Microtubules (MTs) are polarized polymers with highly dynamic plus ends that stochastically switch between growth and shrinkage phases. In eukaryotic cells, a plethora of MT-associated proteins (MAPs) regulate the dynamics and higher-order organization of MTs to mediate distinct cellular functions. Plus-end tracking proteins (+TIPs) are a group of MAPs that specifically accumulate at the growing MT plus ends, where they modulate the behavior of the MT plus ends and mediate interactions with cellular targets. Although several functionally important + TIP proteins have been characterized in yeast and animals, little is known about this group of proteins in plants.

Tektins are insoluble alpha-helical proteins essential for the construction of cilia and flagella and are found throughout the eukaryotes apart from higher plants. Being almost universal but still fairly free to mutate, their coding sequences have proved useful for estimating the evolutionary relationships between closely related species. Their protein molecular structure, typically consisting of four coiled-coil rod segments connected by linkers, resembles that of intermediate filament (IF) proteins and lamins. Tektins assemble into continuous rods 2 nm in diameter that are probably equivalent to subfilaments of the 10 nm diameter IFs. Tektin and IF rod sequences both have a repeating pattern of charged amino acids superimposed on the seven-amino-acid hydrophobic pattern of coiled-coil proteins. The length of the repeat segment matches that of tubulin subunits, suggesting that tektins and tubulins may have coevolved, and that lamins and IFs may have emerged later as modified forms of tektin. Unlike IFs, tektin sequences include one copy of a conserved peptide of nine amino acids that may bind tubulin. The 2 nm filaments associate closely with tubulin in doublet and triplet microtubules of axonemes and centrioles, respectively, and help to stabilize these structures. Their supply restricts the assembled lengths of cilia and flagella. In doublet microtubules, the 2 nm filaments may also help to organize the longitudinal spacing of accessory structures, such as groups of inner dynein arms and radial spokes.

Tubulin posttranslational modifications have been predicted to control cytoskeletal functions by coordinating the molecular interactions between microtubules and their associating proteins. A prominent tubulin modification in neurons is polyglutamylation, the deregulation of which causes neurodegeneration. Yet, the underlying molecular mechanisms have remained elusive. Here, using in-vitro reconstitution, we determine how polyglutamylation generated by the two predominant neuronal polyglutamylases, TTLL1 and TTLL7, specifically modulates the activities of three major microtubule interactors: the microtubule-associated protein Tau, the microtubule-severing enzyme katanin and the molecular motor kinesin-1. We demonstrate that the unique modification patterns generated by TTLL1 and TTLL7 differentially impact those three effector proteins, thus allowing for their selective regulation. Given that our experiments were performed with brain tubulin from mouse models in which physiological levels and patterns of polyglutamylation were altered by the genetic knockout of the main modifying enzymes, our quantitative measurements provide direct mechanistic insight into how polyglutamylation could selectively control microtubule interactions in neurons.

The ends of growing microtubules (MTs) accumulate a set of diverse factors known as MT plus end-tracking proteins (+TIPs), which control microtubule dynamics and organization. In this paper, we identify SLAIN2 as a key component of +TIP interaction networks. We showed that the C-terminal part of SLAIN2 bound to end-binding proteins (EBs), cytoplasmic linker proteins (CLIPs), and CLIP-associated proteins and characterized in detail the interaction of SLAIN2 with EB1 and CLIP-170. Furthermore, we found that the N-terminal part of SLAIN2 interacted with ch-TOG, the mammalian homologue of the MT polymerase XMAP215. Through its multiple interactions, SLAIN2 enhanced ch-TOG accumulation at MT plus ends and, as a consequence, strongly stimulated processive MT polymerization in interphase cells. Depletion or disruption of the SLAIN2-ch-TOG complex led to disorganization of the radial MT array. During mitosis, SLAIN2 became highly phosphorylated, and its interaction with EBs and ch-TOG was inhibited. Our study provides new insights into the molecular mechanisms underlying cell cycle-specific regulation of MT polymerization and the organization of the MT network.

