Direct Regulation of Microtubule Dynamics by KIF17 Motor and Tail Domains

KIF17 is a kinesin-2 family motor that interacts with EB1 at microtubule (MT) plus-ends and contributes to MT stabilization in epithelial cells. The mechanism by which KIF17 affects MTs and how its activity is regulated are not yet known. Here, we show that EB1 and the KIF17 autoinhibitory tail domain (KIF17-Tail) interacted competitively with the KIF17 catalytic motor domain (K370). Both EB1 and KIF17-Tail decreased the K_0.5MT of K370, with opposing effects on MT-stimulated ATPase activity. Importantly, K370 had independent effects on MT dynamic instability, resulting in formation of long MTs without affecting polymerization rate or total polymer mass. K370 also inhibited MT depolymerization induced by dilution in vitro and by nocodazole in cells, suggesting that it acts by protecting MT plus-ends. Interestingly, KIF17-Tail bound MTs and tubulin dimers, delaying initial MT polymerization in vitro and MT regrowth in cells. However, neither EB1 nor KIF17-Tail affected K370-mediated MT polymerization or stabilization significantly in vitro, and EB1 was dispensable for MT stabilization by K370 in cells. Thus, although EB1 and KIF17-Tail may coordinate KIF17 catalytic activity, our data reveal a novel and direct role for KIF17 in regulating MT dynamics.     

One-Dimensional Brownian Motion of Charged Nanoparticles along Microtubules: A Model System for Weak Binding Interactions

Various proteins are known to exhibit one-dimensional Brownian motion along charged rodlike polymers, such as microtubules (MTs), actin, and DNA. The electrostatic interaction between the proteins and the rodlike polymers appears to be crucial for one-dimensional Brownian motion, although the underlying mechanism has not been fully clarified. We examined the interactions of positively-charged nanoparticles composed of polyacrylamide gels with MTs. These hydrophilic nanoparticles bound to MTs and displayed one-dimensional Brownian motion in a charge-dependent manner, which indicates that nonspecific electrostatic interaction is sufficient for one-dimensional Brownian motion. The diffusion coefficient decreased exponentially with an increasing particle charge (with the exponent being 0.10 k_BT per charge), whereas the duration of the interaction increased exponentially (exponent of 0.22 k_BT per charge). These results can be explained semiquantitatively if one assumes that a particle repeats a cycle of binding to and movement along an MT until it finally dissociates from the MT. During the movement, a particle is still electrostatically constrained in the potential valley surrounding the MT. This entire process can be described by a three-state model analogous to the Michaelis-Menten scheme, in which the two parameters of the equilibrium constant between binding and movement, and the rate of dissociation from the MT, are derived as a function of the particle charge density. This study highlights the possibility that the weak binding interactions between proteins and rodlike polymers, e.g., MTs, are mediated by a similar, nonspecific charge-dependent mechanism.     

Post-translational modifications within tau paired helical filament nucleating motifs perturb microtubule interactions and oligomer formation

Post-translationally modified tau is the primary component of tau neurofibrillary tangles, a pathological hallmark of Alzheimer''s disease and other tauopathies. Post-translational modifications (PTMs) within the tau microtubule (MT)-binding domain (MBD), which encompasses two hexapeptide motifs that act as critical nucleating regions for tau aggregation, can potentially modulate tau aggregation as well as interactions with MTs and membranes. Here, we characterize the effects of a recently discovered tau PTM, lysine succinylation, on tau–tubulin interactions and compare these to the effects of two previously reported MBD modifications, lysine acetylation and tyrosine phosphorylation. As generation of site-specific PTMs in proteins is challenging, we used short synthetic peptides to quantify the effects on tubulin binding of three site-specific PTMs located within the PHF6^∗ (paired helical filament [PHF] residues 275–280) and PHF6 (residues 306–311) hexapeptide motifs: K280 acetylation, Y310 phosphorylation, and K311 succinylation. We compared these effects to those observed for MBD PTM-mimetic point mutations K280Q, Y310E, and K311E. Finally, we evaluated the effects of these PTM-mimetic mutations on MBD membrane binding and membrane-induced fibril and oligomer formation. We found that all three PTMs perturb tau MT binding, with Y310 phosphorylation exerting the strongest effect. PTM-mimetic mutations partially recapitulated the effects of the PTMs on MT binding and also disrupted tau membrane binding and membrane-induced oligomer and fibril formation. These results imply that these PTMs, including the novel and Alzheimer''s disease–specific succinylation of tau K311, may influence both the physiological and pathological interactions of tau and thus represent targets for therapeutic intervention.     

