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IC2 participates in the cooperative activation of outer arm dynein densely attached to microtubules.Cell Structure and Function Sep 2023Ciliary outer-arm dynein (OAD) consists of heavy chains (HCs), intermediate chains (ICs), and light chains (LCs), of which HCs are the motor proteins that produce force....
Ciliary outer-arm dynein (OAD) consists of heavy chains (HCs), intermediate chains (ICs), and light chains (LCs), of which HCs are the motor proteins that produce force. Studies using the green alga Chlamydomonas have revealed that ICs and LCs form a complex (IC/LC tower) at the base of the OAD tail and play a crucial role in anchoring OAD to specific sites on the microtubule. In this study, we isolated a novel slow-swimming Chlamydomonas mutant deficient in the IC2 protein. This mutation, E279K, is in the third of the seven WD repeat domains. No apparent abnormality was observed in electron microscope observations of axonemes or in SDS-PAGE analyses of dynein subunits. To explore the reason for the lowered motility in this mutant, in vitro microtubule sliding experiments were performed, which revealed that the motor activity of the mutant OAD was lowered. In particular, a large difference was observed between wild type (WT) and the mutant in the microtubule sliding velocity in microtubule bundles formed with the addition of OAD: ~35.3 μm/sec (WT) and ~4.3 μm/sec (mutant). From this and other results, we propose that IC2 in an OAD interacts with the β HC of the adjacent OAD, and that an OAD-OAD interaction is important for efficient beating of cilia and flagella.Key words: cilia, axoneme, dynein heavy chain, cooperativity.
Topics: Dyneins; Microtubules; Axoneme; Cilia; Flagella; Chlamydomonas; Mutation
PubMed: 37518064
DOI: 10.1247/csf.23044 -
Cells Jul 2023The hypothesis about the role of the cortical cytoskeleton as the primary mechanosensor was tested. oocytes were exposed to simulated microgravity (by 3D clinorotation...
The hypothesis about the role of the cortical cytoskeleton as the primary mechanosensor was tested. oocytes were exposed to simulated microgravity (by 3D clinorotation in random directions with 4 rotations per minute-sµg group) and hypergravity at the 2 g level (by centrifugal force from one axis rotation-hg group) for 30, 90, and 210 min without and with cytochalasin B, colchicine, acrylamide, and calyculin A. Cell stiffness was measured by atomic force microscopy, protein content in the membrane and cytoplasmic fractions by Western blotting, and cellular respiration by polarography. The obtained results indicate that the stiffness of the cortical cytoskeleton of oocytes decreases in simulated micro- (after 90 min) and hypergravity (after 30 min), possibly due to intermediate filaments. The cell stiffness recovered after 210 min in the hg group, but intact microtubules were required for this. Already after 30 min of exposure to sµg, the cross-sectional area of oocytes decreased, which indicates deformation, and the singed protein, which organizes microfilaments into longitudinal bundles, diffused from the cortical cytoskeleton into the cytoplasm. Under hg, after 30 min, the cross-sectional area of the oocytes increased, and the proteins that organize filament networks, alpha-actinin and spectrin, diffused from the cortical cytoskeleton.
Topics: Animals; Drosophila melanogaster; Hypergravity; Cytoskeleton; Oocytes; Mercury
PubMed: 37508484
DOI: 10.3390/cells12141819 -
PNAS Nexus Jul 2023The cytoskeleton is a major focus of physical studies to understand organization inside cells given its primary role in cell motility, cell division, and cell mechanics....
The cytoskeleton is a major focus of physical studies to understand organization inside cells given its primary role in cell motility, cell division, and cell mechanics. Recently, protein condensation has been shown to be another major intracellular organizational strategy. Here, we report that the microtubule crosslinking proteins, MAP65-1 and PRC1, can form phase separated condensates at physiological salt and temperature without additional crowding agents in vitro. The size of the droplets depends on the concentration of protein. MAP65 condensates are liquid at first and can gelate over time. We show that these condensates can nucleate and grow microtubule bundles that form asters, regardless of the viscoelasticity of the condensate. The droplet size directly controls the number of projections in the microtubule asters, demonstrating that the MAP65 concentration can control the organization of microtubules. When gel-like droplets nucleate and grow asters from a shell of tubulin at the surface, the microtubules are able to re-fluidize the MAP65 condensate, returning the MAP65 molecules to solution. This work implies that there is an interplay between condensate formation from microtubule-associated proteins, microtubule organization, and condensate dissolution that could be important for the dynamics of intracellular organization.
