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Journal of Alzheimer's Disease : JAD 2013Inge Grundke-Iqbal and Khalid Iqbal found a connection between microtubule associated tau and Alzheimer's disease. They described that abnormally phosphorylated tau is a...
Inge Grundke-Iqbal and Khalid Iqbal found a connection between microtubule associated tau and Alzheimer's disease. They described that abnormally phosphorylated tau is a component of the paired helical filaments found in the disease. Afterwards they described that tau hyperphosphorylation prevents microtubule assembly. Now trying to complement the relationship between microtubules and tau phosphorylation, we have commented on the effect of microtubule disassembly on tau phosphorylation. In this study, we investigated the role of microtubule depolymerization induced by nocodazole on tau phosphorylation in human neuroblastoma SH-SY5Y cells. Our results indicate that nocodazole provokes tau phosphorylation mediated by GSK3, as determined by using AT-8 or Tau-1 antibodies. Interestingly, total GSK3β and GSK3β phosphorylation on Ser-9 are not altered during nocodazole treatment. In addition, microtubule stabilization with taxol had similar effects, likely because taxol and tau compete for the same binding sites on microtubules, and in the presence of taxol, tau could be detached from microtubules. Thus, unbound tau from microtubles can be phosphorylated by GSK3, even if the activity of GSK3 is not altered, probably because tau unbound to microtubules could be a better substrate for the kinase than microtubule-associated tau. These findings suggest that microtubule depolymerization can be a primary event in neurodegenerative disorders like Alzheimer's disease and that tau phosphorylation takes place afterwards.
Topics: Cell Line, Tumor; Humans; Microtubules; Nocodazole; Phosphorylation; Polymerization; Tubulin Modulators; tau Proteins
PubMed: 23948896
DOI: 10.3233/JAD-130545 -
Current Opinion in Cell Biology Feb 2014Accurate segregation of the replicated genome during cell division depends on dynamic attachments formed between chromosomes and the microtubule polymers of the spindle.... (Review)
Review
Accurate segregation of the replicated genome during cell division depends on dynamic attachments formed between chromosomes and the microtubule polymers of the spindle. Here we review recent advances in mechanistic analysis of microtubule attachment formation and regulation.
Topics: Animals; Chromosome Segregation; Humans; Kinetochores; Microtubules; Nuclear Proteins; Protein Binding
PubMed: 24529253
DOI: 10.1016/j.ceb.2013.12.005 -
Current Biology : CB Mar 2020Microtubule-dependent organization of membranous organelles occurs through motor-based pulling and by coupling microtubule dynamics to membrane remodeling. For example,...
Microtubule-dependent organization of membranous organelles occurs through motor-based pulling and by coupling microtubule dynamics to membrane remodeling. For example, tubules of endoplasmic reticulum (ER) can be extended by kinesin- and dynein-mediated transport and through the association with the tips of dynamic microtubules. The binding between ER and growing microtubule plus ends requires End Binding (EB) proteins and the transmembrane protein STIM1, which form a tip-attachment complex (TAC), but it is unknown whether these proteins are sufficient for membrane remodeling. Furthermore, EBs and their partners undergo rapid turnover at microtubule ends, and it is unclear how highly transient protein-protein interactions can induce load-bearing processive motion. Here, we reconstituted membrane tubulation in a minimal system with giant unilamellar vesicles, dynamic microtubules, an EB protein, and a membrane-bound protein that can interact with EBs and microtubules. We showed that these components are sufficient to drive membrane remodeling by three mechanisms: membrane tubulation induced by growing microtubule ends, motor-independent membrane sliding along microtubule shafts, and membrane pulling by shrinking microtubules. Experiments and modeling demonstrated that the first two mechanisms can be explained by adhesion-driven biased membrane spreading on microtubules. Optical trapping revealed that growing and shrinking microtubule ends can exert forces of ∼0.5 and ∼5 pN, respectively, through attached proteins. Rapidly exchanging molecules that connect membranes to dynamic microtubules can thus bear a sufficient load to induce membrane deformation and motility. Furthermore, combining TAC components and a membrane-attached kinesin in the same in vitro assays demonstrated that they can cooperate in promoting membrane tubule extension.
Topics: Endoplasmic Reticulum; Escherichia coli; Escherichia coli Proteins; Kinesins; Membrane Proteins; Microtubule-Associated Proteins; Microtubules
PubMed: 32032506
DOI: 10.1016/j.cub.2020.01.036 -
Current Biology : CB Dec 2016Microtubule dynamics rely on the properties of tubulin and are regulated by microtubule-associated proteins. GTP-tubulin assembles into hollow polymers, which can...
