Swimming against the Tide: Mobility of the Microtubule ... - CiteSeerX

9916 • The Journal of Neuroscience, September 12, 2007 • 27(37):9916 –9927

Neurobiology of Disease

Swimming against the Tide: Mobility of the MicrotubuleAssociated Protein Tau in Neurons Sven Konzack, Edda Thies, Alexander Marx, Eva-Maria Mandelkow, and Eckhard Mandelkow Max-Planck-Unit for Structural Molecular Biology, 22607 Hamburg, Germany

Long-haul transport along microtubules is crucial for neuronal polarity, and transport defects cause neurodegeneration. Tau protein stabilizes microtubule tracks, but in Alzheimer’s disease it aggregates and becomes missorted into the somatodendritic compartment. Tau can inhibit axonal transport by obstructing motors on microtubules, yet tau itself can still move into axons. We therefore investigated tau movement by live-cell fluorescence microscopy, FRAP (fluorescence recovery after photobleaching), and FSM (fluorescence speckle microscopy). Tau is highly dynamic, with diffusion coefficients of ⬃3 ␮m 2/s and microtubule dwell times of ⬃4 s. This facilitates the entry of tau into axons over distances of millimeters and periods of days. For longer distances and times, two mechanisms of tau transport are observed. At low near-physiological levels, tau is cotransported with microtubule fragments from cell bodies into axons, moving at instantaneous velocities ⬃1 ␮m/s. At high concentrations, tau forms local accumulations moving bidirectionally at ⬃0.3 ␮m/s. These clusters first appear at distal endings of axons and may indicate an early stage of neurite degeneration. Key words: Alzheimer’s disease; axonal transport; microtubules; tau; mitochondria; synapse

Introduction Neurons are polarized cells with two types of cell extensions, dendrites and axons, whose establishment depends on the microtubule cytoskeleton. Microtubules (MTs) are used as tracks for long-distance transport of vesicles, organelles, and nucleoprotein complexes (Baas and Buster, 2004; Hirokawa and Takemura, 2005; Hollenbeck and Saxton, 2005; Miller and Sheetz, 2006). Tau, the main axonal MT-associated protein (MAP), regulates microtubule stability, functions as a spacer between MTs (Chen et al., 1992), and serves as an anchor for enzymes (Sontag et al., 1999). Tau can regulate MT-dependent transport by interfering with motor proteins (Stamer et al., 2002). In Alzheimer’s disease (AD), the hyperphosphorylation of tau, aggregation into neurofibrillary tangles, and missorting into the somatodendritic compartment are hallmarks of the disease (Garcia and Cleveland, 2001; Lee et al., 2001). Tau can be subdivided into a C-terminal “assembly domain,” which promotes microtubule assembly, and an N-terminal “projection domain,” which projects away from the microtubule surface (see Fig. 1a). The human CNS contains mainly six isoforms (352– 411 residues), which are generated by alternative splicing (Andreadis, 2005; Goedert and Jakes, 2005). The microtubulebinding site of tau comprises the repeat domain (three or four Received March 1, 2007; revised July 23, 2007; accepted July 25, 2007. This work was supported in part by the Deutsche Forschungsgemeinschaft. We thank N. Gehrke and F. Baltruschat for cloning and adenovirus production, Dr. J. Biernat for providing many tau constructs, and Dr. G. Steinberg (Max Planck Institute, Marburg, Germany) for providing a 3x-eGFP plasmid. Correspondence should be addressed to Eckhard Mandelkow, Max-Planck-Unit for Structural Molecular Biology, Notkestrasse 85, 22607 Hamburg, Germany. E-mail: [email protected]. S. Konzack’s present address: Olympus Life and Material Science Europa GmbH, Wendenstrasse 14-18, 20097 Hamburg, Germany. DOI:10.1523/JNEUROSCI.0927-07.2007 Copyright © 2007 Society for Neuroscience 0270-6474/07/279916-12$15.00/0

