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Human Molecular Genetics Advance Access originally published online on January 18, 2008
Human Molecular Genetics 2008 17(9):1245-1252; doi:10.1093/hmg/ddn014
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Effect of spastic paraplegia mutations in KIF5A kinesin on transport activity

Bettina Ebbing1,3,*, Klaudiusz Mann1, Agata Starosta1, Johann Jaud3, Ludger Schöls2, Rebecca Schüle2 and Günther Woehlke3,*

1 Institute for Cell Biology, University of Munich, Schillerstr. 42, D-80336 Munich, Germany 2 Hertie-Institute for Clinical Brain Research, Otfried-Müller-Str. 3, 72076 Tübingen, Germany 3 Physics Department E22, Technical University Munich, James-Franck-Str., 85747 Garching, Germany

* To whom correspondence should be addressed. Tel: +49 8928912486; Fax: +49 8928912523; Email: guenther.woehlke{at}lrz.uni-muenchen.de (G.W.); bettina.ebbing{at}lrz.uni-muenchen.de (B.E.)

Received October 30, 2007; Revised October 30, 2007; Accepted January 15, 2008


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Hereditary spastic paraplegia (HSP) is a neurodegenerative disease caused by motoneuron degeneration. It is linked to at least 30 loci, among them SPG10, which causes dominant forms and originates in point mutations in the neuronal Kinesin-1 gene (KIF5A). Here, we investigate the motility of KIF5A and four HSP mutants. All mutations are single amino-acid exchanges and located in kinesin's motor or neck domain. The mutation in the neck (A361V) did not change the gliding properties in vitro, the others either reduced microtubule affinity or gliding velocity or both. In laser-trapping assays, none of the mutants moved more than a few steps along microtubules. Motility assays with mixtures of homodimeric wild-type, homodimeric mutant and heterodimeric wild-type/mutant motors revealed that only one mutant (N256S) reduces the gliding velocity at ratios present in heterozygous patients, whereas the others (K253N, R280C) do not. Attached to quantum dots as artificial cargo, mixtures involving N256S mutants produced slower cargo populations lagging behind in transport, whereas mixtures with the other mutants led to populations of quantum dots that rarely bound to microtubules. These differences indicate that the dominant inheritance of SPG10 is caused by two different mechanisms that both reduce the gross cargo flux, leading to deficient supply of the synapse.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Kinesin superfamily proteins are typically ATP- and microtubule-dependent motor proteins (1). Members of the Kinesin-1 family (formerly conventional kinesins) are involved in intracellular long-distance transport, and especially important in axons and dendrites (2,3). Vertebrate Kinesin-1 motors consist of two heavy chains (KHC, 120 kDa) that are responsible for motor activity, and two light chains (64 kDa, KLC) involved in cargo association (4). The N-terminal motor domains of the heavy chains function as a microtubule-activated ATPase and generate force and motility. Each ATP hydrolysis is coupled to an 8 nm step towards the plus-end of the microtubule (5,6). The characteristic, processive hand-over-hand type motility requires the coordinated activity of two coupled motor heads (7,8).

The slow and the fast anterograde components of axonal transport seem to arise at least in part from Kinesin-1 action (9–11). In vertebrates, one ubiquitous (uKHC or KIF5B), and two neuron-specific forms of KIF5 (KIF5A or nKHC, and KIF5C) are known (12–14). There is strong evidence that KIF5A is the major anterograde motor for the slow axonal transport of neurofilaments (9,15).