Microtubule dynamics rely on the properties of tubulin and are regulated by microtubule-associated proteins. GTP-tubulin assembles into hollow polymers, which can depolymerize upon GTP hydrolysis. Depolymerizing microtubules may stop shrinking and resume growth. Such rescues are regulated by microtubule-associated proteins like CLIP-170 and the CLASPs [1, 2]. Microtubule domains prone to rescues contain discrete regions (previously termed "GTP islands") that retain a GTP-tubulin-like conformation in the main body of the microtubule [3]. However, the exact nature of these domains and the mechanisms controlling their occurrence and distribution are largely unknown. Here we show that collisions between growing microtubules and mechanical obstacles (including other microtubules) in vitro result in the higher abundance of GTP-like islands in stressed microtubule regions. Furthermore, these islands were found to be efficiently generated by both lateral contacts and mechanical constraints applied to the main body of the microtubules. They were also particularly prominent where shifts in the number of protofilaments occur in the microtubule lattice. GTP-like islands and rescues frequently co-occurred at microtubule intersections in vitro and in living cells, both in crossing and in crossed microtubules. We also observed that CLIP-170 recognizes GTP-like islands in vivo and is retained at microtubule crossings. Therefore, we propose that rescues occur via a two-stage mechanism: (1) lattice defects determine potential rescue-promoting islands in the microtubule structure, and (2) CLIP-170 detects these islands to stimulate microtubule rescue. Our results reveal the interplay between rescue-promoting factors and microtubule architecture and organization to control microtubule dynamics.
Topics: Animals; Cell Line; Guanosine Triphosphate; Microtubules; Molecular Dynamics Simulation; Polymers; Protein Conformation
PubMed: 27916523
DOI: 10.1016/j.cub.2016.10.048 -
Biophysical Journal Jan 2023Control of microtubule abundance, stability, and length is crucial to regulate intracellular transport as well as cell polarity and division. How microtubule stability...
Control of microtubule abundance, stability, and length is crucial to regulate intracellular transport as well as cell polarity and division. How microtubule stability depends on tubulin addition or removal at the dynamic ends is well studied. However, microtubule rescue, the event when a microtubule switches from shrinking to growing, occurs at tubulin exchange sites along the shaft. Molecular motors have recently been shown to promote such exchanges. Using a stochastic theoretical description, we study how microtubule stability and length depend on motor-induced tubulin exchange and thus rescue. Our theoretical description matches our in vitro experiments on microtubule dynamics in the presence of kinesin-1 molecular motors. Although the overall dynamics of a population of microtubules can be captured by an effective rescue rate, by assigning rescue to exchange sites, we reveal that the dynamics of individual microtubules within the population differ dramatically. Furthermore, we study in detail a transition from bounded to unbounded microtubule growth. Our results provide novel insights into how molecular motors imprint information of microtubule stability on the microtubule network.
Topics: Tubulin; Microtubules; Kinesins
PubMed: 36502273
DOI: 10.1016/j.bpj.2022.12.010 -
Small GTPases 2021Since the discovery by Madaule and Axel in 1985 of the first Ras homologue (Rho) protein in and its human orthologue RhoB, membership in the Rho GTPase family has grown... (Review)
Review
Since the discovery by Madaule and Axel in 1985 of the first Ras homologue (Rho) protein in and its human orthologue RhoB, membership in the Rho GTPase family has grown to 20 proteins, with representatives in all eukaryotic species. These GTPases are molecular switches that cycle between active (GTP bound) and inactivate (GDP bound) states. The exchange of GDP for GTP on Rho GTPases is facilitated by guanine exchange factors (GEFs). Approximately 80 Rho GEFs have been identified to date, and only a few GEFs associate with microtubules. The guanine nucleotide exchange factor H1, GEF-H1, is a unique GEF that associates with microtubules and is regulated by the polymerization state of microtubule networks. This review summarizes the regulation and functions of GEF-H1 and discusses the roles of GEF-H1 in human diseases.
Topics: Animals; Disease; Gene Expression Regulation; Humans; Microtubules; Rho Guanine Nucleotide Exchange Factors
PubMed: 33126816
DOI: 10.1080/21541248.2020.1840889 -
Journal of Structural Biology Jan 2020Microtubules are polar filaments built from αβ-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged...
Microtubules are polar filaments built from αβ-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of α- and β-tubulin. The determination of the αβ-tubulin register and seam location during image processing is essential for alignment accuracy that enables determination of biologically relevant structures. Here we present a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Microtubule RELION-based Pipeline (MiRP). The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing near-atomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins.
