PRC1 and EB1 Binding Dynamics Reveal a Solidifying Central Spindle during Anaphase Compaction in Human Cells

ABSTRACTDuring mitosis the spindle undergoes considerable morphological and dynamic changes. Particularly the central spindle reorganizes drastically at the onset of anaphase when the antiparallel microtubule bundler PRC1 starts to accumulate and recruit a subset of spindle proteins to the midzone. Little is known about how the dynamic properties of the central spindle change during its morphological changes in human cells. Using CRISPR/Cas9 gene editing, we generated human RPE1 cells that express from their endogenous locus fluorescently tagged versions of the two cytoskeletal network hub proteins PRC1 and the end binding protein EB1 to be able to quantify their spindle distribution and binding/unbinding turnover under native conditions. We find that throughout mitosis EB1 binds central spindle microtubule bundles in a PRC1-dependent manner using a binding mode different from EB1 at growing microtubule ends. Both proteins, PRC1 and EB1, progressively accumulate and bind increasingly strongly to compacting central antiparallel microtubule overlaps. These results show that the central spindle gradually ‘solidifies’ during mitosis, suggesting that the two protein interaction networks around PRC1 and EB1 cooperate to stabilize the shortening central spindle, explaining the importance of both proteins for correct chromosome segregation and cytokinesis.

Download Full-text

Gradual compaction of the central spindle decreases its dynamicity in PRC1 and EB1 gene-edited cells

Life Science Alliance ◽

10.26508/lsa.202101222 ◽

2021 ◽

Vol 4 (12) ◽

pp. e202101222

Author(s):

Jayant Asthana ◽

Nicholas I Cade ◽

Davide Normanno ◽

Wei Ming Lim ◽

Thomas Surrey

Keyword(s):

Gene Editing ◽

Morphological Changes ◽

Dynamic Properties ◽

Human Cells ◽

Dynamic Changes ◽

Central Spindle ◽

Slowing Down ◽

Microtubule Growth ◽

Late Mitosis ◽

Endogenous Locus

During mitosis, the spindle undergoes morphological and dynamic changes. It reorganizes at the onset of the anaphase when the antiparallel bundler PRC1 accumulates and recruits central spindle proteins to the midzone. Little is known about how the dynamic properties of the central spindle change during its morphological changes in human cells. Using gene editing, we generated human cells that express from their endogenous locus fluorescent PRC1 and EB1 to quantify their native spindle distribution and binding/unbinding turnover. EB1 plus end tracking revealed a general slowdown of microtubule growth, whereas PRC1, similar to its yeast orthologue Ase1, binds increasingly strongly to compacting antiparallel microtubule overlaps. KIF4A and CLASP1 bind more dynamically to the central spindle, but also show slowing down turnover. These results show that the central spindle gradually becomes more stable during mitosis, in agreement with a recent “bundling, sliding, and compaction” model of antiparallel midzone bundle formation in the central spindle during late mitosis.

Download Full-text

Functional central spindle assembly requires de novo microtubule generation in the interchromosomal region during anaphase

The Journal of Cell Biology ◽

10.1083/jcb.201004150 ◽

2010 ◽

Vol 191 (2) ◽

pp. 259-267 ◽

Cited By ~ 53

Author(s):

Ryota Uehara ◽

Gohta Goshima

Keyword(s):

Protein Complex ◽

De Novo ◽

Spindle Assembly ◽

Live Imaging ◽

Human Cells ◽

Dependent Manner ◽

Spindle Formation ◽

Subsequent Formation ◽

Central Spindle ◽

New Model

The central spindle forms between segregating chromosomes during anaphase and is required for cytokinesis. Although anaphase-specific bundling and stabilization of interpolar microtubules (MTs) contribute to formation of the central spindle, it remains largely unknown how these MTs are prepared. Using live imaging of MT plus ends and an MT depolymerization and regrowth assay, we show that de novo MT generation in the interchromosomal region during anaphase is important for central spindle formation in human cells. Generation of interchromosomal MTs and subsequent formation of the central spindle occur independently of preanaphase MTs or centrosomal MT nucleation but require augmin, a protein complex implicated in nucleation of noncentrosomal MTs during preanaphase. MTs generated in a hepatoma up-regulated protein (HURP)–dependent manner during anaphase also contribute to central spindle formation redundantly with preanaphase MTs. Based on these results, a new model for central spindle assembly is proposed.