Mutations in KIF5A's motor domain have recently been shown to cause hereditary spastic paraplegia (HSP), a disorder characterized by progressive spastic weakness of the lower extremities (16–19). It is a so-called ‘dying-back’ neuropathy because synapses degrade first (20,21). At least 30 genetic loci are linked to HSP, among them SPG10, which turned out to contain the gene for KIF5A (19). SPG10 patients have point mutations in one copy of KIF5A, and these mutations are responsible for the disease (16–19). Although the genetic cause of SPG10 is thus known today, the mechanism of pathogenesis is unclear. The inheritance is autosomal dominant, and therefore defective KIF5A alleles may lead to dysfunctional kinesin motors that inhibit microtubule-dependent transport directly. Judging from the literature, this mechanism is likely for one of the known lesions in KIF5A, as a similar mutation at the same position in Ncd and Kar3 led to a catalytically inactive, rigor microtubule-binding protein (22). Alternatively, the patients may suffer from haplo-insufficiency, as proposed for SPG4 (23). To find out which mechanism is more likely, we investigate here the properties of KIF5A and four of the known HSP mutants in enzymatic, motility and cargo transport assays.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Location of HSP mutants
We focus here on the three mutants found first [N256S, R280C and A361V; (16,18,19)], and one unpublished mutant found by one of the co-authors (K253N; R.S.). Two of the mutations (K253N and N256S) are located in loop 11, which connects microtubule and ATP-binding sites (Fig. 1). The third mutation (R280C) is located directly in the microtubule-binding site (24,25). To avoid oxidation artifacts in vitro, we introduced a serine residue instead of a cysteine. The A361V mutation is located in the neck. For the characterization of these mutants, we used his-tagged, bacterially expressed proteins comprising the first 391 amino acids of KIF5A (or KIF5A mutant), followed by part of KIF5B's tail. The first coiled-coil of KIF5B's tail is known to bind unspecific to glass and carboxylated surfaces, allowing easy surface attachment for gliding assays (26–28).


Figure 1
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  		Figure 1. Location of HSP mutations. The figure shows a ribbon model of dimeric rat kinesin (3KIN) with stick representations of exchanged amino acids (red). ADP ligands are yellow, structures underlying the microtubule-binding site are green.

 
Motility and processivity
To elucidate the basic properties of KIF5A and its HSP mutant forms, we performed multiple motor gliding assays. At high motor coating densities on glass coverslips the wild-type motor displaced microtubules at 0.73 ± 0.02 µm/s (standard error of the mean, SEM). Except for the A361V mutant that was indistinguishable from wild-type, all other KIF5A mutants were significantly slower (Table 1 and Fig. 2A). Noteworthy, only few microtubules bound to the R280S mutant, and if so the velocities varied widely (6% standard deviation (s.d.) instead of 1% for wild-type), suggesting a low microtubule affinity.


Figure 2
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  		Figure 2. Multiple motor gliding and ATPase assays. (A) Velocities at different motor densities. The bar graph shows the mean microtubule velocities and SEM in standard gliding assays at high (~1 µM) and low (critical concentrations, the lowest density where microtubule attachment occurred) motor surface densities. In contrast to wild-type and the A361V mutant, most mutant velocities slightly dropped at low density. (B) Steady-state ATPase activity. The ATP turnover is plotted against the microtubule concentration and fitted using a Michaelis–Menten kinetics. Maximal turnover rates (kcat) and half-maximal activation constants (K0.5,MT) were deduced from measurements of at least two independent protein preparations (Table 1). (C) Dominance in vitro. The bars and error bars represent average velocities and SEM of mixtures of homodimeric wild-type and mutant motors at different ratios. Only the N256S mutant slowed down the microtubule velocity at equimolar stoichiometry to wild-type, the K253N and R280S mutants interfered only at large excess.

 


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  		Table 1. Properties of HSP kinesin mutants

 
To find out whether the gliding activity of KIF5A mutants was based on a cooperative effect, we measured the velocities in dependence of coating densities on the coverslip. We estimated a critical concentration below which no microtubule binding occurred, and measured the gliding velocities at this concentration. Here, the wild-type motor showed an elevated velocity compared with high coating densities (0.88 ± 0.01 versus 0.73 µm/s) but except for the A361V mutant all mutants were slightly slower (Table 1; Fig. 2A).

The critical concentrations of the mutants were quite different from wild-type, indicating largely altered microtubule affinities. The wild-type protein could be diluted to 19 nM. At this concentration microtubule single point attachments were observed, indicating that microtubules were tethered by only one kinesin motor (29). None of the homodimeric mutants showed microtubules swivelling around a single point of attachment, suggesting that multiple motors are necessary to bind microtubules. The mutants also required up to one order of magnitude higher coating densities to promote microtubule motion, supporting that multiple motors are necessary for microtubule binding (Table 1). The strongest effect was found in the R280S mutant (~15-fold), in agreement with observations in high-density motility assays.

