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BioRxiv : the Preprint Server For... Aug 2023The cytoplasmic dynein-1 (dynein) motor organizes cells by shaping microtubule networks and moving a large variety of cargoes along them. However, dynein's diverse roles...
The cytoplasmic dynein-1 (dynein) motor organizes cells by shaping microtubule networks and moving a large variety of cargoes along them. However, dynein's diverse roles complicate studies of its functions significantly. To address this issue, we have used gene editing to generate a series of missense mutations in Dynein heavy chain (Dhc). We find that mutations associated with human neurological disease cause a range of defects in larval and adult flies, including impaired cargo trafficking in neurons. We also describe a novel mutation in the microtubule-binding domain (MTBD) of Dhc that, remarkably, causes metaphase arrest of mitotic spindles in the embryo but does not impair other dynein-dependent processes. We demonstrate that the mitotic arrest is independent of dynein's well-established roles in silencing the spindle assembly checkpoint. reconstitution and optical trapping assays reveal that the mutation only impairs the performance of dynein under load. all-atom molecular dynamics simulations show that this effect correlates with increased flexibility of the MTBD, as well as an altered orientation of the stalk domain, with respect to the microtubule. Collectively, our data point to a novel role of dynein in anaphase progression that depends on the motor operating in a specific load regime. More broadly, our work illustrates how cytoskeletal transport processes can be dissected by manipulating mechanical properties of motors.
PubMed: 37577480
DOI: 10.1101/2023.08.03.551815 -
PLoS Biology Aug 2023Both the spindle microtubule-organizing centers and the nuclear pore complexes (NPCs) are convoluted structures where many signaling pathways converge to coordinate key...
Both the spindle microtubule-organizing centers and the nuclear pore complexes (NPCs) are convoluted structures where many signaling pathways converge to coordinate key events during cell division. Interestingly, despite their distinct molecular conformation and overall functions, these structures share common components and collaborate in the regulation of essential processes. We have established a new link between microtubule-organizing centers and nuclear pores in budding yeast by unveiling an interaction between the Bfa1/Bub2 complex, a mitotic exit inhibitor that localizes on the spindle pole bodies, and the Nup159 nucleoporin. Bfa1/Bub2 association with Nup159 is reduced in metaphase to not interfere with proper spindle positioning. However, their interaction is stimulated in anaphase and assists the Nup159-dependent autophagy pathway. The asymmetric localization of Bfa1/Bub2 during mitosis raises the possibility that its interaction with Nup159 could differentially promote Nup159-mediated autophagic processes, which might be relevant for the maintenance of the replicative lifespan.
Topics: Cell Cycle Proteins; Nuclear Pore Complex Proteins; Saccharomyces cerevisiae Proteins; Saccharomyces cerevisiae; Spindle Pole Bodies; Cytoskeletal Proteins; Spindle Apparatus; Mitosis
PubMed: 37535687
DOI: 10.1371/journal.pbio.3002224 -
Frontiers in Cell and Developmental... 2023Faithful chromosome segregation during cell division requires accurate mitotic spindle formation. As mitosis occurs rapidly within the cell cycle, the proteins involved...
Faithful chromosome segregation during cell division requires accurate mitotic spindle formation. As mitosis occurs rapidly within the cell cycle, the proteins involved in mitotic spindle assembly undergo rapid changes, including their interactions with other proteins. The proper localization of the HURP protein on the kinetochore fibers, in close proximity to chromosomes, is crucial for ensuring accurate congression and segregation of chromosomes. In this study, we employ photoactivation and FRAP experiments to investigate the impact of alterations in microtubule flux and phosphorylation of HURP at the Ser627 residue on its dynamics. Furthermore, through immunoprecipitations assays, we demonstrate the interactions of HURP with various proteins, such as TPX2, Aurora A, Eg5, Dynein, Kif5B, and Importin , in mammalian cells during mitosis. We also find that phosphorylation of HURP at Ser627 regulates its interaction with these partners during mitosis. Our findings suggest that HURP participates in at least two distinct complexes during metaphase to ensure its proper localization in close proximity to chromosomes, thereby promoting the bundling and stabilization of kinetochore fibers.
