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Nucleic Acids Research Oct 2023Stringent control of centrosome duplication and separation is important for preventing chromosome instability. Structural and numerical alterations in centrosomes are...
Stringent control of centrosome duplication and separation is important for preventing chromosome instability. Structural and numerical alterations in centrosomes are hallmarks of neoplastic cells and contribute to tumorigenesis. We show that a Centrosome Amplification 20 (CA20) gene signature is associated with high expression of the Tripartite Motif (TRIM) family member E3 ubiquitin ligase, TRIM69. TRIM69-ablation in cancer cells leads to centrosome scattering and chromosome segregation defects. We identify Serine/threonine-protein kinase 3 (MST2) as a new direct binding partner of TRIM69. TRIM69 redistributes MST2 to the perinuclear cytoskeleton, promotes its association with Polo-like kinase 1 (PLK1) and stimulates MST2 phosphorylation at S15 (a known PLK1 phosphorylation site that is critical for centrosome disjunction). TRIM69 also promotes microtubule bundling and centrosome segregation that requires PRC1 and DYNEIN. Taken together, we identify TRIM69 as a new proximal regulator of distinct signaling pathways that regulate centrosome dynamics and promote bipolar mitosis.
Topics: Cell Cycle Proteins; Centrosome; Chromosome Segregation; Mitosis; Phosphorylation; Signal Transduction; Spindle Apparatus
PubMed: 37739411
DOI: 10.1093/nar/gkad766 -
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 -
Cytoskeleton (Hoboken, N.J.) 2023Microtubule-associated proteins (MAPs) regulate assembly and stability of microtubules (MTs) during cell cytokinesis, cell migration, neuronal growth, axon guidance, and...
Microtubule-associated proteins (MAPs) regulate assembly and stability of microtubules (MTs) during cell cytokinesis, cell migration, neuronal growth, axon guidance, and synapse formation. Using data mining of the Human Protein Atlas database and experimental screening, we identified nucleosome assembly protein 1 like 1 (NAP1L1) as a new MAP. The Human Protein Atlas and PubMed database screening identified 99 potential new MAPs. Twenty candidate proteins that highly co-localized with MTs were exogenously expressed with green fluorescent protein (GFP) or hemagglutinin (HA) tags in tissue culture cells and MTs were co-stained for immunofluorescent microscopy. We found that NAP1L1 is mainly localized in the cytosol with MTs during interphase. Using bacterially expressed recombinant NAP1L1 fragments and purified MTs, we biochemically mapped the MT-binding site on the N-terminal region (1-72aa) and the central region (164-269aa) of NAP1L1. NAP1L1 dimerizes through the long helix region (73-163aa), and full-length NAP1L1 induces the formation of thick MTs, indicating that NAP1L1 has the ability to bundle MTs in cells. Analysis of publicly available RNA-seq data of NAP1L1 depleted cells suggested that NAP1L1 is involved in cell adhesion and migration in agreement with the function of NAP1L1 as a MAP.
PubMed: 37098731
DOI: 10.1002/cm.21761 -
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 -
PLoS Genetics Jul 2021The formation and maintenance of microtubules requires their polymerisation, but little is known about how this polymerisation is regulated in cells. Focussing on the...
The formation and maintenance of microtubules requires their polymerisation, but little is known about how this polymerisation is regulated in cells. Focussing on the essential microtubule bundles in axons of Drosophila and Xenopus neurons, we show that the plus-end scaffold Eb1, the polymerase XMAP215/Msps and the lattice-binder Tau co-operate interdependently to promote microtubule polymerisation and bundle organisation during axon development and maintenance. Eb1 and XMAP215/Msps promote each other's localisation at polymerising microtubule plus-ends. Tau outcompetes Eb1-binding along microtubule lattices, thus preventing depletion of Eb1 tip pools. The three factors genetically interact and show shared mutant phenotypes: reductions in axon growth, comet sizes, comet numbers and comet velocities, as well as prominent deterioration of parallel microtubule bundles into disorganised curled conformations. This microtubule curling is caused by Eb1 plus-end depletion which impairs spectraplakin-mediated guidance of extending microtubules into parallel bundles. Our demonstration that Eb1, XMAP215/Msps and Tau co-operate during the regulation of microtubule polymerisation and bundle organisation, offers new conceptual explanations for developmental and degenerative axon pathologies.
