed by enzymatic digestion) as powerful experimental material for in vitro manipulations and crop improvement. This technology encompasses two main components: 1) protoplast isolation and 2) protoplast culture and regeneration. Brown algae are a group of mostly marine, photosynthetic organisms that are used in food, animal feed, traditional medicine, the alginate industry, cosmetics, and pharmaceutical applications. Despite their high economic importance, protoplast technology in brown algae lags far behind other multicellular algae (e.g., green and red algae) and higher plants. Also, most protocols rely on crude extracts or non-commercial enzymes for producing protoplasts, which are expensive, time-consuming and/or low reproducible. Thus, protocols with commercial enzymes are needed for properly establishing protoplast technology in brown algae.
In this study, I selected 7 brown algal species (Dictyopteris pacifica, Ecklonia cava, Hecatonema terminale, Petalonia fascia, Scytosiphon lomentaria, Sphacelaria fusca and Undaria pinnatifida) and developed protoplast isolation protocols for them using only commercial enzymes. Among these species, the economic brown alga U. pinnatifida and others 3 (H. terminale, P. fascia and Sp. fusca) were selected for protoplast culture. In U. pinnatifida, I further explored the effect of light-emitting diodes (LED) on protoplast regeneration from the microscopic gametophytes and
macroscopic sporophytes. Finally, I tested the potential of protoplast-derived aposporous filaments (PDAFs) for clonal propagation of U. pinnatifida sporophyte. In all the species, high amount of protoplasts were obtained using a simple mixture of commercial enzymes (cellulase RS and alginate lyase). Protoplasts yields ranged from 104-105 protoplasts g-1 fresh weight (FW) in the filamentous forms H. terminale and Sp. fusca, to 106-107 protoplasts g-1 FW in more complex brown algae (D. pacifica, E. cava, P. fascia, S. lomentaria and U. pinnatifida). Dictyopteris pacifica, E. cava, H. terminale and Sp. fusca represented new reports for protoplast production. The most important factors during isolation were growth, chelation pre-treatment, pH and osmolarity. Successful regeneration was achieved, for the first time, in H. terminale, P. fascia and Sp. fusca. In U. pinnatifida, an improved method for protoplast culture and regeneration was developed. Critical conditions during this step were regeneration medium, initial protoplast density, antibiotics, light exposure, starting time of osmolarity reduction, and temperature. LED experiments in U. pinnatifida showed that dichromatic light (red plus blue, 1:2) enhanced protoplast regeneration and growth from filamentous gametophytes, increasing the formation of normal sporophytes from PDAFs. In green LED, PDAFs could be propagated without formation of sporophytes while keeping a high potential for producing them upon dichromatic light exposure. Subculturing PDAFs with subsequent 6 weeks of re-growth could sustain sporophyte production over time. Regenerated sporophytes were diploid and showed identical genotype with the mother PDAF culture. This suggests that PDAFs could be used for sustained clonal propagation of U. pinnatifida sporophyte, opening a new possibility of protoplasts uses in brown algae.
... The cell periphery was marked in each cell, the mean adjusted fluorescence was quantified, integrated density with background reading was measured using ImageJ software. Quantification of the fluorescence intensity was done using ImageJ software from 200 cells for each sample [37,38]. Two independent measurements were undertaken with two technical replicates. ...
In this study, an enhanced antifungal response of ZnO nanobullets (NBs) against Schizosaccharomyces pombe is reported. The ZnO NBs were prepared by alkali precipitation method and confirmed by microscopic, morphological and optical studies using SEM, EDX, TEM, HRTEM and photoluminescence (PL) spectroscopic techniques. Growth kinetics and MIC studies were conducted following the growth inhibition percentage studies. Colony forming assay, well diffusion, disc diffusion, N-acetyl cysteine (NAC) effect on S. pombe growth, trypan blue study, cellular reactive oxygen species (ROS) quantification using H2DCFDA dye, Bradford assay, DNA fragmentation and all other relevant protocols were performed in antifungal studies. ZnO nanobullets (NBs) were shown by SEM and TEM examinations to have an average size of 50 nm. The hexagonal wurtzite structure of ZnO NBs was confirmed by HRTEM’s lattice fringe findings. Defectrelated visible emissions at 412, 436, 457 and 564 nm were confirmed via PL analysis. It was found that ZnO NBs resulted in complete growth inhibition of S. pombe at 200 μg/mL. When S. pombe was treated with ZnO NBs, the Bradford assay revealed enhanced protein leakage, but the TBARS assay revealed lipid peroxidation brought on by reactive oxygen species (ROS). When S. pombe was exposed to ZnO NBs, the H2DCFDA assay revealed increased ROS generation, whilst the trypan blue assay revealed increased cell membrane fusion and lower viability. According to present study, the treatment with ZnO NBs caused S. pombe to develop damaged cell walls, leaky proteins and DNA breakage.
... readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html; accessed on 1 July 2023), as previously described [61,62]. The GAPDH fluorescent intensity was comparable in the different experiments (Supplementary Table S3). ...
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration of motor neurons (MNs). Astrocytes display a toxic phenotype in ALS, which results in MN damage. Glutamate (Glu)-mediated excitotoxicity and group I metabotropic glutamate receptors (mGluRs) play a pathological role in the disease progression. We previously demonstrated that in vivo genetic ablation or pharmacological modulation of mGluR5 reduced astrocyte activation and MN death, prolonged survival and ameliorated the clinical progression in the SOD1G93A mouse model of ALS. This study aimed to investigate in vitro the effects of mGluR5 downregulation on the reactive spinal cord astrocytes cultured from adult late symptomatic SOD1G93A mice. We observed that mGluR5 downregulation in SOD1G93A astrocytes diminished the cytosolic Ca2+ overload under resting conditions and after mGluR5 simulation and reduced the expression of the reactive glial markers GFAP, S100β and vimentin. In vitro exposure to an anti-mGluR5 antisense oligonucleotide or to the negative allosteric modulator CTEP also ameliorated the altered reactive astrocyte phenotype. Downregulating mGluR5 in SOD1G93A mice reduced the synthesis and release of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α and ameliorated the cellular bioenergetic profile by improving the diminished oxygen consumption and ATP synthesis and by lowering the excessive lactate dehydrogenase activity. Most relevantly, mGluR5 downregulation hampered the neurotoxicity of SOD1G93A astrocytes co-cultured with spinal cord MNs. We conclude that selective reduction in mGluR5 expression in SOD1G93A astrocytes positively modulates the astrocyte reactive phenotype and neurotoxicity towards MNs, further supporting mGluR5 as a promising therapeutic target in ALS.
... Three oval selections were drawn adjacent to all measured nuclei to be used for normalization against background fluorescence. The corrected total nuclear fluorescence (CTNF) = integrated density -(area of selected nucleus × mean fluorescence of background readings) was calculated according to McCloy et al., 2014 using Excel. The number of mCitrine-CENH3 signals in individual nuclei and mitotic figures was manually determined across image stacks using the multi-point tool. ...
During root nodule symbiosis (RNS), cell-division activity is re-initiated and sustained in the root cortex to create a hospitable cellular niche. Such temporary and spatially confined site is required to render host cells compatible with the intracellular progression of rhizobia. Although it has been suggested that early infection events might involve a pre-mitotic cell-cycle arrest, this process has not been dissected with cellular resolution. Here, we show that a dual-colour Medicago histone reporter robustly identifies cells with different mitotic or endoreduplication activities in the root cortex. By imaging deep root tissues, we found that a confined trajectory of cortical cells that are transcellularly passed by infection threads are in a stage of the cell-cycle that is distinct from directly adjacent cells. Distinctive features of infected cells include nuclear widening and large-scale chromatin rearrangements consistent with a cell-cycle exit prior to differentiation. Using a combination of fluorescent reporters demarcating cell-cycle phase progression, we confirmed that a reduced proliferation potential and modulating the G2/M transition, a process possibly controlled by the NF-YA1 transcription factor, mark the success of rhizobial delivery to nodule cells.
... Images were analyzed using FIJI software (NIH). The corrected total cell fluorescence (CTCF) represents the integrated density minus an area of the selected cell multiplied by the mean fluorescence of the background readings [18,62]. ...
Apoptosis of endothelial cells prompts the release of apoptotic exosome-like vesicles (ApoExos), subtype extracellular vesicles secreted by apoptotic cells after caspase-3 activation. ApoExos are different from both apoptotic bodies and classical exosomes in their protein and nucleic acid contents and functions. In contrast to classical apoptotic bodies, ApoExos induce immunogenic responses that can be maladaptive when not tightly regulated. In the present study, we elucidated the mechanisms by which ApoExos are internalized by endothelial cells, which leads to shared specific and functional mRNAs of importance to endothelial function. Using flow cytometry and confocal microscopy, we revealed that ApoExos were actively internalized by endothelial cells. SiRNA-induced inhibition of classical endocytosis pathways with pharmacological inhibitors showed that ApoExos were internalized via phosphatidylserine-dependent macropinocytosis independently of classical endocytosis pathways. An electron microscopy analysis revealed that ApoExos increased the macropinocytosis rate in endothelial cells, setting in motion a positive feedback loop that increased the amount of internalized ApoExos. Deep sequencing of total RNA revealed that ApoExos possessed a unique protein-coding RNA profile, with PCSK5 being the most abundant mRNA. Internalization of ApoExos by cells led to the transfer of this RNA content from the ApoExos to cells. Specifically, PCSK5 mRNA was transferred to cells that had taken up ApoExos, and these cells subsequently expressed PCSK5. Collectively, our findings suggest that macropinocytosis is an effective entry pathway for the delivery of RNAs carried by ApoExos and that these RNAs are functionally expressed by the endothelial cells that internalize them. As ApoExos express a specific mRNA signature, these results suggest new avenues to understand how ApoExos produced at sites of vascular injury impact vascular function.
... Cell fluorescence was studied as total cell fluorescence (TCF) and base cell fluorescence (BCF). Both was calculated using the following equation [corrected total cell fluorescence (CTCF)]: CTCF = IDF-(A cell x X bf ) ; where IDF = the integrated density of fluorescence emitted from cell planes, A cell = cell area and X bf = mean bac kgr ound r eading fluor escence (Measuring cell fluor escence using Ima geJ; The Open Lab Book 2014 , McCloy et al. 2014 ). BCF measur ements wer e obtained using the lar gest cellspreading plane as a reference, including the range from two planes above to two planes below the spreading plane. ...