Human Microtubule-Associated-Protein Tau Regulates the Number of Protofilaments in Microtubules: A Synchrotron X-Ray Scattering Study

Microtubules (MTs), a major component of the eukaryotic cytoskeleton, are 25 nm protein nanotubes with walls comprised of assembled protofilaments built from αβ heterodimeric tubulin. In neural cells, different isoforms of the microtubule-associated-protein (MAP) tau regulate tubulin assembly and MT stability. Using synchrotron small angle x-ray scattering (SAXS), we have examined the effects of all six naturally occurring central nervous system tau isoforms on the assembly structure of taxol-stabilized MTs. Most notably, we found that tau regulates the distribution of protofilament numbers in MTs as reflected in the observed increase in the average radius 〈R^MT〉 of MTs with increasing Φ, the tau/tubulin-dimer molar ratio. Within experimental scatter, the change in 〈R^MT〉 seems to be isoform independent. Significantly, 〈R^MT〉 was observed to rapidly increase for 0 < Φ < 0.2 and saturate for Φ between 0.2-0.5. Thus, a local shape distortion of the tubulin dimer on tau binding, at coverages much less than a monolayer, is spread collectively over many dimers on the scale of protofilaments. This implies that tau regulates the shape of protofilaments and thus the spontaneous curvature C_o^MT of MTs leading to changes in the curvature C^MT (=1/R^MT). An important biological implication of these findings is a possible allosteric role for tau where the tau-induced shape changes of the MT surface may effect the MT binding activity of other MAPs present in neurons. Furthermore, the results, which provide insight into the regulation of the elastic properties of MTs by tau, may also impact biomaterials applications requiring radial size-controlled nanotubes.     

Reconstitution of dynamic microtubules with Drosophila XMAP215, EB1, and Sentin

Dynamic microtubules (MTs) are essential for various intracellular events, such as mitosis. In Drosophila melanogaster S2 cells, three MT tip-localizing proteins, Msps/XMAP215, EB1, and Sentin (an EB1 cargo protein), have been identified as being critical for accelerating MT growth and promoting catastrophe events, thus resulting in the formation of dynamic MTs. However, the molecular activity of each protein and the basis of the modulation of MT dynamics by these three factors are unknown. In this paper, we showed in vitro that XMAP215^msps had a potent growth-promoting activity at a wide range of tubulin concentrations, whereas Sentin, when recruited by EB1 to the growing MT tip, accelerated growth and also increased catastrophe frequency. When all three factors were combined, the growth rate was synergistically enhanced, and rescue events were observed most frequently, but frequent catastrophes restrained the lengthening of the MTs. We propose that MT dynamics are promoted by the independent as well as the cooperative action of XMAP215^msps polymerase and the EB1–Sentin duo.     

Modulating Microtubules: A Molecular Perspective on the Effects of Tail Modifications

Microtubules (MTs), an essential component of the eukaryotic cytoskeleton, are a lattice of polymerized tubulin dimers and are crucial for various cellular processes. The genetic and chemical diversity of tubulin and their disordered tails gives rise to a “tubulin code”. The functional role of tubulin post-translational modifications (PTMs), which contribute to the chemical diversity of the tubulin code, is gradually being unraveled. However, variation in the length and spatial organization of tubulin poly-modifications leads to an enormous combinatorial PTM space, which is difficult to study experimentally. Hence, the impact of the combinatorial tubulin PTM space on the biophysical properties of tubulin tails and their interactions with other proteins remains elusive.Here, we combine all-atom and coarse-grained molecular dynamics simulations to elucidate the biophysical implications of the large combinatorial tubulin PTM space in the context of an MT lattice. We find that tail–body interactions are more dominant in the tubulin dimer than in an MT lattice, and are more significant for the tails of α compared with β tubulin. In addition, polyglutamylation, but not polyglycylation, expands the dimensions of the tubulin tails. Polyglutamylation also leads to a decrease in the diffusion rate of MT-associated protein EB1 on MTs, while polyglycylation often increases diffusion rate. These observations are generally not sensitive to the organization of the polymodifications. The effect of PTMs on MT charge density and tail dynamics are also discussed. Overall, this study presents a molecular quantification of the biophysical properties of tubulin tails and their polymodifications, and provides predictions on the functional importance of tubulin PTMs.     