Download Full-text

Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms22157906 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7906

Author(s):

Alexey A. Komissarov ◽

Maria A. Karaseva ◽

Marina P. Roschina ◽

Andrey V. Shubin ◽

Nataliya A. Lunina ◽

...

Keyword(s):

Cell Death ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Proteolytic Enzymes ◽

Morphological Changes ◽

Human Cells ◽

Mitochondrial Potential ◽

3C Protease ◽

Wide Range

Regulated cell death (RCD) is a fundamental process common to nearly all living beings and essential for the development and tissue homeostasis in animals and humans. A wide range of molecules can induce RCD, including a number of viral proteolytic enzymes. To date, numerous data indicate that picornaviral 3C proteases can induce RCD. In most reported cases, these proteases induce classical caspase-dependent apoptosis. In contrast, the human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause caspase-independent cell death accompanied by previously undescribed features. Here, we expressed 3Cpro in HEK293, HeLa, and A549 human cell lines to characterize 3Cpro-induced cell death morphologically and biochemically using flow cytometry and fluorescence microscopy. We found that dead cells demonstrated necrosis-like morphological changes including permeabilization of the plasma membrane, loss of mitochondrial potential, as well as mitochondria and nuclei swelling. Additionally, we showed that 3Cpro-induced cell death was efficiently blocked by ferroptosis inhibitors and was accompanied by intense lipid peroxidation. Taken together, these results indicate that 3Cpro induces ferroptosis upon its individual expression in human cells. This is the first demonstration that a proteolytic enzyme can induce ferroptosis, the recently discovered and actively studied type of RCD.

Download Full-text

Structural and dynamic mechanisms of CBF3-guided centromeric nucleosome formation

Nature Communications ◽

10.1038/s41467-021-21985-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ruifang Guan ◽

Tengfei Lian ◽

Bing-Rui Zhou ◽

Emily He ◽

Carl Wu ◽

...

Keyword(s):

Dna Sequence ◽

Chromosome Segregation ◽

Budding Yeast ◽

Dna Conformation ◽

Dependent Manner ◽

Dynamic Interactions ◽

Nucleosome Core ◽

Dynamic Mechanisms ◽

Nucleosome Formation ◽

Core Histones

AbstractAccurate chromosome segregation relies on the specific centromeric nucleosome–kinetochore interface. In budding yeast, the centromere CBF3 complex guides the deposition of CENP-A, an H3 variant, to form the centromeric nucleosome in a DNA sequence-dependent manner. Here, we determine the structures of the centromeric nucleosome containing the native CEN3 DNA and the CBF3core bound to the canonical nucleosome containing an engineered CEN3 DNA. The centromeric nucleosome core structure contains 115 base pair DNA including a CCG motif. The CBF3core specifically recognizes the nucleosomal CCG motif through the Gal4 domain while allosterically altering the DNA conformation. Cryo-EM, modeling, and mutational studies reveal that the CBF3core forms dynamic interactions with core histones H2B and CENP-A in the CEN3 nucleosome. Our results provide insights into the structure of the budding yeast centromeric nucleosome and the mechanism of its assembly, which have implications for analogous processes of human centromeric nucleosome formation.

Download Full-text

Docosahexaenoic Acid, a Peroxisome Proliferator-Activated Receptor-α Activator, Induces Apoptosis in Vascular Smooth Muscle Cells by Activation of P38 Mitogen-Activated Protein Kinase

Hypertension ◽

10.1161/hyp.36.suppl_1.679-e ◽

2000 ◽

Vol 36 (suppl_1) ◽

pp. 679-679

Author(s):

Quy N Diep ◽

Rhian M Touyz ◽

Ernesto L Schiffrin

Keyword(s):

