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The Journal of Cell Biology Jan 2016The understanding of cytoskeleton dynamics has benefited from the capacity to generate fluorescent fiducial marks on cytoskeleton components. Here we show that... (Comparative Study)
Comparative Study
The understanding of cytoskeleton dynamics has benefited from the capacity to generate fluorescent fiducial marks on cytoskeleton components. Here we show that light-induced imprinting of three-dimensional (3D) fluorescent speckles significantly improves speckle signal and contrast relative to classic (random) fluorescent speckle microscopy. We predict theoretically that speckle imprinting using photobleaching is optimal when the laser energy and fluorophore responsivity are related by the golden ratio. This relation, which we confirm experimentally, translates into a 40% remaining signal after speckle imprinting and provides a rule of thumb in selecting the laser power required to optimally prepare the sample for imaging. This inducible speckle imaging (ISI) technique allows 3D speckle microscopy to be performed in readily available libraries of cell lines or primary tissues expressing fluorescent proteins and does not preclude conventional imaging before speckle imaging. As a proof of concept, we use ISI to measure metaphase spindle microtubule poleward flux in primary cells and explore a scaling relation connecting microtubule flux to metaphase duration.
Topics: Animals; Cell Line, Transformed; Drosophila; Humans; Imaging, Three-Dimensional; Metaphase; Microscopy, Fluorescence; Microtubules; Spindle Apparatus
PubMed: 26783303
DOI: 10.1083/jcb.201506128 -
Chromosoma Mar 2023Topoisomerase IIα (Topo IIα) and the centromere-specific histone H3 variant CENH3 are key proteins involved in chromatin condensation and centromere determination,...
Topoisomerase IIα (Topo IIα) and the centromere-specific histone H3 variant CENH3 are key proteins involved in chromatin condensation and centromere determination, respectively. Consequently, they are required for proper chromosome segregation during cell divisions. We combined two super-resolution techniques, structured illumination microscopy (SIM) to co-localize Topo IIα and CENH3, and photoactivated localization microscopy (PALM) to determine their molecule numbers in barley metaphase chromosomes. We detected a dispersed Topo IIα distribution along chromosome arms but an accumulation at centromeres, telomeres, and nucleolus-organizing regions. With a precision of 10-50 nm, we counted ~ 20,000-40,000 Topo IIα molecules per chromosome, 28% of them within the (peri)centromere. With similar precision, we identified ~13,500 CENH3 molecules per centromere where Topo IIα proteins and CENH3-containing chromatin intermingle. In short, we demonstrate PALM as a useful method to count and localize single molecules with high precision within chromosomes. The ultrastructural distribution and the detected amount of Topo IIα and CENH3 are instrumental for a better understanding of their functions during chromatin condensation and centromere determination.
Topics: Hordeum; Metaphase; Microscopy; Centromere; Chromatin
PubMed: 36719450
DOI: 10.1007/s00412-023-00785-8 -
Cytoskeleton (Hoboken, N.J.) Nov 2019Chromosome segregation is mediated by spindle microtubules that attach to the kinetochore via dynamic protein complexes, such as Ndc80, Ska, Cdt1 and ch-TOG during...
Chromosome segregation is mediated by spindle microtubules that attach to the kinetochore via dynamic protein complexes, such as Ndc80, Ska, Cdt1 and ch-TOG during mitotic metaphase. While experimental studies have previously shown that these proteins and protein complexes are all essential for maintaining a stable kinetochore-microtubule (kMT) interface, their exact roles in the mitotic metaphase remains elusive. In this study, we employed experimental and computational methods in order to characterize how these proteins can strengthen kMT attachments in both nonload-bearing and load-bearing conditions, typical of prometaphase and metaphase, respectively. Immunofluorescence staining of HeLa cells showed that the levels of Ska and Cdt1 significantly increased from prometaphase to metaphase, while levels of the Ndc80 complex remained unchanged. Our new computational model showed that by incorporating binding and unbinding of each protein complex coupled with a biased diffusion mechanism, the displacement of a possible complex formed by Ndc80-Ska-Cdt1 is significantly higher than that of Ndc80 alone or Ndc80-Ska. In addition, when we incorporate Ndc80/ch-TOG in the model, rupture force and time of attachment of the kMT interface increases. These results support the hypothesis that Ndc80-associated proteins strengthen kMT attachments, and that the interplay between kMT protein complexes in metaphase ensures stable attachments.