Identification of microtubule growth deceleration and its regulation by conserved and novel proteins

Microtubules (MTs) are cytoskeletal polymers that participate in diverse cellular functions, including cell division, intracellular trafficking, and templating of cilia and flagella. MTs undergo dynamic instability, alternating between growth and shortening via catastrophe and rescue events. The rates and frequencies of MT dynamic parameters appear to be characteristic for a given cell type. We recently reported that all MT dynamic parameters vary throughout differentiation of a smooth muscle cell type in intact Caenorhabditis elegans. Here we describe local differences in MT dynamics and a novel MT behavior: an abrupt change in growth rate (deceleration) of single MTs occurring in the cell periphery of these cells. MT deceleration occurs where there is a decrease in local soluble tubulin concentration at the cell periphery. This local regulation of tubulin concentration and MT deceleration are dependent on two novel homologues of human cylicin. These novel ORFs, which we name cylc-1 and -2, share sequence homology with stathmins and encode small, very basic proteins containing several KKD/E repeats. The TOG domain–containing protein ZYG-9^TOGp is responsible for the faster polymerization rate within the cell body. Thus we have defined two contributors to the molecular regulation for this novel MT behavior.     

Probing Interactions between CLIP-170, EB1, and Microtubules

Cytoplasmic linker protein 170 (CLIP-170) is a microtubule (MT) plus-end tracking protein (+ TIP) that dynamically localizes to the MT plus end and regulates MT dynamics. The mechanisms of these activities remain unclear because the CLIP-170-MT interaction is poorly understood, and even less is known about how CLIP-170 and other + TIPs act together as a network. CLIP-170 binds to the acidic C-terminal tail of α-tubulin. However, the observation that CLIP-170 has two CAP-Gly (cytoskeleton-associated protein glycine-rich) motifs and multiple serine-rich regions suggests that a single CLIP-170 molecule has multiple tubulin binding sites, and that these sites might bind to multiple parts of the tubulin dimer. Using a combination of chemical cross-linking and mass spectrometry, we find that CLIP-170 binds to both α-tubulin and β-tubulin, and that binding is not limited to the acidic C-terminal tails. We provide evidence that these additional binding sites include the H12 helices of both α-tubulin and β-tubulin and are significant for CLIP-170 activity. Previous work has shown that CLIP-170 binds to end-binding protein 1 (EB1) via the EB1 C-terminus, which mimics the acidic C-terminal tail of tubulin. We find that CLIP-170 can utilize its multiple tubulin binding sites to bind to EB1 and MT simultaneously. These observations help to explain how CLIP-170 can nucleate MTs and alter MT dynamics, and they contribute to understanding the significance and properties of the + TIP network.     

The Vip1 Inositol Polyphosphate Kinase Family Regulates Polarized Growth and Modulates the Microtubule Cytoskeleton in Fungi

Microtubules (MTs) are pivotal for numerous eukaryotic processes ranging from cellular morphogenesis, chromosome segregation to intracellular transport. Execution of these tasks requires intricate regulation of MT dynamics. Here, we identify a new regulator of the Schizosaccharomyces pombe MT cytoskeleton: Asp1, a member of the highly conserved Vip1 inositol polyphosphate kinase family. Inositol pyrophosphates generated by Asp1 modulate MT dynamic parameters independent of the central +TIP EB1 and in a dose-dependent and cellular-context-dependent manner. Importantly, our analysis of the in vitro kinase activities of various S. pombe Asp1 variants demonstrated that the C-terminal phosphatase-like domain of the dual domain Vip1 protein negatively affects the inositol pyrophosphate output of the N-terminal kinase domain. These data suggest that the former domain has phosphatase activity. Remarkably, Vip1 regulation of the MT cytoskeleton is a conserved feature, as Vip1-like proteins of the filamentous ascomycete Aspergillus nidulans and the distantly related pathogenic basidiomycete Ustilago maydis also affect the MT cytoskeleton in these organisms. Consistent with the role of interphase MTs in growth zone selection/maintenance, all 3 fungal systems show aspects of aberrant cell morphogenesis. Thus, for the first time we have identified a conserved biological process for inositol pyrophosphates.     

