Microtubule Surface Research Articles

Measurement Of The Protein Friction Between The Yeast Kinesin-8 Kip3p And Microtubules

Several proteins have been shown to undergo 'one-dimensional' diffusion along the surface of microtubules. Diffusion is thought to enhance the rate of targeting of proteins to the microtubule end for the depolymerizing kinesin-13 and the polymerase XMAP215, or to increase the processivity of kinesin-1 and dynein. According to the Einstein-Smolukowski relation, the diffusion coefficient, D, is related to the friction coefficient, gamma, according to D = kT/gamma. This relation, however, has not been experimentally tested for individual bio-molecules. We measured both the diffusional and frictional properties of single yeast kinesin-8 motor proteins, Kip3p, interacting with microtubule filaments in the ADP state. Using single molecule fluorescence we found that that the diffusion coefficient was 5400±1400 nm2/s with an average lifetime on the microtubule lattice of 8 s. Using an optical trap to drag a microsphere coated with Kip3p along microtubules, we measured a single molecule drag coefficient of 790±230 nNs/m at low speeds. Thus we verified the Einstein-Smolukowski relation. For larger speeds and drag forces, we measured a non-linear force-velocity relation which was well fit by a model in which Kip3p is diffusing in a periodic potential with an 8-nm periodicity and a barrier height between binding sites of 14±2 kT. This finding of an 8-nm periodicity is supported by an analysis of the positional fluctuations. Our measurements are a step towards resolving the molecular mechanism underlying protein friction an important parameter for active protein locomotion limiting the efficiency.

Microtubule Binding and Rotation of the Kinesin-14 Stalk

Movement of motors along cytoskeletal filaments is thought to be driven by small structural changes that are amplified by a large rotation of the alpha-helical coiled coil. The coiled-coil stalk of the kinesin-14 motor, Ncd, has been visualized in a rotated conformation in a crystal structure and proposed to act like a lever to amplify force produced by the motor, resulting in a working stroke that directs the motor to the microtubule minus end. We show here that an Ncd mutant that is trapped in a stalk-rotated conformation binds tightly to microtubules and shows fluorescence resonance energy transfer between the end of the stalk and microtubule, indicating that rotation of the stalk towards the microtubule is coupled to motor binding to microtubules. A mutant blocked in stalk rotation binds weakly to microtubules and shows no energy transfer, demonstrating that binding by the motor to microtubules requires movement of the stalk. Energy transfer assays show that wild-type Ncd binds to microtubules without added nucleotide with the end of the stalk more than ∼9 nm from the microtubule, rotating less than 50 degrees from a position perpendicular to the microtubule. Upon binding with a non-hydrolysable ATP analogue, the stalk lies within ∼6 nm of the microtubule surface, representing a rotation of ∼70 degrees. These findings are consistent with previous reports by cryoEM that the Ncd stalk rotates when the microtubule-bound motor binds ATP. However, our results indicate that stalk rotation is initiated by filament binding and ADP release, and completed upon ATP binding, rather than triggered by ATP binding. Initiation of the Ncd stalk rotation by microtubule binding and ADP release, and its completion on ATP binding is reminiscent of the two-step working stroke of myosin I, revealing an unexpected similarity between the motors.

Alzheimer disease specific phosphoepitopes of Tau interfere with assembly of tubulin but not binding to microtubules

In Alzheimer disease (AD)-affected neurons, the Tau protein is found in an aggregated and hyperphosphorylated state. A common hypothesis is that Tau hyperphosphorylation causes its dissociation from the microtubular surface, with consequently a breakdown of the microtubules (MTs) and aggregation of the unbound Tau. We evaluated the effect of Tau phosphorylation on both tubulin assembly and MT binding. We show that the cyclin-dependent kinase 2/cyclin A3 kinase complex can generate the AT8 and AT180 AD-specific phospho-epitopes and use NMR spectroscopy to validate qualitatively and quantitatively the phospho content of our samples. The simultaneous presence of both epitopes disables the tubulin assembly capacity of Tau in conditions whereby Tau is the driving force for the assembly process but does not, however, inhibit MT assembly when the latter is driven by an increased tubulin concentration. When compared to the isolated MT binding repeats (K(d)=0.3 microM), the phospho-Tau retains a substantial affinity for preformed MTs (K(d)=11 nM), suggesting that the phosphorylated proline-rich region still participates in the binding event. Our results hence indicate that the sole phosphorylation at the AT8/AT180 epitopes, although leading to a functional defect for Tau, is not sufficient for its dissociation from the MT surface and subsequent aggregation as observed in AD.