semiconserved sequences of ⬃31 residues) and the flanking proline-rich domains (Butner and Kirschner, 1991; Gustke et al., 1994). Phosphorylation of tau, especially of the KXGS motifs in the repeats, tends to detach tau from microtubules and thus destabilizes them (Biernat et al., 1993; Stoothoff and Johnson, 2005). Tau mutants, which cause neurodegeneration in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), have a reduced ability to bind to microtubules in vitro and in cells (Hong et al., 1998; Barghorn et al., 2000; Lu and Kosik, 2001). The basis for the sorting of tau into axons (or missorting into dendrites during neurodegeneration) is still not well understood. In embryonic brain, fetal tau (the smallest isoform) and other MAPs are initially homogeneously distributed. Tau expression increases sharply during brain development, concomitant with neurite outgrowth, diversification of tau (from one isoform to six in humans) and sorting into axons (Drubin and Kirschner, 1986; Kosik and Finch, 1987). Several mechanisms could contribute to axonal sorting, e.g., tau protein in axons could bind more tightly to microtubules than in dendrites, where it might be degraded more easily; tau protein could be targeted into axons but not into dendrites; and tau mRNA could be targeted into axons for local translation (Hirokawa et al., 1996; Aronov et al., 2002; Utton et al., 2002). Tau is transported along the axon with the rate of slow axonal transport, similar to tubulin and most other cytoskeletal proteins (0.1– 8 mm/d, or ⬃0.001– 0.08 ␮m/s), whereas vesicles move at rates of fast axonal transport (50 – 400 mm/d, ⬃0.5– 4 ␮m/s) (Mercken et al., 1995). In the case of MTs and neurofilaments, the slow axonal transport rate is the result of fast, bidirectional, and intermittent transport along axons (Roy et al., 2000; Wang et al., 2000; Wang and Brown, 2002). The transport mechanism of MAPs and cytosolic proteins in the axon is still a matter of debate (Baas and Buster, 2004). For vesicles, mitochondria,

Konzack et al. • Mobility of Tau in Neurons

J. Neurosci., September 12, 2007 • 27(37):9916 –9927 • 9917

ported along the axon and what changes occur when tau becomes missorted, we analyzed diffusion, MT binding, and the transport behavior of different cyan fluorescent protein (CFP)-tagged tau mutants in primary neurons by live cell microscopy, fluorescence recovery after photobleaching (FRAP), and fluorescence speckle microscopy (FSM). The results show that tau diffuses unexpectedly fast, considering its nature as an MT-bound protein. This is explained by the highly dynamic interaction between tau and MT in the axon. The high exchange rate of tau is observed even when tau is not phosphorylated. The fast diffusion of tau contributes to its dispersion along the axon. The main transport mechanism in near-physiological conditions appears to be the piggybacking of tau on MT fragments, which are transported along the axon rapidly at typical velocities of motor proteins and with saltatory characteristics. At elevated tau concentrations, reminiscent of an ADlike situation, globular accumulations of tau appear, which move bidirectionally along the axon and build up from the distal end of axons.