PubMed: 37484914
DOI: 10.3389/fcell.2023.981425 -
Biochemical and Biophysical Research... Oct 2023We previously identified a cell cycle-dependent periodic subcellular distribution of cancer metastasis-associated antigen 1 (MTA1) and unraveled a novel role of MTA1 in...
We previously identified a cell cycle-dependent periodic subcellular distribution of cancer metastasis-associated antigen 1 (MTA1) and unraveled a novel role of MTA1 in inhibiting spindle damage-induced spindle assembly checkpoint (SAC) activation in cancer cells. However, the more detailed subcellular localization of MTA1 in mitotic cells and its copartner in SAC regulation in cancer cells are still poorly understood. Here, through immunofluorescent colocalization analysis of MTA1 and alpha-tubulin in mitotic cancer cells, we reveal that MTA1 is dynamically localized to the spindle apparatus throughout the entire mitotic process. We also demonstrated a reversible upregulation of MTA1 expression upon spindle damage-induced SAC activation, and time-lapse imaging assays indicated that MTA1 silencing delayed the mitotic metaphase-anaphase transition in cancer cells. Further investigation revealed that MTA1 interacts and colocalizes with Translocated Promoter Region (TPR) on spindle microtubules in mitotic cells, and this interaction is attenuated on SAC activation. TPR is well-implicated in SAC regulation via binding the MAD1-MAD2 complex, however, no interactions between MTA1 and MAD1 or MAD2 were detected in our coimmunoprecipitation (co-IP) assays, suggesting that the MTA1-TPR may represent a distinct SAC-associated complex separate from the previously reported TPR-MAD1/MAD2 complex. Our data provide new insights into the subcellular localization and molecular function of MTA1 in SAC regulation in cancer, and indicate that intervention of the MTA1-TPR interaction may be effective to modulate SAC and hence chromosomal instability (CIN) in tumorigenesis.
Topics: Cell Cycle Proteins; M Phase Cell Cycle Checkpoints; Nuclear Proteins; Spindle Apparatus; Cell Cycle Checkpoints; Mad2 Proteins; Kinetochores
PubMed: 37467663
DOI: 10.1016/j.bbrc.2023.07.021 -
Centrosome linker diversity and its function in centrosome clustering and mitotic spindle formation.The EMBO Journal Sep 2023The centrosome linker joins the two interphase centrosomes of a cell into one microtubule organizing center. Despite increasing knowledge on linker components, linker...
The centrosome linker joins the two interphase centrosomes of a cell into one microtubule organizing center. Despite increasing knowledge on linker components, linker diversity in different cell types and their role in cells with supernumerary centrosomes remained unexplored. Here, we identified Ninein as a C-Nap1-anchored centrosome linker component that provides linker function in RPE1 cells while in HCT116 and U2OS cells, Ninein and Rootletin link centrosomes together. In interphase, overamplified centrosomes use the linker for centrosome clustering, where Rootletin gains centrosome linker function in RPE1 cells. Surprisingly, in cells with centrosome overamplification, C-Nap1 loss prolongs metaphase through persistent activation of the spindle assembly checkpoint indicated by BUB1 and MAD1 accumulation at kinetochores. In cells lacking C-Nap1, the reduction of microtubule nucleation at centrosomes and the delay in nuclear envelop rupture in prophase probably cause mitotic defects like multipolar spindle formation and chromosome mis-segregation. These defects are enhanced when the kinesin HSET, which normally clusters multiple centrosomes in mitosis, is partially inhibited indicating a functional interplay between C-Nap1 and centrosome clustering in mitosis.
Topics: Centrosome; Cell Cycle; Cell Cycle Proteins; Interphase; Mitosis; Spindle Apparatus
PubMed: 37401899
DOI: 10.15252/embj.2021109738 -
BioRxiv : the Preprint Server For... Jun 2023Myosin 10 (Myo10) has the ability to link actin filaments to integrin-based adhesions and to microtubules by virtue of its integrin-binding FERM domain and...
