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Biomolecules Jun 2023Fluorescently labeled proteins absorb and emit light, appearing as Gaussian spots in fluorescence imaging. When fluorescent tags are added to cytoskeletal polymers such...
Fluorescently labeled proteins absorb and emit light, appearing as Gaussian spots in fluorescence imaging. When fluorescent tags are added to cytoskeletal polymers such as microtubules, a line of fluorescence and even non-linear structures results. While much progress has been made in techniques for imaging and microscopy, image analysis is less well-developed. Current analysis of fluorescent microtubules uses either manual tools, such as kymographs, or automated software. As a result, our ability to quantify microtubule dynamics and organization from light microscopy remains limited. Despite the development of automated microtubule analysis tools for in vitro studies, analysis of images from cells often depends heavily on manual analysis. One of the main reasons for this disparity is the low signal-to-noise ratio in cells, where background fluorescence is typically higher than in reconstituted systems. Here, we present the Toolkit for Automated Microtubule Tracking (TAMiT), which automatically detects, optimizes, and tracks fluorescent microtubules in living yeast cells with sub-pixel accuracy. Using basic information about microtubule organization, TAMiT detects linear and curved polymers using a geometrical scanning technique. Images are fit via an optimization problem for the microtubule image parameters that are solved using non-linear least squares in Matlab. We benchmark our software using simulated images and show that it reliably detects microtubules, even at low signal-to-noise ratios. Then, we use TAMiT to measure monopolar spindle microtubule bundle number, length, and lifetime in a large dataset that includes several mutants that affect microtubule dynamics and bundling. The results from the automated analysis are consistent with previous work and suggest a direct role for CLASP/Cls1 in bundling spindle microtubules. We also illustrate automated tracking of single curved astral microtubules in , with measurement of dynamic instability parameters. The results obtained with our fully-automated software are similar to results using hand-tracked measurements. Therefore, TAMiT can facilitate automated analysis of spindle and microtubule dynamics in yeast cells.
Topics: Saccharomyces cerevisiae; Microscopy, Fluorescence; Microtubules; Software
PubMed: 37371519
DOI: 10.3390/biom13060939 -
Animals : An Open Access Journal From... Jun 2023The spermatozoon ultrastructure of (Stossich, 1886) (Digenea: Opecoelidae), an intestinal parasite of the sheephead bream (Walbaum, 1792) (Sparidae), is described by...
The spermatozoon ultrastructure of (Stossich, 1886) (Digenea: Opecoelidae), an intestinal parasite of the sheephead bream (Walbaum, 1792) (Sparidae), is described by means of transmission electron microscopy (TEM). The mature spermatozoon possesses two axonemes of the 9+'1' trepaxonematan pattern, an anterior electron-dense material, two mitochondria, a nucleus and parallel cortical microtubules distributed in two bundles. The absence of external ornamentation of the plasma membrane and spine-like bodies are the noteworthy characters that distinguish the spermatozoon of from those of most opecoelids. In fact, only lacks external ornamentation in the spermatozoon. A comparative study with the remaining opecoelids described so far reveals similarities in the ultrastructural organization of their sperm cells. In addition, the current data on sperm ultrastructure in species of the recognized opecoelid subfamilies are compared, namely the Hamacreadiinae, Helicometrinae, Opecoelinae, Opistholebetinae and Plagioporinae.
PubMed: 37370463
DOI: 10.3390/ani13121953 -
Cell Jun 2023Inside sperm flagella, there are nine doublet microtubules composed of A and B tubules. In this issue of Cell, Leung et al. and Zhou et al. present high-resolution...
Inside sperm flagella, there are nine doublet microtubules composed of A and B tubules. In this issue of Cell, Leung et al. and Zhou et al. present high-resolution cryo-EM structures of doublet microtubules from mammalian sperms and show unprecedented structures of the A tubules, which are almost entirely occupied with tektin bundles.
Topics: Animals; Male; Semen; Microtubules; Microtubule Proteins; Sperm Tail; Flagella; Mammals
PubMed: 37352832
DOI: 10.1016/j.cell.2023.05.018 -
Cell Jun 2023Sperm motility is crucial to reproductive success in sexually reproducing organisms. Impaired sperm movement causes male infertility, which is increasing globally. Sperm...