Materials and Methods Plasmid construction, transfection, and adenovirus production. For expression in Vero cells and adenovirus production, the SfiI–NheI fragments containing parts of the cDNAs of htau40-⌬K280, htau40-P301L (both FTDP-17 mutants), pseudophosphorylated tau-KXGE (Ser-to-Glu at all four KXGS motifs, positions 262, 293, 324, 356), and eight repeat mutants of tau (tau without N-terminal inserts and including a duplicated insertion of the four repeats) Figure 1. Tau expression inhibits anterograde transport of organelles and vesicles but not of tau itself. a, Diagram of tau441, (Gustke et al., 1994) were subcloned into the the longest isoform in human CNS, with repeat domain (R1–R4), the flanking proline-rich regions (which together constitute the respective restriction sites of the pShuttle cytomicrotubule-binding domain), and the N-terminal projection domain (alternatively spliced inserts in black). Phosphorylation sites megalovirus (CMV) vector containing CFPin the repeat domain that control MT binding are indicated. b–f, Cortical neurons 20 h after transfection with CFP-hTau40. htau40. A fusion of three enhanced green fluoCFP-hTau40 is shown in blue (b); mitochondria are labeled with MitoTracker Red (c). In the overlay (d), two neurites are boxed in rescent protein (EGFP) molecules to the N green (left box, with tau expression; right, without tau). The movements of mitochondria during a 1000 s period are visualized in terminus of htau40 in the pShuttle vector was kymographs derived from the boxed neurites with Tau expression (e) or without (f ). The anterograde direction is indicated by an performed by PCR TOPO cloning (Invitrogen, arrow from left to right (“ant”). g–i, Cortical neurons expressing CFP-hTau40 for 21 h. CFP-hTau40 is pseudocolored in blue (g), Carlsbad, CA) of three EGFPs from the 3GFPand mitochondria are pseudocolored in red (h). In the overlay, mitochondria are visible in the axon without tau expression but not myo5 plasmid (kindly provided by G. Steinin the axon with tau (i). j, k, Quantitation of tau distribution along an axon (same as in g; highlighted by dotted line) shown by a berg, Max-Planck Institute, Marburg, Gercolor gradient (j) and plotted versus axon length (k; plot extends between positions marked by diamond and arrowhead). Note many), followed by subcloning into CMV-tauthat the highest apparent level is in the cell body (red, blue edges) and extends into the axon (blue, proximal; green, distal). pShuttle. For transfection into Vero cells, DNA Occasional shifts in intensity (e.g., around L ⫽ 280 ␮m) are caused by stronger background intensity in this area. Scale bars, 20 was prepared by an endotoxin-free Maxiprep kit (Qiagen, Hilden, Germany) and 1 ␮g of ␮m. DNA was transfected into 10 6 cells with an electroporation system (Amaxa, Cologne, Gerand neurofilaments, the long-haul transport uses mainly MTs as many) according to the manual. After transfection, the cells were directly tracks and motor proteins kinesin for anterograde and dynein for plated in self-made glass-bottom chambers and incubated for 6 –24 h retrograde movement. In contrast, actin filaments and their corbefore imaging. Recombinant adenoviruses for transfection of primary responding myosin motors are used for short-haul transport of neurons were generated according to the study by He et al. (1998). mitochondria and vesicles to achieve exact positioning and memTransfection of retinal ganglion and cortical cultured neurons. Cortical brane fusion (Hollenbeck and Saxton, 2005). neurons were obtained from embryonic day 18 (E18) rat embryos and The present study was motivated by the apparent contradiccultured according to the study by Banker and Goslin (1988). Cells were tion between the ability of tau to inhibit anterograde transport of transfected 6 – 8 d after plating. For adenoviral transfection of primary vesicles and organelles, whereas tau itself is able to distribute cortical and retinal neurons with the vectors encoding the tau fusion along axons (Stamer et al., 2002). To understand how tau is transproteins a 100-fold multiplicity of infection was used. Retinal ganglion