Cutaneous leishmaniasis is an infectious disease that may lead to a single or multiple disseminated cutaneous lesions. The mechanisms involved in Leishmania dissemination to different areas of the skin and the internal organs remain poorly understood. Evidence shows that Very Late Antigen-4 (VLA-4)-dependent phagocyte adhesion is impaired by Leishmania infection, which may be related to the mechanisms of parasite dissemination. We investigated factors potentially associated with decreased VLA-4-mediated adhesion in Leishmania-infected macrophages, including lipid raft-mediated VLA-4 mobilization along the cellular membrane, integrin cluster formation at the cell base (adhesion site) and focal adhesion complex assembly. Phagocytes treated with Methyl-β-Cyclodextrin (MβCD) demonstrated reduced adhesion, similarly to Leishmania amazonensis-infected J774 cells. Infected and MβCD-treated macrophages presented decreased VLA-4 mobilization to the adhesion plane, as well as reduced integrin clustering. Leishmania amazonensis-infected cells exhibited talin depletion, as well as a decreased mobilization of adhesion complex proteins, such as talin and viculin, which were associated with lower VLA-4 concentrations at the adhesion site and limited cell-spreading. Our results suggest that Leishmania infection may modulate the firm adhesion phase of the cell-spreading process, which could contribute to the bloodstream dissemination of infected cells.
... The total corrected cellular fluorescence (TCCF) = Integrated density-(area of selected cell × mean fluorescence) was calculated. (23) Data normalisation was made using the TCFF against the number of cells (using DAPI) per field. ...
Background:
Cerebral malaria (CM) is a severe immunovasculopathy caused for Plasmodium falciparum infection, which is characterised by the sequestration of parasitised red blood cells (pRBCs) in brain microvessels. Previous studies have shown that some terpenes, such as perillyl alcohol (POH), exhibit a marked efficacy in preventing cerebrovascular inflammation, breakdown of the brain-blood barrier (BBB) and brain leucocyte accumulation in experimental CM models.
Objective:
To analyse the effects of POH on the endothelium using human brain endothelial cell (HBEC) monolayers co-cultured with pRBCs.
Methodology:
The loss of tight junction proteins (TJPs) and features of endothelial activation, such as ICAM-1 and VCAM-1 expression were evaluated by quantitative immunofluorescence. Microvesicle (MV) release by HBEC upon stimulation by P. falciparum was evaluated by flow cytometry. Finally, the capacity of POH to revert P. falciparum-induced HBEC monolayer permeability was examined by monitoring trans-endothelial electrical resistance (TEER).
Findings:
POH significantly prevented pRBCs-induced endothelial adhesion molecule (ICAM-1, VCAM-1) upregulation and MV release by HBEC, improved their trans-endothelial resistance, and restored their distribution of TJPs such as VE-cadherin, Occludin, and JAM-A.
Conclusions:
POH is a potent monoterpene that is efficient in preventing P. falciparum-pRBCs-induced changes in HBEC, namely their activation, increased permeability and alterations of integrity, all parameters of relevance to CM pathogenesis.
Objective
This study was undertaken to explore manipulation of the Myc protein interactome, members of an oncogene group, in enhancing the intrinsic growth of injured peripheral adult postmitotic neurons and the nerves they supply. New approaches to enhance adult neuron growth properties are a key strategy in improving nerve regeneration.
Methods
Expression and impact of Myc interactome members c‐Myc, N‐Myc, Mad1, and Max were evaluated within naive and “preconditioned” adult sensory neurons and Schwann cells (SCs), using siRNA and transfection of CRISPR/Cas9 or luciferase reporter in vitro. Morphological, behavioral, and electrophysiological indices of nerve regeneration were analyzed in vivo.
Results
c‐Myc, N‐Myc, Max, and Mad were expressed in adult sensory neurons and in partnering SCs. In vitro knockdown (KD) of either Mad1 or Max, competitive inhibitors of Myc, unleashed heightened neurite outgrowth in both naive uninjured or preconditioned adult neurons. In contrast, KD or inhibition of both isoforms of Myc was required to suppress growth. In SCs, Mad1 KD not only enhanced migratory behavior but also conditioned increased outgrowth in separately cultured adult sensory neurons. In vivo, local Mad1 KD improved electrophysiological, behavioral, and structural indices of nerve regeneration out to 60 days of follow‐up.
Interpretation
Members of the Myc interactome, specifically Mad1, are novel targets for improving nerve regeneration. Unleashing of Myc growth signaling through Mad1 KD enhances the regrowth of both peripheral neurons and SCs to facilitate better regrowth of nerves. ANN NEUROL 2024
Background
TP53, the most frequently mutated gene in human cancers, orchestrates a complex transcriptional program crucial for cancer prevention. While certain TP53-dependent genes have been extensively studied, others, like the recently identified RNF144B, remained poorly understood. This E3 ubiquitin ligase has shown potent tumor suppressor activity in murine Eμ Myc -driven lymphoma, emphasizing its significance in the TP53 network. However, little is known about its targets and its role in cancer development, requiring further exploration. In this work, we investigate RNF144B's impact on tumor suppression beyond the hematopoietic compartment in human cancers.
Methods
Employing TP53 wild-type cells, we generated models lacking RNF144B in both non-transformed and cancerous cells of human and mouse origin. By using proteomics, transcriptomics, and functional analysis, we assessed RNF144B's impact in cellular proliferation and transformation. Through in vitro and in vivo experiments, we explored proliferation, DNA repair, cell cycle control, mitotic progression, and treatment resistance. Findings were contrasted with clinical datasets and bioinformatics analysis.
Results
Our research underscores RNF144B's pivotal role as a tumor suppressor, particularly in lung adenocarcinoma. In both human and mouse oncogene-expressing cells, RNF144B deficiency heightened cellular proliferation and transformation. Proteomic and transcriptomic analysis revealed RNF144B's novel function in mediating protein degradation associated with cell cycle progression, DNA damage response and genomic stability. RNF144B deficiency induced chromosomal instability, mitotic defects, and correlated with elevated aneuploidy and worse prognosis in human tumors. Furthermore, RNF144B-deficient lung adenocarcinoma cells exhibited resistance to cell cycle inhibitors that induce chromosomal instability.
Conclusions
Supported by clinical data, our study suggests that RNF144B plays a pivotal role in maintaining genomic stability during tumor suppression.
Graphical Abstract
Plant defense responses to the soil-borne fungus Verticillium longisporum causing stem stripe disease on oilseed rape (Brassica napus) are poorly understood. In this study, a population of recombinant inbred lines (RILs) using the Arabidopsis thaliana accessions Sei-0 and Can-0 was established. Composite interval mapping, transcriptome data and T-DNA mutant screening identified the NITRATE/PEPTIDE TRANSPORTER FAMILY 5.12 gene (AtNPF5.12) as being associated with disease susceptibility in Can-0. Coimmunoprecipitation revealed interaction between AtNPF5.12 and the MAJOR LATEX PROTEIN family member AtMLP6, and fluorescence microscopy confirmed interaction in the plasma membrane and endoplasmic reticulum. CRISPR/Cas9 technology was applied to mutate the NPF5.12 and MLP6 genes in B. napus. Elevated fungal growth in the npf5.12 mlp6 double mutant of both oilseed rape and Arabidopsis demonstrated their importance in defense against V. longisporum. Colonization of this fungus depends also on available nitrates in the host root. Accordingly, the negative effect of nitrate depletion on fungal growth was less pronounced in Atnpf5.12 plants with impaired nitrate transport. In addition, suberin staining revealed involvement of the NPF5.12 and MLP6 genes in suberin barrier formation. Together, these results demonstrate a dependency of multiple plant factors that lead to successful V. longisporum root infection.
The efficiency of motor skill acquisition is age-dependent, making it increasingly challenging to learn complex maneuvers later in life. Zebra finches, for instance, acquire a complex vocal motor program during a developmental critical period after which the learned song is essentially impervious to modification. Although inhibitory interneurons are implicated in critical period closure, it is unclear whether manipulating them can reopen heightened motor plasticity windows. Using pharmacology and a novel cell-type specific optogenetic approach, we manipulated inhibitory neuron activity in a premotor area of adult zebra finches beyond their critical period. When exposed to auditory stimulation in the form of novel song, manipulated birds added new vocal syllables to their stable song sequence. By lifting inhibition in a premotor area during sensory experience, we reintroduced vocal plasticity, promoting an expansion of the syllable repertoire without compromising pre-existing song production. Our findings provide insights into motor skill learning capacities, offer potential for motor recovery after injury, and suggest avenues for treating neurodevelopmental disorders involving inhibitory dysfunctions.
Adolescent idiopathic scoliosis (AIS), a sideways curvature of the spine, is sexually dimorphic, with increased incidence in females. A genome-wide association study identified a female-specific AIS susceptibility locus near the PAX1 gene. Here, we use mouse enhancer assays, three mouse enhancer knockouts, and subsequent phenotypic analyses to characterize this region. Using mouse enhancer assays, we characterize a sequence, PEC7, which overlaps the AIS-associated variant, and find it to be active in the tail tip and intervertebral disc. Removal of PEC7 or Xe1, a known sclerotome enhancer nearby, or deletion of both sequences lead to a kinky tail phenotype only in the Xe1 and combined (Xe1+PEC7) knockouts, with only the latter showing a female sex dimorphic phenotype. Extensive phenotypic characterization of these mouse lines implicates several differentially expressed genes and estrogen signaling in the sex dimorphic bias. In summary, our work functionally characterizes an AIS-associated locus and dissects the mechanism for its sexual dimorphism.
Cancer cachexia is a tumour-induced wasting syndrome, characterised by extreme loss of skeletal muscle. Defective mitochondria can contribute to muscle wasting; however, the underlying mechanisms remain unclear. Using a Drosophila larval model of cancer cachexia, we observed enlarged and dysfunctional muscle mitochondria. Morphological changes were accompanied by upregulation of beta-oxidation proteins and depletion of muscle glycogen and lipid stores. Muscle lipid stores were also decreased in Colon-26 adenocarcinoma mouse muscle samples, and expression of the beta-oxidation gene CPT1A was negatively associated with muscle quality in cachectic patients. Mechanistically, mitochondrial defects result from reduced muscle insulin signalling, downstream of tumour-secreted insulin growth factor binding protein (IGFBP) homologue ImpL2. Strikingly, muscle-specific inhibition of Forkhead box O (FOXO), mitochondrial fusion, or beta-oxidation in tumour-bearing animals preserved muscle integrity. Finally, dietary supplementation with nicotinamide or lipids, improved muscle health in tumour-bearing animals. Overall, our work demonstrates that muscle FOXO, mitochondria dynamics/beta-oxidation and lipid utilisation are key regulators of muscle wasting in cancer cachexia.
