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Scientific Reports Jun 2024The challenge of in-situ handling and high-resolution low-dose imaging of intact, sensitive and wet samples in their native state at nanometer scale, including live...
The challenge of in-situ handling and high-resolution low-dose imaging of intact, sensitive and wet samples in their native state at nanometer scale, including live samples is met by Advanced Environmental Scanning Electron Microscopy (A-ESEM). This new generation of ESEM utilises machine learning-based optimization of thermodynamic conditions with respect to sample specifics to employ a low temperature method and an ionization secondary electron detector with an electrostatic separator. A modified electron microscope was used, equipped with temperature, humidity and gas pressure sensors for in-situ and real-time monitoring of the sample. A transparent ultra-thin film of ionic liquid is used to increase thermal and electrical conductivity of the samples and to minimize sample damage by free radicals. To validate the power of the new method, we analyze condensed mitotic metaphase chromosomes to reveal new structural features of their perichromosomal layer, and the organization of chromatin fibers, not observed before by any microscopic technique. The ability to resolve nano-structural details of chromosomes using A-ESEM is validated by measuring gold nanoparticles with achievable resolution in the lower nanometre units.
Topics: Microscopy, Electron, Scanning; Humans; Gold; Metal Nanoparticles; Mitosis; Chromosomes
PubMed: 38844535
DOI: 10.1038/s41598-024-63515-9 -
Open Biology May 2024The precise spatial and temporal control of histone phosphorylations is important for the ordered progression through the different phases of mitosis. The...
The precise spatial and temporal control of histone phosphorylations is important for the ordered progression through the different phases of mitosis. The phosphorylation of H2B at S6 (H2B S6ph), which is crucial for chromosome segregation, reaches its maximum level during metaphase and is limited to the inner centromere. We discovered that the temporal and spatial regulation of this modification, as well as its intensity, are governed by the scaffold protein RepoMan and its associated catalytically active phosphatases, PP1α and PP1γ. Phosphatase activity is inhibited at the area of maximal H2B S6 phosphorylation at the inner centromere by site-specific Aurora B-mediated inactivation of the PP1/RepoMan complex. The motor protein Mklp2 contributes to the relocalization of Aurora B from chromatin to the mitotic spindle during anaphase, thus alleviating Aurora B-dependent repression of the PP1/RepoMan complex and enabling dephosphorylation of H2B S6. Accordingly, dysregulation of Mklp2 levels, as commonly observed in tumour cells, leads to the lack of H2B S6 dephosphorylation during early anaphase, which might contribute to chromosomal instability.
Topics: Aurora Kinase B; Phosphorylation; Humans; Histones; Mitosis; Protein Phosphatase 1; Cell Cycle Proteins; HeLa Cells; Spindle Apparatus; Centromere; Nuclear Proteins
PubMed: 38806145
DOI: 10.1098/rsob.230460 -
Mathematical Biosciences May 2024This paper develops a theory for anaphase in cells. After a brief description of microtubules, the mitotic spindle and the centrosome, a mathematical model for anaphase...
This paper develops a theory for anaphase in cells. After a brief description of microtubules, the mitotic spindle and the centrosome, a mathematical model for anaphase is introduced and developed in the context of the cell cytoplasm and liquid crystalline structures. Prophase, prometaphase and metaphase are then briefly described in order to focus on anaphase, which is the main study of this paper. The entities involved are modelled in terms of liquid crystal defects and microtubules are represented as defect flux lines. The mathematical techniques employed make extensive use of energy considerations based on the work that was developed by Dafermos (1970) from the classical Frank-Oseen nematic liquid crystal energy (Frank, 1958; Oseen, 1933). With regard to liquid crystal theory we introduce the concept of regions of influence for defects which it is believed have important implications beyond the subject of this paper. The results of this paper align with observed biochemical phenomena and are explored in application to HeLa cells and Caenorhabditis elegans. This unified approach offers the possibility of gaining insight into various consequences of mitotic abnormalities which may result in Down syndrome, Hodgkin lymphoma, breast, prostate and various other types of cancer.