Smooth Muscle ◽

Cytochrome C ◽

Docosahexaenoic Acid ◽

Vascular Smooth Muscle ◽

Morphological Changes ◽

Mitogen Activated Protein Kinase ◽

Peroxisome Proliferator ◽

Dependent Manner ◽

Mitogen Activated Protein ◽

P38 Mapks

9 Omega-3 fatty acids (n-3 FAs) exert a blood pressure-lowering effect in hypertension, possibly by influencing vascular structure. We previously demonstrated that n-3 FAs might induce vascular smooth muscle cell (VSMC) apoptosis, which could exert an effect on structure of blood vessels. This study investigated signaling pathways through which n-3 FAs mediate apoptosis in VSMCs. Cultured Mesenteric VSMCs from Sprague Dawley rats were stimulated with docosahexaenoic acid (DHA), a representative n-3 FA. Morphological changes of apoptosis and DNA fragmentation were examined by phase-contrast microscopy and fluorescence microscopy with Hoechst 33342 staining. To clarify possible pathways of apoptosis, expression of phosphorylated p38 mitogen-activated protein kinases (p38 MAPKs), bax, bcl-2, cytochrome C and peroxisome proliferator-activated receptors-α (PPARs-α) was evaluated by Western blot analysis. DHA treatment induced cell shrinkage, cell membrane blebbing and apoptotic bodies in VSMCs. DHA increased apoptosis (%) in a time-dependent manner to 1.5±0.1, 3.6±0.5, 7.1±0.4, 22.5±0.6, 50.8±1.8 and 61.4±0.9 after 0, 1, 3, 6, 17, and 24 h, respectively. DHA time-dependently activated p38 MAPKs, bax, PPARs-α and cytochrome C with maximal effects obtained after 5, 30 min, 1 h and 3 h, respectively to 551±42, 245±55, 310±12 and 407±14.7 % of controls, respectively. SB-203580 (10 -5 M) and SB-202190 (10 -5 M), selective p38 inhibitors, reduced DHA-elicited apoptosis and expression of PPARs-α, but had no effect on expression of bax or cytochrome C. The present results indicate that DHA induces apoptosis in VSMCs through at least two distinct mechanisms: (i) a p38-dependent pathway that regulates PPAR-α and (ii) a p38-independent pathway via dissipation of mitochondrial transmembrane potential. The death-signaling pathway mediated by DHA may involve an integration of these multiple pathways. By triggering VSMC apoptosis, DHA could play a pathophysiological role in vascular remodeling in cardiovascular disease.

Download Full-text

Morphology and behaviour of dinoflagellate chromosomes during the cell cycle and mitosis

Journal of Cell Science ◽

10.1242/jcs.113.7.1231 ◽

2000 ◽

Vol 113 (7) ◽

pp. 1231-1239 ◽

Cited By ~ 3

Author(s):

Y. Bhaud ◽

D. Guillebault ◽

J. Lennon ◽

H. Defacque ◽

M.O. Soyer-Gobillard ◽

...

Keyword(s):

Electron Microscopy ◽

Cell Cycle ◽

Confocal Microscopy ◽

Nuclear Envelope ◽

Chromosome Segregation ◽

Morphological Changes ◽

S Phase ◽

Chromosome Behaviour ◽

Double Number ◽

Do So

The morphology and behaviour of the chromosomes of dinoflagellates during the cell cycle appear to be unique among eukaryotes. We used synchronized and aphidicolin-blocked cultures of the dinoflagellate Crypthecodinium cohnii to describe the successive morphological changes that chromosomes undergo during the cell cycle. The chromosomes in early G(1) phase appeared to be loosely condensed with numerous structures protruding toward the nucleoplasm. They condensed in late G(1), before unwinding in S phase. The chromosomes in cells in G(2) phase were tightly condensed and had a double number of arches, as visualised by electron microscopy. During prophase, chromosomes elongated and split longitudinally, into characteristic V or Y shapes. We also used confocal microscopy to show a metaphase-like alignment of the chromosomes, which has never been described in dinoflagellates. The metaphase-like nucleus appeared flattened and enlarged, and continued to do so into anaphase. Chromosome segregation occurred via binding to the nuclear envelope surrounding the cytoplasmic channels and microtubule bundles. Our findings are summarized in a model of chromosome behaviour during the cell cycle.

Download Full-text

Effect of methyl farnesoate and farnesoic acid during 5th zoea and megalopa metamorphosis in the mud crab Scylla paramamosain Estampador, 1950 (Decapoda, Brachyura, Portunidae)

Crustaceana ◽

10.1163/15685403-bja10133 ◽

2021 ◽

Vol 94 (7) ◽

pp. 855-863

Author(s):

Ming Zhao ◽

Fengying Zhang ◽

Wei Wang ◽

Zhiqiang Liu ◽

Lingbo Ma

Keyword(s):