The mitotic spindle is resilient to perturbation due to the concerted, and sometimes redundant, action of motors and microtubule-associated proteins. Here, we utilize an inducible ectopic microtubule nucleation site in the nucleus of Saccharomyces cerevisiae to study three necessary steps in the formation of a bipolar array: the recruitment of the γ-tubulin complex, nucleation and elongation of microtubules (MTs), and the organization of MTs relative to each other. This novel tool, an Spc110 chimera, reveals previously unreported roles of the microtubule-associated proteins Stu2, Bim1, and Bik1, and the motors Vik1 and Kip3. We report that Stu2 and Bim1 are required for nucleation and that Bik1 and Kip3 promote nucleation at the ectopic site. Stu2, Bim1, and Kip3 join their homologs XMAP215, EB1 and kinesin-8 as promoters of microtubule nucleation, while Bik1 promotes MT nucleation indirectly via its role in SPB positioning. Furthermore, we find that the nucleation activity of Stu2 in vivo correlates with its polymerase activity in vitro. Finally, we provide the first evidence that Vik1, a subunit of Kar3/Vik1 kinesin-14, promotes microtubule minus end focusing at the ectopic site.

Microtubule-associated proteins (MAPs) of the MAP2/Tau family include the vertebrate proteins MAP2, MAP4, and Tau and homologs in other animals. All three vertebrate members of the family have alternative splice forms; all isoforms share a conserved carboxy-terminal domain containing microtubule-binding repeats, and an amino-terminal projection domain of varying size. MAP2 and Tau are found in neurons, whereas MAP4 is present in many other tissues but is generally absent from neurons. Members of the family are best known for their microtubule-stabilizing activity and for proposed roles regulating microtubule networks in the axons and dendrites of neurons. Contrary to this simple, traditional view, accumulating evidence suggests a much broader range of functions, such as binding to filamentous (F) actin, recruitment of signaling proteins, and regulation of microtubule-mediated transport. Tau is also implicated in Alzheimer's disease and other dementias. The ability of MAP2 to interact with both microtubules and F-actin might be critical for neuromorphogenic processes, such as neurite initiation, during which networks of microtubules and F-actin are reorganized in a coordinated manner. Various upstream kinases and interacting proteins have been identified that regulate the microtubule-stabilizing activity of MAP2/Tau family proteins.

Cilia are ubiquitous, hair-like appendages found in eukaryotic cells that carry out functions of cell motility and sensory reception. Cilia contain an intriguing cytoskeletal structure, termed the axoneme that consists of nine doublet microtubules radially interlinked and longitudinally organized in multiple specific repeat units. Little is known, however, about how the axoneme allows cilia to be both actively bendable and sturdy or how it is assembled. To answer these questions, we used cryo-electron microscopy to structurally analyse several of the repeating units of the doublet at sub-nanometre resolution. This structural detail enables us to unambiguously assign α- and β-tubulins in the doublet microtubule lattice. Our study demonstrates the existence of an inner sheath composed of different kinds of microtubule inner proteins inside the doublet that likely stabilizes the structure and facilitates the specific building of the B-tubule.

We have studied the occurrence, stage specificity and cellular location of key molecules associated with microtubules in Plasmodium falciparum merozoites. Antibodies to gamma tubulin, conventional kinesin and cytoplasmic dynein were used to determine the polarity of merozoite microtubules (mt), the stage specificity of the motor proteins and their location during merozoite development. We conclude that the minus ends of the mts are located at their apical pole. Kinesin was present throughout the lifecycle, appearing as a distinct crescent at the apex of developing merozoites. The vast majority of cytoplasmic dynein reactivity occurred in late merogony, also appearing at the merozoite apex. Destruction of mt with dinitroanilines did not affect the cellular location of kinesin or dynein. In invasion assays, dynein inhibitors reduced the number of ring stage parasites. Our results show that both conventional kinesin and cytoplasmic dynein are abundant, located at the negative pole of the merozoite mt and, intriguingly, appear there only in very late merogony, prior to merozoite release and invasion.