PubMed: 38795952
DOI: 10.1016/j.mbs.2024.109219 -
Biosensors Apr 2024Separase is a key cysteine protease in the separation of sister chromatids through the digestion of the cohesin ring that inhibits chromosome segregation as a trigger of...
Separase is a key cysteine protease in the separation of sister chromatids through the digestion of the cohesin ring that inhibits chromosome segregation as a trigger of the metaphase-anaphase transition in eukaryotes. Its activity is highly regulated by binding with securin and cyclinB-CDK1 complex. These bindings prevent the proteolytic activity of separase until the onset of anaphase. Chromosome missegregation and aneuploidy are frequently observed in malignancies. However, there are some difficulties in biochemical examinations due to the instability of separase in vitro and the fact that few spatiotemporal resolution approaches exist for monitoring live separase activity throughout mitotic processes. Here, we have developed FRET-based molecular sensors, including GFP variants, with separase-cleavable sequences as donors and covalently attached fluorescent dyes as acceptor molecules. These are applicable to conventional live cell imaging and flow cytometric analysis because of efficient live cell uptake. We investigated the performance of equivalent molecular sensors, either localized or not localized inside the nucleus under cell cycle control, using flow cytometry. Synchronized cell cycle progression rendered significant separase activity detections in both molecular sensors. We obtained consistent outcomes with localized molecular sensor introduction and cell cycle control by fluorescent microscopic observations. We thus established live cell separase activity monitoring systems that can be used specifically or statistically, which could lead to the elucidation of separase properties in detail.
Topics: Separase; Fluorescence Resonance Energy Transfer; Humans; Chromosome Segregation; Cell Cycle; Biosensing Techniques; HeLa Cells
PubMed: 38667185
DOI: 10.3390/bios14040192 -
BioRxiv : the Preprint Server For... Apr 2024Accurate positioning of the mitotic spindle within the rounded cell body is critical to physiological maintenance. Adherent mitotic cells encounter confinement from...
Accurate positioning of the mitotic spindle within the rounded cell body is critical to physiological maintenance. Adherent mitotic cells encounter confinement from neighboring cells or the extracellular matrix (ECM), which can cause rotation of mitotic spindles and, consequently, titling of the metaphase plate (MP). To understand the positioning and orientation of mitotic spindles under confinement by fibers (ECM-confinement), we use flexible ECM-mimicking nanofibers that allow natural rounding of the cell body while confining it to differing levels. Rounded mitotic bodies are anchored in place by actin retraction fibers (RFs) originating from adhesion clusters on the ECM-mimicking fibers. We discover the extent of ECM-confinement patterns RFs in 3D: triangular and band-like at low and high confinement, respectively. A stochastic Monte-Carlo simulation of the centrosome (CS), chromosome (CH), membrane interactions, and 3D arrangement of RFs on the mitotic body recovers MP tilting trends observed experimentally. Our mechanistic analysis reveals that the 3D shape of RFs is the primary driver of the MP rotation. Under high ECM-confinement, the fibers can mechanically pinch the cortex, causing the MP to have localized deformations at contact sites with fibers. Interestingly, high ECM-confinement leads to low and high MP tilts, which mechanistically depend upon the extent of cortical deformation, RF patterning, and MP position. We identify that cortical deformation and RFs work in tandem to limit MP tilt, while asymmetric positioning of MP leads to high tilts. Overall, we provide fundamental insights into how mitosis may proceed in fibrous ECM-confining microenvironments in vivo.
PubMed: 38659898
DOI: 10.1101/2024.04.12.589246 -
BioRxiv : the Preprint Server For... Apr 2024Microtubule-based spindle formation is essential to faithful chromosome segregation during cell division. In many animal species, the oocyte meiotic spindle forms...