As these observations suggested that none of the mutants was able to promote processive single-molecule motility, we investigated their motility in optical laser trap assays (27,30). To this end, microtubules were immobilized on the coverslip, motors attached to carboxylated polystyrene beads, and finally trapped in a laser beam. Single wild-type KIF5A motors showed long processive runs (≥1µm) and single 8 nm steps could be distinguished (Supplementary Material, Fig. S1). In stark contrast, none of the mutants showed displacements of more than a few nanometers. This type of bead motility was only observed at high kinesin densities at which steps could not be discerned. These data suggest that the SPG10 mutants are no longer processive.

Enzymatic activity
To measure altered microtubule affinities quantitatively, and to determine enzymatic velocities, we measured the microtubule-dependent ATP turnover in steady-state assays (31,32). As in motility assays, the N256S mutant was slowest but its microtubule affinity was only slightly decreased (Fig. 2B and Table 1). The K253N mutant reached two-thirds of the wild-type kcat, and showed significantly increased microtubule half-maximal activation constants (K0.5,MT). The R280S mutant had such a low microtubule affinity that the assay did not allow adding saturating microtubule concentrations. Therefore, the extrapolated kcat and, as a consequence, K0.5,MT, are inaccurate. Still, they indicate that kcat is not significantly affected but that K0.5,MT is at least one order of magnitude higher than wild-type.

As in gliding assays, the A361V mutant did not show any significant difference to wild-type. We suspected that it might be a thermosensitive mutant but prolonged incubation at 37°C did not lead to any defects in gliding. Why this mutation causes HSP is therefore unclear from our in vitro studies. Its disease causing effect remains unclear also from clinical grounds since segregation with the disease was not shown (16). Possibly, the mRNA stability is affected, or the mutation represents an accidental polymorphism. For these reasons we did not investigate this mutant further.

Heterozygous patients
The above gliding and enzymatic assays suggested that KIF5A mutant motors involved in HSP either are slower (N256S), or have a reduced microtubule affinity (R280S), or both (K253N). However, these conclusions were based on observations of homodimeric mutant motors. In reality patients are heterozygous and possess one mutated and one intact KIF5A allele. If both alleles were expressed equally—and we did not find any reason why this should not be the case (33)—one expects three populations of kinesin dimers: homodimeric wild-type motors, homodimeric mutant motors and heterodimeric motors with one wild-type and one mutant subunit at a stoichiometry of 1:1:2. The question therefore is why the inheritance of SPG10 is dominant and patients develop the disorder despite one intact copy of the KIF5A gene.

To address this question, we started using simple gliding assays with mixtures of wild-type and homodimeric mutant motors. We therefore mixed wild-type protein with homodimeric mutants at ratios from 50% wild-type to 0.2% wild-type, and observed the microtubule gliding velocity (Fig. 2C). One explanation for the dominant inheritance could be that the mutant motor slows down the microtubule gliding velocity in gliding assays of mixed motors. Only the N256S mutant behaved this way, and reduced the microtubule gliding velocity more than 2-fold at a 50:50 mixture of wild-type and mutant motor (mixture: 0.39 ± 0.01 µm/s; wild-type: 0.88 ± 0.01 µm/s). The K253N mutant did not affect the velocity significantly below 99-fold excess, the R280S mutant interfered only slightly with the assay at a 500-fold excess (0.2% wild-type; Fig. 2C). These mixed gliding assays showed that only the N256S mutant has a dominating effect, and that the mutations K253N and R280S do not interfere with wild-type motility in vitro.

Cargo transport assays
To answer the question whether heterodimeric mutants (one wild-type, one mutant subunit) are dominant over wild-type motors, we did not use the simplistic multi-motor gliding assay but a modified bead assay to mimic the cellular situation more realistically. We coated quantum dots (serving as an artificial, fluorescent cargo) with a 1:2:1 mixture of mutant homodimer: heterodimer:wild-type homodimer. We added motor in a stoichiometric excess of five (total motor dimers over quantum dots), based on previous reports on vesicle transport in vivo (34–36). Assuming a Poisson distribution, the majority of quantum dots are expected to be coated with 3–6 motors, 3–4% with only one motor (Supplementary Material, Fig. S2).