Sperm motility is crucial to reproductive success in sexually reproducing organisms. Impaired sperm movement causes male infertility, which is increasing globally. Sperm are powered by a microtubule-based molecular machine-the axoneme-but it is unclear how axonemal microtubules are ornamented to support motility in diverse fertilization environments. Here, we present high-resolution structures of native axonemal doublet microtubules (DMTs) from sea urchin and bovine sperm, representing external and internal fertilizers. We identify >60 proteins decorating sperm DMTs; at least 15 are sperm associated and 16 are linked to infertility. By comparing DMTs across species and cell types, we define core microtubule inner proteins (MIPs) and analyze evolution of the tektin bundle. We identify conserved axonemal microtubule-associated proteins (MAPs) with unique tubulin-binding modes. Additionally, we identify a testis-specific serine/threonine kinase that links DMTs to outer dense fibers in mammalian sperm. Our study provides structural foundations for understanding sperm evolution, motility, and dysfunction at a molecular level.
Topics: Male; Animals; Cattle; Sperm Tail; Sperm Motility; Semen; Microtubules; Axoneme; Spermatozoa; Mammals
PubMed: 37327785
DOI: 10.1016/j.cell.2023.05.026 -
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 -
Current Protocols May 2023Microtubules, polymers of α, β-tubulin heterodimers, are organized into multi-microtubule arrays for diverse cellular functions. The dynamic properties of microtubule...
Microtubules, polymers of α, β-tubulin heterodimers, are organized into multi-microtubule arrays for diverse cellular functions. The dynamic properties of microtubule arrays govern their structural and functional properties. While numerous insights into the biophysical mechanisms underlying microtubule organization have been gleaned from in vitro reconstitution studies, the assays are largely restricted to visualization of single or pairs of microtubules. Thus, the dynamic processes underlying the remodeling of multi-microtubule arrays remain poorly understood. Recent work shows that Atomic Force Microscopy (AFM) enables the visualization of nanoscale dynamics within multi-microtubule 2D arrays. In this assay, electrostatic interactions permit the non-specific adsorption of microtubule arrays to mica. AFM imaging in tapping mode, a gentle method of imaging, allows the visualization of microtubules and protofilaments without sample damage. The height information captured by AFM imaging enables the tracking of structural changes in microtubules and protofilaments within multi-microtubule arrays over time. The experimental data from the method described here reveal previously unseen modes of nanoscale dynamics in microtubule bundles formed by the microtubule-crosslinking protein PRC1 in the presence of the depolymerase MCAK. The observations demonstrate the potential of AFM imaging in transforming our understanding of the fundamental cellular process by which multi-microtubule arrays are dynamically assembled and disassembled. © 2023 Wiley Periodicals LLC. Basic Protocol: Sample preparation and real-time visualization of microtubule arrays by atomic force microscopy Alternate Protocol: Protocol for coating surface with poly-L-lysine and immobilizing microtubules.
Topics: Microscopy, Atomic Force; Cytoskeleton; Microtubules; Tubulin; Adsorption
PubMed: 37227098
DOI: 10.1002/cpz1.779 -
IScience May 2023Cell centers their division apparatus to ensure symmetric cell division, a challenging task when the governing dynamics is stochastic. Using fission yeast, we show that...
Cell centers their division apparatus to ensure symmetric cell division, a challenging task when the governing dynamics is stochastic. Using fission yeast, we show that the patterning of nonequilibrium polymerization forces of microtubule (MT) bundles the precise localization of spindle pole body (SPB), and hence the division septum, at the onset of mitosis. We define two cellular objectives, , the mean SPB position relative to the geometric center, and , the variance of the SPB position, which are sensitive to genetic perturbations that change cell length, MT bundle number/orientation, and MT dynamics. We show that simultaneous control of reliability and robustness is required to minimize septum positioning error achieved by the wild type (WT). A stochastic model for the MT-based nucleus centering, with parameters measured directly or estimated using Bayesian inference, recapitulates the maximum fidelity of WT. Using this, we perform a sensitivity analysis of the parameters that control nuclear centering.
PubMed: 37182105
DOI: 10.1016/j.isci.2023.106665 -
Centralspindlin proteins Pavarotti and Tumbleweed along with WASH regulate nuclear envelope budding.The Journal of Cell Biology Aug 2023Nuclear envelope (NE) budding is a nuclear pore-independent nuclear export pathway, analogous to the egress of herpesviruses, and required for protein quality control,...