PubMed: 37497046
DOI: 10.1093/pnasnexus/pgad231 -
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 -
Molecular Biology of the Cell Sep 2023During anaphase, antiparallel-overlapping midzone microtubules elongate and form bundles, contributing to chromosome segregation and the location of contractile ring... (Comparative Study)
Comparative Study
During anaphase, antiparallel-overlapping midzone microtubules elongate and form bundles, contributing to chromosome segregation and the location of contractile ring formation. Midzone microtubules are dynamic in early but not late anaphase; however, the kinetics and mechanisms of stabilization are incompletely understood. Using photoactivation of cells expressing PA-EGFP-α-tubulin we find that immediately after anaphase onset, a single highly dynamic population of midzone microtubules is present; as anaphase progresses, both dynamic and stable populations of midzone microtubules coexist. By mid-cytokinesis, only static, non-dynamic microtubules are detected. The velocity of microtubule sliding also decreases as anaphase progresses, becoming undetectable by late anaphase. Following depletion of PRC1, midzone microtubules remain highly dynamic in anaphase and fail to form static arrays in telophase despite furrowing. Cells depleted of Kif4a contain elongated PRC1 overlap zones and fail to form static arrays in telophase. Cells blocked in cytokinesis form short PRC1 overlap zones that do not coalesce laterally; these cells also fail to form static arrays in telophase. Together, our results demonstrate that dynamic turnover and sliding of midzone microtubules is gradually reduced during anaphase and that the final transition to a static array in telophase requires both lateral and longitudinal compaction of PRC1 containing overlap zones.
Topics: Humans; Anaphase; Cell Cycle Proteins; Cytokinesis; Microtubules; Spindle Apparatus; Tubulin
PubMed: 37467037
DOI: 10.1091/mbc.E23-02-0049 -
Prilozi (Makedonska Akademija Na... Jul 2023The glomerulopathies associated with the deposition of extracellular fibrils in the glomeruli are subdivided into Congo red positive (amyloidosis) and Congo red negative...
The glomerulopathies associated with the deposition of extracellular fibrils in the glomeruli are subdivided into Congo red positive (amyloidosis) and Congo red negative (non-amyloidotic glomerulopathies) based on Congo red staining. The non-amyloidotic glomerulopathies are divided into immunoglobulin-derived and non-immunoglobulin-derived glomerulopathies. The immunoglobulin-derived glomerulopathies: fibrillary glomerulopathy (FGn) and immunotactoid glomerulopathy (ITG) are rare glomerulopathies. The diagnosis of fibrillary-immunotactoid glomerulopathy depends on electron microscopy, which shows the presence of microfibrils in the glomeruli. The microfibrils in FGn are randomly arranged with diameters less than 30 nm. The microfibrils in ITG are larger than 30 nm with a visible lumen (microtubules), focally arranged in parallel bundles. Patients with fibrillary-immunotactoid glomerulopathy present with proteinuria (usually in the nephrotic range), microscopic hematuria, arterial hypertension, and chronic kidney disease that progresses to kidney failure over months to years. Currently, there are no guidelines for the treatment of fibrillary-immunotactoid glomerulopathy, although immunotactoid glomerulopathy could be associated with underlying hematologic disorders with the need for clone-directed therapy.
Topics: Humans; Congo Red; Kidney Diseases; Kidney Glomerulus; Glomerulonephritis; Proteinuria
PubMed: 37453107
DOI: 10.2478/prilozi-2023-0030 -
PLoS Genetics Jul 2023Spermatozoa in animal species are usually highly elongated cells with a long motile tail attached to a head that contains the haploid genome in a compact and often...
Spermatozoa in animal species are usually highly elongated cells with a long motile tail attached to a head that contains the haploid genome in a compact and often elongated nucleus. In Drosophila melanogaster, the nucleus is compacted two hundred-fold in volume during spermiogenesis and re-modeled into a needle that is thirty-fold longer than its diameter. Nuclear elongation is preceded by a striking relocalization of nuclear pore complexes (NPCs). While NPCs are initially located throughout the nuclear envelope (NE) around the spherical nucleus of early round spermatids, they are later confined to one hemisphere. In the cytoplasm adjacent to this NPC-containing NE, the so-called dense complex with a strong bundle of microtubules is assembled. While this conspicuous proximity argued for functional significance of NPC-NE and microtubule bundle, experimental confirmation of their contributions to nuclear elongation has not yet been reported. Our functional characterization of the spermatid specific Mst27D protein now resolves this deficit. We demonstrate that Mst27D establishes physical linkage between NPC-NE and dense complex. The C-terminal region of Mst27D binds to the nuclear pore protein Nup358. The N-terminal CH domain of Mst27D, which is similar to that of EB1 family proteins, binds to microtubules. At high expression levels, Mst27D promotes bundling of microtubules in cultured cells. Microscopic analyses indicated co-localization of Mst27D with Nup358 and with the microtubule bundles of the dense complex. Time-lapse imaging revealed that nuclear elongation is accompanied by a progressive bundling of microtubules into a single elongated bundle. In Mst27D null mutants, this bundling process does not occur and nuclear elongation is abnormal. Thus, we propose that Mst27D permits normal nuclear elongation by promoting the attachment of the NPC-NE to the microtubules of the dense complex, as well as the progressive bundling of these microtubules.