Aluminum (Al) is used in everyday life and present in food drugs, packaging, industry, and agriculture. Although it is the most common metal in the Earth crust, a correlation has been demonstrated between its presence and various pathologies, even serious ones, especially of a neurological type. However, there is a histological gap regarding the role Al can have in contact with the covering and secreting epithelia. The alterations of the ventral and dorsal foot mucocytes and their secretions of the snail Eobania vermiculata caused by Al were investigated in situ by histochemical and lectin‐histochemical techniques. Administration to different experimental groups took place for 3 and 9 days with 50 and 200 μM of AlCl 3 . Several types of mucocytes were detected with a prevalent secretion of acid glycans in the foot of E. vermiculata . Sulfated glycans prevail in the dorsal region, with one type showing only fucosylated residues and another also having galactosaminylated and glycosaminylated residues. Carboxylated glycans prevail in the ventral region, with presence of galactosaminylated, glycosaminylated, and fucosylated residuals in both cells. Snails treated presented a general decrease of mucin amount in the secreting cells and affected the mucus composition. These changes could alter the rheological and functional properties of the mucus with possible implications for the health of the treated animals.
Research Highlights
Snails were fed with Al‐contaminated lettuce at different concentrations.
In the foot mucocytes produced mucus with prevailing acidic glycans.
In the treated resulted a reduction in the amount of mucus and an alteration of glycan composition.
Inflammatory bowel diseases (IBDs), which include Crohn's disease (CD) and ulcerative colitis (UC), are chronic inflammatory diseases of the gastrointestinal tract and are characterized by chronic recurrent ulceration of the bowels. Colon-targeted drug delivery systems (DDS) have received significant attention for their potential to treat IBD by improving the inflamed tissue selectivity. Herein, antiMUC5AC-decorated drug loaded nanoparticles (NP) are suggested for active epithelial targeting and selective adhesion to the inflamed tissue in experimental colitis. NPs conjugated with antiMUC5AC (anti-MUC5) were tested for their degree of bioadhesion with HT29-MTX cells by comparison with non-targeted BSA-NP conjugates. In vivo, the selectivity of bioadhesion and the influence of ligand density in bioadhesion efficiency as well as the therapeutic benefit for glucocorticoid loaded anti-MUC5-NP were studied in a murine colitis model. Quantitative adhesion analyses showed that anti-MUC5-conjugated NP exhibited a much higher binding and selectivity to inflamed tissue compared to PNA-, IgG1- and BSA-NP conjugates used as controls. This bioadhesion efficiency was found to be dependent on the ligand density, present at the NP surface. The binding specificity between anti-MUC5 ligand and inflamed tissues was confirmed by fluorescence imaging. Both anti-MUC5-NP and all other glucocorticoid containing formulations led to a significant mitigation of the experimental colitis, as became evident from the substantial reduction of myeloperoxidase activity and pro-inflammatory cytokine concentrations (TNF-α, IL-1β). Targeted NP by using anti-MUC5 appears to be a very promising tool in future treatment of various types of local disorders affecting the gastro-intestinal tract but not limited to colitis.
PHOX2B is a transcription factor essential for the development of the autonomic nervous system. Heterozygous mutations in the PHOX2B coding region are responsible for the occurrence of Congenital Central Hypoventilation Syndrome (CCHS), a rare neurological disorder characterised by inadequate chemosensitivity and life-threatening sleep-related hypoventilation. Animal studies suggest that chemoreflex defects are caused in part by the improper development or function of PHOX2B expressing neurons in the retrotrapezoid nucleus (RTN), a central hub for CO 2 chemosensitivity. Although the function of PHOX2B in rodents during development is well established, its role in the adult respiratory network remains unknown. In this study, we investigated whether reduction in PHOX2B expression in chemosensitive neuromedin-B (NMB) expressing neurons in the RTN altered respiratory function. Four weeks following local RTN injection of a lentiviral vector expressing the short hairpin RNA (shRNA) targeting Phox2b mRNA, a reduction of PHOX2B expression was observed in Nmb neurons compared to both naïve rats and rats injected with the non-target shRNA. PHOX2B knockdown did not affect breathing in room air or under hypoxia, but ventilation was significantly impaired during hypercapnia. PHOX2B knockdown did not alter Nmb expression but reduced the expression of both Task2 and Gpr4 , two CO 2 sensors in the RTN. We conclude that PHOX2B in the adult brain has an important role in CO 2 chemoreception and reduced PHOX2B expression in CCHS beyond the developmental period may contribute to the impaired central chemoreflex function.
Pancreatic cancer is more prevalent in older individuals and often carries a poorer prognosis for them. The relationship between the microenvironment and pancreatic cancer is multifactorial, and age-related changes in nonmalignant cells in the tumor microenvironment may play a key role in promoting cancer aggressiveness. Because fibroblasts have profound impacts on pancreatic cancer progression, we investigated whether age-related changes in pancreatic fibroblasts influence cancer growth and metastasis. Proteomics analysis revealed that aged fibroblasts secrete different factors than young fibroblasts, including increased growth/differentiation factor 15 (GDF-15). Treating young mice with GDF-15 enhanced tumor growth, whereas aged GDF-15 knockout mice showed reduced tumor growth. GDF-15 activated AKT, rendering tumors sensitive to AKT inhibition in an aged but not young microenvironment. These data provide evidence for how aging alters pancreatic fibroblasts and promotes tumor progression, providing potential therapeutic targets and avenues for studying pancreatic cancer while accounting for the effects of aging.
Significance
Aged pancreatic fibroblasts secrete GDF-15 and activate AKT signaling to promote pancreatic cancer growth, highlighting the critical role of aging-mediated changes in the pancreatic cancer microenvironment in driving tumor progression.
See related commentary by Isaacson et al., p. 1185
Migratory cells – either individually or in cohesive groups – are critical for spatiotemporally-regulated processes such as embryonic development and wound healing. Their dysregulation is the underlying cause of formidable health problems such as birth defects and metastatic cancers. Border cell behavior during Drosophila oogenesis provides an effective model to study temporally-regulated, collective cell migration in vivo. Developmental timing in flies is primarily controlled by the steroid hormone ecdysone, which acts through a well-conserved, nuclear hormone receptor complex. Ecdysone signaling determines the timing of border cell migration but the molecular mechanisms governing this remain obscure. We found that border cell clusters expressing a dominant negative form of ecdysone receptor extend ineffective protrusions. Additionally, these clusters have aberrant spatial distributions of E-cadherin, apical domain markers and activated myosin that do not overlap. Remediating their expression or activity individually in ecdysone signaling-mutant clusters did not restore proper migration. We propose that ecdysone signaling synchronizes the functional distribution of E-cadherin, atypical protein kinase C, Discs large, and activated myosin post-transcriptionally to coordinate adhesion, polarity, and contractility and temporally control collective cell migration.
Selfish DNA modules like transposable elements (TEs) are particularly active in the germline, the lineage that passes genetic information across generations. New TE insertions can disrupt genes and impair the functionality and viability of germ cells. However, we find that in P-M hybrid dysgenesis in Drosophila, a sterility syndrome triggered by the P-element DNA transposon, germ cells harbour unexpectedly few new TE insertions, despite accumulating DNA double-strand breaks (DSBs) and inducing cell cycle arrest. Using an engineered CRISPR-Cas9 system, we show that generating DSBs at silenced P-elements or other non-coding sequences is sufficient to induce germ cell loss independently of gene disruption. Indeed, we demonstrate that both developing and adult mitotic germ cells are sensitive to DSBs in a dosage-dependent manner. Following the mitotic-to-meiotic transition, however, germ cells become more tolerant to DSBs, completing oogenesis regardless of the accumulated genome damage. Our findings establish DNA damage tolerance thresholds as crucial safeguards of genome integrity during germline development.
The development of therapies to combat neurodegenerative diseases is widely recognised as a research priority, with conditions like Alzheimers, Amyotrophic lateral sclerosis (ALS) and Parkinsons set to place an ever-heavier burden on healthcare systems in the near future. Despite recent advances in understanding their molecular basis, there is a lack of suitable early biomarkers to test selected compounds and accelerate their translation to clinical trials. We have investigated the utility of in vivo reporters of cytoprotective pathways (e.g. NRF2, p53) as surrogate early biomarkers of the ALS degenerative disease progression. We hypothesized that cellular stress observed in a model of ALS may precede overt cellular damage and could activate our cytoprotective pathway reporters. To test this hypothesis, we generated novel ALS-reporter mice by crossing the hTDP-43tg model into our oxidative stress/inflammation (Hmox1; NRF2 pathway) and DNA damage (p21; p53 pathway) stress reporter models. Histological analysis of reporter expression in a homozygous hTDP-43tg background demonstrated a time-dependent and tissue-specific activation of the reporters in tissues directly associated with ALS. The activation occurs in Purkinje neurons and other parvalbumin-positive (PV+) cells within the cerebellum of mice, before moderate clinical signs are observed. In addition, reporter expression in hTDP-43tg hom peripheral tissues was not observed at the tested mouse ages (15 and 17 days postnatally). Further work is warranted to determine the specific mechanisms by which TDP-43 accumulation leads to reporter activation and whether therapeutic intervention modulates reporters expression. Our current studies suggest that these reporters may represent a powerful approach to accelerate preclinical studies targeting TDP-43 pathologies. We anticipate the reporter strategy could be of great value in developing treatments for a range of degenerative disorders.
Background
In this study, we designed a novel genetic circuit sensitive to Cd²⁺, Zn²⁺ and Pb²⁺ by mimicking the CadA/CadR operon system mediated heavy metal homeostasis mechanism of Pseudomonas aeruginosa. The regular DNA motifs on natural operon were reconfigured and coupled with the enhanced Green Fluorescent Protein (eGFP) reporter to develop a novel basic NOT type logic gate CadA/CadR-eGFP to respond metal ions mentioned above. A Genetically Engineered Microbial (GEM)-based biosensor (E.coli-BL21:pJET1.2-CadA/CadR-eGFP) was developed by cloning the chemically synthesised CadA/CadR-eGFP gene circuit into pJET1.2-plasmid and transforming into Escherichia coli (E. coli)-BL21 bacterial cells.
Results
The GEM-based biosensor cells indicated the reporter gene expression in the presence of Cd²⁺, Zn²⁺ and Pb²⁺ either singly or in combination. Further, the same biosensor cells calibrated for fluorescent intensity against heavy metal concentration generated linear graphs for Cd²⁺, Zn²⁺ and Pb²⁺ with the R² values of 0.9809, 0.9761 and 0.9758, respectively as compared to non-specific metals, Fe³⁺ (0.0373), AsO4³⁻ (0.3825) and Ni²⁺ (0.8498) making our biosensor suitable for the detection of low concentration of the former metal ions in the range of 1–6 ppb. Furthermore, the GEM based biosensor cells were growing naturally within the concentration range of heavy metals, at 37 oC and optimum pH = 7.0 in the medium, resembling the characteristics of wildtype E.coli.
Conclusion
Finally, the novel GEM based biosensor cells developed in this study can be applied for detection of targeted heavy metals in low concentration ranges (1–6 ppb) at normal bacterial physiological conditions.