Nuclear envelope (NE) budding is a nuclear pore-independent nuclear export pathway, analogous to the egress of herpesviruses, and required for protein quality control, synapse development, and mitochondrial integrity. The physical formation of NE buds is dependent on the Wiskott-Aldrich Syndrome protein, Wash, its regulatory complex (SHRC), and Arp2/3, and requires Wash's actin nucleation activity. However, the machinery governing cargo recruitment and organization within the NE bud remains unknown. Here, we identify Pavarotti (Pav) and Tumbleweed (Tum) as new molecular components of NE budding. Pav and Tum interact directly with Wash and define a second nuclear Wash-containing complex required for NE budding. Interestingly, we find that the actin-bundling activity of Pav is required, suggesting a structural role in the physical and/or organizational aspects of NE buds. Thus, Pav and Tum are providing exciting new entry points into the physical machineries of this alternative nuclear export pathway for large cargos during cell differentiation and development.
Topics: Actins; Active Transport, Cell Nucleus; Cell Nucleus; Nuclear Envelope; Drosophila; Microtubule-Associated Proteins; GTPase-Activating Proteins; Drosophila Proteins
PubMed: 37163553
DOI: 10.1083/jcb.202211074 -
Frontiers in Neuroscience 2023Tubby-like proteins are membrane-associated adaptors that mediate directional trafficking into primary cilia. In inner ear sensory epithelia, cilia-including the hair...
Tubby-like proteins are membrane-associated adaptors that mediate directional trafficking into primary cilia. In inner ear sensory epithelia, cilia-including the hair cell's kinocilium-play important roles as organizers of polarity, tissue architecture and cellular function. However, auditory dysfunction in tubby mutant mice was recently found to be related to a non-ciliary function of tubby, the organization of a protein complex in sensory hair bundles of auditory outer hair cells (OHCs). Targeting of signaling components into cilia in the cochlea might therefore rather rely on closely related tubby-like proteins (TULPs). In this study, we compared cellular and subcellular localization of tubby and TULP3 in the mouse inner ear sensory organs. Immunofluorescence microscopy confirmed the previously reported highly selective localization of tubby in the stereocilia tips of OHCs and revealed a previously unnoticed transient localization to kinocilia during early postnatal development. TULP3 was detected in the organ of Corti and vestibular sensory epithelium, where it displayed a complex spatiotemporal pattern. TULP3 localized to kinocilia of cochlear and vestibular hair cells in early postnatal development but disappeared subsequently before the onset of hearing. This pattern suggested a role in targeting ciliary components into kinocilia, possibly related to the developmental processes that shape the sensory epithelia. Concurrent with loss from kinocilia, pronounced TULP3 immunolabeling progressively appeared at microtubule bundles in non-sensory Pillar (PCs) and Deiters cells (DC). This subcellular localization may indicate a novel function of TULP proteins associated with the formation or regulation of microtubule-based cellular structures.
PubMed: 37144094
DOI: 10.3389/fnins.2023.1162937 -
International Journal of Molecular... Apr 2023Bacterial pathogens have evolved intricate ways to manipulate the host to support infection. Here, we systematically assessed the importance of the microtubule...
Bacterial pathogens have evolved intricate ways to manipulate the host to support infection. Here, we systematically assessed the importance of the microtubule cytoskeleton for infection by , which are obligate intracellular bacteria that are of great importance for human health. The elimination of microtubules in human HEp-2 cells prior to infection profoundly attenuated the infection efficiency, demonstrating the need for microtubules for the early infection processes. To identify microtubule-modulating proteins, a screen in the model yeast was performed. Unexpectedly, among 116 selected chlamydial proteins, more than 10%, namely, 13 proteins, massively altered the yeast interphase microtubule cytoskeleton. With two exceptions, these proteins were predicted to be inclusion membrane proteins. As proof of principle, we selected the conserved CPn0443 protein, which caused massive microtubule instability in yeast, for further analysis. CPn0443 bound and bundled microtubules in vitro and co-localized partially with microtubules in vivo in yeast and human cells. Furthermore, CPn0443-transfected U2OS cells had a significantly reduced infection rate by EBs. Thus, our yeast screen identified numerous proteins encoded using the highly reduced genome that modulated microtubule dynamics. Hijacking of the host microtubule cytoskeleton must be a vital part of chlamydial infection.
Topics: Humans; Chlamydophila pneumoniae; Saccharomyces cerevisiae; Chlamydia trachomatis; Cytoskeleton; Microtubules; Schizosaccharomyces; Microtubule-Associated Proteins
PubMed: 37108781
DOI: 10.3390/ijms24087618