Multiple mechanisms determine ER network morphology during the cell cycle in Xenopus egg extracts

In metazoans the endoplasmic reticulum (ER) changes during the cell cycle, with the nuclear envelope (NE) disassembling and reassembling during mitosis and the peripheral ER undergoing extensive remodeling. Here we address how ER morphology is generated during the cell cycle using crude and fractionated Xenopus laevis egg extracts. We show that in interphase the ER is concentrated at the microtubule (MT)-organizing center by dynein and is spread by outward extension of ER tubules through their association with plus ends of growing MTs. Fusion of membranes into an ER network is dependent on the guanosine triphosphatase atlastin (ATL). NE assembly requires fusion by both ATL and ER-soluble N-ethyl-maleimide–sensitive factor adaptor protein receptors. In mitotic extracts, the ER converts into a network of sheets connected by ER tubules and loses most of its interactions with MTs. Together, these results indicate that fusion of ER membranes by ATL and interaction of ER with growing MT ends and dynein cooperate to generate distinct ER morphologies during the cell cycle.     

A Highlights from MBoC Selection: MAP65/Ase1 promote microtubule flexibility

Microtubules (MTs) are dynamic cytoskeletal elements involved in numerous cellular processes. Although they are highly rigid polymers with a persistence length of 1–8 mm, they may exhibit a curved shape at a scale of few micrometers within cells, depending on their biological functions. However, how MT flexural rigidity in cells is regulated remains poorly understood. Here we ask whether MT-associated proteins (MAPs) could locally control the mechanical properties of MTs. We show that two major cross-linkers of the conserved MAP65/PRC1/Ase1 family drastically decrease MT rigidity. Their MT-binding domain mediates this effect. Remarkably, the softening effect of MAP65 observed on single MTs is maintained when MTs are cross-linked. By reconstituting physical collisions between growing MTs/MT bundles, we further show that the decrease in MT stiffness induced by MAP65 proteins is responsible for the sharp bending deformations observed in cells when they coalign at a steep angle to create bundles. Taken together, these data provide new insights into how MAP65, by modifying MT mechanical properties, may regulate the formation of complex MT arrays.     

Nudel/NudE and Lis1 promote dynein and dynactin interaction in the context of spindle morphogenesis

Lis1, Nudel/NudE, and dynactin are regulators of cytoplasmic dynein, a minus end–directed, microtubule (MT)-based motor required for proper spindle assembly and orientation. In vitro studies have shown that dynactin promotes processive movement of dynein on MTs, whereas Lis1 causes dynein to enter a persistent force-generating state (referred to here as dynein stall). Yet how the activities of Lis1, Nudel/NudE, and dynactin are coordinated to regulate dynein remains poorly understood in vivo. Working in Xenopus egg extracts, we show that Nudel/NudE facilitates the binding of Lis1 to dynein, which enhances the recruitment of dynactin to dynein. We further report a novel Lis1-dependent dynein–dynactin interaction that is essential for the organization of mitotic spindle poles. Finally, using assays for MT gliding and spindle assembly, we demonstrate an antagonistic relationship between Lis1 and dynactin that allows dynactin to relieve Lis1-induced dynein stall on MTs. Our findings suggest the interesting possibility that Lis1 and dynactin could alternately engage with dynein to allow the motor to promote spindle assembly.     

Evaluating the microtubule cytoskeleton and its interacting proteins in monocots by mining the rice genome

Background

Microtubules (MTs) are assembled by heterodimers of α- and β-tubulins, which provide tracks for directional transport and frameworks for the spindle apparatus and the phragmoplast. MT nucleation and dynamics are regulated by components such as the γ-tubulin complex which are conserved among eukaryotes, and other components which are unique to plants. Following remarkable progress made in the model plant Arabidopsis thaliana toward revealing key components regulating MT activities, the completed rice (Oryza sativa) genome has prompted a survey of the MT cytoskeleton in this important crop as a model for monocots.