Microtubule-Driven Multimerization Recruits ase1p onto Overlapping Microtubules

Microtubule (MT) crosslinking proteins of the ase1p/PRC1/Map65 family play a major role in the construction of MT networks such as the mitotic spindle. Most homologs in this family have been shown to localize with a remarkable specificity to sets of MTs that overlap with an antiparallel relative orientation [1-4]. Regulatory proteins bind to ase1p/PRC1/Map65 and appear to use the localization to set up precise spatial signals [5-10]. Here, we present evidence for a mechanism of localized protein multimerization underlying the specific targeting of ase1p, the fision yeast homolog. In controlled in vitro experiments, dimers of ase1-GFP diffused along the surface of single MTs and, at concentrations above a certain threshold, assembled into static multimeric structures. We observed that this threshold was significantly lower on overlapping MTs. We also observed diffusion and multimerization of ase1-GFP on MTs inside living cells, suggesting that a multimerization-driven localization mechanism is relevant in vivo. The domains responsible for MT binding and multimerization were identified via a series of ase1p truncations. Our findings show that cells use a finely tuned cooperative localization mechanism that exploits differences in the geometry and concentration of ase1p binding sites along single and overlapping MTs.

S2-03-04: Role of aminopeptidase NPPS in tauopathy

Abstract not available.

S2-03-06: The role of tau in axonal transport and neurodegeneration

Abnormal changes in the properties of Tau protein are closely correlated with the progression of Alzheimer disease. They include aggregation into neurofibrillary tangles, hyperphosphorylation, missorting from the axons into the somatodendritic compartments of neurons. It is currently not well understood what causes the abnormal changes, and how they are related to the toxicity of Abeta, the decay of synapses, and the death of neurons. We have used different modes of live cell microscopy of primary neurons, adenoviral transfection of neurons with different variants of tau, and inducible transgenic mouse models of tauopathy. The study mainly focussed on three facets of Tau: (1) By obstructing the surface of microtubules, Tau is capable, in principle, of slowing down axonal transport by inhibiting the attachment of motor proteins. This is particularly visible in the form of reduced mitochondrial movement, which in turn causes an energy crisis in the neuron by reducing the ATP level. As a result, synapses disintegrate, and synaptic markers disappear. (2) Although Tau is mostly a “microtubule-associated protein” (MAP), its residence time on microtubules is only a few seconds, and thus Tau is able to move through cytosolic space by diffusion with surprising speed. This may be one of the factors that enables Tau to enter the somatodendritic compartment, once the normal sorting mechanisms break down during Alzheimer disease. (3) There is a debate on whether the aggregation of Tau as such is toxic to neurons, or whether tau-induced toxicity depends on other factors. Using inducible cell and transgenic mouse models we find that the toxicity of Tau to neurons is tightly correlated with its ability to form beta-structure and to aggregate. Thus mutants of Tau that have a high beta-propensity show rapid aggregation and high toxicity, whereas mutants that are unable to aggregate are not toxic. The findings argue that the toxicity of Tau to neurons requires a particular pathological conformation which promotes beta-structure and tau-tau interactions. Using small molecule inhibitors it may be possible to prevent the pathological conformation and thus to keep Tau functional in the neuron without aggregation.