Topics: Animals; Axons; Drosophila Proteins; Drosophila melanogaster; Microtubule-Associated Proteins; Microtubules; Neurons; Polymerization; Xenopus Proteins; Xenopus laevis; tau Proteins
PubMed: 34228717
DOI: 10.1371/journal.pgen.1009647 -
Journal of Veterinary Diagnostic... Nov 2023An 11-y-old hembra alpaca was admitted because of cerebellar and vestibular signs, dysphagia, and aspiration pneumonia; without clinical improvement following empirical...
An 11-y-old hembra alpaca was admitted because of cerebellar and vestibular signs, dysphagia, and aspiration pneumonia; without clinical improvement following empirical therapy, the patient was euthanized. On autopsy, a neoplasm was found incorporating the right vestibulocochlear nerve at the level of the acoustic meatus. Histologically, the mass was composed of a multiphasic primitive cell population associated with a dense fibrous stroma and enveloping a remnant ganglion and nerve bundles. Patterns included dense ribbons and cords of embryonal neuroepithelial cells admixed with loosely defined interlacing spindle cells. The embryonal cells had angular cell profiles with variable amounts of lightly basophilic cytoplasm, ovoid-to-irregular nuclei, and an open chromatin pattern with a typically inapparent nucleolus. Necrosis was not evident, and there was 1 mitotic figure per 2.37 mm. The entire mass was infiltrated by small numbers of lymphocytes and plasma cells. Immunohistochemistry (IHC) revealed strong and diffuse cytoplasmic immunolabeling for vimentin, microtubule-associated protein-2, protein gene product 9.5, and synaptophysin; ~50% immunolabeling for cytokeratin AE1/3; sporadic OLIG2 and S100 immunolabeling; and absent glial fibrillary acidic protein immunolabeling. Based on the histologic pattern and the IHC results, our diagnosis was a poorly differentiated embryonal tumor with ependymal differentiation associated with the vestibulocochlear nerve.
Topics: Animals; Camelids, New World; Neoplasms, Germ Cell and Embryonal
PubMed: 37638696
DOI: 10.1177/10406387231195611 -
Endocrinology Oct 2022Microtubule affinity-regulating kinases (MARKs) are nonreceptor Ser/Thr protein kinases known to regulate cell polarity and microtubule dynamics in Caenorhabditis...
Microtubule affinity-regulating kinases (MARKs) are nonreceptor Ser/Thr protein kinases known to regulate cell polarity and microtubule dynamics in Caenorhabditis elegans, Drosophila, invertebrates, vertebrates, and mammals. An earlier study has shown that MARK4 is present at the ectoplasmic specialization and blood-testis barrier (BTB) in the seminiferous epithelium of adult rat testes. Here, we report the function of MARK4 and another isoform MARK2 in Sertoli cells at the BTB. Knockdown of MARK2, MARK4, or MARK2 and MARK4 by RNAi using the corresponding siRNA duplexes without apparent off-target effects was shown to impair tight junction (TJ)-permeability barrier at the Sertoli cell BTB. It also disrupted microtubule (MT)- and actin-based cytoskeletal organization within Sertoli cells. Although MARK2 and MARK4 were shown to share sequence homology, they likely regulated the Sertoli cell BTB and MT cytoskeleton differently. Disruption of the TJ-permeability barrier following knockdown of MARK4 was considerably more severe than loss of MARK2, though both perturbed the barrier. Similarly, loss of MARK2 affected MT organization in a different manner than the loss of MARK4. Knockdown of MARK2 caused MT bundles to be arranged around the cell periphery, whereas knockdown of MARK4 caused MTs to retract from the cell edge. These differences in effects on the TJ-permeability barrier are likely from the unique roles of MARK2 and MARK4 in regulating the MT cytoskeleton of the Sertoli cell.
Topics: Actin Cytoskeleton; Actins; Animals; Blood-Testis Barrier; Male; Microtubules; Protein Serine-Threonine Kinases; RNA, Small Interfering; Rats; Rats, Sprague-Dawley; Sertoli Cells; Spermatogenesis; Tight Junctions
PubMed: 35971301
DOI: 10.1210/endocr/bqac130 -
International Journal of Biological... Jul 2019In specialized cell types such as neurons, microtubules maintain the integrity of axons by forming stable bundles and facilitate the transport of synaptic vesicles. The... (Review)
Review
In specialized cell types such as neurons, microtubules maintain the integrity of axons by forming stable bundles and facilitate the transport of synaptic vesicles. The cells regulate the stability and dynamics of microtubules using a diverse array of mechanisms. One of the mechanisms involves the interaction of microtubules with its associated proteins. Microtubule-associated proteins (MAPs) may have either stabilizing or destabilizing effects on the microtubules. Tau, a neuronal MAP, promotes the assembly and bundling of microtubules and suppresses microtubule dynamics. Abnormal functioning of tau is implicated in several neuronal disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD), wherein tau forms insoluble aggregates and causes neurotoxicity. In this review, we focus on the effects of tau on neuronal microtubule stability and dynamics. We also discuss the factors that lead to tau aggregation and the resulting destabilization of microtubules and the implications of this phenomenon in the AD and other tauopathies.