Glioblastomas (GBMs) are highly aggressive, infiltrative, and heterogeneous brain tumors driven by complex driver mutations and glioma stem cells (GSCs). The neurodevelopmental transcription factors ASCL1 and OLIG2 are co-expressed in GBMs, but their role in regulating the heterogeneity and hierarchy of GBM tumor cells is unclear. Here, we show that oncogenic driver mutations lead to dysregulation of ASCL1 and OLIG2, which function redundantly to initiate brain tumor formation in a mouse model of GBM. Subsequently, the dynamic levels and reciprocal binding of ASCL1 and OLIG2 to each other and to downstream target genes then determine the cell types and degree of migration of tumor cells. Single-cell RNA sequencing (scRNA-seq) reveals that a high level of ASCL1 is key in defining GSCs by upregulating a collection of ribosomal protein, mitochondrial, neural stem cell (NSC), and cancer metastasis genes, all essential for sustaining the high proliferation, migration, and therapeutic resistance of GSCs.
Uveal melanoma is an ocular tumor with a high risk of developing metastases. The endo‐lysosomal system can affect the melanoma progression by accelerating and facilitating invasion or metastasis. This study aims to conduct comparative analysis of normal choroidal melanocytes and uveal melanoma cells ultrastructure with a focus on intracellular transport system, and to examine the patterns of autophagy‐ and vesicular trafficking‐related proteins expression in a case series of uveal melanomas. Transmission electron microscopy was used to assess the ultrastructure of normal choroidal melanocytes and uveal melanoma cells. The expression levels of autophagy‐ and vesicular trafficking‐related proteins in three histological types of uveal melanoma were analyzed by immunofluorescence staining. Electron microscopy results showed that the autophagic vacuoles were more abundant in normal choroidal melanocytes, than in uveal melanoma cells. The normal choroidal melanocytes were characterized by active intracellular vesicular trafficking; however, the proportion of caveolae was higher in uveal melanoma cells. The spindle type of tumor was characterized by a high expression levels of LC3 beta, while Rab7 and Rab11 proteins expression was significantly up‐regulated in the mixed‐type tumor cells. The results indicate that uveal melanoma cells probably have lower basal levels of autophagy and higher receptor‐mediated endocytic trafficking‐associated with caveolae than normal choroidal melanocytes.
Research Highlights
The autophagic vacuoles are abundant in normal choroidal melanocytes.
Uveal melanoma cells are characterized by a high proportion of caveolae.
The high expression levels of LC3 beta were revealed in a spindle type of tumor, while Rab7 and Rab11 proteins expression was up‐regulated in the mixed‐type tumor cells.
Current cancer studies focus on molecular-targeting diagnostics and interactions with surroundings; however, there are still gaps in characterization based on topological differences and elemental composition. Glioblastoma (GBM cells; GBMCs) is an astrocytic aggressive brain tumor. At the molecular level, GBMCs and astrocytes may differ, and cell elemental/topological analysis is critical for identifying potential new cancer targets. Here, we used U87 MG cells for GBMCS. U87 MG cell lines, which are frequently used in glioblastoma research, are an important tool for studying the various features and underlying mechanisms of this aggressive brain tumor. For the first time, atomic force microscopy (AFM), scanning electron microscopy (SEM) accompanied by energy-dispersive X-ray spectroscopy (EDS), and X-ray photoelectron spectroscopy (XPS) are used to report the topology and chemistry of cancer (U87 MG) and healthy (SVG p12) cells. In addition, F-actin staining and cytoskeleton-based gene expression analyses were performed. The degree of gene expression for genes related to the cytoskeleton was similar; however, the intensity of F-actin, anisotropy values, and invasion-related genes were different. Morphologically, GBMCs were longer and narrower while astrocytes were shorter and more disseminated based on AFM. Furthermore, the roughness values of these cells differed slightly between the two call types. In contrast to the rougher astrocyte surfaces in the lamellipodial area, SEM–EDS analysis showed that elongated GBMCs displayed filopodial protrusions. Our investigation provides considerable further insight into rapid cancer cell characterization in terms of a combinatorial spectroscopic and microscopic approach.
Numerous roles for the Alk receptor tyrosine kinase have been described in Drosophila, including functions in the CNS, however the molecular details are poorly understood. To gain mechanistic insight, we employed Targeted DamID (TaDa) transcriptional profiling to identify targets of Alk signaling in the larval CNS. TaDa was employed in larval CNS tissues, while genetically manipulating Alk signaling output. The resulting TaDa data were analysed together with larval CNS scRNA-seq datasets performed under similar conditions, identifying a role for Alk in the transcriptional regulation of neuroendocrine gene expression. Further integration with bulk/scRNA seq and protein datasets from larval brains in which Alk signaling was manipulated, identified a previously uncharacterized Drosophila neuropeptide precursor encoded by CG4577 as an Alk signaling transcriptional target. CG4577, which we named Sparkly (Spar), is expressed in a subset of Alk-positive neuroendocrine cells in the developing larval CNS, including circadian clock neurons. In agreement with our TaDa analysis, overexpression of the Drosophila Alk ligand Jeb resulted in increased levels of Spar protein in the larval CNS. We show that Spar protein is expressed in circadian (Clock) neurons, and Spar mutant flies exhibit defects in sleep and circadian rhythm control. In summary, we report a novel activity regulating neuropeptide precursor gene that is regulated by Alk signaling in the Drosophila CNS.
The transcriptional machinery is thought to dissociate from DNA during replication. Certain proteins, termed epigenetic marks, must be transferred from parent to daughter DNA strands in order to maintain the memory of transcriptional states1,2. These proteins are believed to re-initiate rebuilding of chromatin structure, which ultimately recruits RNA polymerase II (Pol II) to the newly replicated daughter strands. It is believed that Pol II is recruited back to active genes only after chromatin is rebuilt3,4. However, there is little experimental evidence addressing the central questions of when and how Pol II is recruited back to the daughter strands and resumes transcription. Here we show that immediately after passage of the replication fork, Pol II in complex with other general transcription proteins and immature RNA re-associates with active genes on both leading and lagging strands of nascent DNA, and rapidly resumes transcription. This suggests that the transcriptionally active Pol II complex is retained in close proximity to DNA, with a Pol II–PCNA interaction potentially underlying this retention. These findings indicate that the Pol II machinery may not require epigenetic marks to be recruited to the newly synthesized DNA during the transition from DNA replication to resumption of transcription.
The DNA damage response (DDR), also known as the DNA damage checkpoint (DNADC) acts to prevent cells with damaged DNA from entering mitosis. Kinds of DNA damage that can trigger the checkpoint include single-strand breaks, double-strand breaks, stalled replication forks, and premature chain termination. Many tumours have mutations or deletions of one or more components of the DDR. For this reason, tumours are often repair-deficient, so that DNA-damaging drugs tend to have antitumour selectivity. Inhibitors of components of the DDR, such as PARP, or chk-1, will potentiate the antitumour activity of DNA-damaging drugs, or of radiation. Where a tumour has a defective DDR, inhibitors of the pathway may result in synthetic lethality; e.g. olaparib, a PARP inhibitor, is highly selective against BRCA-deficient tumours. There is cross-talk between the DDR and the SAC, resulting in regulatory coordination. Even if a cell with unrepaired DNA damage enters mitosis, it may be unable to progress to anaphase, in which case it will undergo apoptosis. A model of the DDR can be used to design treatment with specific activity against tumours with particular mutation profiles.
Nanoplastics smaller than 1 µm accumulate as anthropogenic material in the food chain, but only little is known about their uptake and possible effects on potentially strongly exposed cells of the small intestine. The aim of the study was to observe the uptake of 100 nm polystyrene nanoplastics into a non-tumorigenic small intestine cell culture model (IPEC-J2 cells) and to monitor the effects on cell growth and gene regulation, compared to a 100 nm non-plastic silica nanoparticle reference. The intracellular uptake of both types of nanoparticles was proven via (confocal) fluorescence microscopy and complemented with transmission electron microscopy. Fluorescence microscopy showed a growth phase-dependent uptake of nanoparticles into the cells, hence further experiments included different time points related to epithelial closure, determined via electric cell substrate impedance sensing. No retardations in epithelial closure of cells after treatment with polystyrene nanoparticles could be found. In contrast, epithelial cell closure was partly negatively influenced by silica nanoparticles. An increased production of organic nanoparticles, like extracellular vesicles, was not measurable via nanoparticle tracking analysis. An assessment of messenger RNA by next generation sequencing and subsequent pathway analysis revealed that the TP53 pathway was influenced significantly by the polystyrene nanoparticle treatment. In both treatments, dysregulated mRNAs were highly enriched in the NOTCH signaling pathway compared to the non-particle control.
The formation of the mitotic spindle is an essential prerequisite for successful mitosis. The dramatic changes in the level of microtubule (Mt) nucleation at the centrosomes and Mt dynamics that occur in prophase are presumed to be initiated through the activity of cdc2/cyclin B. Here we present data that the cdc25B isoform functions to activate the cytoplasmic pool of cdc2/cyclin B responsible for these events. In contrast to cdc25C, cdc25B is present at low levels in HeLa cells during interphase, but sharply increases in prophase, when cdc25B accumulation in the cytoplasm correlates with prophase spindle formation. Overexpression of wild type and dominant negative mutants of cdc25B and cdc25C shows that prophase Mt nucleation is a consequence of cytoplasmic cdc25B activity, and that cdc25C regulates nuclear G2/M events. Our data also suggest that the functional status of the centrosome can regulate nuclear mitotic events.
When vertebrate cells exit mitosis various cellular structures are re-organized to build functional interphase cells. This depends on Cdk1 (cyclin dependent kinase 1) inactivation and subsequent dephosphorylation of its substrates. Members of the protein phosphatase 1 and 2A (PP1 and PP2A) families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we use a live-cell imaging assay and RNAi knockdown to screen a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identify a trimeric PP2A-B55alpha complex as a key factor in mitotic spindle breakdown and postmitotic reassembly of the nuclear envelope, Golgi apparatus and decondensed chromatin. Using a chemically induced mitotic exit assay, we find that PP2A-B55alpha functions downstream of Cdk1 inactivation. PP2A-B55alpha isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate, histone H1, and was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor importin-beta1, and RNAi depletion of importin-beta1 delayed mitotic exit synergistically with PP2A-B55alpha. This demonstrates that PP2A-B55alpha and importin-beta1 cooperate in the regulation of postmitotic assembly mechanisms in human cells.
