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Developmental Biology Apr 2019Eggs have developed their own strategies for early development. Amphibian, teleost fish, and ascidian eggs show cortical rotation and an accompanying structure, a... (Review)
Review
Eggs have developed their own strategies for early development. Amphibian, teleost fish, and ascidian eggs show cortical rotation and an accompanying structure, a cortical parallel microtubule (MT) array, during the one-cell embryonic stage. Cortical rotation is thought to relocate maternal deposits to a certain compartment of the egg and to polarize the embryo. The common features and differences among chordate eggs as well as localized maternal proteins and mRNAs that are related to the organization of MT structures are described in this review. Furthermore, recent studies report progress in elucidating the molecular nature and functions of the noncentrosomal MT organizing center (ncMTOC). The parallel array of MT bundles is presumably organized by ncMTOCs; therefore, the mechanism of ncMTOC control is likely inevitable for these species. Thus, the molecules related to the ncMTOC provide clues for understanding the mechanisms of early developmental systems, which ultimately determine the embryonic axis.
Topics: Animals; Biological Transport; Centrosome; Chordata; Embryonic Development; Microtubules; Zygote
PubMed: 30521810
DOI: 10.1016/j.ydbio.2018.11.019 -
Proceedings of the National Academy of... Dec 2022Cellular morphogenesis and processes such as cell division and migration require the coordination of the microtubule and actin cytoskeletons. Microtubule-actin crosstalk...
Cellular morphogenesis and processes such as cell division and migration require the coordination of the microtubule and actin cytoskeletons. Microtubule-actin crosstalk is poorly understood and largely regarded as the capture and regulation of microtubules by actin. Septins are filamentous guanosine-5'-triphosphate (GTP) binding proteins, which comprise the fourth component of the cytoskeleton along microtubules, actin, and intermediate filaments. Here, we report that septins mediate microtubule-actin crosstalk by coupling actin polymerization to microtubule lattices. Superresolution and platinum replica electron microscopy (PREM) show that septins localize to overlapping microtubules and actin filaments in the growth cones of neurons and non-neuronal cells. We demonstrate that recombinant septin complexes directly crosslink microtubules and actin filaments into hybrid bundles. In vitro reconstitution assays reveal that microtubule-bound septins capture and align stable actin filaments with microtubules. Strikingly, septins enable the capture and polymerization of growing actin filaments on microtubule lattices. In neuronal growth cones, septins are required for the maintenance of the peripheral actin network that fans out from microtubules. These findings show that septins directly mediate microtubule interactions with actin filaments, and reveal a mechanism of microtubule-templated actin growth with broader significance for the self-organization of the cytoskeleton and cellular morphogenesis.
Topics: Septins; Actins; Microtubules
PubMed: 36475946
DOI: 10.1073/pnas.2202803119 -
Oxidative Medicine and Cellular... 2022Microtubules (MTs) are highly dynamic polymers essential for a wide range of cellular physiologies, such as acting as directional railways for intracellular transport... (Review)
Review
Microtubules (MTs) are highly dynamic polymers essential for a wide range of cellular physiologies, such as acting as directional railways for intracellular transport and position, guiding chromosome segregation during cell division, and controlling cell polarity and morphogenesis. Evidence has established that maintaining microtubule (MT) stability in neurons is vital for fundamental cellular and developmental processes, such as neurodevelopment, degeneration, and regeneration. To fulfill these diverse functions, the nervous system employs an arsenal of microtubule-associated proteins (MAPs) to control MT organization and function. Subsequent studies have identified that the disruption of MT function in neurons is one of the most prevalent and important pathological features of traumatic nerve damage and neurodegenerative diseases and that this disruption manifests as a reduction in MT polymerization and concomitant deregulation of the MT cytoskeleton, as well as downregulation of microtubule-associated protein (MAP) expression. A variety of MT-targeting agents that reverse this pathological condition, which is regarded as a therapeutic opportunity to intervene the onset and development of these nervous system abnormalities, is currently under development. Here, we provide an overview of the MT-intrinsic organization process and how MAPs interact with the MT cytoskeleton to promote MT polymerization, stabilization, and bundling. We also highlight recent advances in MT-targeting therapeutic agents applied to various neurological disorders. Together, these findings increase our current understanding of the function and regulation of MT organization in nerve growth and regeneration.