Within cells, motor and non-motor microtubule-associated proteins (MAPs) simultaneously converge on the microtubule. How the binding activities of non-motor MAPs are coordinated and how they contribute to the balance and distribution of motor transport is unknown. Here, we examine the relationship between MAP7 and tau owing to their antagonistic roles in vivo. We find that MAP7 and tau compete for binding to microtubules, and determine a mechanism by which MAP7 displaces tau from the lattice. MAP7 promotes kinesin-based transport in vivo and strongly recruits kinesin-1 to the microtubule in vitro, providing evidence for direct enhancement of motor motility by a MAP. Both MAP7 and tau strongly inhibit kinesin-3 and have no effect on cytoplasmic dynein, demonstrating that MAPs differentially control distinct classes of motors. Overall, these results reveal a general principle for how MAP competition dictates access to the microtubule to determine the correct distribution and balance of motor activity.

End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.

The role of plus end-tracking proteins in regulating microtubule (MT) dynamics was investigated by expressing a dominant negative mutant that removed endogenous cytoplasmic linker proteins (CLIPs) from MT plus ends. In control CHO cells, MTs exhibited asymmetric behavior: MTs persistently grew toward the plasma membrane and displayed frequent fluctuations of length near the cell periphery. In the absence of CLIPs, the microtubule rescue frequency was reduced by sevenfold. MT behavior became symmetrical, consisting of persistent growth and persistent shortening. Removal of CLIPs also caused loss of p150Glued but not CLIP-associating protein (CLASP2) or EB1. This result raised the possibility that the change in dynamics was a result of the loss of either CLIPs or p150Glued. To distinguish between these possibilities, we performed rescue experiments. Normal MT dynamics were restored by expression of the CLIP-170 head domain, but p150Glued was not recruited back to MT plus ends. Expression of p150Glued head domain only partially restored MT dynamics. We conclude that the CLIP head domain is sufficient to alter MT dynamics either by itself serving as a rescue factor or indirectly by recruiting a rescue factor. By promoting a high rescue frequency, CLIPs provide a mechanism by which MT plus ends may be concentrated near the cell margin.

Microtubule-binding proteins (MBPs) are structurally and functionally diverse regulators of microtubule-mediated cellular processes. Alteration of MBPs has been implicated in the pathogenesis of human diseases, including cancer. MBPs can stabilize or destabilize microtubules or move along microtubules to transport various cargoes. In addition, MBPs can control microtubule dynamics through direct interaction with microtubules or coordination with other proteins. To better understand microtubule structure and function, it is necessary to identify additional MBPs. In this study, we isolated microtubules and MBPs from mammalian cells by a taxol-based method and then profiled a panel of MBPs by mass spectrometry. We discovered a number of previously uncharacterized MBPs, including several membrane-associated proteins and proteins involved in post-translational modifications, in addition to several structural components. These results support the notion that microtubules have a wide range of functions and may undergo more exquisite regulation than previously recognized.

MAP2 and tau are the two most prominent neuron-specific microtubule-associated proteins. They have been implicated in the stabilization of microtubules and consequently of neurite morphology. To investigate their influence on microtubule dynamics, we have tagged both proteins with green fluorescent protein and expressed them in non-neuronal cells. Time-lapse recordings of living cells showed that MAP2 and tau did not significantly affect the rates of microtubule growth and shrinkage. Longer recordings revealed the growth and disappearance of MAP-induced microtubule bundles coinciding with changes in cell shape. This supports the idea that microtubule dynamics are influenced by the cortical cytoskeleton. The dynamics-preserving stabilization of microtubules by MAP2 and tau thus provides a molecular basis for the morphological plasticity reported to exist in established neurites.