Topics: Animals; Humans; Microtubules; Mutation; Neurons; Protein Processing, Post-Translational; Tauopathies; tau Proteins
PubMed: 31004638
DOI: 10.1016/j.ijbiomac.2019.04.120 -
Cells Feb 2020The sensing, integrating, and coordinating features of the eukaryotic cells are achieved by the complex ultrastructural arrays and multifarious functions of the... (Review)
Review
The sensing, integrating, and coordinating features of the eukaryotic cells are achieved by the complex ultrastructural arrays and multifarious functions of the cytoskeleton, including the microtubule network. Microtubules play crucial roles achieved by their decoration with proteins/enzymes as well as by posttranslational modifications. This review focuses on the Tubulin Polymerization Promoting Protein (TPPP/p25), a new microtubule associated protein, on its "regulatory functions by day and pathological functions at night". Physiologically, the moonlighting TPPP/p25 modulates the dynamics and stability of the microtubule network by bundling microtubules and enhancing the tubulin acetylation due to the inhibition of tubulin deacetylases. The optimal endogenous TPPP/p25 level is crucial for its physiological functions, to the differentiation of oligodendrocytes, which are the major constituents of the myelin sheath. Pathologically, TPPP/p25 forms toxic oligomers/aggregates with α-synuclein in neurons and oligodendrocytes in Parkinson's disease and Multiple System Atrophy, respectively; and their complex is a potential therapeutic drug target. TPPP/p25-derived microtubule hyperacetylation counteracts uncontrolled cell division. All these issues reveal the anti-mitotic and α-synuclein aggregation-promoting potency of TPPP/p25, consistent with the finding that Parkinson's disease patients have reduced risk for certain cancers.
Topics: Animals; Humans; Microtubule-Associated Proteins; Models, Biological; Neoplasms; Nervous System Diseases; Photoperiod; Tubulin
PubMed: 32033023
DOI: 10.3390/cells9020357 -
BMC Biology Apr 2020Upon water uptake and release of seed dormancy, embryonic plant cells expand, while being mechanically constrained by the seed coat. Cortical microtubules (CMTs) are key...
BACKGROUND
Upon water uptake and release of seed dormancy, embryonic plant cells expand, while being mechanically constrained by the seed coat. Cortical microtubules (CMTs) are key players of cell elongation in plants: their anisotropic orientation channels the axis of cell elongation through the guidance of oriented deposition of load-bearing cellulose microfibrils in the cell wall. Interestingly, CMTs align with tensile stress, and consistently, they reorient upon compressive stress in growing hypocotyls. How CMTs first organise in germinating embryos is unknown, and their relation with mechanical stress has not been investigated at such an early developing stage.
RESULTS
Here, we analysed CMT dynamics in dormant and non-dormant Arabidopsis seeds by microscopy of fluorescently tagged microtubule markers at different developmental time points and in response to abscisic acid and gibberellins. We found that CMTs first appear as very few thick bundles in dormant seeds. Consistently, analysis of available transcriptome and translatome datasets show that limiting amounts of tubulin and microtubule regulators initially hinder microtubule self-organisation. Seeds imbibed in the presence of gibberellic acid or abscisic acid displayed altered microtubule organisation and transcriptional regulation. Upon the release of dormancy, CMTs then self-organise into multiple parallel transverse arrays. Such behaviour matches the tensile stress patterns in such mechanically constrained embryos. This suggests that, as CMTs first self-organise, they also align with shape-derived tensile stress patterns.
CONCLUSIONS
Our results provide a scenario in which dormancy release in the embryo triggers microtubule self-organisation and alignment with tensile stress prior to germination and anisotropic growth.
Topics: Arabidopsis; Germination; Microtubules; Seeds
PubMed: 32354334
DOI: 10.1186/s12915-020-00774-8