P2-135: Mechanisms of neuronal traffic inhibition by tau protein

The missorting of Tau protein from the axonal to the somatodendritic compartment and loss of synapses are hallmarks of Alzheimer's disease which precede neuronal loss and the pathological aggregation of tau protein into neurofibrillary tangles. We are interested in the mechanisms by which Tau can affect the integrity of synapses. One major factor is the inhibition of movement of mitochondria by Tau which leads to mitochondrial dysfunction and energy deprivation (loss of ATP production). We have now studied the pathway of Tau's interference with mitochondrial transport. Primary hippocampal neurons were transfected with CFP-Tau, mitochondrial traffic and synaptic integrity was observed by time-resolved confocal microscopy, followed by thin-sectioning electron microscopy to visualize the state of mitochondria, synapses, and the cytoskeleton. The results show that Tau operates on two distinct levels. One is a direct inhibition mechanism whereby bound Tau covers the microtubule surface and consequently interferes with the attachment of motor proteins. The second is an indirect inhibition mechanism which is based on the higher stability of microtubules when Tau is increased. This leads to a reduced level of tubulin subunits in the cytosol, which in turn causes the upregulation of tubulin synthesis (Cleveland et al., Nature 1983). This causes an increase in microtubule density, the microtubules become tightly bundled so that mitochondria can no longer move freely along axons and dendrites. The example illustrates how an improvement of microtubule stability can cause traffic deficits in neurons, rather than curing them. Research supported by MPG and DFG.

Tuning Microtubule‐Based Transport Through Filamentous MAPs: The Problem of Dynein

We recently proposed that regulating the single-to-multiple motor transition was a likely strategy for regulating kinesin-based transport in vivo. In this study, we use an in vitro bead assay coupled with an optical trap to investigate how this proposed regulatory mechanism affects dynein-based transport. We show that tau's regulation of kinesin function can proceed without interfering with dynein-based transport. Surprisingly, at extremely high tau levels--where kinesin cannot bind microtubules (MTs)--dynein can still contact MTs. The difference between tau's effects on kinesin- and dynein-based motility suggests that tau can be used to tune relative amounts of plus-end and minus-end-directed transport. As in the case of kinesin, we find that the 3RS isoform of tau is a more potent inhibitor of dynein binding to MTs. We show that this isoform-specific effect is not because of steric interference of tau's projection domains but rather because of tau's interactions with the motor at the MT surface. Nonetheless, we do observe a modest steric interference effect of tau away from the MT and discuss the potential implications of this for molecular motor structure.

Neuronal Microtubule-associated Proteins: Insights into Their Structures and Functions

Two families of microtubule-associated proteins (MAPs), the MAP1 family and the MAP2/MAP4/tau superfamily, are found in neurons. This review describes the cellular and subcellular localizations, structural features, and in vitro properties of these MAP families. The MAP1 family consists of three members, MAP1A, MAP1B, and MAP1S. They not only show partial amino acid sequence homology, but also share a common structural property: each MAP1 polypeptide translated from the corresponding mRNA is proteolytically cleaved into two chains, which assemble into the mature MAP1 complex. MAP1A and MAP1B expression is complementary during neuronal development, and MAP1B malfunction disturbs axonal growth. The MAP1 family proteins not only bind to microtubules but also interact with other cellular components. The MAP2/MAP4/tau superfamily also consists of three members, MAP2, MAP4, and tau. They share highly homologous microtubule-binding sequences in their microtubule-binding domains, while the other domains that protrude from the microtubule surfaces are totally distinct among the family members. MAP2 is mainly localized in cell bodies and dendrites, while tau is concentrated in axons. A low content of MAP4 is evenly distributed in cell bodies, dendrites and axons. Multiple variants of MAP2, MAP4, and tauare generated through alternative splicing, which is regulated in developmental stage- and tissue-specific manners. The MAP2/MAP4/tau superfamily proteins are highly active in forming, stabilizing, and stiffening microtubules. The minimal essential domain common among the family members was deduced to include the carboxyl terminal part of the Pro-rich region and a couple of microtubule-binding sequence repeats.

Conformational Dynamics and Thermal Cones of C Terminal Tubulin Tails in Neuronal Microtubules

In this paper we present a model for estimation of the C-terminal tubulin tail (CTT) dynamics in cytoskeletal microtubules of nerve cells. We show that the screened Coulomb interaction between a target CTT and the negatively charged microtubule surface as well as its immediate CTT neighbours results in confinement of the CTT motion within a restricted volume referred to as a thermal cone. Within the thermal cone the CTT motion is driven by the thermal fluctuations, while outside the thermal cone the CTT interaction energy with its environment is above the thermal energy solely due to repulsion from the negatively charged microtubule surface. Computations were performed for different CTT geometries and we have found that the CTT conformation with lowest energy is perpendicular to the microtubule surface. Since the coupling between a target CTT with its neighbour CTTs is 8 orders of magnitude below the thermal energy and considering the extremely short cytosolic Debye length of 0.79 nm, our results rule out generation and propagation of CTT conformational waves along the protofilament as a result of local CTT perturbations. The results as presented support a model in which the cytosolic electric fields and ionic currents generated by the neuronal excitations are “projected” onto the CTTs of underlying microtubules thus affecting their regulatory function of kinesin motion and MAP attachment/detachment.