Myosin 10 (Myo10) has the ability to link actin filaments to integrin-based adhesions and to microtubules by virtue of its integrin-binding FERM domain and microtubule-binding MyTH4 domain, respectively. Here we used Myo10 knockout cells to define Myo10's contribution to the maintenance of spindle bipolarity, and complementation to quantitate the relative contributions of its MyTH4 and FERM domains. Myo10 knockout HeLa cells and mouse embryo fibroblasts (MEFs) both exhibit a pronounced increase in the frequency of multipolar spindles. Staining of unsynchronized metaphase cells showed that the primary driver of spindle multipolarity in knockout MEFs and knockout HeLa cells lacking supernumerary centrosomes is pericentriolar material (PCM) fragmentation, which creates γ-tubulin-positive acentriolar foci that serve as additional spindle poles. For HeLa cells possessing supernumerary centrosomes, Myo10 depletion further accentuates spindle multipolarity by impairing the clustering of the extra spindle poles. Complementation experiments show that Myo10 must interact with both integrins and microtubules to promote PCM/pole integrity. Conversely, Myo10's ability to promote the clustering of supernumerary centrosomes only requires that it interact with integrins. Importantly, images of Halo-Myo10 knock-in cells show that the myosin localizes exclusively within adhesive retraction fibers during mitosis. Based on these and other results, we conclude that Myo10 promotes PCM/pole integrity at a distance, and that it facilitates supernumerary centrosome clustering by promoting retraction fiber-based cell adhesion, which likely provides an anchor for the microtubule-based forces driving pole focusing.
PubMed: 37398378
DOI: 10.1101/2023.06.15.545002 -
Microscopy Research and Technique Aug 2023Industrial activities and unconscious consumption of natural resources cause environmental pollution with the rapid increase in the world population. As a result of the...
Industrial activities and unconscious consumption of natural resources cause environmental pollution with the rapid increase in the world population. As a result of the widespread use of iron oxide nanoparticles (Fe O NP) with nano-industrial activities, it is predicted that this NP will accumulate in the air, water, and soil. In the present study, the purpose was to find out the genotoxic effects on root meristem cells of the Triticum aestivum L. plant, which is an indicator organism exposed to 20-40 nm Fe O NPs at different concentrations (100, 200, and 400 ppm). The amount of Fe O NP accumulated in T. aestivum used in the study was determined with x-ray diffraction (XRD) spectroscopy, scanning electron microscopy (SEM), SEM element map, and EDS characteristic spectrum. All concentrations of Fe O NP caused significant decreases in the mitotic index. Fe O NPs significantly increased the frequency of mitotic abnormalities in T. aestivum root tip cells at all treatment times and all concentrations when compared to the control. Fe O NPs were formed by various mitotic abnormalities such as loss of genetic material, deconstructed prophase, adhesion, chromosome groupings in metaphase, deconstructed metaphase, C-Metaphase, chromosomal loss, chromosomal fracture, polyploidy, deconstructed anaphase, lagging chromosome, fragment, polar deviation, bridging, propagation, asynchronous division, star anaphase, multipolarity, and deconstructed telophase. All these results show that Fe O NPs are genotoxic and clastogenic and may also cause DNA damage. Briefly, these data show that Fe O NPs taken by organisms may pose a danger to the organism and the upper consumer. These findings also show that the production and use of Fe O NPs, which affect organisms, must be controlled, and ultimately, be safely disposed of to reduce their bioaccumulation. RESEARCH HIGHLIGHTS: In the present study, the purpose was to find out the genotoxic effects on root meristem cells of the Triticum aestivum L. (bread wheat) plant, which is an indicator organism exposed to 20-40 nm Fe O NPs at different concentrations (100, 200, and 400 ppm). The amount of Fe O NP accumulated in T. aestivum used in the study was determined with x-ray diffraction (XRD) spectroscopy, scanning electron microscopy (SEM), SEM element map, and EDS characteristic spectrum. The mitotic index was calculated to reveal the genotoxic effect. "Loss of genetic material, deconstructed prophase, adhesion, chromosome groupings in metaphase, deconstructed metaphase, C-Metaphase, chromosomal loss, chromosomal fracture, polyploidy, deconstructed anaphase, lagging chromosome, fragment, polar deviation, bridging, propagation, asynchronous division, star Chromosomal abnormalities such as anaphase, multipolarity, and deconstructed telophase" were visualized. Fe O NPs are genotoxic and clastogenic and may also cause DNA damage.