To understand the effect of defective motors on quantum dot transport we first measured the velocities of quantum dots. We added motor-coated quantum dots to a flow cell that had been prepared to contain surface-attached microtubules. We then recorded the quantum dot motility and analyzed it using kymograms (Fig. 3A and Supplementary Material, Movie). The velocities were determined from the slopes of phases of uninterrupted movement and plotted into a histogram. In these assays, wild-type KIF5A transported quantum dots at a velocity of 0.79 ± 0.02 µm/s, comparable with velocities in conventional gliding assays. The K253S mutant displaced the cargo at a similar mean velocity (0.78 ± 0.01 µm/s) but showed a second peak in the histogram at lower velocities (Fig. 3B). The N256S mutant slowed down the mean velocity modestly to 0.74 ± 0.07 µm/s. Here, the fraction of slowly moving quantum dots was much more pronounced and led to a clear increase of the standard deviation (0.26 µm/s instead of 0.21 µm/s). From a theoretical point of view, we expect three overlapping Gaussian distributions originating from three dimer populations. As indicated by curve fitting (data not shown), the variances of the velocities are too wide to distinguish the populations clearly. In contrast to the other two mutants, the R280S mutants showed a slightly faster mean velocity in the cargo transport assay (0.83 ± 0.03 µm/s). Possibly, the extremely low microtubule affinity of the mutant reduces the effective density of motor heads on the surface of the cargo. As in multiple motor gliding assays, this may have led to a higher velocity. These considerations suggest that cargo velocities in patients with the R280C mutation are not significantly altered. Therefore, other defects have to cause the disease, possibly the amount or rate of cargo particles arriving at the synapse.


Figure 3
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  		Figure 3. Cargo transport assay. (A) Kymograms of quantum dot motility. Video sequences were reduced to one dimension along the length of a microtubule (y-axis; scale bar 5 µm), and resolved in time along the x-axis (1 min in total). Bright lines are moving quantum dots, their angles reflect the velocity. As an example, quantum dots coated with the N256S mutant move visibly slower than wild-type-coated dots. (B) Histograms of velocities and transport frequencies. The histograms show velocity distributions of quantum dots coated with mixtures of wild-type and mutant KIF5A. The quantum dots were incubated with a 5-fold stoichiometric excess of motors. The mutants were tested as mixtures of 25% wild-type, 50% heterodimeric and 25% homodimeric mutant motors, the ratio expected in patients. The velocities of wild-type KIF5A were distributed nearly Gaussian with a mean velocity of 0.79 µm/s and a standard deviation of 0.21 µm/s. Mutant mixtures led to similar maximal velocities, but the K253N and N256S mutants caused more frequent slow events. The distribution of K253N velocities is clearly bimodal, that of N256S blurrier but clearly extended to slow velocities (standard deviation 0.26 µm/s). The R280S mutant did not show an altered distribution because mutant-coated quantum dots rarely bound to microtubules. Next to the histograms bars indicate the fraction of motile quantum dots of all binding-competent quantum dots. This number was determined by comparison of microtubule-bound quantum dots in the presence of ATP and AMP-PNP, and standardized with regard to wild-type (see Material and Methods). The error bars are standard errors from assays on two independent protein preparations.

 
A reduced arrival rate of cargo particles could also result from a larger fraction of cargo particles unable to bind to microtubules. For this reason we added AMP-PNP to the quantum dot motility assay after three movies, and then counted the number of immobilized quantum dots per microtubule length. We then compared this number with the number of previously moving quantum dots per microtubule length and time of the same sample, and finally calculated a relative binding ratio. We set the ratio to 100% for wild-type, and compared the mutants with wild-type. The relative binding ratio of K253N- and R280S-coated quantum dots dropped to <60% (Fig. 3B; mean of two independent protein preparations). The N256S mutant did not differ significantly from wild-type, suggesting that the N256S mutant does not reduce the cargo-microtubule binding frequency, but the K253N and R280S mutants do so.