Scope

The rice genome contains three α-tubulin genes, eight β-tubulin genes and a single γ-tubulin gene. A functional γ-tubulin ring complex is expected to form in rice as genes encoding all components of the complex are present. Among proteins that interact with MTs, compared with A. thaliana, rice has more genes encoding some members such as the MAP65/Ase1p/PRC1 family, but fewer for the motor kinesins, the end-binding protein EB1 and the mitotic kinase Aurora. Although most known MT-interacting factors have apparent orthologues in rice, no orthologues of arabidopsis RIC1 and MAP18 have been identified in rice. Among all proteins surveyed here, only a few have had their functions characterized by genetic means in rice. Elucidating functions of proteins of the rice MT cytoskeleton, aided by recent technical advances made in this model monocot, will greatly advance our knowledge of how monocots employ their MTs to regulate their growth and form.Key words: Cytoskeleton, kinesins, microtubules (MTs), microtubule-associated proteins (MAPs), motors, rice, Oryza sativa     

Metallothionein functions and structural characteristics

Metallothioneins (MTs) are low molecular weight proteins characterized by a high cysteine content and give rise to metal-thiolate clusters. Most MTs have two metal clusters containing three and four bivalent metal ions, respectively. The MT gene family in mammals consists of four subfamilies designated MT-1 through MT-4. MT-3 is expressed predominantly in brain and MT-4 in differentiating stratified squamous epithelial cells. Many reports have addressed MT structure and function, but despite the increasing experimental data several topics remain to be clarified, and the true function of this elusive protein has yet to be disclosed. Owing to their induction by a variety of stimuli, MTs are considered valid biomarkers in medicine and environmental studies. Here, we will discuss only a few topics taken from the latest literature. Special emphasis will be placed on MT antioxidant functions, the related oxidation of cysteines, which can give rise to intra/intermolecular bridges, and the relations between MTs and diseases which could be originated by metal dysregulation.     

Bile and liver metallothionein behavior in copper-exposed fish

The present study analyzed metallothionein (MT) excretion from liver to bile in Nile Tilapia (Oreochromis niloticus) exposed to sub-lethal copper concentrations (2 mg L⁻¹) in a laboratory setting. MTs in liver and bile were quantified by spectrophotometry after thermal incubation and MT metal-binding profiles were characterized by size exclusion high performance liquid chromatography coupled to ICP-MS (SEC-HPLC–ICP-MS). Results show that liver MT is present in approximately 250-fold higher concentrations than bile MT in non-exposed fish. Differences between the MT profiles from the control and exposed group were observed for both matrices, indicating differential metal-binding behavior when comparing liver and bile MT. This is novel data regarding intra-organ MT comparisons, since differences between organs are usually present only with regard to quantification, not metal-binding behavior. Bile MT showed statistically significant differences between the control and exposed group, while the same did not occur with liver MT. This indicates that MTs synthesized in the liver accumulate more slowly than MTs excreted from liver to bile, since the same fish presented significantly higher MT levels in liver when compared to bile. We postulate that bile, although excreted in the intestine and partially reabsorbed by the same returning to the liver, may also release MT-bound metals more rapidly and efficiently, which may indicate an efficient detoxification route. Thus, we propose that the analysis of bile MTs to observe recent metal exposure may be more adequate than the analysis of liver MTs, since organism responses to metals are more quickly observed in bile, although further studies are necessary.     

Building the Microtubule Cytoskeleton Piece by Piece

The microtubule (MT) cytoskeleton gives cells their shape, organizes the cellular interior, and segregates chromosomes. These functions rely on the precise arrangement of MTs, which is achieved by the coordinated action of MT-associated proteins (MAPs). We highlight the first and most important examples of how different MAP activities are combined in vitro to create an ensemble function that exceeds the simple addition of their individual activities, and how the Xenopus laevis egg extract system has been utilized as a powerful intermediate between cellular and purified systems to uncover the design principles of self-organized MT networks in the cell.     