Accurate chromosome segregation relies on microtubule end conversion, the ill-understood ability of kinetochores to transit from lateral microtubule attachment to durable association with dynamic microtubule plus-ends. The molecular requirements for this conversion and the underlying biophysical mechanisms are elusive. We reconstituted end conversion in vitro using two kinetochore components: the plus end-directed kinesin CENP-E and microtubule-binding Ndc80 complex, combined on the surface of a microbead. The primary role of CENP-E is to ensure close proximity between Ndc80 complexes and the microtubule plus-end, whereas Ndc80 complexes provide lasting microtubule association by diffusing on the microtubule wall near its tip. Together, these proteins mediate robust plus-end coupling during several rounds of microtubule dynamics, in the absence of any specialized tip-binding or regulatory proteins. Using a Brownian dynamics model, we show that end conversion is an emergent property of multimolecular ensembles of microtubule wall-binding proteins with finely tuned force-dependent motility characteristics.

MARK phosphorylates the microtubule-associated proteins tau, MAP2, and MAP4 on their microtubule-binding domain, causing their dissociation from microtubules and increased microtubule dynamics. We describe the molecular cloning, distribution, activation mechanism, and overexpression of two MARK proteins from rat that arise from distinct genes. They encode Ser/Thr kinases of 88 and 81 kDa, respectively, and show similarity to the yeast kin1+ and C. elegans par-1 genes that are involved in the establishment of cell polarity. Expression of both isoforms is ubiquitous, and homologous genes are present in humans. Catalytic activity depends on phosphorylation of two residues in subdomain VIII. Overexpression of MARK in cells leads to hyperphosphorylation of MAPs on KXGS motifs and to disruption of the microtubule array, resulting in morphological changes and cell death.

Dendrite microtubules are polarized with minus-end-out orientation in Drosophila neurons. Nucleation sites concentrate at dendrite branch points, but how they localize is not known. Using Drosophila, we found that canonical Wnt signaling proteins regulate localization of the core nucleation protein γTubulin (γTub). Reduction of frizzleds (fz), arrow (low-density lipoprotein receptor-related protein [LRP] 5/6), dishevelled (dsh), casein kinase Iγ, G proteins, and Axin reduced γTub-green fluorescent protein (GFP) at branch points, and two functional readouts of dendritic nucleation confirmed a role for Wnt signaling proteins. Both dsh and Axin localized to branch points, with dsh upstream of Axin. Moreover, tethering Axin to mitochondria was sufficient to recruit ectopic γTub-GFP and increase microtubule dynamics in dendrites. At dendrite branch points, Axin and dsh colocalized with early endosomal marker Rab5, and new microtubule growth initiated at puncta marked with fz, dsh, Axin, and Rab5. We propose that in dendrites, canonical Wnt signaling proteins are housed on early endosomes and recruit nucleation sites to branch points.

The γ-tubulin complex acts as the predominant microtubule (MT) nucleator that initiates MT formation and is therefore an essential factor for cell proliferation. Nonetheless, cellular MTs are formed after experimental depletion of the γ-tubulin complex, suggesting that cells possess other factors that drive MT nucleation. Here, by combining gene knockout, auxin-inducible degron, RNA interference, MT depolymerization/regrowth assay, and live microscopy, we identified four microtubule-associated proteins (MAPs), ch-TOG, CLASP1, CAMSAPs, and TPX2, which are involved in γ-tubulin-independent MT generation in human colon cancer cells. In the mitotic MT regrowth assay, nucleated MTs organized noncentriolar MT organizing centers (ncMTOCs) in the absence of γ-tubulin. Depletion of CLASP1 or TPX2 substantially delayed ncMTOC formation, suggesting that these proteins might promote MT nucleation in the absence of γ-tubulin. In contrast, depletion of ch-TOG or CAMSAPs did not affect the timing of ncMTOC appearance. CLASP1 also accelerates γ-tubulin-independent MT regrowth during interphase. Thus, MT generation can be promoted by MAPs without the γ-tubulin template.