Topics: Triticum; Nanoparticles; DNA Damage; Chromosome Aberrations; Polyploidy
PubMed: 37357999
DOI: 10.1002/jemt.24377 -
Cell Cycle (Georgetown, Tex.) Jul 2023Tightly controlled fluctuations in kinase and phosphatase activity play important roles in regulating M-phase transitions. Protein Phosphatase 1 (PP1) is one of these...
Tightly controlled fluctuations in kinase and phosphatase activity play important roles in regulating M-phase transitions. Protein Phosphatase 1 (PP1) is one of these phosphatases, with oscillations in PP1 activity driving mitotic M-phase. Evidence from a variety of experimental systems also points to roles in meiosis. Here, we report that PP1 is important for M-phase transitions through mouse oocyte meiosis. We employed a unique small-molecule approach to inhibit or activate PP1 at distinct phases of mouse oocyte meiosis. These studies show that temporal control of PP1 activity is essential for the G2/M transition, metaphase I/anaphase I transition, and the formation of a normal metaphase II oocyte. Our data also reveal that inappropriate activation of PP1 is more deleterious at the G2/M transition than at prometaphase I-to-metaphase I, and that an active pool of PP1 during prometaphase is vital for metaphase I/anaphase I transition and metaphase II chromosome alignment. Taken together, these results establish that loss of oscillations in PP1 activity causes a range of severe meiotic defects, pointing to essential roles for PP1 in female fertility, and more broadly, M-phase regulation.
Topics: Female; Mice; Animals; Meiosis; Oocytes; Metaphase; Anaphase; Mitosis; Protein Phosphatase 1; Mammals
PubMed: 37340734
DOI: 10.1080/15384101.2023.2225924 -
Methods in Molecular Biology (Clifton,... 2023Despite more than a century of intensive study of mitotic chromosomes, their three-dimensional organization remains enigmatic. The last decade established Hi-C as a...
Despite more than a century of intensive study of mitotic chromosomes, their three-dimensional organization remains enigmatic. The last decade established Hi-C as a method of choice for study of spatial genome-wide interactions. Although its utilization has been focused mainly on studying genomic interactions in interphase nuclei, the method can be also successfully applied to study 3D architecture and genome folding in mitotic chromosomes. However, obtaining sufficient number of mitotic chromosomes as an input material and effective coupling with Hi-C method is challenging in plant species. An elegant way to overcome hindrances with obtaining a pure mitotic chromosome fraction is their isolation via flow cytometric sorting. This chapter presents a protocol describing plant sample preparation for chromosome conformation studies, for flow-sorting of plant mitotic metaphase chromosomes and for the Hi-C procedure.
Topics: Chromatin; Chromosomes; Cell Nucleus; Genomics; Molecular Conformation; Plants
PubMed: 37335495
DOI: 10.1007/978-1-0716-3226-0_29 -
Methods in Molecular Biology (Clifton,... 2023Recently developed bulked oligo-FISH is a highly versatile method, which is applicable in any plant species with an assembled genome sequence. This technique allows in...
Recently developed bulked oligo-FISH is a highly versatile method, which is applicable in any plant species with an assembled genome sequence. This technique allows in situ identification of individual chromosomes, large chromosomal rearrangements, comparative karyotype analysis, or even the reconstruction of the three-dimensional organization of the genome. The method is based on the identification of thousands of short oligonucleotides, unique to specific genome regions, which are synthesized in parallel, fluorescently labeled and used as probes for FISH. In this chapter, we propose a detailed protocol for amplification and labeling of single-stranded oligo-based painting probes from so-called MYtags immortal libraries, the preparation of mitotic metaphase and meiotic pachytene chromosome spreads, and a protocol for the fluorescence in situ hybridization procedure using the synthetic oligo probes. The proposed protocols are demonstrated for banana (Musa spp.).
Topics: Chromosome Painting; In Situ Hybridization, Fluorescence; Chromosomes, Plant; Karyotype; Karyotyping
PubMed: 37335493
DOI: 10.1007/978-1-0716-3226-0_27