Topics: Cytoskeleton; Humans; Microtubules
PubMed: 35295719
DOI: 10.1155/2022/1623181 -
Nucleus (Austin, Tex.) 2014The nucleus is a cellular compartment that hosts several macro-molecular machines displaying a highly complex spatial organization. This tight architectural... (Review)
Review
The nucleus is a cellular compartment that hosts several macro-molecular machines displaying a highly complex spatial organization. This tight architectural orchestration determines not only DNA replication and repair but also regulates gene expression. In budding yeast microtubules play a key role in structuring the nucleus since they condition the Rabl arrangement in G1 and chromosome partitioning during mitosis through their attachment to centromeres via the kinetochore proteins. Recently, we have shown that upon quiescence entry, intranuclear microtubules emanating from the spindle pole body elongate to form a highly stable bundle that spans the entire nucleus. Here, we examine some molecular mechanisms that may underlie the formation of this structure. As the intranuclear microtubule bundle causes a profound re-organization of the yeast nucleus and is required for cell survival during quiescence, we discuss the possibility that the assembly of such a structure participates in quiescence establishment.
Topics: Animals; Cell Cycle; Cell Nucleus; Centromere; Gene Expression Regulation; Humans; Microtubules
PubMed: 24637834
DOI: 10.4161/nucl.28538 -
BMC Biology Oct 2018Upon maturation in the bone marrow, polyploid megakaryocytes elongate very long and thin cytoplasmic branches called proplatelets. Proplatelets enter the sinusoids blood...
BACKGROUND
Upon maturation in the bone marrow, polyploid megakaryocytes elongate very long and thin cytoplasmic branches called proplatelets. Proplatelets enter the sinusoids blood vessels in which platelets are ultimately released. Microtubule dynamics, bundling, sliding, and coiling, drive these dramatic morphological changes whose regulation remains poorly understood. Microtubule properties are defined by tubulin isotype composition and post-translational modification patterns. It remains unknown whether microtubule post-translational modifications occur in proplatelets and if so, whether they contribute to platelet formation.
RESULTS
Here, we show that in proplatelets from mouse megakaryocytes, microtubules are both acetylated and polyglutamylated. To bypass the difficulties of working with differentiating megakaryocytes, we used a cell model that allowed us to test the functions of these modifications. First, we show that α2bβ3integrin signaling in D723H cells is sufficient to induce β1tubulin expression and recapitulate the specific microtubule behaviors observed during proplatelet elongation and platelet release. Using this model, we found that microtubule acetylation and polyglutamylation occur with different spatio-temporal patterns. We demonstrate that microtubule acetylation, polyglutamylation, and β1tubulin expression are mandatory for proplatelet-like elongation, swelling formation, and cytoplast severing. We discuss the functional importance of polyglutamylation of β1tubulin-containing microtubules for their efficient bundling and coiling during platelet formation.
CONCLUSIONS
We characterized and validated a powerful cell model to address microtubule behavior in mature megakaryocytes, which allowed us to demonstrate the functional importance of microtubule acetylation and polyglutamylation for platelet release. Furthermore, we bring evidence of a link between the expression of a specific tubulin isotype, the occurrence of microtubule post-translational modifications, and the acquisition of specific microtubule behaviors. Thus, our findings could widen the current view of the regulation of microtubule behavior in cells such as osteoclasts, spermatozoa, and neurons, which express distinct tubulin isotypes and display specific microtubule activities during differentiation.
Topics: Acetylation; Animals; Blood Platelets; Megakaryocytes; Mice; Microtubules; Protein Processing, Post-Translational; Tubulin
PubMed: 30336771
DOI: 10.1186/s12915-018-0584-6 -
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 -
Journal of Visualized Experiments : JoVE May 2022Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as specialized arrays...
Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as specialized arrays during mitosis to regulate chromosome segregation. Proteins that interact with microtubules include motors such as kinesins and dynein, which can generate active forces and directional motion, as well as non-motor proteins that crosslink filaments into higher-order networks or regulate filament dynamics. To date, biophysical studies of microtubule-associated proteins have overwhelmingly focused on the role of single motor proteins needed for vesicle transport, and significant progress has been made in elucidating the force-generating properties and mechanochemical regulation of kinesins and dyneins. However, for processes in which microtubules act both as cargo and track, such as during filament sliding within the mitotic spindle, much less is understood about the biophysical regulation of ensembles of the crosslinking proteins involved. Here, we detail our methodology for directly probing force generation and response within crosslinked microtubule minimal networks reconstituted from purified microtubules and mitotic proteins. Microtubule pairs are crosslinked by proteins of interest, one microtubule is immobilized to a microscope coverslip, and the second microtubule is manipulated by an optical trap. Simultaneous total internal reflection fluorescence microscopy allows for multichannel visualization of all the components of this microtubule network as the filaments slide apart to generate force. We also demonstrate how these techniques can be used to probe pushing forces exerted by kinesin-5 ensembles and how viscous braking forces arise between sliding microtubule pairs crosslinked by the mitotic MAP PRC1. These assays provide insights into the mechanisms of spindle assembly and function and can be more broadly adapted to study dense microtubule network mechanics in diverse contexts, such as the axon and dendrites of neurons and polar epithelial cells.