9918 • J. Neurosci., September 12, 2007 • 27(37):9916 –9927


neurons were obtained from white leghorn chicken eyes at E6 and cultured in glass-bottom dishes. The dishes were previously incubated with 4 ␮g/ml laminin overnight (SigmaAldrich, St. Louis, MO) and washed twice. Retina explants were centered in the dishes and incubated for 24 h in DME-F-12 media (Invitrogen) with 10% FCS and 0.4% methyl cellulose at 37°C, 5% CO2, and 100% relative humidity. The transfection procedure was performed as described above. Live-cell imaging and FSM. For live-cell imaging and FSM, cells were imaged in glassbottom dishes with an Axiovert 200 (Zeiss, Oberkochen, Germany) equipped with a planapochromatic 63⫻ (numerical aperture, 1.4) oil-immersion objective lens and a 50 ms filterwheel (Sutter Instrument, Novato, CA) for fast dual-color imaging. The complete microscope was integrated in an incubation system (Harnischmacher, Hannover, Germany) to image cells at 37°C and 5% CO2. Images were captured with a CCD camera (CoolSNAP HQ; Roper Scientific, Tucson, AZ) and MetaMorph imaging software (Universal Imaging, Buck- Figure 2. Tau binds to MTs and stains the axon smoothly. a, Time-lapse images showing the distribution of mRFP-htau40 in inghamshire, UK). Time-lapse series were ob- cortical neurons. b, Intensity versus time at different positions. c, d, Chicken RGC neurons transfected with CFP-htau40. Tau tained at 0.5–3 s intervals. Image processing localization along the axon is visualized in a black and white image (c). d, Tau localization on single MTs in the growth cone, and measurements were done with Meta- extending from the central domain to the left and to the bottom (MTs in the shaft are not resolved). e, In mouse cortical neurons Morph, ImageJ, and Photoshop (Adobe Sys- expressing CFP-htau40, tau stains MTs in the cell body, axon, and dendrites smoothly. f, In Vero cells, tau stains the MTs. g, h, In tems, San Jose, CA). For FSM, the cells were contrast, the tau-KXGE mutant lacks strong axonal localization (g) and shows no MT binding in Vero cells (h). Scale bars, 20 ␮m. only incubated for 4 – 6 h before imaging instead of 1–2 d. Mitochondria were visualized by areas of the same cell. If general bleaching appeared, it was subtracted as changing the medium against fresh prewarmed medium with a final explained above. The intensity was plotted as a function of time and fitted concentration of 12 nM MitoTracker Red (Invitrogen, Karlsruhe, Gerwith a single-exponential function to generate the half-time of microtumany) for 2 h, followed by exchange of MitoTracker medium for normal bule binding (Bulinski et al., 2001). medium before imaging. CFP quantification was performed by reference to calibrated solutions. Photobleaching. Two different types of bleaching experiments were Results done: (1) bleaching of CFP-tau in a large axonal region to obtain the Tau moves along the axon, whereas anterograde transport of diffusion coefficient and (2) bleaching of small regions of CFP-tau in organelles and vesicles is inhibited neurons and on microtubules in Vero cells to study the microtubule The adenovirus vector encoding CFP-htau40 fusion protein binding behavior. In the first method, an LSM510 confocal microscope (CFP-tau) was added to 8-d-old rat cortical neurons. Twenty (Zeiss) was used to bleach 30- to 50-␮m-long axonal regions of retinal hours after transfection CFP-tau became visible in the cell body ganglion cell (RGC) axons with 100% laser intensity of the 458 nm line of and in proximal parts of the neurites (Fig. 1b). Time-lapse imaga 100 mW argon laser. The microscope was inside an incubation system ing of CFP-tau-expressing neurons showed a strong decrease in (Harnischmacher) to image cells at 37°C and 5% CO2. For imaging, the the number of moving mitochondria, in particular the net ansame laser was used with 10% laser intensity. To calculate the apparent diffusion coefficient, the signals of the central region of the bleached area terograde transport was reduced (Fig. 1c–f ). Similar observations containing the whole width of the axon were averaged. The background applied to other axonally transported vesicles and organelles obtained from areas outside the fluorescent axon was subtracted. Bleach(data not shown). When the expression rate was increased, the ing by imaging was nearly avoided by applying a low laser power and long CFP-tau signals moved further along the axon in the same time imaging intervals; it was corrected by calculating the loss of signals in period (20 h) (Fig. 1g–i), indicating that the entry of tau into unbleached axons and from the signal intensities in the image series neurites is concentration dependent. At the same time, extended before bleaching. The apparent diffusion coefficient, Dapp, was calculated axonal regions were already cleared of mitochondria because of from the half-time of recovery, t1/2, at the center of the bleached region 2 the strong inhibition of anterograde transport by tau. Therefore, using the equation Dapp ⫽ 0.275 L /t1/2, valid for diffusion in a narrow the question arises how tau manages to travel down the axons cylinder open at both ends, with L equaling the length of the bleached despite its negative effect on microtubule-based traffic in general. area. For the second photobleaching procedure, a confocal microscope Olympus (Tokyo, Japan) FV1000 was used. To obtain faster bleaching and faster images after bleaching, a second scanner for simultaneous CFP-tau expression leads to a gradient of tau distribution bleaching was used, equipped with a 405 nm UV-laser diode (Melles along the axon Griot, Irvine, CA). A 2- to 4-␮m-diameter circle was bleached with 50% To understand the movement of tau along the axon, series of laser intensity, with the Tornado scanning function and imaging of the z-stack images were collected from cortical neurons. Ten to bleached region started directly after bleaching ended (bleaching time, twenty z-stacks from the cell body to the distal end of a given 0.1– 0.3 s). To avoid bleaching by imaging the time interval was adapted neuron were obtained. The images with highest signal intensities to the half-time of the analyzed protein (0.3–2 s). The signals of a small were joined up and pseudocolored to show the variations in the linear region along the microtubule containing the whole width of the CFP-tau concentrations. CFP-tau clearly shows a smoothly demicrotubule and the length of the bleached area were averaged and backcreasing concentration from the cell body toward the distal end of ground subtracted. The background was measured in microtubule-free