Regulation of microtubule dynamics by TOG-domain proteins XMAP215/Dis1 and CLASP

The molecular mechanisms by which microtubule-associated proteins (MAPs) regulate the dynamic properties of microtubules (MTs) are still poorly understood. We review recent advances in our understanding of two conserved families of MAPs, the XMAP215/Dis1 and CLASP family of proteins. In vivo and in vitro studies show that XMAP215 proteins act as microtubule polymerases at MT plus ends to accelerate MT assembly, and CLASP proteins promote MT rescue and suppress MT catastrophe events. These are structurally related proteins that use conserved TOG domains to recruit tubulin dimers to MTs. We discuss models for how these proteins might use these individual tubulin dimers to regulate dynamic behavior of MT plus ends.     

Post-translational modifications of tubulin in the nervous system

Many studies have shown that microtubules (MTs) interact with MT-associated proteins and motor proteins. These interactions are essential for the formation and maintenance of the polarized morphology of neurons and have been proposed to be regulated in part by highly diverse, unusual post-translational modifications (PTMs) of tubulin, including acetylation, tyrosination, detyrosination, Δ2 modification, polyglutamylation, polyglycylation, palmitoylation, and phosphorylation. However, the precise mechanisms of PTM generation and the properties of modified MTs have been poorly understood until recently. Recent PTM research has uncovered the enzymes mediating tubulin PTMs and provided new insights into the regulation of MT-based functions. The identification of tubulin deacetylase and discovery of its specific inhibitors have paved the way to understand the roles of acetylated MTs in kinesin-mediated axonal transport and neurodegenerative diseases such as Huntington's disease. Studies with tubulin tyrosine ligase (TTL)-null mice have shown that tyrosinated MTs are essential in normal brain development. The discovery of TTL-like genes encoding polyglutamylase has led to the finding that polyglutamylated MTs which accumulate during brain development are involved in synapse vesicle transport or neurite outgrowth through interactions with motor proteins or MT-associated proteins, respectively. Here we review current exciting topics that are expected to advance MT research in the nervous system.     

Polyglutamylated Tubulin Binding Protein C1orf96/CSAP Is Involved in Microtubule Stabilization in Mitotic Spindles

The centrosome-associated C1orf96/Centriole, Cilia and Spindle-Associated Protein (CSAP) targets polyglutamylated tubulin in mitotic microtubules (MTs). Loss of CSAP causes critical defects in brain development; however, it is unclear how CSAP association with MTs affects mitosis progression. In this study, we explored the molecular mechanisms of the interaction of CSAP with mitotic spindles. Loss of CSAP caused MT instability in mitotic spindles and resulted in mislocalization of Nuclear protein that associates with the Mitotic Apparatus (NuMA), with defective MT dynamics. Thus, CSAP overload in the spindles caused extensive MT stabilization and recruitment of NuMA. Moreover, MT stabilization by CSAP led to high levels of polyglutamylation on MTs. MT depolymerization by cold or nocodazole treatment was inhibited by CSAP binding. Live-cell imaging analysis suggested that CSAP-dependent MT-stabilization led to centrosome-free MT aster formation immediately upon nuclear envelope breakdown without γ-tubulin. We therefore propose that CSAP associates with MTs around centrosomes to stabilize MTs during mitosis, ensuring proper bipolar spindle formation and maintenance.     

Stabilization of overlapping microtubules by fission yeast CLASP

Many microtubule (MT) structures contain dynamic MTs that are bundled and stabilized in overlapping arrays. CLASPs are conserved MT-binding proteins implicated in the regulation of MT plus ends. Here, we show that the Schizosaccharomyces pombe CLASP, cls1p/peg1p, mediates the stabilization of overlapping MTs within the mitotic spindle and interphase bundles. cls1p localizes to these regions but not to interphase MT plus ends. Inactivation of cls1p leads to the rapid depolymerization of spindle midzone MTs. cls1p also stabilizes a subset of MTs within interphase bundles. cls1p prevents disassembly of the entire microtubule, while still allowing for plus-end growth. It has no measurable effects on MT nucleation, polymerization, catastrophe, or bundling. A direct interaction with ase1p (PRC1/MAP65) targets cls1p to regions of antiparallel MT overlap. These findings show how a MT-stabilizing factor attached to specific sites on MTs can help to generate MT structures that have both dynamic and stable components.