Larval Development ◽

Morphological Changes ◽

Scylla Paramamosain ◽

Dependent Manner ◽

Mud Crab ◽

Methyl Farnesoate ◽

Concentration Dependent Manner ◽

Low Levels ◽

Juvenile Crab ◽

Farnesoic Acid

Abstract The mud crab Scylla paramamosain is one of the economically important aquaculture species in China. The larval development of the mud crab is characterized by two significant morphological changes, from the 5th zoea (Z5) to the megalopa (M) stage and from the M to the first juvenile crab (C1) stage. In this study, we found that methyl farnesoate (MF) could prohibit the Z5 to M metamorphosis in a concentration-dependent manner, and that a concentration of 10 μM MF could completely prohibit the Z5 metamorphosis. Farnesoic acid (FA) could also prohibit the Z5 metamorphosis, but its effects seemed to be concentration-independent. In addition, MF could delay rather than prohibit the M to C1 metamorphosis, while FA had no effect on the M to C1 metamorphosis at all. To summarize, it is hypothesized that either absence of MF and FA, or at least very low levels of these substances, might be necessary for a successful Z5 to M metamorphosis.

Download Full-text

Xanthohumol Induces Growth Inhibition and Apoptosis in Ca Ski Human Cervical Cancer Cells

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2015/921306 ◽

2015 ◽

Vol 2015 ◽

pp. 1-10 ◽

Cited By ~ 13

Author(s):

Wai Kuan Yong ◽

Sri Nurestri Abd Malek

Keyword(s):

Cell Cycle ◽

Cervical Cancer ◽

Cancer Cells ◽

Cancer Cell ◽

Morphological Changes ◽

S Phase ◽

Phase Cell ◽

Cervical Cancer Cell Line ◽

Dependent Manner ◽

Cervical Cancer Cells

We investigate induction of apoptosis by xanthohumol on Ca Ski cervical cancer cell line. Xanthohumol is a prenylated chalcone naturally found in hop plants, previously reported to be an effective anticancer agent in various cancer cell lines. The present study showed that xanthohumol was effective to inhibit proliferation of Ca Ski cells based on IC50values using sulforhodamine B (SRB) assay. Furthermore, cellular and nuclear morphological changes were observed in the cells using phase contrast microscopy and Hoechst/PI fluorescent staining. In addition, 48-hour long treatment with xanthohumol triggered externalization of phosphatidylserine, changes in mitochondrial membrane potential, and DNA fragmentation in the cells. Additionally, xanthohumol mediated S phase arrest in cell cycle analysis and increased activities of caspase-3, caspase-8, and caspase-9. On the other hand, Western blot analysis showed that the expression levels of cleaved PARP, p53, and AIF increased, while Bcl-2 and XIAP decreased in a dose-dependent manner. Taken together, these findings indicate that xanthohumol-induced cell death might involve intrinsic and extrinsic apoptotic pathways, as well as downregulation of XIAP, upregulation of p53 proteins, and S phase cell cycle arrest in Ca Ski cervical cancer cells. This work suggests that xanthohumol is a potent chemotherapeutic candidate for cervical cancer.

Download Full-text

Oestrogen-Dependent Oxytocin Dynamics in the Hypothalamus of Female Rats

10.21203/rs.3.rs-319589/v1 ◽

2021 ◽

Author(s):

Kazuaki Nishimura ◽

Kiyoshi Yoshino ◽

Naofumi Ikeda ◽

Kazuhiko Baba ◽

Kenya Sanada ◽

...

Keyword(s):

Systemic Circulation ◽

Female Rats ◽

Dependent Manner ◽

Transgenic Rats ◽

Dynamic Changes ◽

Hypothalamic Nuclei ◽

The Central Nervous System ◽

Posterior Pituitary Gland ◽

Dose Dependent ◽

Ovariectomised Rats

Abstract Oxytocin (OXT) is produced in the hypothalamic nuclei and is secreted into systemic circulation from the posterior pituitary gland (PP). In the central nervous system, OXT regulates behaviours including maternal and feeding behaviours. Our aim was to evaluate whether oestrogen regulates hypothalamic OXT dynamics. Herein, we provide the first evidence that OXT dynamics in the hypothalamus vary with sex and that oestrogen may modulate dynamic changes in OXT levels, using OXT-mRFP1 transgenic rats. The fluorescence intensity of OXT-mRFP1 in the hypothalamic nuclei and PP was most strongly expressed during the oestrus stage in female rats and decreased significantly in ovariectomised rats. Oestrogen replacement caused significant increases in the fluorescent intensities in the hypothalamic nuclei and PP in a dose-dependent manner. This was also demonstrated in feeding behaviour and hypothalamic Fos neurons using immunohistochemistry. Hypothalamic OXT expression was oestrogen dependent and could be enhanced centrally by the administration of oestrogen.