understand how the appearance of tau depends on active transport and/or diffusion, we analyzed diffusion, MT binding, and transport of tau in this system using FRAP and FSM (Waterman-Storer et al., 1998). Axonal tau accumulation depends on MT binding We generated adenoviruses encoding CFP-tagged fusion proteins of different tau mutants and analyzed their localization in primary cortical neurons, RGC neurons, and Vero cells. Because high levels of tau can cause cell defects, we expressed CFP-tau only briefly (⬍24 h) and chose cells with low signal intensities, barely observable at sensitive settings. Chicken RGC neurons (Fig. 2c) showed smoothly decaying tau gradients along axons from the proximal to the distal end without major discontinuities (gaps or local clusters). In rat cortical neurons and Vero cells, CFP-tau also localized to microtubules, as indicated by the pattern in the growth cone and the smooth staining along the axon (Fig. 2d–f ). A strong localization to the axon and on MTs in Vero cells was also observed for a tau mutant with a duplicated repeat domain (CFPtau-8R) (data not shown). Two FTDP-17 mutants of full-length four-repeat tau were studied as well, CFP-tau⌬K280 and Figure 3. Quantification of CFP-tau in neurons. a, CFP-tau image with two transfected axons. b, Same axons as in a in color CFP-tauP301L, which bind to MT with a spectrum to visualize the different signal intensities in the two axons. The protein concentration was calculated from a calibration somewhat reduced affinity (Barghorn et against a CFP solution. c, Tau concentrations along two representative axons. Gray, Background intensity; blue, the tau concen- al., 2000; Lu and Kosik, 2001). In both tration in an axon with low and uniformly dispersed tau, red, axon containing inhomogeneities. The line shows the moving cases, there was a smooth axonal localizaaverage (50 pixels). Note the “bumpy” trace and higher tau concentration in an axon containing tau clusters. d, Scheme of a tion, analogous to wild-type tau, and the neuron. The concentration of smoothly distributed tau (red) decreases from the cell body to the growth cone, whereas local tau proteins were localized on microtubules in accumulations emanate from the distal end of the axon first. Scale bar, 10 ␮m. Vero cells (data not shown). These mutants did not differ in their localization on the axon (Fig. 1j– k). Over the distances and time periods of our microtubules in non-neuronal cells or in neuronal cell bodies and experiments, we did not observe a sharply defined advancing along axons (first 100 ␮m), compared with wild-type tau. Of particular interest was the question of tau phosphorylation front, nor did we observe vesicle-like movements. The decreasing at the KXGS motifs of the repeats because this causes the detachsignal intensity up to 400 ␮m away from the cell body can be explained by diffusion, without the need to assume active transment of tau from microtubules (Biernat et al., 1993). In normal port. Likewise, in primary neurons, tubulin showed no moving conditions, tau showed a low extent of phosphorylation at these front or vesicle-like localization along the axon, although it is sites, consistent with its microtubule-bound state. Likewise, the actively transported by motor proteins (He et al., 2005). One CFP-tau-4KXGA mutant showed the same localization as explanation of this observation is that diffusion is a major deterthe wild-type protein (data not shown), as expected because the minant of the distribution, at least over distances of up to ⬃1, and phosphorylation sites were eliminated in all KXGS motifs. In that tau is not transported in well defined packets. contrast, the mutant CFP-tau-4KXGE is in a pseudophosphoryWe investigated the formation of the tau gradient by timelated state; it showed weak microtubule binding and a weaker lapse analysis. For long-time imaging, we used an adenovirus axonal localization (Fig. 2g– h). In other words, tau mutants that expressing mono-red fluorescent protein (mRFP)-tau instead of bind less tightly to MTs are less localized in the axon. These data CFP-tau to reduce bleaching and photodamage. In strongly exsuggest that spreading of tau into the axon depends not only on pressing cells, the first mRFP-tau signals were visible 3 h after its diffusion but also on the equilibration between free and transfection and were followed by neurodegeneration within microtubule-bound tau. 12 h. A milder expressing neuron is illustrated in Figure 2a– b. To verify differences between wild-type and mutants, the difThe signal intensity developed at the same rate in the cell body fusion coefficients and MT-binding kinetics were analyzed and a and the initial part of the axon, indicating rapid distribution by mathematical model of the dispersion of the different tau mudiffusion. At 100 ␮m into the axon, the tau signal increased more tants was calculated and will be described below (Fig. 6d and slowly because the protein has to move out along the axon. To supplemental material, available at www.jneurosci.org).