Cytokinesis follows separase activation and chromosome segregation. This order is ensured in budding yeast by the mitotic exit network (MEN), where Cdc14p dephosphorylates key conserved Cdk1-substrates exemplified by the anaphase spindle-elongation protein Ase1p. However, in metazoans, MEN and Cdc14 function is not conserved. Instead, the PP2A-B55α/ENSA/Greatwall (BEG) pathway controls the human Ase1p ortholog PRC1. In this pathway, PP2A-B55 inhibition is coupled to Cdk1-cyclin B activity, whereas separase inhibition is maintained by cyclin B concentration. This creates two cyclin B thresholds during mitotic exit. Simulation and experiments using PRC1 as a model substrate show that the first threshold permits separase activation and chromosome segregation, and the second permits PP2A-B55 activation and initiation of cytokinesis. Removal of the ENSA/Greatwall (EG) timer module eliminates this second threshold, as well as associated delay in PRC1 dephosphorylation and initiation of cytokinesis, by uncoupling PP2A-B55 from Cdk1-cyclin B activity. Therefore, temporal order during mitotic exit is promoted by the metazoan BEG pathway.
Significance
Nuclear envelope breakdown (NEB) leads to the exposure of nuclear structures to cytoplasmic activities. Greatwall is a kinase able to inhibit PP2A phosphatases that counteract Cdk-dependent phosphorylation required for mitosis. Here we show that Greatwall, an essential protein in mammals, is exported to the cytoplasm in a Cdk-dependent manner before NEB, thus protecting mitotic phosphates from phosphatase activity.
The spindle assembly checkpoint (SAC) is essential in mammalian mitosis to ensure the equal segregation of sister chromatids. The SAC generates a mitotic checkpoint complex (MCC) to prevent the anaphase-promoting complex/cyclosome (APC/C) from targeting key mitotic regulators for destruction until all of the chromosomes have attached to the mitotic apparatus. A single unattached kinetochore can delay anaphase for several hours, but how it is able to block the APC/C throughout the cell is not understood. Present concepts of the SAC posit that either it exhibits an all-or-nothing response or there is a minimum threshold sufficient to block the APC/C (ref. ). Here, we have used gene targeting to measure SAC activity, and find that it does not have an all-or-nothing response. Instead, the strength of the SAC depends on the amount of MAD2 recruited to kinetochores and on the amount of MCC formed. Furthermore, we show that different drugs activate the SAC to different extents, which may be relevant to their efficacy in chemotherapy.
Mitotic division is induced by protein phosphorylation. For a long time the supported hypothesis was that mitotic entry and exit were the exclusive result of cyclin B-Cdk1 kinase activation and inactivation, whereas the phosphatase activity required to dephosphorylate mitotic substrates was thought to be constant during mitosis. Recent data demonstrate that phosphatase activity must also be tightly regulated to promote correct cell division. Here we describe the new pathway involved in phosphatase regulation and the questions that this discovery raises concerning the classic view of cell cycle regulation.
Polo-like kinase 1 (PLK1) critically regulates mitosis through its dynamic localization to kinetochores, centrosomes and the midzone. The polo-box domain (PBD) and activity of PLK1 mediate its recruitment to mitotic structures, but the mechanisms regulating PLK1 dynamics remain poorly understood. Here, we identify PLK1 as a target of the cullin 3 (CUL3)-based E3 ubiquitin ligase, containing the BTB adaptor KLHL22, which regulates chromosome alignment and PLK1 kinetochore localization but not PLK1 stability. In the absence of KLHL22, PLK1 accumulates on kinetochores, resulting in activation of the spindle assembly checkpoint (SAC). CUL3-KLHL22 ubiquitylates Lys 492, located within the PBD, leading to PLK1 dissociation from kinetochore phosphoreceptors. Expression of a non-ubiquitylatable PLK1-K492R mutant phenocopies inactivation of CUL3-KLHL22. KLHL22 associates with the mitotic spindle and its interaction with PLK1 increases on chromosome bi-orientation. Our data suggest that CUL3-KLHL22-mediated ubiquitylation signals degradation-independent removal of PLK1 from kinetochores and SAC satisfaction, which are required for faithful mitosis.
Historically, the analysis of DNA replication in mammalian tissue culture cells has been limited to static time points, and the use of nucleoside analogues to pulse-label replicating DNA. Here we characterize for the first time a novel Chromobody cell line that specifically labels endogenous PCNA. By combining this with high-resolution confocal time-lapse microscopy, and with a simplified analysis workflow, we were able to produce highly detailed, reproducible, quantitative 4D data on endogenous DNA replication. The increased resolution allowed accurate classification and segregation of S phase into early-, mid-, and late-stages based on the unique subcellular localization of endogenous PCNA. Surprisingly, this localization was slightly but significantly different from previous studies, which utilized over-expressed GFP tagged forms of PCNA. Finally, low dose exposure to Hydroxyurea caused the loss of mid- and late-S phase localization patterns of endogenous PCNA, despite cells eventually completing S phase. Taken together, these results indicate that this simplified method can be used to accurately identify and quantify DNA replication under multiple and various experimental conditions.
Cdk2 promotes DNA replication and is a promising cancer therapeutic target, but its functions appear redundant with Cdk1, an essential Cdk affected by most Cdk2 inhibitors. Here, we present an integrated multidisciplinary approach to address Cdk redundancy. Mathematical modeling of enzymology data predicted conditions allowing selective chemical Cdk2 inhibition. Together with experiments in Xenopus egg extracts, this supports a rate-limiting role for Cdk2 in DNA replication. To confirm this we designed inhibitor-resistant (ir)-Cdk2 mutants using a novel bioinformatics approach. Bypassing inhibition with ir-Cdk2 or with Cdk1 shows that Cdk2 is rate-limiting for replication in this system because Cdk1 is insufficiently active. Additionally, crystal structures and kinetics reveal alternative binding modes of Cdk1-selective and Cdk2-selective inhibitors and mechanisms of Cdk2 inhibitor resistance. Our approach thus provides insight into structure, functions, and biochemistry of a cyclin-dependent kinase.
Polo-like kinase-1 (Plk1) is a highly conserved kinase with multiple mitotic functions. Plk1 localizes to prometaphase kinetochores and is reduced at metaphase kinetochores, similar to many checkpoint signaling proteins, but Plk1 is not required for spindle checkpoint function. Plk1 is also implicated in stabilizing kinetochore-microtubule attachments, but these attachments are most stable when kinetochore Plk1 levels are low at metaphase. Therefore, it is unclear how Plk1 function at kinetochores can be understood in the context of its dynamic localization. In this paper, we show that Plk1 activity suppresses kinetochore-microtubule dynamics to stabilize initial attachments in prometaphase, and Plk1 removal from kinetochores is necessary to maintain dynamic microtubules in metaphase. Constitutively targeting Plk1 to kinetochores maintained high activity at metaphase, leading to reduced interkinetochore tension and intrakinetochore stretch, a checkpoint-dependent mitotic arrest, and accumulation of microtubule attachment errors. Together, our data show that Plk1 dynamics at kinetochores control two critical mitotic processes: initially establishing correct kinetochore-microtubule attachments and subsequently silencing the spindle checkpoint.
Most of the current drugs used to treat cancer can be classified as anti-proliferative drugs. These drugs perturb the proliferative cycle of tumor cells at diverse stages of the cell cycle. Examples of such drugs are DNA-damaging agents and inhibitors of cyclin-dependent kinases that arrest cell cycle progression at different stages of interphase. Another class of anti-proliferative drugs is the so-called anti-mitotic drugs, which selectively perturb progression through mitosis. Mitosis is the shortest and final stage in the cell cycle and has evolved to accurately divide the duplicated genome over the two daughter cells. This review deals with the different strategies that are currently considered to perturb mitotic progression in the treatment of cancer.
Mitosis requires precise coordination of multiple global reorganizations of the nucleus and cytoplasm. Cyclin-dependent kinase 1 (Cdk1) is the primary upstream kinase that directs mitotic progression by phosphorylation of a large number of substrate proteins. Cdk1 activation reaches the peak level due to positive feedback mechanisms. By inhibiting Cdk chemically, we showed that, in prometaphase, when Cdk1 substrates approach the peak of their phosphorylation, cells become capable of proper M-to-G1 transition. We interfered with the molecular components of the Cdk1-activating feedback system through use of chemical inhibitors of Wee1 and Myt1 kinases and Cdc25 phosphatases. Inhibition of Wee1 and Myt1 at the end of the S phase led to rapid Cdk1 activation and morphologically normal mitotic entry, even in the absence of G2. Dampening Cdc25 phosphatases simultaneously with Wee1 and Myt1 inhibition prevented Cdk1/cyclin B kinase activation and full substrate phosphorylation and induced a mitotic "collapse," a terminal state characterized by the dephosphorylation of mitotic substrates without cyclin B proteolysis. This was blocked by the PP1/PP2A phosphatase inhibitor, okadaic acid. These findings suggest that the positive feedback in Cdk activation serves to overcome the activity of Cdk-opposing phosphatases and thus sustains forward progression in mitosis.
Entry into mitosis in eukaryotes requires the activity of cyclin-dependent kinase 1 (Cdk1). Cdk1 is opposed by protein phosphatases
in two ways: They inhibit activation of Cdk1 by dephosphorylating the protein kinases Wee1 and Myt1 and the protein phosphatase
Cdc25 (key regulators of Cdk1), and they also antagonize Cdk1’s own phosphorylation of downstream targets. A particular form
of protein phosphatase 2A (PP2A) containing a B55δ subunit (PP2A- B55δ) is the major protein phosphatase that acts on model
CDK substrates in Xenopus egg extracts and has antimitotic activity. The activity of PP2A-B55δ is high in interphase and low in mitosis, exactly opposite
that of Cdk1. We report that inhibition of PP2A-B55δ results from a small protein, known as α-endosulfine (Ensa), that is
phosphorylated in mitosis by the protein kinase Greatwall (Gwl). This converts Ensa into a potent and specific inhibitor of
PP2A-B55δ. This pathway represents a previously unknown element in the control of mitosis.
Greatwall (Gwl) was originally discovered in Drosophila as an essential kinase for correct chromosome condensation and mitotic progression. In Xenopus, Gwl may influence the positive-feedback loop that directs cyclin B1-Cdk1 activation and the mitotic state by inhibiting the phosphatase PP 2A. Here, we describe the human orthologue of Gwl called microtubule-associated serine/threonine kinase-like (MASTL). We found that MASTL localizes to the nucleus in interphase and re-localizes in part to centrosomes in mitosis, when it is active. Cells strongly depleted of MASTL by RNAi delay in G(2) phase and reveal slow chromosome condensation. MASTL RNAi cells that enter and progress through mitosis often fail to completely separate their sister chromatids in anaphase. This causes chromatin to be trapped in the cleavage furrow, which may lead to the formation of 4N G(1) cells by cytokinesis failure. Further, our experiments indicate that MASTL supports the phosphorylation state of mitotic phospho-proteins downstream of cyclin B1-Cdk1, including the APC/C. Cyclin B1 destruction is incomplete when mitotic cells that are strongly depleted of MASTL exit mitosis. We propose that MASTL enhances cyclin B1-Cdk1-dependent mitotic phosphorylation events, directing mitotic entry, anaphase and cytokinesis in human cells.