Topics: Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Computer Simulation; Cytoskeletal Proteins; HeLa Cells; Humans; Kinetochores; Metaphase; Microtubules; Mitosis; Protein Binding
PubMed: 31525284
DOI: 10.1002/cm.21562 -
Cellular Physiology and Biochemistry :... 2016Ras dexamethasone-induced protein (RASD1) is a member of Ras superfamily of small GTPases. RASD1 regulates various signaling pathways involved in iron homeostasis,...
BACKGROUND
Ras dexamethasone-induced protein (RASD1) is a member of Ras superfamily of small GTPases. RASD1 regulates various signaling pathways involved in iron homeostasis, growth hormone secretion, and circadian rhythm. However, RASD1 function in oocyte remains unknown.
METHODS
Using immunohistochemistry, immunofluorescence, and quantitative real-time RT-PCR, RASD1 expression in mouse ovary and RASD1 role in oocyte maturation-related gene expression, spindle formation, and chromosome alignment were analyzed. RNAi microinjection and time-lapse video microscopy were used to examine the effect of Rasd1 knockdown on oocyte maturation.
RESULTS
RASD1 was highly detected in oocytes transitioning from primordial to secondary follicles. Rasd1 was highly expressed in germinal vesicle (GV), during GV breakdown, and in metaphase I (MI) stage as oocytes mature, and its expression was significantly downregulated in MII stage. With knockdown of Rasd1, maturation in GV oocytes was arrested at MI stage, showing disrupted meiotic spindling and chromosomal misalignment. In addition, Obox4 and Arp2/3, engaged in MI-MII transition and cytokinesis, respectively, were misregulated in GV oocytes by Rasd1 knockdown.
CONCLUSION
These findings suggest that RASD1 is a novel factor in MI-MII oocyte transition and may be involved in regulating the progression of cytokinesis and spindle formation, controlling related signaling pathways during oocyte maturation.
Topics: Animals; Cell Differentiation; Chromosomes, Mammalian; Cytokinesis; Female; Gene Expression Profiling; Gene Expression Regulation; Gene Knockdown Techniques; Metaphase; Mice, Inbred ICR; Oocytes; Organ Specificity; RNA Interference; Spindle Apparatus; ras Proteins
PubMed: 27997888
DOI: 10.1159/000453182 -
The Journal of Biological Chemistry Sep 2020The spindle apparatus segregates bi-oriented sister chromatids during mitosis but mono-oriented homologous chromosomes during meiosis I. It has remained unclear if...
The spindle apparatus segregates bi-oriented sister chromatids during mitosis but mono-oriented homologous chromosomes during meiosis I. It has remained unclear if similar molecular mechanisms operate to regulate spindle dynamics during mitosis and meiosis I. Here, we employed live-cell microscopy to compare the spindle dynamics of mitosis and meiosis I in fission yeast cells and demonstrated that the conserved kinesin-14 motor Klp2 plays a specific role in maintaining metaphase spindle length during meiosis I but not during mitosis. Moreover, the maintenance of metaphase spindle stability during meiosis I requires the synergism between Klp2 and the conserved microtubule cross-linker Ase1, as the absence of both proteins causes exacerbated defects in metaphase spindle stability. The synergism is not necessary for regulating mitotic spindle dynamics. Hence, our work reveals a new molecular mechanism underlying meiotic spindle dynamics and provides insights into understanding differential regulation of meiotic and mitotic events.
Topics: Metaphase; Microtubule-Associated Proteins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Spindle Apparatus
PubMed: 32723864
DOI: 10.1074/jbc.RA120.012905 -
Journal of Biomedical Optics Jun 2024Preparation of a recipient cytoplast by oocyte enucleation is an essential task for animal cloning and assisted reproductive technologies in humans. The femtosecond...