Kinetochores connect centromeric chromatin to spindle microtubules during mitosis. Neurons are postmitotic, so it was surprising to identify transcripts of structural kinetochore (KT) proteins and regulatory chromosome passenger complex (CPC) and spindle assembly checkpoint (SAC) proteins in Drosophila neurons after dendrite injury. To test whether these proteins function during dendrite regeneration, postmitotic RNA interference (RNAi) was performed and dendrites or axons were removed using laser microsurgery. Reduction of KT, CPC, and SAC proteins decreased dendrite regeneration without affecting axon regeneration. To understand whether neuronal functions of these proteins rely on microtubules, we analyzed microtubule behavior in uninjured neurons. The number of growing plus, but not minus, ends increased in dendrites with reduced KT, CPC, and SAC proteins, while axonal microtubules were unaffected. Increased dendritic microtubule dynamics was independent of dual leucine zipper kinase (DLK)-mediated stress but was rescued by concurrent reduction of γ-tubulin, the core microtubule nucleation protein. Reduction of γ-tubulin also rescued dendrite regeneration in backgrounds containing kinetochore RNAi transgenes. We conclude that kinetochore proteins function postmitotically in neurons to suppress dendritic microtubule dynamics by inhibiting nucleation.

Microtubules are inherently dynamic cytoskeletal polymers whose length and organization can be altered to perform essential functions in eukaryotic cells, such as providing tracks for intracellular trafficking and forming the mitotic spindle. Microtubules can be bundled to create more stable structures that collectively propagate force, such as in the flagellar axoneme, which provides motility. The subpellicular microtubule array of the protist parasite Trypanosoma brucei, the causative agent of African sleeping sickness, is a remarkable example of a highly specialized microtubule bundle. It is comprised of a single layer of microtubules that are crosslinked to each other and to the overlying plasma membrane. The array microtubules appear to be highly stable and remain intact throughout the cell cycle, but very little is known about the pathways that tune microtubule properties in trypanosomatids. Here, we show that the subpellicular microtubule array is organized into subdomains that consist of differentially localized array-associated proteins at the array posterior, middle, and anterior. The array-associated protein PAVE1 stabilizes array microtubules at the cell posterior and is essential for maintaining its tapered shape. PAVE1 and the newly identified protein PAVE2 form a complex that binds directly to the microtubule lattice, demonstrating that they are a true kinetoplastid-specific MAP. TbAIR9, which localizes to the entirety of the subpellicular array, is necessary for maintaining the localization of array-associated proteins within their respective subdomains of the array. The arrangement of proteins within the array likely tunes the local properties of array microtubules and creates the asymmetric shape of the cell, which is essential for parasite viability.

The wide functional impacts of microtubules are unleashed and controlled by a battery of microtubule-associated proteins (MAPs). Specialists in the field appreciate the diversity of known MAPs and propel the identifications of novel MAPs. By contrast, there is neither specific database to record known MAPs, nor MAP predictor that can facilitate the discovery of potential MAPs. We here report the establishment of a MAP-centered online analysis tool MAPanalyzer, which consists of a MAP database and a MAP predictor. In the database, a core MAP dataset, which is fully manually curated from the literature, is further enriched by MAP information collected via automated pipeline. The core dataset, on the other hand, enables the building of a novel MAP predictor which combines specialized machine learning classifiers and the BLAST homology searching tool. Benchmarks on the curated testing dataset and the Arabidopsis thaliana whole genome dataset have shown that the proposed predictor outperforms not only its own components (i.e. the machine learning classifiers and BLAST), but also another popular homology searching tool, PSI-BLAST. Therefore, MAPanalyzer will serve as a promising computational resource for the investigations of MAPs. Database URL: http://systbio.cau.edu.cn/mappred/.