Download Full-text

Complex CSF3R Protein Isoform Interactions Dictate Cytokine Responsiveness

Blood ◽

10.1182/blood-2020-143107 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 35-35

Author(s):

Sara L. Seegers ◽

Amanda Lance ◽

Lawrence J Druhan ◽

Belinda R Avalos

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Splice Variant ◽

Splice Variants ◽

Polyclonal Antibodies ◽

Human Cells ◽

Dependent Manner ◽

Epitope Tag ◽

Proliferative Signaling ◽

Primary Human Cells

CSF3R, the receptors for granulocyte colony stimulating factor, is a critical regulator of neutrophil production. Multiple CSF3R mRNA transcripts have been identified and are annotated in Genbank. The expression and function of the different CSF3R proteins have not been fully elucidated. We generated antibodies specific for two of the identified and annotated isoforms, V3 and V4. CSF3R-V4 is a truncated variant of V1 with a unique C-terminal 34 amino acids and this variant confers enhanced growth signals. Changes in the ratio of V1:V4 isoforms have been implicated in chemotherapy resistance and relapse of AML. CSF3R-V3 is a variant of V1 with a 27 amino acid insertion between two conserved domains in the cytoplasmic portion of the receptor involved in JAK/STAT activation, termed the box 1 and box 2. CSF3R-V3 produces reduced proliferative signaling in response to G-CSF. When V3 is co-expressed with V1, proliferative signaling is reduced in a concentration dependent manner. In order to generate custom rabbit polyclonal antibodies specific for CSF3R-V3 and CSF3R-V4 we used either a peptide that corresponds to a unique amino acid sequence present only in CSF3R-V3 or a peptide specific for a portion of the C-terminal amino acid sequence unique to the CSF3R-V4 isoform conjugated to an immunogenic carrier protein. These immunogens both produced robust immune responses, and the polyclonal antibodies were subsequently purified from bulk sera. Immunoblot analysis of lysates from Ba/F3 cells expressing CSF3R-V1 (V1), CSF3R-V3 (V3), or CSF3R-V4 (V4) demonstrated that both the custom generated anti-CSF3R-V3 and anti-CSF3R-V4 antibodies were very specific, recognizing only the appropriate CSF3R receptor isoform. All three CSF3R splice variants are recognized by commercially available anti-CSF3R (clone LMM741 to CD114), while the anti-CSF3R-V4 custom antibody and the custom anti-CSF3R-V3 antibody recognizes only the CSF3R-V4 and CSF3R-V3 isoforms, respectively. We next sought to detect the CSF3R receptor isoforms in primary human cells. Using our custom antibodies, we detected for the first time, both the CSF3R-V3 and CSF3R-V4 receptor forms in primary neutrophils isolated from healthy donors. Each of the CSF3R isoforms produce unique signaling, and we hypothesized that the observed differences in G-CSF-dependent signaling is produced by the expression level of each receptor isoform via both homodimerization and by heterodimerization of the receptor splice variant proteins. To investigate the potential for heterodimerization of the CSF3R-V1 with the V3 and V4 isoforms, we generated a CSF3R-V1 with a c-terminal epitope tag and co-expressed this construct with both CSF3R-V3 or CSF3R-V4. Immunoprecipitation with an antibody to the epitope tag (recognizing the V1 variant) followed by immunoblotting with the custom anti-V3 or anti-V4 antibodies demonstrated that both CSF3R-V3 and CSF3R-V4 co-immunoprecipitated with CSF3R-V1, in agreement with our hypothesis that the splice variants form receptor heterodimers. Of note, the CSF3R receptor heterodimers are detected even in the absence of G-CSF, thus demonstrating that CSF3R exist as a preformed receptor dimer in an inactive state. In conclusion, we have generated antibodies that specifically detect the CSF3R-V3 and the CSF3R-V4 receptor proteins. These are the first studies to demonstrate the expression of the CSF3R splice variants at the protein level, in both cell lines and primary human cells. In addition, these are the first studies to demonstrate the formation of heterodimers of the CSF3R splice variants, providing a mechanism for the observed alteration in ligand-dependent signaling produced under conditions of altered splice variant expression. Disclosures Avalos: Juno: Membership on an entity's Board of Directors or advisory committees; Best Practice-Br Med J: Patents & Royalties: receives royalties from a coauthored article on evaluation of neutropenia.

Download Full-text