Quantification of CFP-tau in neurons To understand how the tau transport in axons is correlated with the level of tau expression, we quantified the amount of CFP-tau in the axons. Recombinant CFP was cloned and purified, and a calibration curve of intensity versus concentration was generated using confocal microscopy. We compared the signal intensity of CFP in solution directly with the fluorescence intensity of CFP-tau in the axon to define the CFP-tau concentration. Image stacks of 16 different RGC axons expressing different amounts of CFP-tau in four separate experiments were imaged under the same microscope settings. The concentration of CFP-tau was calculated by correlating the signal intensity of up to 50-␮mlong areas of axons with the intensities of purified CFP in solution. We found two different modes of tau localization in axons, one with a smooth tau distribution Figure 4. FRAP of RGC neurons expressing CFP-tau. a, Time-lapse fluorescence images of axon (t ⫽ 0 at end of bleaching). a, (type-1 axons) and axons with clusters or b, Red vertical lines indicate the bleached region. The boxed area (a) defines the region used for the kymograph (b). Scale bars, 10 inhomogeneities in the tau distribution ␮m. c, Normalized signal intensities along the axon before bleaching (black), directly after bleaching (red), and 194 s after (type-2 axons). The mean tau concentra- bleaching (blue), showing ⬃50% recovery. Red lines in center delineate the area for measuring the intensity recovery and hence tions in type-1 axons were relatively low the diffusion coefficient, shown in d as a percentage of initial level (half time of recovery, ⬃180 s). e, Apparent diffusion (⬃1–2 ␮M), whereas type-2 axons had coefficients of GFP, CFP-tau, and 3 CFP-tau mutants derived from FRAP. higher tau concentrations (up to 4 ␮M). In (Fig. 4e). Both proteins diffuse freely because their observed difthe example of Figure 3, the tau concentrations along two axons fusion coefficients are consistent with theoretical expectations were plotted over 40 ␮m to visualize the large variation of tau calculated from their molecular weights and hydrodynamic radii concentrations in a type-2 axon, compared with the more even (see supplemental material, available at www.jneurosci.org) distribution along type-1 axons (Fig. 3c). The transition from a (GFP, Dcalc ⬃ 28; CFP-tau-4KXGE, Dcalc⬃12 ␮m 2/s). In consmooth to an inhomogeneous distribution begins at a mean tau trast, CFP-tau diffuses approximately four times more slowly concentration of approximately ⬃1 ␮M. (D ⬃ 3 ␮m 2/s) than CFP-tau-4KXGE because ⬃75% of it is MT-bound (Table 1 and supplemental material, available at wwFRAP experiments of CFP-tau reveals rapid diffusion w.jneurosci.org). The enhancement of the microtubule affinity of To determine tau distribution at near-physiological conditions of the eight-repeat mutant results in a 12-fold reduced diffusion low tau expression (type-1 axons), we performed FRAP expericompared with CFP-tau-4KXGE (D ⬃ 1 ␮m 2/s). ments on primary RGC axons growing from an explant of a chick The relatively fast recovery of CFP-tau after bleaching is sugretina. The bleaching eliminates the background of total tau gestive of a highly dynamic interaction with microtubules. How(mostly microtubule-bound) in the bleached zone, and CFP-tau ever, theoretically one might argue that a similar result could be diffuses into the zone after bleaching. Thirty- to fiftyachieved by phosphorylation of tau at sites which influence MT micrometer-long areas of axons were bleached and observed by binding. To test this hypothesis, the diffusion of the nonphosconfocal microscopy. The fluorescence in the bleached zone was phorylatable mutant CFP-tau-4KXGA was measured. Its diffufilled up rapidly from both ends so that in ⬃3 min, ⬃50% of the sion (D ⬃ 3 ␮m 2/s) did not differ significantly from that of initial fluorescence was recovered at the center (Fig. 4). We did CFP-tau, arguing that the exchange rate of tau on the microtunot detect a preferential direction of tau recovery through the bule surface does not require phosphorylation. bleached zone, either in the form of a moving front or as tau particles. The recovery of the signal intensity could be fitted to a Tau–microtubule interaction is highly dynamic in the axon curve consistent with rapid diffusion in a thin axonal cylinder. In Although the above FRAP experiments over long axonal segthis model, the recovery time to 50% at the midpoint is given by ments report on the overall diffusion of tau, FRAPping a small t50% ⫽ 0.27 L 2/D, yielding an apparent diffusion coefficient D ⬇ 3 ␮m 2/s for CFP-tau. This high value was unexpected considervolume (4 ␮m long) reveals the dynamics of the tau–microtubule ing that most tau is bound to MTs. In contrast, bleaching and interaction because in this case the diffusion of tau is fast, and photoactivation experiments with labeled tubulin lead to longrecovery of the fluorescence signal is mainly determined by the lived gaps or activated areas (200 – 400 min), indicating that tuoff-rate koff (Bulinski et al., 2001) (see supplemental material, available at www.jneurosci.org). Vero cells were transfected eibulin does not diffuse readily (Lim et al., 1989; Sabry et al., 1995). ther with CFP-tau, CFP-8Rtau, or yellow fluorescent protein To understand the fast diffusion of tau, we performed FRAP (YFP)-tubulin. Areas of 2– 4 ␮m length along single microtuexperiments to define the diffusion coefficients of different CFPbules were bleached, and the recovery of the fluorescence was tau mutants and GFP as a control. The experiments were done at measured (Fig. 5). The recovery time of CFP-tau was ⬃3 s. By a distance of at least 300 ␮m from the cell body and the growth cone. GFP diffuses at D ⬃ 25 ␮m 2/s, approximately twice as fast comparison, the tight binding CFP-8Rtau has a recovery halfas CFP-tau-4KXGE, which cannot bind to MT (D ⬃11 ␮m 2/s) time of 20 s, whereas YFP-tubulin requires a long time to recover