Microtubule-based spindle formation is essential to faithful chromosome segregation during cell division. In many animal species, the oocyte meiotic spindle forms without centrosomes, unlike most mitotic cells. Even in mitotic cells, centrosomes are sometimes dispensable for bipolar spindle formation. In some systems, Ran-GEF on chromatin initiates spindle assembly. We found that in oocytes, endogenously-tagged Ran-GEF dissociates from chromatin during spindle assembly but re-associates during meiotic anaphase. Meiotic spindle assembly was normal after auxin-induced degradation of Ran-GEF but anaphase I was faster than controls and extrusion of the first polar body frequently failed. In search of a possible alternative pathway for spindle assembly, we found that soluble tubulin concentrates in the nuclear volume during germinal vesicle breakdown as well as in the spindle region during metaphase I and metaphase II. Through light and electron microscopy we found that the concentration of soluble tubulin in the metaphase II spindle region is enclosed by ER sheets which exclude cytoplasmic organelles including mitochondria and yolk granules from the meiotic spindle. We suggest that this concentration of soluble tubulin may be a redundant mechanism promoting spindle assembly near chromosomes. We present data supporting a model in which cytoplasmic organelles exclude cytoplasmic volume to drive concentration of tubulin within the nuclear/spindle envelope.
PubMed: 38659754
DOI: 10.1101/2024.04.19.590357 -
Development (Cambridge, England) May 2024The planar orientation of cell division (OCD) is important for epithelial morphogenesis and homeostasis. Here, we ask how mechanics and antero-posterior (AP) patterning...
The planar orientation of cell division (OCD) is important for epithelial morphogenesis and homeostasis. Here, we ask how mechanics and antero-posterior (AP) patterning combine to influence the first divisions after gastrulation in the Drosophila embryonic epithelium. We analyse hundreds of cell divisions and show that stress anisotropy, notably from compressive forces, can reorient division directly in metaphase. Stress anisotropy influences the OCD by imposing metaphase cell elongation, despite mitotic rounding, and overrides interphase cell elongation. In strongly elongated cells, the mitotic spindle adapts its length to, and hence its orientation is constrained by, the cell long axis. Alongside mechanical cues, we find a tissue-wide bias of the mitotic spindle orientation towards AP-patterned planar polarised Myosin-II. This spindle bias is lost in an AP-patterning mutant. Thus, a patterning-induced mitotic spindle orientation bias overrides mechanical cues in mildly elongated cells, whereas in strongly elongated cells the spindle is constrained close to the high stress axis.
Topics: Animals; Metaphase; Stress, Mechanical; Epithelial Cells; Spindle Apparatus; Drosophila melanogaster; Cell Division; Cell Polarity; Body Patterning; Myosin Type II; Embryo, Nonmammalian; Drosophila Proteins; Gastrulation
PubMed: 38639390
DOI: 10.1242/dev.202862 -
MBio May 2024Condensin I is a pentameric complex that regulates the mitotic chromosome assembly in eukaryotes. The kleisin subunit CAP-H of the condensin I complex acts as a linchpin...
UNLABELLED
Condensin I is a pentameric complex that regulates the mitotic chromosome assembly in eukaryotes. The kleisin subunit CAP-H of the condensin I complex acts as a linchpin to maintain the structural integrity and loading of this complex on mitotic chromosomes. This complex is present in all eukaryotes and has recently been identified in spp. However, how this complex is assembled and whether the kleisin subunit is critical for this complex in these parasites are yet to be explored. To examine the role of PfCAP-H during cell division within erythrocytes, we generated an inducible PfCAP-H knockout parasite. We find that PfCAP-H is dynamically expressed during mitosis with the peak expression at the metaphase plate. PfCAP-H interacts with PfCAP-G and is a non-SMC member of the condensin I complex. Notably, the absence of PfCAP-H does not alter the expression of PfCAP-G but affects its localization at the mitotic chromosomes. While mitotic spindle assembly is intact in PfCAP-H-deficient parasites, duplicated centrosomes remain clustered over the mass of unsegmented nuclei with failed karyokinesis. This failure leads to the formation of an abnormal nuclear mass, while cytokinesis occurs normally. Altogether, our data suggest that PfCAP-H plays a crucial role in maintaining the structural integrity of the condensin I complex on the mitotic chromosomes and is essential for the asexual development of malarial parasites.