Successful completion of cytokinesis requires the spatio-temporal regulation of protein phosphorylation and the coordinated activity of protein kinases and phosphatases. Many mitotic protein kinases are well characterized while mitotic phosphatases are largely unknown. Here, we show that the Ca(2+)- and calmodulin-dependent phosphatase, calcineurin (CaN), is required for cytokinesis in mammalian cells, functioning specifically at the abscission stage. CaN inhibitors induce multinucleation in HeLa cells and prolong the time cells spend connected via an extended intracellular bridge. Upon Ca(2+) influx during cytokinesis, CaN is activated, targeting a set of proteins for dephosphorylation, including dynamin II (dynII). At the intracellular bridge, phospho-dynII and CaN are co-localized to dual flanking midbody rings (FMRs) that reside on either side of the central midbody ring. CaN activity and disassembly of the FMRs coincide with abscission. Thus, CaN activity at the midbody plays a key role in regulating the completion of cytokinesis in mammalian cells.
The cyclin B-Cdk1 kinase triggers mitosis in most eukaryotes. In animal cells, cyclin B shuttles between the nucleus and cytoplasm in interphase before rapidly accumulating in the nucleus at prophase, which promotes disassembly of the nuclear lamina and nuclear envelope breakdown (NEBD). What triggers the nuclear accumulation of cyclin B1 is presently unclear, although the prevailing view is that the Plk1 kinase inhibits its nuclear export. In this study, we use a biosensor specific for cyclin B1-Cdk1 activity to show that activating cyclin B1-Cdk1 immediately triggers its rapid accumulation in the nucleus through a 40-fold increase in nuclear import that remains dependent on Cdk1 activity until NEBD. Nevertheless, a substantial proportion of cyclin B1-Cdk1 remains in the cytoplasm. The increase in nuclear import is driven by changes in the nuclear import machinery that require neither Plk1 nor inhibition of nuclear export. Thus, the intrinsic link between cyclin B1-Cdk1 activation and its rapid nuclear import inherently coordinates the reorganization of the nucleus and the cytoplasm at mitotic entry.
We have previously shown that Greatwall kinase (Gwl) is required for M phase entry and maintenance in Xenopus egg extracts. Here, we demonstrate that Gwl plays a crucial role in a novel biochemical pathway that inactivates, specifically during M phase, "antimitotic" phosphatases directed against phosphorylations catalyzed by cyclin-dependent kinases (CDKs). A major component of this phosphatase activity is heterotrimeric PP2A containing the B55delta regulatory subunit. Gwl is activated during M phase by Cdk1/cyclin B (MPF), but once activated, Gwl promotes PP2A/B55delta inhibition with no further requirement for MPF. In the absence of Gwl, PP2A/B55delta remains active even when MPF levels are high. The removal of PP2A/B55delta corrects the inability of Gwl-depleted extracts to enter M phase. These findings support the hypothesis that M phase requires not only high levels of MPF function, but also the suppression, through a Gwl-dependent mechanism, of phosphatase(s) that would otherwise remove MPF-driven phosphorylations.
To explore the mechanisms and evolution of cell-cycle control, we analyzed the position and conservation of large numbers
of phosphorylation sites for the cyclin-dependent kinase Cdk1 in the budding yeast Saccharomyces cerevisiae. We combined specific chemical inhibition of Cdk1 with quantitative mass spectrometry to identify the positions of 547 phosphorylation
sites on 308 Cdk1 substrates in vivo. Comparisons of these substrates with orthologs throughout the ascomycete lineage revealed
that the position of most phosphorylation sites is not conserved in evolution; instead, clusters of sites shift position in
rapidly evolving disordered regions. We propose that the regulation of protein function by phosphorylation often depends on
simple nonspecific mechanisms that disrupt or enhance protein-protein interactions. The gain or loss of phosphorylation sites
in rapidly evolving regions could facilitate the evolution of kinase-signaling circuits.
To determine why the duration of mitosis (DM) is less in Taxol than in nocodazole or Eg5 inhibitors we studied the relationship between Taxol concentration, the DM, and the mitotic checkpoint. We found that unlike for other spindle poisons, in Taxol the DM becomes progressively shorter as the concentration surpasses approximately 0.5 microM. Studies on RPE1 and PtK2 expressing GFP/cyclin B or YFP/Mad2 revealed that cells ultimately satisfy the checkpoint in Taxol and do so faster at concentrations >0.5 microM. Inhibiting the aurora-B kinase in Taxol-treated RPE1 cells accelerates checkpoint satisfaction by stabilizing syntelic kinetochore attachments and reduces the DM to approximately 1.5 h regardless of drug concentration. A similar stabilization of syntelic attachments by Taxol itself appears responsible for accelerated checkpoint satisfaction at concentrations >0.5 microM. Our results provide a novel conceptual framework for how Taxol prolongs mitosis and caution against using it in checkpoint studies. They also offer an explanation for why some cells are more sensitive to lower versus higher Taxol concentrations.
Entry into mitosis depends on the activity of cyclin-dependent kinases (CDKs). Conversely, exit from mitosis occurs when mitotic cyclins are degraded, thereby extinguishing CDK activity. Exit from mitosis must also require mitotic phosphoproteins to revert to their interphase hypophosphorylated forms, but there is a controversy about which phosphatase(s) is/are responsible for dephosphorylating the CDK substrates. We find that PP2A associated with a B55 delta subunit is relatively specific for a model mitotic CDK substrate in Xenopus egg extracts. The phosphatase activity measured by this substrate is regulated during the cell cycle--high in interphase and suppressed during mitosis. Depletion of PP2A-B55 delta (in interphase) from 'cycling' frog egg extracts accelerated their entry into mitosis and kept them indefinitely in mitosis. When PP2A-B55 delta was depleted from mitotic extracts, however, exit from mitosis was hardly delayed, showing that other phosphatase(s) are also required for mitotic exit. Increasing the concentration of PP2A-B55 delta in extracts by adding recombinant enzyme inhibited the entry into mitosis. This form of PP2A seems to be a key regulator of entry into and exit from mitosis.
After a decade of extensive work on gene knockout mouse models of cell-cycle regulators, the classical model of cell-cycle regulation was seriously challenged. Several unexpected compensatory mechanisms were uncovered among cyclins and Cdks in these studies. The most astonishing observation is that Cdk2 is dispensable for the regulation of the mitotic cell cycle with both Cdk4 and Cdk1 covering for Cdk2's functions. Similar to yeast, it was recently discovered that Cdk1 alone can drive the mammalian cell cycle, indicating that the regulation of the mammalian cell cycle is highly conserved. Nevertheless, cell-cycle-independent functions of Cdks and cyclins such as in DNA damage repair are still under investigation. Here we review the compensatory mechanisms among major cyclins and Cdks in mammalian cell-cycle regulation.
The decision to enter mitosis is mediated by a network of proteins that regulate activation of the cyclin B-Cdk1 complex. Within this network, several positive feedback loops can amplify cyclin B-Cdk1 activation to ensure complete commitment to a mitotic state once the decision to enter mitosis has been made. However, evidence is accumulating that several components of the feedback loops are redundant for cyclin B-Cdk1 activation during normal cell division. Nonetheless, defined feedback loops become essential to promote mitotic entry when normal cell cycle progression is perturbed. Recent data has demonstrated that at least three Plk1-dependent feedback loops exist that enhance cyclin B-Cdk1 activation at different levels. In this review, we discuss the role of various feedback loops that regulate cyclin B-Cdk1 activation under different conditions, the timing of their activation, and the possible identity of the elusive trigger that controls mitotic entry in human cells.
The formation of the mitotic spindle is an essential prerequisite for successful mitosis. The dramatic changes in the level of microtubule (Mt) nucleation at the centrosomes and Mt dynamics that occur in prophase are presumed to be initiated through the activity of cdc2/cyclin B. Here we present data that the cdc25B isoform functions to activate the cytoplasmic pool of cdc2/cyclin B responsible for these events. In contrast to cdc25C, cdc25B is present at low levels in HeLa cells during interphase, but sharply increases in prophase, when cdc25B accumulation in the cytoplasm correlates with prophase spindle formation. Overexpression of wild type and dominant negative mutants of cdc25B and cdc25C shows that prophase Mt nucleation is a consequence of cytoplasmic cdc25B activity, and that cdc25C regulates nuclear G2/M events. Our data also suggest that the functional status of the centrosome can regulate nuclear mitotic events.
Spinning disc confocal microscopy of LLCPK1 cells expressing GFP-tubulin was used to demonstrate that microtubules (MTs) rapidly elongate to the cell cortex after anaphase onset. Concurrently, individual MTs are released from the centrosome and the centrosome fragments into clusters of MTs. Using cells expressing photoactivatable GFP-tubulin to mark centrosomal MT minus ends, a sevenfold increase in MT release in anaphase is documented as compared with metaphase. Transport of both individually released MTs and clusters of MTs is directionally biased: motion is directed away from the equatorial region. Clusters of MTs retain centrosomal components at their focus and the capacity to nucleate MTs. Injection of mRNA encoding nondegradable cyclin B blocked centrosome fragmentation and the stimulation of MT release in anaphase despite allowing anaphase-like chromosome segregation. Biased MT release may provide a mechanism for MT-dependent positioning of components necessary for specifying the site of contractile ring formation.
We have examined the role of PRC1, a midzone-associated, microtubule bundling, Cdk substrate protein, in regulating the spatiotemporal formation of the midzone in HeLa cells. Cdk-mediated phosphorylation of PRC1 in early mitosis holds PRC1 in an inactive monomeric state. During the metaphase-to-anaphase transition, PRC1 is dephosphorylated, promoting PRC1 oligomerization. Using time-lapse video microscopy, RNA interference, 3D immunofluorescence reconstruction imaging, and rescue experiments, we demonstrate that the dephosphorylated form of PRC1 is essential for bundling antiparallel, nonkinetochore, interdigitating microtubules to establish the midzone that is necessary for cytokinesis. Our results thus indicate that PRC1 is an essential factor in controlling the spatiotemporal formation of the midzone in human cells.
• Cdk phosphorylation
• mitosis/cytokinesis
• microtubule-associated proteins
• microtubule bundling
A guiding hypothesis for cell-cycle regulation asserts that regulated proteolysis constrains the directionality of certain cell-cycle transitions. Here we test this hypothesis for mitotic exit, which is regulated by degradation of the cyclin-dependent kinase 1 (Cdk1) activator, cyclin B. Application of chemical Cdk1 inhibitors to cells in mitosis induces cytokinesis and other normal aspects of mitotic exit, including cyclin B degradation. However, chromatid segregation fails, resulting in entrapment of chromatin in the midbody. If cyclin B degradation is blocked with a proteasome inhibitor or by expression of non-degradable cyclin B, Cdk inhibitors will nonetheless induce mitotic exit and cytokinesis. However, if after mitotic exit, the Cdk1 inhibitor is washed free from cells in which cyclin B degradation is blocked, the cells can revert back to M phase. This reversal is characterized by chromosome recondensation, nuclear envelope breakdown, assembly of microtubules into a mitotic spindle, and in most cases, dissolution of the midbody, reopening of the cleavage furrow, and realignment of chromosomes at the metaphase plate. These findings demonstrate that proteasome-dependent degradation of cyclin B provides directionality for the M phase to G1 transition.