Mechanical Properties of Inner-Arm Dynein-F (Dynein I1) Studied With In Vitro Motility Assays

Inner-arm dynein-f of Chlamydomonas flagella is a heterodimeric dynein. We performed conventional in vitro motility assays showing that dynein-f translocates microtubules at the comparatively low velocity of ∼1.2 μm/s. From the dependence of velocity upon the surface density of dynein-f, we estimate its duty ratio to be 0.6–0.7. The relation between microtubule landing rate and surface density of dynein-f are well fitted by the first-power dependence, as expected for a processive motor. At low dynein densities, progressing microtubules rotate erratically about a fixed point on the surface, at which a single dynein-f molecule is presumably located. We conclude that dynein-f has high processivity. In an axoneme, however, slow and processive dynein-f could impede microtubule sliding driven by other fast dyneins (e.g., dynein-c). To obtain insight into the in vivo roles of dynein-f, we measured the sliding velocity of microtubules driven by a mixture of dyneins -c and -f at various mixing ratios. The velocity is modulated as a function of the ratio of dynein-f in the mixture. This modulation suggests that dynein-f acts as a load in the axoneme, but force pushing dynein-f molecules forward seems to accelerate their dissociation from microtubules.

Protein Arms in the Kinetochore-Microtubule Interface of the Yeast DASH Complex

The yeast DASH complex is a heterodecameric component of the kinetochore necessary for accurate chromosome segregation. DASH forms closed rings around microtubules with a large gap between the DASH ring and the microtubule cylinder. We characterized the microtubule-binding properties of limited proteolysis products and subcomplexes of DASH, thus identifying candidate polypeptide extensions involved in establishing the DASH-microtubule interface. The acidic C-terminal extensions of tubulin subunits are not essential for DASH binding. We also measured the molecular mass of DASH rings on microtubules with scanning transmission electron microscopy and found that approximately 25 DASH heterodecamers assemble to form each ring. Dynamic association and relocation of multiple flexible appendages of DASH may allow the kinetochore to translate along the microtubule surface.

NMR Investigation of the Interaction between the Neuronal Protein Tau and the Microtubules

Whereas the interaction between Tau and the microtubules has been studied in great detail both by macroscopic techniques (cosedimentation, cryo-electron microscopy, and fluorescence spectroscopy) using the full-length protein or by peptide mapping assays, no detailed view at the level of individual amino acids has been presented when using the full-length protein. Here, we present a nuclear magnetic resonance (NMR) study of the interaction between the full-length neuronal protein Tau and paclitaxel-stabilized microtubules (MTs). As signal disappearance in the heteronuclear 1H-15N correlation spectra of isotope-labeled Tau in complex with MTs is due to direct association of the corresponding residue with the solid-like MT wall, we can map directly the fragment in interaction with the MT surface, and obtain a molecular picture of the precise interaction zones. The N-terminal region projects from the microtubule surface, and the lack of chemical shift variations when compared with free Tau proves that this region can regulate microtubular separation without adopting a stable conformation. Amino acids in the four microtubule binding repeats (MTBRs) lose all of their intensity, underscoring their immobilization upon binding to the MTs. The same loss of NMR intensity was observed for the proline-rich region starting at Ser214, underscoring its importance in the Tau:MT interaction. Fluorescence resonance energy transfer (FRET) experiments were used to obtain thermodynamic binding parameters, and led to the conclusion that the NMR defined fragment indeed is the major player in the interaction. When the same Ser214 is phosphorylated by the PKA kinase, the Tau:MT interaction strength decreases by 2 orders of magnitude, but the proline-rich region including the phospho-Ser214 does not gain sufficient mobility in the complex to make it observable by NMR spectroscopy. The presence of an intramolecular disulfide bridge, on the contrary, does lead to a partial detachment of the C-terminus of Tau, and decreases significantly the overloading of Tau on the MT surface.