Is this conclusion also valid for motor/cargo stoichiometries other than 5:1, as chosen in our assays? Most likely, the effect is even more pronounced at lower coating densities, and decreases if more than five motors bind to one cargo particle. This follows from Poisson statistics, which applies if wild-type/mutant dimers form randomly, and attach to quantum dots randomly (Supplementary Material, Fig. S2). Our motility assays show that the slower moving quantum dot population causes ~10–30% of the motility events, in agreement with a Poisson distribution. As the cargo-binding site is unaffected in HSP mutants, a Poisson distribution is also likely for the cargo-attachment of microtubule affinity mutants.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Spastic paraplegia patients belonging to the SPG10 group develop the disorder due to mutations in the KIF5A gene (16–19). As the degeneration starts from synapses of long motoneurons, and KIF5A is one of the major anterograde motors of neuronal transport, the lack of kinesin cargo delivery at the axon tip is the likely reason for degeneration. There are two possible explanations why mutated KIF5A decreases the arrival rate of cargo at axon tips. Either, mutated kinesins act as brakes for cargo particles or even block microtubule tracks, or they are microtubule-binding incompetent and compete for binding sites at the cargo that then are inaccessible for intact motors. Both models could explain the dominant inheritance of SPG10. Our data suggest that the second model is likely for two of the mutants (N253K and R280C) because they do not interfere significantly with wild-type KIF5A in several different types of motility assays. They have low microtubule affinities, indicating that they do not act as roadblocks (Fig. 4A). The first model may apply to a third mutant (N256S), which shows similar microtubule affinities as wild-type and slows down microtubule gliding velocities at equimolar amounts to wild-type (Fig. 4A). Noteworthy, this mutant does not bind microtubules in rigor, as expected from similar mutations in Kar3 and Ncd (22). Here, the N to K exchange at this position decoupled microtubule binding and catalytic activity, leading to a constitutively tight microtubule-binding mutant.


Figure 4
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  		Figure 4. Model for HSP kinesin mutant defects. (A) Motility defects in HSP mutant kinesins. The four investigated HSP mutant KIF5A motors show different defects in vitro. The velocity defect was deduced from conventional in vitro gliding assays on homodimeric mutants, the affinity defect from landing rates of microtubules to coated glass surfaces and ATPase assays, the dominance from the ability of the mutant to reduce the gross gliding velocity in mixed motor gliding assays, the transport frequencies from the ratio of quantum dots that bind to microtubules in the presence of AMP-PNP but not attach with ATP. n/d, not determined. (B) Model for emergence of transport defects. According to theoretical models the anterograde axonal transport rate is determined by cargo binding and detachment frequencies, and the velocity and runlength of microtubule-attached cargo (38,39). The mutants K253N and R280S diminish cargo attachment rates (shorter arrow), the mutants K253N and N256S the velocity of cargo.

 
How is axonal transport affected by these molecular defects? In our experiments we addressed this question in transport assays with artificial cargo. We mimicked the situation in patients—all known patients are heterozygous—by mixing wild-type motors with homo- and heterodimeric mutants. These assays revealed that the presence of mutant motors leads either to slow moving cargo populations, or populations that almost never bind to microtubules, or both. As a result, the anterograde transport frequency will be lower, and the arrival of the cargo transported by mutant motors at the synapse largely delayed. This effect might be enhanced by the presence of retrograde motors at the cargo that could dominate the gross transport.

As discussed earlier, this explanation holds true even if cargo particles were transported by more or less than five motors, the number used in our assays. With fewer motors per cargo particle, the fraction of poorly transported cargo would even be larger because kinesins seem to attach to quantum dots randomly, leading to a Poisson distribution of the numbers of motors per cargo. Accordingly, a lower coating density makes it more likely to find cargo particles attached to mutant motors only, leading to a more pronounced phenotype.

It is not exactly known how many motors are involved in the transport of cellular cargo in vivo. Electron microscopy indicates that several motor proteins are present at one cargo vesicle, and the excess of five chosen in our assays is realistic (34–36). Such vesicles have been suspected to be the cargo in fast axonal transport (9). Molecular evidence suggests that Kinesin-1 is a major player in fast anterograde axonal transport, and therefore our experiments might be directly applicable to neurons (9,11). The K253N and R280S mutants would diminish the fraction of transported cargo, the N256S heterodimer mutant would slow down each cargo vesicle it is attached to.

Alternatively, axonal degeneration in HSP patients might be induced by altered slow axonal transport. The most likely cargo of slow transport are neurofilaments, and there is strong evidence that KIF5A is the major anterograde motor for transport of neurofilament precursors (9,15). It is unknown, however, how many motors are attached to one neurofilament precursor complex. Still, the lack of neurofilament supply to the synapse is a plausible reason for neuro-degeneration, as the mouse knockout mutants of several neurofilament components and the conditional mouse KIF5A knockout lead to neurodegeneration (37).

