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Journal of Alzheimer's Disease : JAD 2019The defining pathological hallmarks of Alzheimer's disease (AD) are proteinopathies marked by the amyloid-β (Aβ) peptide and hyperphosphorylated tau. In addition,... (Review)
Review
The defining pathological hallmarks of Alzheimer's disease (AD) are proteinopathies marked by the amyloid-β (Aβ) peptide and hyperphosphorylated tau. In addition, Hirano bodies and cofilin-actin rods are extensively found in AD brains, both of which are associated with the actin cytoskeleton. The actin-binding protein cofilin known for its actin filament severing, depolymerizing, nucleating, and bundling activities has emerged as a significant player in AD pathogenesis. In this review, we discuss the regulation of cofilin by multiple signaling events impinging on LIM kinase-1 (LIMK1) and/or Slingshot homolog-1 (SSH1) downstream of Aβ. Such pathophysiological signaling pathways impact actin dynamics to regulate synaptic integrity, mitochondrial translocation of cofilin to promote neurotoxicity, and formation of cofilin-actin pathology. Other intracellular signaling proteins, such as β-arrestin, RanBP9, Chronophin, PLD1, and 14-3-3 also impinge on the regulation of cofilin downstream of Aβ. Finally, we discuss the role of activated cofilin as a bridge between actin and microtubule dynamics by displacing tau from microtubules, thereby destabilizing tau-induced microtubule assembly, missorting tau, and promoting tauopathy.
Topics: Alzheimer Disease; Animals; Brain; Cofilin 1; Cytoskeleton; Humans; Lim Kinases; Phosphoprotein Phosphatases; Tauopathies
PubMed: 31594228
DOI: 10.3233/JAD-190585 -
Cell Reports Apr 2022Chromosome segregation in mammals relies on the maturation of a thick bundle of kinetochore-attached microtubules known as k-fiber. How k-fibers mature from initial...
Chromosome segregation in mammals relies on the maturation of a thick bundle of kinetochore-attached microtubules known as k-fiber. How k-fibers mature from initial kinetochore microtubule attachments remains a fundamental question. By combining molecular perturbations and phenotypic analyses in Indian muntjac fibroblasts containing the lowest known diploid chromosome number in mammals (2N = 6) and distinctively large kinetochores, with fixed/live-cell super-resolution coherent-hybrid stimulated emission depletion (CH-STED) nanoscopy and laser microsurgery, we demonstrate a key role for augmin in kinetochore microtubule self-organization and maturation, regardless of pioneer centrosomal microtubules. In doing so, augmin promotes kinetochore and interpolar microtubule turnover and poleward flux. Tracking of microtubule growth events within individual k-fibers reveals a wide angular dispersion, consistent with augmin-mediated branched microtubule nucleation. Augmin depletion reduces the frequency of kinetochore microtubule growth events and hampers efficient repair after acute k-fiber injury by laser microsurgery. Together, these findings underscore the contribution of augmin-mediated microtubule amplification for k-fiber self-organization and maturation in mammals.
Topics: Animals; Chromosome Segregation; Kinetochores; Mammals; Microtubules; Mitosis; Spindle Apparatus
PubMed: 35385739
DOI: 10.1016/j.celrep.2022.110610 -
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 -
Nature Cell Biology Oct 2016The dynamic instability of microtubules is characterized by slow growth phases stochastically interrupted by rapid depolymerizations called catastrophes. Rescue events...
The dynamic instability of microtubules is characterized by slow growth phases stochastically interrupted by rapid depolymerizations called catastrophes. Rescue events can arrest the depolymerization and restore microtubule elongation. However, the origin of these rescue events remains unexplained. Here we show that microtubule lattice self-repair, in structurally damaged sites, is responsible for the rescue of microtubule growth. Tubulin photo-conversion in cells revealed that free tubulin dimers can incorporate along the shafts of microtubules, especially in regions where microtubules cross each other, form bundles or become bent due to mechanical constraints. These incorporation sites appeared to act as effective rescue sites ensuring microtubule rejuvenation. By securing damaged microtubule growth, the self-repair process supports a mechanosensitive growth by specifically promoting microtubule assembly in regions where they are subjected to physical constraints.
Topics: Animals; Cell Membrane; Cells, Cultured; Focal Adhesion Kinase 1; Microtubule-Associated Proteins; Microtubules; Photolysis; Rats; Tubulin
PubMed: 27617929
DOI: 10.1038/ncb3406 -
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 -
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 -
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 -
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