IMPORTANCE
Mitosis is a fundamental process for parasites, which plays a vital role in their survival within two distinct hosts-human and mosquitoes. Despite its great significance, our comprehension of mitosis and its regulation remains limited. In eukaryotes, mitosis is regulated by one of the pivotal complexes known as condensin complexes. The condensin complexes are responsible for chromosome condensation, ensuring the faithful distribution of genetic material to daughter cells. While condensin complexes have recently been identified in spp., our understanding of how this complex is assembled and its precise functions during the blood stage development of remains largely unexplored. In this study, we investigate the role of a central protein, PfCAP-H, during the blood stage development of . Our findings reveal that PfCAP-H is essential and plays a pivotal role in upholding the structure of condensin I and facilitating karyokinesis.
Topics: Humans; Adenosine Triphosphatases; DNA-Binding Proteins; Erythrocytes; Gene Knockout Techniques; Mitosis; Multiprotein Complexes; Plasmodium falciparum; Protozoan Proteins; Cell Nucleus Division
PubMed: 38564676
DOI: 10.1128/mbio.02850-23 -
Materials (Basel, Switzerland) Mar 2024Hyaluronic acid (HA) has attracted much attention in tumor-targeted drug delivery due to its ability to specifically bind to the CD44 cellular receptor, which is widely...
Hyaluronic acid (HA) has attracted much attention in tumor-targeted drug delivery due to its ability to specifically bind to the CD44 cellular receptor, which is widely expressed on cancer cells. We present HA-capped magnetic nanoparticles (HA-MNPs) obtained via the co-precipitation method, followed by the electrostatic adsorption of HA onto the nanoparticles' surfaces. A theoretical study carried out with the PM3 method evidenced a dipole moment of 3.34 D and negatively charged atom groups able to participate in interactions with nanoparticle surface cations and surrounding water molecules. The ATR-FTIR spectrum evidenced the hyaluronic acid binding to the surface of the ferrophase, ensuring colloidal stability in the water dispersion. To verify the success of the synthesis and stabilization, HA-MNPs were also characterized using other investigation techniques: TEM, EDS, XRD, DSC, TG, NTA, and VSM. The results showed that the HA-MNPs had a mean physical size of 9.05 nm (TEM investigation), a crystallite dimension of about 8.35 nm (XRD investigation), and a magnetic core diameter of about 8.31 nm (VSM investigation). The HA-MNPs exhibited superparamagnetic behavior, with the magnetization curve showing saturation at a high magnetic field and a very small coercive field, corresponding to the net dominance of single-domain magnetic nanoparticles that were not aggregated with reversible magnetizability. These features satisfy the requirement for magnetic nanoparticles with a small size and good dispersibility for long-term stability. We performed some preliminary tests regarding the nanotoxicity in the environment, and some chromosomal aberrations were found to be induced in corn root meristems, especially in the anaphase and metaphase of mitotic cells. Due to their properties, HA-MNPs also seem to be suitable for use in the biomedical field.
PubMed: 38541384
DOI: 10.3390/ma17061229 -
Toxics Mar 2024Indigo carmine has a variety of uses in foods, textiles, medicine, pharmaceuticals, and cosmetics. There are studies reporting the toxic potential of indigo carmine on...
Indigo carmine has a variety of uses in foods, textiles, medicine, pharmaceuticals, and cosmetics. There are studies reporting the toxic potential of indigo carmine on human health and the environment. In this study, we investigated the cytogenotoxic effects of indigo carmine using apical root cells of . bulbs were subjected to four treatments with indigo carmine (0.0032, 0.0064, 0.0125, and 0.2 mg/mL) and to ultrapure water as a control. After 5 days, root growth, root length, mitotic index, mitotic inhibition, chromosomal anomalies, and cell morphology were analyzed. According to our results, a decrease in root length and mitotic index was observed at all concentrations of indigo carmine. Additionally, several types of chromosomal abnormalities were observed, such as disturbed metaphase, sticky chain metaphase, anaphase bridge, and laggard chromosomes. Moreover, histological observation indicated that indigo carmine induces alterations in various components of root tip tissue, such as deformation and alteration of the cell wall, progressive condensation of chromatin, shrinkage of the nuclei, and an increase in the number of irregularly shaped nuclei and nuclear fragments. Our results indicate that the tested concentrations of indigo carmine may have toxic effects and raise concerns about its intensive use in many fields.
PubMed: 38535927
DOI: 10.3390/toxics12030194