The velocity of the slow axonal transport component is much slower than the velocity of KIF5A, and seems to emerge due to frequent, long pauses of the cargo. They make up 97% of the time and are likely to involve a number of regulatory steps (38,39). We imagine that the HSP KIF5A mutants N253K and R280C still possess the regulatory mechanisms of wild-type but rarely bind to microtubules in the activated state (Fig. 4B). The K256S mutant will bind as frequently as wild-type but move slower. This, again, would lead to infrequent arrival of cargo at the synapse and would be enhanced by the presence of normal retrograde motors.

Regardless of which component is affected the gross effect of the investigated HSP mutations on cargo transport is amazingly mild. SPG10 patients still have 25% wild-type KIF5A dimers, and mixed motor assays indicate that the majority of cargo particles still move at wild-type rates, even in the presence of mutant motors. This may explain the late onset of the disease in patients with the K253N (R.S., unpublished data) and the R280S mutation at ages between the second and the fourth life decade (16–19). The N256S mutation leads to more severe effects and has a dominant effect in vitro, correlating with an early onset of clinical symptoms before the age of 20 in patients carrying the mutation. Interestingly, SPG10 patients show no clinical signs of peripheral neuropathy although lower motoneurons have axons almost as long as upper motoneurons of the corticospinal tract. Still, KIF5A is clearly expressed in both cell types, and therefore differences in the cellular transport machinery might explain why defects in SPG10 almost exclusively affect the corticospinal tract. It will be interesting to investigate SPG10 mutants in a cellular environment that includes regulatory mechanisms for kinesin activity. The basic effects of the K253N, R280S and N256S mutations on transport are clear from our experiments and may open ways to suppress the progress of neurodegeneration. In principle, the effect of the first two mutants could be suppressed by introducing excess KIF5A wild-type motor, whereas that of the third mutant could only be defeated by more sophisticated approaches, such as specific inhibitors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Cloning, protein expression and purification
All constructs are based on a synthetic gene encoding the N-terminal 391 amino acids of KIF5A and its mutants (Sloning GmbH, Puchheim, Germany), cloned into a pET 24a vector (Merck, Darmstadt, Germany). One hundred and sixty amino acids of the human kinesin (uKHC or KIF5B) tail were added by cloning and followed by a Strep- or his-tag sequence (Qiagen, Düren, Germany). For the expression of heterodimers the inserts of Strep-tag mutant vectors were cloned into the his-tag wildtype vector.

Homodimeric protein was expressed, lysed and purified in a phosphate buffer (50 mM Na2HPO4, 250 mM NaCl, 2 mM MaCl2) over Ni-NTA-columns, followed by a Q-Sepharose anion exchange column in pipes buffer (25 mM Na-Pipies, 2 mM MaCl2, 1 mM EGTA, pH 6.9). Heterodimers were tandem purified by a Ni-NTA-column, followed by a Strep-Tactin column. Protein concentrations were determined by a Bradford test (BioRad, Hercules, USA) or from an SDS–Gel with a BSA concentration series (Analysis with ImageJ). Microtubules were prepared from pig brain tubulin and polymerized as described (40), the Atto488 (AttoTec, Siegen, Germany), tetramethylrhodamine and Biotin labelled tubulin was obtained as published (41).

ATPase assay
Microtubule activated steady-state ATPase rates were determined in a coupled enzymatic assay (28,31). The assay was performed in 12A25+ buffer (12.5 mM Aces·KOH, 25 mM potassium acetate, 5 mM MgCl2, 0.5 M EGTA, pH 6.8) at 22°C. The measurements were done at increasing microtubule concentrations, the kcat was obtained from the extrapolated turnover at infinite microtubule concentration. K0.5,MT was determined at an ATP concentration of 1 mM. All values were determined on two independent protein preparations.