Topics: Male; Animals; Nuclear Pore; Drosophila; Drosophila melanogaster; Microtubules; Spermatogenesis; Nuclear Envelope; Microtubule-Associated Proteins; Drosophila Proteins
PubMed: 37428798
DOI: 10.1371/journal.pgen.1010837 -
Science Advances Jul 2023A continuum description is essential for understanding a variety of collective phenomena in active matter. However, building quantitative continuum models of active...
A continuum description is essential for understanding a variety of collective phenomena in active matter. However, building quantitative continuum models of active matter from first principles can be extremely challenging due to both the gaps in our knowledge and the complicated structure of nonlinear interactions. Here, we use a physically informed data-driven approach to construct a complete mathematical model of an active nematic from experimental data describing kinesin-driven microtubule bundles confined to an oil-water interface. We find that the structure of the model is similar to the Leslie-Ericksen and Beris-Edwards models, but there are appreciable and important differences. Rather unexpectedly, elastic effects are found to play no role in the experiments considered, with the dynamics controlled entirely by the balance between active stresses and friction stresses.
PubMed: 37406118
DOI: 10.1126/sciadv.abq6120 -
The FEBS Journal Oct 2023FKBP25 (FKBP3 gene) is a dual-domain PPIase protein that consists of a C-terminal PPIase domain and an N-terminal basic tilted helix bundle (BTHB). The PPIase domain of...
FKBP25 (FKBP3 gene) is a dual-domain PPIase protein that consists of a C-terminal PPIase domain and an N-terminal basic tilted helix bundle (BTHB). The PPIase domain of FKBP25 has been shown to bind to microtubules, which has impacts upon microtubule polymerisation and cell cycle progression. Using quantitative proteomics, it was recently found that FKBP25 was expressed in the top 10% of the mouse skeletal muscle proteome. However, to date there have been few studies investigating the role of FKBP25 in non-transformed systems. As such, this study aimed to investigate potential roles for FKBP25 in myoblast viability, migration and differentiation and in adaptation of mature skeletal muscle. Doxycycline-inducible FKBP25 knockdown in C2C12 myoblasts revealed an increase in cell accumulation/viability and migration in vitro that was independent of alterations in tubulin dynamics; however, FKBP25 knockdown had no discernible impact on myoblast differentiation into myotubes. Finally, a series of in vivo models of muscle adaptation were assessed, where it was observed that FKBP25 protein expression was increased in hypertrophy and regeneration conditions (chronic mechanical overload and the mdx model of Duchenne muscular dystrophy) but decreased in an atrophy model (denervation). Overall, the findings of this study establish FKBP25 as a regulator of myoblast viability and migration, with possible implications for satellite cell proliferation and migration and muscle regeneration, and as a potential regulator of in vivo skeletal muscle adaptation.
Topics: Mice; Animals; Mice, Inbred mdx; Muscle, Skeletal; Muscle Fibers, Skeletal; Myoblasts; Cell Differentiation; Peptidylprolyl Isomerase
PubMed: 37345229
DOI: 10.1111/febs.16894 -
Genetics Aug 2023Gamete formation is essential for sexual reproduction in metazoans. Meiosis in males gives rise to spermatids that must differentiate and individualize into mature...
Gamete formation is essential for sexual reproduction in metazoans. Meiosis in males gives rise to spermatids that must differentiate and individualize into mature sperm. In Drosophila melanogaster, individualization of interconnected spermatids requires the formation of individualization complexes that synchronously move along the sperm bundles. Here, we show that Mob4, a member of the Mps-one binder family, is essential for male fertility but has no detectable role in female fertility. We show that Mob4 is required for proper axonemal structure and its loss leads to male sterility associated with defective spermatid individualization and absence of mature sperm in the seminal vesicles. Transmission electron micrographs of developing spermatids following mob4RNAi revealed expansion of the outer axonemal microtubules such that the 9 doublets no longer remained linked to each other and defective mitochondrial organization. Mob4 is a STRIPAK component, and male fertility is similarly impaired upon depletion of the STRIPAK components, Strip and Cka. Expression of the human Mob4 gene rescues all phenotypes of Drosophila mob4 downregulation, indicating that the gene is evolutionarily and functionally conserved. Together, this suggests that Mob4 contributes to the regulation of the microtubule- and actin-cytoskeleton during spermatogenesis through the conserved STRIPAK complex. Our study advances the understanding of male infertility by uncovering the requirement for Mob4 in sperm individualization.
Topics: Animals; Female; Humans; Male; Adaptor Proteins, Signal Transducing; Drosophila; Drosophila melanogaster; Drosophila Proteins; Infertility, Male; Nerve Tissue Proteins; Semen; Spermatids; Spermatogenesis; Testis
PubMed: 37259670
DOI: 10.1093/genetics/iyad104