An Isoform of Microtubule-associated Protein 4 Inhibits Kinesin-driven Microtubule Gliding

Recently, we revealed that microtubule-associated protein (MAP) 4 isoforms, which differ in the number of repeat sequences, alter the microtubule surface properties, and we proposed a hypothesis stating that the change in the surface properties may regulate the movements of microtubule motors [Tokuraku et al. (2003) J Biol Chem 278: 29609-29618]. In this study, we examined whether MAP4 isoforms affect the kinesin motor activity. When the MAP4 isoforms were present in an in vitro gliding assay, the five-repeat isoform but not the three- and four-repeat isoforms inhibited the movement of the microtubules in a concentration-dependent manner. The observation of individual microtubules revealed that in the presence of the five-repeat isoform, the microtubules completely stopped their movements or recurrently paused and resumed their movements, with no deceleration in the moving phase. The result can be explained by assuming that kinesin stops its movement when it encounters a microtubular region whose properties are altered by the MAPs. A sedimentation assay demonstrated that the MAP4 isoforms did not compete with kinesin for binding to microtubules, indicating that kinesin can bind to the MAP-bound microtubules, although it cannot move on them.

Kinesin as an Electrostatic Machine

Kinesin and related motor proteins utilize ATP fuel to propel themselves along the external surface of microtubules in a processive and directional fashion. We show that the observed step-like motion is possible through time-varying charge distributions furnished by the ATP hydrolysis cycle while the static charge configuration on the microtubule provides the guide for motion. Thus, while the chemical hydrolysis energy induces appropriate local conformational changes, the motor translational energy is fundamentally electrostatic. Numerical simulations of the mechanical equations of motion show that processivity and directionality are direct consequences of the ATP-dependent electrostatic interaction between the different charge distributions of kinesin and the microtubule.

The Schizosaccharomyces pombe EB1 Homolog Mal3p Binds and Stabilizes the Microtubule Lattice Seam

End binding 1 (EB1) proteins are highly conserved regulators of microtubule dynamics. Using electron microscopy (EM) and high-resolution surface shadowing we have studied the microtubule-binding properties of the fission yeast EB1 homolog Mal3p. This allowed for a direct visualization of Mal3p bound on the surface of microtubules. Mal3p particles usually formed a single line on each microtubule along just one of the multiple grooves that are formed by adjacent protofilaments. We provide structural data showing that the alignment of Mal3p molecules coincides with the microtubule lattice seam as well as data suggesting that Mal3p not only binds but also stabilizes this seam. Accordingly, Mal3p stabilizes microtubules through a specific interaction with what is potentially the weakest part of the microtubule in a way not previously demonstrated. Our findings further suggest that microtubules exhibit two distinct reaction platforms on their surface that can independently interact with target structures such as microtubule-associated proteins, motors, kinetochores, or membranes.

The distance that kinesin-1 holds its cargo from the microtubule surface measured by fluorescence interference contrast microscopy

Kinesin-1 is a motor protein that carries cellular cargo such as membrane-bounded organelles along microtubules (MTs). The homodimeric motor molecule contains two N-terminal motor domains (the motor "heads"), a long coiled-coil domain (the "rod" or "stalk"), and two small globular "tail" domains. Much has been learned about how kinesin's heads step along a MT and how the tail is involved in cargo binding and autoinhibition. However, little is known about the role of the rod. Here, we investigate the extension of the rod during active transport by measuring the height at which MTs glide over a kinesin-coated surface in the presence of ATP. To perform height measurements with nanometer precision, we used fluorescence interference contrast microscopy, which is based on the self-interference of fluorescent light from objects near a reflecting surface. Using an in situ calibrating method, we determined that kinesin-1 molecules elevate gliding MTs 17 +/- 2 nm (mean +/- SEM) above the surface. When varying the composition of the surrounding nucleotides or removing the negatively charged -COOH termini of the MTs by subtilisin digestion, we found no significant changes in the measured distance. Even though this distance is significantly shorter than the contour length of the motor molecule ( approximately 60 nm), it may be sufficient to prevent proteins bound to the MTs or prevent the organelles from interfering with transport.