SIGNIFICANCE
Preparation of a recipient cytoplast by oocyte enucleation is an essential task for animal cloning and assisted reproductive technologies in humans. The femtosecond laser is a precise and low-invasive tool for oocyte enucleation, and it should be an appropriate alternative to traditional enucleation by a microneedle aspiration. However, until recently, the laser enucleation was performed only with applying a fluorescent dye.
AIM
This work is aimed to (1) achieve femtosecond laser oocyte enucleation without applying a fluorescent dye and (2) to study the effect of laser destruction of chromosomes on the structure and dynamics of the spindle.
APPROACH
We applied polarized light microscopy for spindle visualization and performed stain-free mouse and human oocyte enucleation with a 1033 nm femtosecond laser. Also, we studied transformation of a spindle after metaphase plate elimination by a confocal microscopy.
RESULTS
We demonstrated a fundamental possibility of inactivating the metaphase plate in mouse and human oocytes by 1033 nm femtosecond laser radiation without applying a fluorescent dye. Irradiation of the spindle area, visualized by polarized light microscopy, resulted in partly or complete metaphase plate destruction but avoided the microtubules impairment. After the metaphase plate elimination, the spindle reorganized, however, it was not a complete depolymerization.
CONCLUSIONS
This method of recipient cytoplast preparation is expected to be useful for animal cloning and assisted reproductive technologies.
Topics: Animals; Mice; Oocytes; Humans; Female; Lasers; Spindle Apparatus; Microscopy, Confocal; Metaphase; Microscopy, Polarization
PubMed: 38812963
DOI: 10.1117/1.JBO.29.6.065002 -
Methods in Cell Biology 2018Cell division involves mechanical processes, such as chromosome transport and centrosome separation. Quantitative micromanipulation-based approaches have been central to...
Cell division involves mechanical processes, such as chromosome transport and centrosome separation. Quantitative micromanipulation-based approaches have been central to dissecting the forces driving these processes. We highlight two biophysical assays that can be employed for such analyses. First, an in vitro "mini-spindle" assay is described that can be used to examine the collective mechanics of mitotic motor proteins cross-linking two microtubules. In the spindle, motor proteins (e.g., kinesin-5, kinesin-14, and dynein) can localize to overlapping microtubules that slide relative to each other, work as an ensemble, and equilibrate between cytoplasm and the microtubules. The "mini-spindle" assay can recapitulate these features and allows measurements of forces generated between adjacent microtubules and their dependence on filament orientation, sliding speed, overlap length, and motor protein density. Second, we describe a force-calibrated microneedle-based "whole-spindle" micromechanics assay. Microneedle-based micromanipulation can be a useful technique to examine cellular scale mechanics, but its use has been restricted by the difficulty in getting probes to penetrate the plasma membrane without disrupting cell physiology. As detailed here, the use of cell-free extracts prepared from metaphase-arrested Xenopus eggs can address this limitation. These micromanipulation studies also benefit from the use of frozen stocks of Xenopus egg extract. Together, these approaches can be used to decipher how micromechanics and biochemical activities ensure successful cell division.
Topics: Animals; Cell Division; Cell Membrane; Dyneins; Kinesins; Metaphase; Micromanipulation; Microtubules; Spindle Apparatus; Xenopus Proteins; Xenopus laevis
PubMed: 29957203
DOI: 10.1016/bs.mcb.2018.03.022 -
Molecular Biology of the Cell Oct 2023During mitosis, the budding yeast, kinetochores remain attached to microtubules, except for a brief period during S phase. Sister-kinetochores separate into two clusters...
During mitosis, the budding yeast, kinetochores remain attached to microtubules, except for a brief period during S phase. Sister-kinetochores separate into two clusters (bilobed organization) upon stable end-on attachment to microtubules emanating from opposite spindle poles. However, in meiosis, the outer kinetochore protein (Ndc80) reassembles at the centromeres much later after prophase I, establishing new kinetochore-microtubule attachments. Perhaps due to this, despite homolog bi-orientation, we observed that the Ndc80 are linearly dispersed between spindle poles during metaphase I of meiosis. The presence of end-on attachment marker Dam1 as a cluster near each pole suggests one of the other possibilities that the pole-proximal and pole-distal kinetochores are attached end-on and laterally to the microtubules, respectively. Colocalization studies of kinetochores and kinesin motors suggest that budding yeast kinesin 5, Cin8, and Kip1 perhaps localize to the end-on attached kinetochores while kinesin 8 and Kip3 resides at all the kinetochores. Our findings, including kinesin 5 and Ndc80 coappearance after prophase I and reduced Ndc80 levels in null mutant, suggest that kinesin motors are crucial for kinetochore reassembly and stability during early meiosis. Thus, this work reports yet another meiosis specific function of kinesin motors.