Gliding assays
A flow cell was incubated for 5 min with hKtail-tagged motors in dilution buffer [10 mM ATP, 1 mg/ml BSA, 150 mM NaCl in BRB80+ (80 mM PIPES·KOH, pH 6.8, 5 mM MgCl2, 1 mM EGTA)]. After washing with blocking buffer (1 mg/ml BSA in BRB80+), the flow chamber was filled with atto488-labeled microtubules in motility buffer (2 mM ATP, 20µM taxol, 1 mM DTT, 0.1 mg/ml glucose oxides, 0.37 mg/ml casein, 0.02 mg/ml catalase, 2.25 mg/ml glucose). All assays were performed at 22°C. Gliding microtubules was observed by total internal reflection microscope (see below) and the velocity microtubules (at least 20) was measured using the manufacturers' software (Olympus Biosystems GmbH, Planegg, Germany). All measurements were based on two independent protein preparations.

Laser trapping assay
Optical trapping experiments were performed in a custom built optical trap described (42). Beads were captured in the beam of an 8 W Nd:YAG laser (Coherent Deutschland GmbH, Germany) focused through a high numerical aperture objective (NA = 1.45, Olympus Deutschland GmbH, Germany). The position of trapped beads was detected by bright field imaging onto a quadrant diode (SPOT4D, UDT Sensors Inc., CA, USA). Data were acquired by an A/D converter board (NI-PCI-6259, National Instruments, Germany) with a sampling frequency of 40 kHz per channel and stored without prior filtering. Fluorescently labeled microtubules were fixed to the glass surface of a flow chamber by the use of the biotin–streptavidin system (discussed earlier) and imaged with TIRF. Kinesin molecules were allowed to adsorb to carboxylated polystyrene beads (532 nm, Polysciences Inc., USA) as described (27). Data from optical trapping experiments were analyzed using IGOR Pro 4.01 (WaveMetrics, Portland, OR, USA).

Quantum dot assays
Biotin- and tetramethylrhodamine-labeled microtubules were attached to the surface of a flow chamber that had been incubated with 2 mg/ml BSA-biotin (Sigma-Aldrich Co., St. Louis, MO, USA) and 1 mg/ml streptavidin in BRB80+ buffer with 20 µM paclitaxel. 160 nM carboxylated Qdots525 (Invitrogen, Carlsbad, USA) were mixed with 800 nM kinesin in BRB80+, 1 mM ATP and 1 mg/ml casein. The solution was stored on ice for at least 5 min. After washing the flow chamber with 1 mg/ml BSA in BRB80+ motility mix (1 µl quantum dots coated with kinesins, 2 mM ATP, oxygen scavenger (see above), 0.2 mg/ml casein, 1 mM DTT in BRB80+) was flushed in. The gliding activity was observed in an Olympus IX71 TIRF microscope with an excitation wavelength of 488 nm and a Hamamatsu C-9100 front-illuminated CCD camera. The optical resolution was 160 nm per 2x2-binned pixel, the integration time 200 ms.

To determine the number of binding-competent quantum dots in the flow chamber, 5 µl of 100 mM AMP-PNP was added to the edge of the flow chamber after recording of three movie sequences in the presence of 2 mM ATP. When all quantum dots were static (~3 min) images were recorded. The number of quantum dots per microtubule length was counted, and compared with the number of moving quantum dots per time and microtubule length in the presence of ATP. Relative values were calculated by setting wild-type to 100%. All measurements were confirmed by a second independent protein preparation.

Data analysis
Movies taken from the quantum dot assay were analysed with ImageJ. The ‘Brightest Point Projection’ command showed moving quantum dots as a line, which were then converted to time-space plots (kymograms). From these kymograms velocities were calculated in areas of constant velocity. In parallel, the number of events was counted. The length of microtubules and number of attached quantum dots after AMP-PNP addition were also analysed in ImageJ. For statistical analysis SigmaPlot 2000 and Sigma Stat 3.1 Software (Systat, Point Richmont, CA, USA) was used.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
FUNDING
 REFERENCES
 
Supplementary Material is available at HMG Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
REFERENCES
 
This work was supported by the Deutsche Forschungsgemeinschaft, the BMBF (GeNEMove), the Center for Integrated Protein Sciences Munich (CiPS), and the Friedrich-Baur-Foundation. B.E. acknowledges the financial support by the Elite Network of Bavaria.


   ACKNOWLEDGEMENTS
 
Our special thanks go to Till Bretschneider who developed the ImageJ macro. We also thank Sven Leier and Renate Dombi for excellent technical support, Zeynep Ökten for fruitful discussions and Manfred Schliwa for continuous support and discussions.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 

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