Microtubule depolymerization can drive poleward chromosome motion in fission yeast

Prometaphase kinetochores interact with spindle microtubules (MTs) to establish chromosome bi-orientation. Before becoming bi-oriented, chromosomes frequently exhibit poleward movements (P-movements), which are commonly attributed to minus end-directed, MT-dependent motors. In fission yeast there are three such motors: dynein and two kinesin-14s, Pkl1p and Klp2p. None of these enzymes is essential for viability, and even the triple deletion grows well. This might be due to the fact that yeasts kinetochores are normally juxtapolar at mitosis onset, removing the need for poleward chromosome movement during prometaphase. Anaphase P-movement might also be dispensable in a spindle that elongates significantly. To test this supposition, we have analyzed kinetochore dynamics in cells whose kinetochore-pole connections have been dispersed. In cells recovering from this condition, the maximum rate of poleward kinetochore movement was unaffected by the deletion of any or all of these motors, strongly suggesting that other factors, like MT depolymerization, can cause such movements in vivo. However, Klp2p, which localizes to kinetochores, contributed to the effectiveness of P-movement by promoting the shortening of kinetochore fibers.

Microtubule Binding and Clustering of Human Tau-4R and Tau-P301L Proteins Isolated from Yeast Deficient in Orthologues of Glycogen Synthase Kinase-3β or cdk5

Phosphorylation of Tau protein and binding to microtubules is complex in neurons and was therefore studied in the less complicated model of humanized yeast. Human Tau was readily phosphorylated at pathological epitopes, but in opposite directions regulated by kinases Mds1 and Pho85, orthologues of glycogen synthase kinase-3beta and cdk5, respectively (1). We isolated recombinant Tau-4R and mutant Tau-P301L from wild type, Delta mds1 and Delta pho85 yeast strains and measured binding to Taxol-stabilized mammalian microtubules in relation to their phosphorylation patterns. Tau-4R isolated from yeast lacking mds1 was less phosphorylated and bound more to microtubules than Tau-4R isolated from wild type yeast. Paradoxically, phosphorylation of Tau-4R isolated from kinase Pho85-deficient yeast was dramatically increased resulting in very poor binding to microtubules. Dephosphorylation promoted binding to microtubules to uniform high levels, excluding other modifications. Isolated hyperphosphorylated, conformationally altered Tau-4R completely failed to bind microtubules. In parallel to Tau-4R, we expressed, isolated, and analyzed mutant Tau-P301L. Total dephosphorylated Tau-4R and Tau-P301L bound to microtubules very similarly. Surprisingly, Tau-P301L isolated from all yeast strains bound to microtubules more extensively than Tau-4R. Atomic force microscopy demonstrated, however, that the high apparent binding of Tau-P301L was due to aggregation on the microtubules, causing their deformation and bundling. Our data explain the pathological presence of granular Tau aggregates in neuronal processes in tauopathies.

The evolution of the structure of tubulin and its potential consequences for the role and function of microtubules in cells and embryos

This paper discusses the results of homology modeling and resulting calculation of key structural and physical properties for close to 300 tubulin sequences, including alpha, beta, gamma, delta and epsilon -tubulins. The basis for our calculations was the structure of the tubulin dimer published several years ago by Nogales et al. (1998), later refined to 3.5 resolution by Lowe et al. (2001). While, it appears that the alpha, beta and gamma-tubulins segregate into distinct structural families, we have found several differences in the physical properties within each group. Each of the alpha, beta and gamma- tubulin groups exhibit major differences in their net electric charge, dipole moments and dipole vector orientations. These properties could influence functional characteristics such as microtubule stability and assembly kinetics, due to their effects on the strength of protein-protein interactions. In addition to the general structural trends between tubulin isoforms, we have observed that the carboxy-termini of alpha and beta-tubulin exists in at least two stable configurations, either projecting away from the tubulin (or microtubule) surface, or collapsed onto the surface. In the latter case, the carboxy-termini form a lattice distinctly different from that of the well-known A and B lattices formed by the tubulin subunits. However, this C-terminal lattice is indistinguishable from the lattice formed when the microtubule-associated protein tau binds to the microtubule surface. Finally, we have discussed how tubulin sequence diversity arose in evolution giving rise to its particular phylogeny and how it may be used in cell- and tissue-specific expression including embryonal development.