CDK1 is a nonredundant cyclin-dependent kinase (CDK) with an essential role in mitosis, but its multiple functions still are poorly understood at a molecular level. Here we identify a selective small-molecule inhibitor of CDK1 that reversibly arrests human cells at the G2/M border of the cell cycle and allows for effective cell synchronization in early mitosis. Inhibition of CDK1 during cell division revealed that its activity is necessary and sufficient for maintaining the mitotic state of the cells, preventing replication origin licensing and premature cytokinesis. Although CDK1 inhibition for up to 24 h is well tolerated, longer exposure to the inhibitor induces apoptosis in tumor cells, suggesting that selective CDK1 inhibitors may have utility in cancer therapy.
• apoptosis
• cancer therapy
• cell cycle
• cytokinesis
• replication origin
Spatial and temporal coordination of polo-like kinase 1 (Plk1) activity is necessary for mitosis and cytokinesis, and this is achieved through binding to phosphorylated docking proteins with distinct subcellular localizations. Although cyclin-dependent kinase 1 (Cdk1) creates these phosphorylated docking sites in metaphase, a general principle that explains how Plk1 activity is controlled in anaphase after Cdk1 inactivation is lacking. Here, we show that the microtubule-associated protein regulating cytokinesis (PRC1) is an anaphase-specific binding partner for Plk1, and that this interaction is required for cytokinesis. In anaphase, Plk1 creates its own docking site on PRC1, whereas in metaphase Cdk1 phosphorylates PRC1 adjacent to this docking site and thereby prevents binding of Plk1. Mutation of these Cdk1-sites results in a form of PRC1 that prematurely recruits Plk1 to the spindle during prometaphase and blocks mitotic progression. The activation state of Cdk1, therefore, controls the switch of Plk1 localization from centrosomes and kinetochores during metaphase, to the central spindle during anaphase.
The formation of the mitotic spindle is an essential prerequisite for successful mitosis. This process is initiated at prophase when changes in microtubule dynamics and an increase in the number of microtubules nucleated from the centrosome, results in the conversion of the centrosome from an interphase to a mitotic microtubule organising centre (MTOC). This dramatic change in microtubule nucleation and dynamics is presumed to be initiated through the activity of cdc2/cyclin complexes. We have shown that the cdc25B isoform functions to activate the cytoplasmic pool of cdc2/cyclin complexes responsible for these events [1]. In contrast to cdc25C, cdc25B is present at low levels in HeLa cells during interphase, but sharply increases in prophase, when cdc25B accumulation in the cytoplasm correlates with spindle formation. The overexpression of catalytically inactive cdc25B in HeLa cells resulted in an accumulation of cells in G2/M, which appeared to be due to a dominant negative effect on prophase microtubule nucleation. HeLa cells overexpressing wild type cdc25B often displayed striking "mini-spindles" which were likely to have formed as a consequence of the inappropriate activation of cdc2/cyclin complexes. In contrast, overexpression of mutant or wild type cdc25C did not influence prophase microtubule nucleation. The data also suggests that the status of the centrosome can regulate nuclear mitotic events.
Polyploid giant cancer cells (PGCCs) have been observed by pathologists for over a century. PGCCs contribute to solid tumor heterogeneity, but their functions are largely undefined. Little attention has been given to these cells, largely because PGCCs have been generally thought to originate from repeated failure of mitosis/cytokinesis and have no capacity for long-term survival or proliferation. Here we report our successful purification and culture of PGCCs from human ovarian cancer cell lines and primary ovarian cancer. These cells are highly resistant to oxygen deprivation and could form through endoreduplication or cell fusion, generating regular-sized cancer cells quickly through budding or bursting similar to simple organisms like fungi. They express normal and cancer stem cell markers, they divide asymmetrically and they cycle slowly. They can differentiate into adipose, cartilage and bone. A single PGCC formed cancer spheroids in vitro and generated tumors in immunodeficient mice. These PGCC-derived tumors gained a mesenchymal phenotype with increased expression of cancer stem cell markers CD44 and CD133 and become more resistant to treatment with cisplatin. Taken together, our results reveal that PGCCs represent a resistant form of human cancer using an ancient, evolutionarily conserved mechanism in response to hypoxia stress; they can contribute to the generation of cancer stem-like cells, and also play a fundamental role in regulating tumor heterogeneity, tumor growth and chemoresistance in human cancer.Oncogene advance online publication, 25 March 2013; doi:10.1038/onc.2013.96.
During mitosis and meiosis, the spindle assembly checkpoint acts to maintain genome stability by delaying cell division until accurate chromosome segregation can be guaranteed. Accuracy requires that chromosomes become correctly attached to the microtubule spindle apparatus via their kinetochores. When not correctly attached to the spindle, kinetochores activate the spindle assembly checkpoint network, which in turn blocks cell cycle progression. Once all kinetochores become stably attached to the spindle, the checkpoint is inactivated, which alleviates the cell cycle block and thus allows chromosome segregation and cell division to proceed. Here we review recent progress in our understanding of how the checkpoint signal is generated, how it blocks cell cycle progression and how it is extinguished.
Therapy-induced cellular senescence (TCS), characterized by prolonged cell cycle arrest, is an in vivo response of human cancers to chemotherapy and radiation. Unfortunately, TCS is reversible for a subset of senescent cells, leading to cellular re-proliferation and ultimately tumor progression. This invariable consequence of TCS recapitulates the clinical treatment experience of patients with advanced cancer. We report the findings of a clinicopathological study in patients with locally advanced non-small cell lung cancer demonstrating that marker of in vivo TCS following neoadjuvant therapy prognosticate adverse clinical outcome. In our efforts to elucidate key molecular pathways underlying TCS and cell cycle escape, we have previously shown that the deregulation of mitotic kinase Cdk1 and its downstream effectors are important mediators of survival and cell cycle reentry. We now report that aberrant expression of Cdk1 interferes with apoptosis and promotes the formation of polyploid scenescent cells during TCS. These polyploid senescent cells represent important transition states through which escape preferentially occurs. The Cdk1 pathway is in part modulated differentially by p21 and p27 two members of the KIP cyclin-dependent kinase inhibitor family during TCS. Altogether, these studies underscore the importance of TCS in cancer therapeutics. © 2012 Wiley Periodicals, Inc.
The cell cycle is a tightly regulated series of events that governs cell replication and division. Deregulation of cell cycle kinases, e.g., cyclin-dependent kinases (CDKs), can initiate a hyper-proliferative cell phenotype and cause genomic instability, thus facilitating malignant transformation. Pharmacological agents targeting CDKs have been developed as potential anti-cancer agents for over 20 years, evolving from early pan-CDK inhibitors to second-generation inhibitors with much greater specificity and selectivity. Despite these advances in drug design and highly successful preclinical investigations, CDK inhibitors have yet to achieve their expected efficacy in clinical trials. In addition, inhibitors of other cell cycle kinases are currently progressing through clinical trials. Recent biochemical and genetic studies might be used to improve the effectiveness of cell cycle kinase inhibitors as anti-cancer agents through better drug design, therapeutic combinations, and patient selection.
In animal cells, formation of the cytokinetic furrow requires activation of the GTPase RhoA by the guanine nucleotide exchange factor Ect2. How Ect2, which is associated with the spindle midzone, controls RhoA activity at the equatorial cortex during anaphase is not understood. Here, we show that Ect2 concentrates at the equatorial membrane during cytokinesis in live cells. Ect2 membrane association requires a pleckstrin homology domain and a polybasic cluster that bind to phosphoinositide lipids. Both guanine nucleotide exchange function and membrane targeting of Ect2 are essential for RhoA activation and cleavage furrow formation in human cells. Membrane localization of Ect2 is spatially confined to the equator by centralspindlin, Ect2's spindle midzone anchor complex, and is temporally coordinated with chromosome segregation through the activation state of CDK1. We propose that targeting of Ect2 to the equatorial membrane represents a key step in the delivery of the cytokinetic signal to the cortex.
The spindle assembly checkpoint (SAC) restricts mitotic exit to cells that have completed chromosome-microtubule attachment. Cdc20 is a bifunctional protein. In complex with SAC proteins Mad2, BubR1, and Bub3, Cdc20 forms the mitotic checkpoint complex (MCC), which binds the anaphase-promoting complex (APC/C) and inhibits its mitotic exit-promoting activity. When devoid of SAC proteins, Cdc20 serves as an APC/C coactivator and promotes mitotic exit. During mitotic arrest, Cdc20 is continuously degraded via ubiquitin-dependent proteolysis and resynthesized. It is believed that this cycle keeps the levels of Cdc20 below a threshold above which Cdc20 would promote mitotic exit. We report that p31(comet), a checkpoint antagonist, is necessary for mitotic destabilization of Cdc20. p31(comet) depletion stabilizes the MCC, super-inhibits the APC/C, and delays mitotic exit, indicating that Cdc20 proteolysis in prometaphase opposes the checkpoint. Our studies reveal a homeostatic network in which checkpoint-sustaining and -repressing forces oppose each other during mitotic arrest and suggest ways for enhancing the sensitivity of cancer cells to antitubulin chemotherapeutics.
Accurate chromosome segregation relies on activity of the spindle assembly checkpoint, a surveillance mechanism that prevents premature anaphase onset until all chromosomes are properly attached to the mitotic spindle apparatus and aligned at the metaphase plate. Defects in this mechanism contribute to chromosome instability and aneuploidy, a hallmark of malignant cells. Here, we review the molecular mechanisms of activation and silencing of the spindle assembly checkpoint and its relationship to tumourigenesis.
Drugs targeting the mitotic spindle are used extensively during chemotherapy, but surprisingly, little is known about how they kill tumor cells. This is largely because many of the population-based approaches are indirect and lead to vague and confusing interpretations. Here, we use a high-throughput automated time-lapse light microscopy approach to systematically analyze over 10,000 single cells from 15 cell lines in response to three different classes of antimitotic drug. We show that the variation in cell behavior is far greater than previously recognized, with cells within any given line exhibiting multiple fates. We present data supporting a model wherein cell fate is dictated by two competing networks, one involving caspase activation, the other protecting cyclin B1 from degradation.