Topics: Cryoelectron Microscopy; Databases, Factual; Humans; Image Processing, Computer-Assisted; Microtubule-Associated Proteins; Microtubules
PubMed: 31610239
DOI: 10.1016/j.jsb.2019.10.004 -
Traffic (Copenhagen, Denmark) Mar 2009Cross-talk between microtubule networks and sites of cell-matrix and cell-cell adhesion has profound impact on these structures and is essential for proper cell... (Review)
Review
Cross-talk between microtubule networks and sites of cell-matrix and cell-cell adhesion has profound impact on these structures and is essential for proper cell organization, polarization and motility. Components of adhesion sites can interact directly with microtubules or with proteins that specifically associate with microtubule plus ends and minus ends and in this way capture, stabilize or destabilize microtubules. In their turn, microtubules can serve as routes for delivery of structural and regulatory factors that control adhesion site turnover. In addition, the microtubule lattice or growing microtubule plus ends can serve as diffusional sinks that accumulate and scaffold regulatory molecules, thereby affecting their activity in the vicinity of adhesions. Combination of these mechanisms underlies the functional co-operation between microtubules and adhesion sites and defines their dynamic behavior.
Topics: Animals; Biological Transport; Cell Adhesion; Microtubules; Models, Biological; Substrate Specificity
PubMed: 19175539
DOI: 10.1111/j.1600-0854.2008.00869.x -
Proceedings of the National Academy of... Jul 2020Microtubules are tubular polymers with essential roles in numerous cellular activities. Structures of microtubules have been captured at increasing resolution by...
Microtubules are tubular polymers with essential roles in numerous cellular activities. Structures of microtubules have been captured at increasing resolution by cryo-EM. However, dynamic properties of the microtubule are key to its function, and this behavior has proved difficult to characterize at a structural level due to limitations in existing structure determination methods. We developed a high-resolution cryo-EM refinement method that divides an imaged microtubule into its constituent protofilaments, enabling deviations from helicity and other sources of heterogeneity to be quantified and corrected for at the single-subunit level. We demonstrate that this method improves the resolution of microtubule 3D reconstructions and substantially reduces anisotropic blurring artifacts, compared with methods that utilize helical symmetry averaging. Moreover, we identified an unexpected, discrete behavior of the m-loop, which mediates lateral interactions between neighboring protofilaments and acts as a flexible hinge between them. The hinge angle adopts preferred values corresponding to distinct conformations of the m-loop that are incompatible with helical symmetry. These hinge angles fluctuate in a stochastic manner, and perfectly cylindrical microtubule conformations are thus energetically and entropically penalized. The hinge angle can diverge further from helical symmetry at the microtubule seam, generating a subpopulation of highly distorted microtubules. However, the seam-distorted subpopulation disappears in the presence of Taxol, a microtubule stabilizing agent. These observations provide clues into the structural origins of microtubule flexibility and dynamics and highlight the role of structural polymorphism in defining microtubule behavior.
Topics: Animals; Cattle; Cryoelectron Microscopy; Microtubules; Molecular Dynamics Simulation
PubMed: 32636254
DOI: 10.1073/pnas.2001546117 -
FEBS Letters Nov 2020Malformations of cortical development (MCDs) are a group of severe brain malformations associated with intellectual disability and refractory childhood epilepsy. Human... (Review)
Review
Malformations of cortical development (MCDs) are a group of severe brain malformations associated with intellectual disability and refractory childhood epilepsy. Human missense heterozygous mutations in the 9 α-tubulin and 10 β-tubulin isoforms forming the heterodimers that assemble into microtubules (MTs) were found to cause MCDs. However, how a single mutated residue in a given tubulin isoform can perturb the entire microtubule population in a neuronal cell remains a crucial question. Here, we examined 85 MCD-associated tubulin mutations occurring in TUBA1A, TUBB2, and TUBB3 and their location in a three-dimensional (3D) microtubule cylinder. Mutations hitting residues exposed on the outer microtubule surface are likely to alter microtubule association with partners, while alteration of intradimer contacts may impair dimer stability and straightness. Other types of mutations are predicted to alter interdimer and lateral contacts, which are responsible for microtubule cohesion, rigidity, and dynamics. MCD-associated tubulin mutations surprisingly fall into all categories, thus providing unexpected insights into how a single mutation may impair microtubule function and elicit dominant effects in neurons.
Topics: Animals; Humans; Microtubules; Mutation; Neurodevelopmental Disorders; Tubulin
PubMed: 33064843
DOI: 10.1002/1873-3468.13958