Topics: Kinesins; Kinetochores; Spindle Apparatus; Meiosis; Metaphase; Microtubules; Mitosis; Chromosome Segregation
PubMed: 37556230
DOI: 10.1091/mbc.E22-12-0569 -
Chromosoma Sep 2019Cyclins, as regulatory partners of cyclin-dependent kinases (CDKs), control the switch-like cell cycle transitions that orchestrate orderly duplication and segregation...
Cyclins, as regulatory partners of cyclin-dependent kinases (CDKs), control the switch-like cell cycle transitions that orchestrate orderly duplication and segregation of genomes. Compared to mitosis, relatively little is known about how cyclin-CDK complexes control meiosis, the specialized cell division that generates gametes for sexual production. Mouse cyclin B3 was previously shown to have expression restricted to the beginning of meiosis, making it a candidate to regulate meiotic events. Indeed, female mice lacking cyclin B3 are sterile because oocytes arrest at the metaphase-to-anaphase transition of meiosis I. However, whether cyclin B3 functions during spermatogenesis was untested. Here, we found that males lacking cyclin B3 are fertile and show no detectable defects in spermatogenesis based on histological analysis of seminiferous tubules. Cytological analysis further showed no detectable defects in homologous chromosome synapsis or meiotic progression, and suggested that recombination is initiated and completed efficiently. Moreover, absence of cyclin B3 did not exacerbate previously described meiotic defects in mutants deficient for cyclin E2, suggesting a lack of redundancy between these cyclins. Thus, unlike in females, cyclin B3 is not essential for meiosis in males despite its prominent meiosis-specific expression.
Topics: Alleles; Amino Acid Sequence; Animals; Cyclin B; Gene Editing; Gene Expression; Immunohistochemistry; Male; Meiosis; Metaphase; Mice; Prophase; Protein Domains; Recombination, Genetic; Spermatogenesis
PubMed: 31446450
DOI: 10.1007/s00412-019-00725-5 -
Science Advances Mar 2020The meiotic prophase I to metaphase I (PI/MI) transition requires chromosome desynapsis and metaphase competence acquisition. However, control of these major meiotic...
The meiotic prophase I to metaphase I (PI/MI) transition requires chromosome desynapsis and metaphase competence acquisition. However, control of these major meiotic events is poorly understood. Here, we identify an essential role for SKP1, a core subunit of the SKP1-Cullin-F-box (SCF) ubiquitin E3 ligase, in the PI/MI transition. SKP1 localizes to synapsed chromosome axes and evicts HORMAD proteins from these regions in meiotic spermatocytes. SKP1-deficient spermatocytes display premature desynapsis, precocious pachytene exit, loss of PLK1 and BUB1 at centromeres, but persistence of HORMAD, γH2AX, RPA2, and MLH1 in diplonema. Strikingly, SKP1-deficient spermatocytes show sharply reduced MPF activity and fail to enter MI despite treatment with okadaic acid. SKP1-deficient oocytes exhibit desynapsis, chromosome misalignment, and progressive postnatal loss. Therefore, SKP1 maintains synapsis in meiosis of both sexes. Furthermore, our results support a model where SKP1 functions as the long-sought intrinsic metaphase competence factor to orchestrate MI entry during male meiosis.
Topics: Alleles; Animals; Gene Expression Regulation; Male; Meiosis; Meiotic Prophase I; Mesothelin; Metaphase; Mice; Mice, Transgenic; Oocytes; S-Phase Kinase-Associated Proteins; Sex Factors
PubMed: 32232159
DOI: 10.1126/sciadv.aaz2129