One does not often look to analytic cubism for insights into the control of the cell cycle, but Pablo Picasso beautifully encapsulated the fundamentals when he said that "every act of creation is, first of all, an act of destruction". The rapid destruction of specific cell cycle regulators at just the right moment in the cell cycle ensures that daughter cells receive an equal and identical set of chromosomes from their mother and that DNA replication always follows mitosis. Remarkably, one protein complex is responsible for this surgical precision, the APC/C (anaphase-promoting complex, also known as the cyclosome). The APC/C is tightly regulated by its co-activators and by the spindle assembly checkpoint.
Pericentriolar satellites are electron-dense granules that are concentrated around the centrosome. They are involved in the recruitment of centrosomal proteins and microtubule organization in interphase cells, but their mitotic functions are largely unknown. In this study, we characterize CEP90 as a component of pericentriolar satellites. CEP90 is present both in the centrosome and in the cytoplasm, but is transiently concentrated at the centrosome once cells enter mitosis. Depletion of CEP90 caused mitotic arrest with misaligned chromosomes. Spindle pole fragmentation was the most characteristic phenotype in CEP90-depleted cells. Spindle poles were fragmented as soon as the spindles attached, suggesting that the mechanical forces of spindle microtubules physically stress the structure of CEP90-depleted spindle poles. Based on these results, we propose that CEP90 is crucial for maintaining the integrity of spindle poles during mitosis.
Control of eukaryotic cell proliferation involves an extended regulatory network, the complexity of which has made it difficult to understand the basic principles of the cell cycle. To investigate the core engine of the mitotic cycle we have generated a minimal control network in fission yeast that efficiently sustains cellular reproduction. Here we demonstrate that orderly progression through the major events of the cell cycle can be driven by oscillation of an engineered monomolecular cyclin-dependent protein kinase (CDK) module lacking much of the canonical regulation. We show further that the CDK oscillator acts as the primary organizer of the cell cycle, imposing timing and directionality to a system of two CDK activity thresholds that define independent cell cycle phases. We propose that this simple core architecture forms the basic control of the eukaryotic cell cycle.
Initiation and maintenance of mitosis require the activation of protein kinase cyclin B–Cdc2 and the inhibition of protein
phosphatase 2A (PP2A), which, respectively, phosphorylate and dephosphorylate mitotic substrates. The protein kinase Greatwall
(Gwl) is required to maintain mitosis through PP2A inhibition. We describe how Gwl activation results in PP2A inhibition.
We identified cyclic adenosine monophosphate–regulated phosphoprotein 19 (Arpp19) and α-Endosulfine as two substrates of Gwl
that, when phosphorylated by this kinase, associate with and inhibit PP2A, thus promoting mitotic entry. Conversely, in the
absence of Gwl activity, Arpp19 and α-Endosulfine are dephosphorylated and lose their capacity to bind and inhibit PP2A. Although
both proteins can inhibit PP2A, endogenous Arpp19, but not α-Endosulfine, is responsible for PP2A inhibition at mitotic entry
in Xenopus egg extracts.
Here we show that the functional human ortholog of Greatwall protein kinase (Gwl) is the microtubule-associated serine/threonine kinase-like protein, MAST-L. This kinase promotes mitotic entry and maintenance in human cells by inhibiting protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates cyclin B-Cdc2 substrates. The complete depletion of Gwl by siRNA arrests human cells in G2. When the levels of this kinase are only partially depleted, however, cells enter into mitosis with multiple defects and fail to inactivate the spindle assembly checkpoint (SAC). The ability of cells to remain arrested in mitosis by the SAC appears to be directly proportional to the amount of Gwl remaining. Thus, when Gwl is only slightly reduced, cells arrest at prometaphase. More complete depletion correlates with the premature dephosphorylation of cyclin B-Cdc2 substrates, inactivation of the SAC, and subsequent exit from mitosis with severe cytokinesis defects. These phenotypes appear to be mediated by PP2A, as they could be rescued by either a double Gwl/PP2A knockdown or by the inhibition of this phosphatase with okadaic acid. These results suggest that the balance between cyclin B-Cdc2 and PP2A must be tightly regulated for correct mitotic entry and exit and that Gwl is crucial for mediating this regulation in somatic human cells.
The CyclinB1-Cdk1 kinase is the catalytic activity at the heart of mitosis-promoting factor (MPF), yet fundamental questions concerning its role in mitosis remained unresolved. It is not known when and how rapidly CyclinB1-Cdk1 is activated in mammalian cells, nor how its activation coordinates the substantial changes in the cell at mitosis. Here, we have developed a FRET biosensor specific for CyclinB1-Cdk1 that enables us to assay its activity with very high temporal precision in living human cells. We show that CyclinB1-Cdk1 is inactive in G2 phase and activated at a set time before nuclear envelope breakdown, thereby initiating the events of prophase. CyclinB1-Cdk1 levels rise to their maximum extent over the course of approximately 30 min, and we demonstrate that different levels of CyclinB1-Cdk1 kinase activity trigger different mitotic events, thus revealing how the remarkable reorganization of the cell is coordinated at mitotic entry.
Greatwall (GW) is a new kinase that has an important function in the activation and the maintenance of cyclin B-Cdc2 activity. Although the mechanism by which it induces this effect is unknown, it has been suggested that GW could maintain cyclin B-Cdc2 activity by regulating its activation loop. Using Xenopus egg extracts, we show that GW depletion promotes mitotic exit, even in the presence of a high cyclin B-Cdc2 activity by inducing dephosphorylation of mitotic substrates. These results indicate that GW does not maintain the mitotic state by regulating the cyclin B-Cdc2 activation loop but by regulating a phosphatase. This phosphatase is PP2A; we show that (1) PP2A binds GW, (2) the inhibition or the specific depletion of this phosphatase from mitotic extracts rescues the phenotype induced by GW inactivation and (3) the PP2A-dependent dephosphorylation of cyclin B-Cdc2 substrates is increased in GW-depleted Xenopus egg extracts. These results suggest that mitotic entry and maintenance is not only mediated by the activation of cyclin B-Cdc2 but also by the regulation of PP2A by GW.
Cell death induced by agents that disrupt microtubules can kill cells by inducing a prolonged mitotic block. This mitotic block is dependent on the spindle assembly checkpoint, a surveillance system that ensures the bipolar attachment of chromosomes to the mitotic spindle before the onset of anaphase. Under some conditions, the spindle assembly checkpoint can become weakened, allowing cells to exit mitosis despite the presence of chromosomes that are not properly attached to the mitotic spindle. Here, we use an Aurora kinase inhibitor to drive mitotic exit and test the effect of mitotic arrest length on death in the subsequent interphase. Cells that are blocked in mitosis for >15 h die shortly after exiting from mitosis, whereas cells that exit after being blocked for <15 h show variable fates, with some living for days after exiting mitosis. Cells blocked in mitosis by either Taxol or epothilone B are acutely sensitive to the death ligand tumor necrosis factor-related apoptosis-inducing ligand, suggesting that prolonged mitosis allows the gradual accumulation of internal death signals, rendering cells hypersensitive to additional prodeath cues. Death under these conditions is initiated while cyclin B1 is still present, indicating that cells are in mitosis. Our experiments suggest that there is a point of no return during prolonged mitotic block after which mitotic exit can no longer block death.
The connections between cancer and the basic machinery of the cell cycle have taken a surprisingly long time to become apparent. However, the past 2 years has seen a dramatic increase in the number of cell cycles regulators that have been implicated as either protooncogenes or as tumour suppressor genes. In this review I will attempt to show how perturbations in the known cell cycle regulators may play a part in the process of oncogenesis.
Uncontrolled cell proliferation is the hallmark of cancer, and tumor cells have typically acquired damage to genes that directly
regulate their cell cycles. Genetic alterations affecting p16INK4a and cyclin D1, proteins that govern phosphorylation of the retinoblastoma protein (RB) and control exit from the G1 phase of the cell cycle, are so frequent in human cancers that inactivation of this pathway may well be necessary for tumor
development. Like the tumor suppressor protein p53, components of this “RB pathway,” although not essential for the cell cycle
per se, may participate in checkpoint functions that regulate homeostatic tissue renewal throughout life.
The mitotic spindle assembly checkpoint delays anaphase until all chromosomes achieve bipolar attachment to the spindle microtubules. The spindle assembly checkpoint protein BubR1 is thought to act by forming an inhibitory complex with Cdc20. We here identify two Cdc20 binding sites on BubR1. A strong Cdc20 binding site is located between residues 490 and 560, but mutations that disrupt Cdc20 binding to this region have no effect upon checkpoint function. A second Cdc20 binding site present between residues 1 and 477 is highly specific for Cdc20 already bound to Mad2. Mutation of a conserved lysine in this region weakened Cdc20 binding and correspondingly reduced checkpoint function. Our results indicate that there may be more than one checkpoint complex containing BubR1, Mad2, and Cdc20. They also lead us to propose that in vivo checkpoint inhibition of Cdc20 is a two-step process in which prior binding of Mad2 to Cdc20 is required to make Cdc20 sensitive to inhibition by BubR1. Thus, Mad2 and BubR1 must cooperate to inhibit Cdc20 activity.
Chemical agents for cell cycle synchronization have greatly facilitated the study of biochemical events driving cell cycle progression. G1, S and M phase inhibitors have been developed and used widely in cell cycle research. However, currently there are no effective G2 phase inhibitors and synchronization of cultured cells in G2 phase has been challenging. Recently, a selective CDK1 inhibitor, RO-3306, has been identified that reversibly arrests proliferating human cells at the G2/M phase border and provides a novel means for cell cycle synchronization. A single-step protocol using RO-3306 permits the synchronization of >95% of cycling cancer cells in G2 phase. RO-3306 arrested cells enter mitosis rapidly after release from the G2 block thus allowing for isolation of mitotic cells without microtubule poisons. RO-3306 represents a new molecular tool for studying CDK1 function in human cells.
Natural product extracts have proven to be a rich source of small molecules that potently inhibit the catalytic activity of certain PPP-family ser/thr protein phosphatases. To date, the list of inhibitors includes okadaic acid (produced by marine dinoflagelates, Prorocentrum sp. and Dinophysis sp.), calyculin A, dragmacidins (isolated from marine sponges), microcystins, nodularins (cyanobacteria, Microcystis sp. and Nodularia sp.), tautomycin, tautomycetin, cytostatins, phospholine, leustroducsins, phoslactomycins, fostriecin (soil bacteria, Streptomyces sp.), and cantharidin (blister beetles, approx 1500 species). Many of these compounds share structural similarities, and several have become readily available for research purposes. Here we will review the specificity of available inhibitors and present methods for their use in studying sensitive phosphatases. Common mistakes in the employment of these compounds will also be addressed briefly, notably the widespread misconception that they only inhibit the activity of PP1 and PP2A. Inhibitors of PP2B (calcineurin) will only be mentioned in passing, except to state that, in our hands, cypermethrin, deltamethrin, and fenvalerate, which are sold as potent inhibitors of PP2B, do not inhibit the catalytic activity of PP2B.