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International Journal of Biological... 2024Cysteine-rich angiogenic inducer 61 (CYR61), also called CCN1, has long been characterized as a secretory protein. Nevertheless, the intracellular function of CYR61...
Cysteine-rich angiogenic inducer 61 (CYR61), also called CCN1, has long been characterized as a secretory protein. Nevertheless, the intracellular function of CYR61 remains unclear. Here, we found that CYR61 is important for proper cell cycle progression. Specifically, CYR61 interacts with microtubules and promotes microtubule polymerization to ensure mitotic entry. Moreover, CYR61 interacts with PLK1 and accumulates during the mitotic process, followed by degradation as mitosis concludes. The proteolysis of CYR61 requires the PLK1 kinase activity, which directly phosphorylates two conserved motifs on CYR61, enhancing its interaction with the SCF E3 complex subunit FBW7 and mediating its degradation by the proteasome. Mutations of phosphorylation sites of Ser167 and Ser188 greatly increase CYR61's stability, while deletion of CYR61 extends prophase and metaphase and delays anaphase onset. In summary, our findings highlight the precise control of the intracellular CYR61 by the PLK1-FBW7 pathway, accentuating its significance as a microtubule-associated protein during mitotic progression.
Topics: Protein Serine-Threonine Kinases; Humans; Polo-Like Kinase 1; Mitosis; Cell Cycle Proteins; Proto-Oncogene Proteins; Cysteine-Rich Protein 61; Microtubules; F-Box-WD Repeat-Containing Protein 7; HeLa Cells; Phosphorylation; Ubiquitin-Protein Ligases; Microtubule-Associated Proteins
PubMed: 38904029
DOI: 10.7150/ijbs.93335 -
Developmental Cell Jun 2024The forces that orient the spindle in human cells remain poorly understood due to a lack of direct mechanical measurements in mammalian systems. We use magnetic tweezers...
The forces that orient the spindle in human cells remain poorly understood due to a lack of direct mechanical measurements in mammalian systems. We use magnetic tweezers to measure the force on human mitotic spindles. Combining the spindle's measured resistance to rotation, the speed at which it rotates after laser ablating astral microtubules, and estimates of the number of ablated microtubules reveals that each microtubule contacting the cell cortex is subject to ∼5 pN of pulling force, suggesting that each is pulled on by an individual dynein motor. We find that the concentration of dynein at the cell cortex and extent of dynein clustering are key determinants of the spindle's resistance to rotation, with little contribution from cytoplasmic viscosity, which we explain using a biophysically based mathematical model. This work reveals how pulling forces on astral microtubules determine the mechanics of spindle orientation and demonstrates the central role of cortical dynein clustering.
PubMed: 38866013
DOI: 10.1016/j.devcel.2024.05.022 -
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 -
International Journal of Radiation... May 2024To analyze the effects of extending lymphocyte cultivation time on the Mitotic Index, frequency of first-division cells, and dose estimation after irradiating blood...
PURPOSE
To analyze the effects of extending lymphocyte cultivation time on the Mitotic Index, frequency of first-division cells, and dose estimation after irradiating blood samples with different doses of radiation.
MATERIALS AND METHODS
Blood samples from two healthy male volunteers were separately irradiated with three doses (3, 5, and 6 Gy) using a Co gamma source (average dose rate: 1.48 kGy.h) and cultivated for conventional (48 h) and extended (56, 68, and 72 h) amounts of time. Colcemid (0.01 µg.mL) was added at the beginning of the culture period. Cells were fixed, stained with (FPG), and analyzed under a light microscope. The effects of prolonged culture duration on the Mitotic Index (MI), frequency of first-division cells (M1 cells), and the First-Division Mitotic Index (FDMI) were investigated. The estimation of delivered doses was conducted using a conventional 48h-culture calibration curve.
RESULTS
Overall, cells presented higher MI (up to 12-fold) with the extension of culture, while higher radiation doses led to lower MI values (up to 80% reduction at 48 h). Cells irradiated with higher doses (5 and 6 Gy) had the most significant increase (5- to 12-fold) of MI as the cultivation was prolonged. The frequency of M cells decreased with the prolongation of culture for all doses (up to 75% reduction), while irradiated cells presented higher frequencies of M cells than non-irradiated ones. FDMI increased for all irradiated cultures but most markedly in those irradiated with higher doses (up to 10-fold). The conventional 48h-culture calibration curve proved adequate for assessing the delivered dose based on dicentric frequency following a 72-hour culture.
CONCLUSION
Compared to the conventional 48-hour protocol, extending the culture length to 72 hours significantly increased the Mitotic Index and the number of first-division metaphases of irradiated lymphocytes, providing slides with a better scorable metaphase density. Extending the culture time to 72 hours, combined with FPG staining to score exclusively first-division metaphases, improved the counting of dicentric chromosomes. The methodology presented and discussed in this study can be a powerful tool for dicentric-based biodosimetry, especially when exposure to high radiation doses is involved.
PubMed: 38787719
DOI: 10.1080/09553002.2024.2356545 -
The Journal of Cell Biology Aug 2024Accurate chromosome segregation requires sister kinetochores to biorient, attaching to opposite spindle poles. To this end, the mammalian kinetochore destabilizes...
Accurate chromosome segregation requires sister kinetochores to biorient, attaching to opposite spindle poles. To this end, the mammalian kinetochore destabilizes incorrect attachments and stabilizes correct ones, but how it discriminates between these is not yet clear. Here, we test the model that kinetochore tension is the stabilizing cue and ask how chromosome size impacts that model. We live image PtK2 cells, with just 14 chromosomes, widely ranging in size, and find that long chromosomes align at the metaphase plate later than short chromosomes. Enriching for errors and imaging error correction live, we show that long chromosomes exhibit a specific delay in correcting attachments. Using chromokinesin overexpression and laser ablation to perturb polar ejection forces, we find that chromosome size and force on arms determine alignment order. Thus, we propose a model where increased force on long chromosomes can falsely stabilize incorrect attachments, delaying their biorientation. As such, long chromosomes may require compensatory mechanisms for correcting errors to avoid chromosomal instability.
Topics: Animals; Cell Line; Chromosome Segregation; Chromosomes, Mammalian; Kinetochores; Mitosis; Spindle Apparatus; Potoroidae
PubMed: 38727808
DOI: 10.1083/jcb.202310010 -
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