Topics: Dyneins; Kinesins; Microtubule-Associated Proteins; Microtubules; Spindle Apparatus
PubMed: 35635475
DOI: 10.3791/63819 -
Scientific Reports Mar 2015Several strategies for controlling microtubule patterns are developed because of the rigidity determined from the molecular structure and the geometrical structure. In...
Several strategies for controlling microtubule patterns are developed because of the rigidity determined from the molecular structure and the geometrical structure. In contrast to the patterns in co-operation with motor proteins or associated proteins, microtubules have a huge potential for patterns via their intrinsic flexural rigidity. We discover that a microtubule teardrop pattern emerges via self-assembly under hydrodynamic flow from the parallel bundles without motor proteins. In the growth process, the bundles ultimately bend according to the critical bending curvature. Such protein pattern formation utilizing the intrinsic flexural rigidity will provide broad understandings of self-assembly of rigid rods, not only in biomolecules, but also in supramolecules.
Topics: Algorithms; Animals; Microscopy, Fluorescence; Microtubules; Models, Theoretical; Rhodamines; Tubulin
PubMed: 25823414
DOI: 10.1038/srep09581 -
Cells Mar 2019γ-Tubulin is a conserved member of the tubulin superfamily with a function in microtubule nucleation. Proteins of γ-tubulin complexes serve as nucleation templates as... (Review)
Review
γ-Tubulin is a conserved member of the tubulin superfamily with a function in microtubule nucleation. Proteins of γ-tubulin complexes serve as nucleation templates as well as a majority of other proteins contributing to centrosomal and non-centrosomal nucleation, conserved across eukaryotes. There is a growing amount of evidence of γ-tubulin functions besides microtubule nucleation in transcription, DNA damage response, chromatin remodeling, and on its interactions with tumor suppressors. However, the molecular mechanisms are not well understood. Furthermore, interactions with lamin and SUN proteins of the LINC complex suggest the role of γ-tubulin in the coupling of nuclear organization with cytoskeletons. γ-Tubulin that belongs to the clade of eukaryotic tubulins shows characteristics of both prokaryotic and eukaryotic tubulins. Both human and plant γ-tubulins preserve the ability of prokaryotic tubulins to assemble filaments and higher-order fibrillar networks. γ-Tubulin filaments, with bundling and aggregating capacity, are suggested to perform complex scaffolding and sequestration functions. In this review, we discuss a plethora of γ-tubulin molecular interactions and cellular functions, as well as recent advances in understanding the molecular mechanisms behind them.
Topics: Animals; Cell Cycle; Cell Nucleus; Humans; Microtubules; Nuclear Envelope; Nuclear Proteins; Tubulin
PubMed: 30893853
DOI: 10.3390/cells8030259 -
Yeast (Chichester, England) Oct 2006During the cell cycle of the fission yeast Schizosaccharomyces pombe, striking changes in the organization of the cytoplasmic microtubule cytoskeleton take place. These... (Review)
Review
During the cell cycle of the fission yeast Schizosaccharomyces pombe, striking changes in the organization of the cytoplasmic microtubule cytoskeleton take place. These may serve as a model for understanding the different modes of microtubule organization that are often characteristic of differentiated higher eukaryotic cells. In the last few years, considerable progress has been made in our understanding of the organization and behaviour of fission yeast cytoplasmic microtubules, not only in the identification of the genes and proteins involved but also in the physiological analysis of function using fluorescently-tagged proteins in vivo. In this review we discuss the state of our knowledge in three areas: microtubule nucleation, regulation of microtubule dynamics and the organization and polarity of microtubule bundles. Advances in these areas provide a solid framework for a more detailed understanding of cytoplasmic microtubule organization.
Topics: Anaphase; Cell Polarity; Cytoplasm; Cytoskeleton; Kinetics; Microtubule-Organizing Center; Microtubules; Schizosaccharomyces; Tubulin
PubMed: 17072892
DOI: 10.1002/yea.1404