Table 1. Diffusion coefficients and microtubule-binding parameters Fraction of free tau in axon Tau construct

Dapp (␮m2/s)

Dtheor (␮m2/s)

observed

Dapp/Dtheor

Rate of detachment [koff (s⫺1)]

MT dwell time [t1/2 (s)]

GFP CFP-htau40 CFP-htau40 – 4KXGA CFP-htau40 – 4KXGE CFP-htau40 – 8R YFP-tubulin GFP-tau23a GFP-tau24a GFP-ensconsinb

25 3 3 11 1 n.d.

28 12 12 12 9 14

25% n.d. 100% n.d. n.d.

25% 25% 92% 11% n.d.

0.17 0.17 1.4 0.046 0.008 0.25 0.28 0.195

4 4 0.5 15 90 2.8 2.5 3.56

n.d., Not determined. a Samsonov et al., 2004 (values for axonal shaft, single MT). b Bulinski et al., 2001.

these conditions, the fast dynamics of tau–MT binding is not dependent on tau phosphorylation. The t1/2 values of the FTDP-17 mutants ⌬K280 and P301L (3.1 and 3.0 s) are marginally smaller than the t1/2 of tau, but these differences are hardly significant. Thus the FTDP-17 pathology of these tau mutants cannot be explained by large differences in the MT binding behavior in axons. Furthermore, the diffusion properties of tau were homogeneous throughout the axons, i.e., the microtubule binding in proximal axon segments, far away at the growth cone or in between were similar. By comparison, in the bleaching experiment of tau on MTs in glia cells of RGC explants, tau shows a recovery time t1/2 of 2.7 s, close to the recovery in Vero cells. The t1/2 values are somewhat shorter than in axons (3.8 s), indicating that diffusion cannot be neglected completely in these experiments, especially in the case of axons (see supplemental mateFigure 5. Photobleaching and recovery of CFP-tau bound to microtubules. a, FRAP of CFP-tau8R on MT in a Vero cell. Fluoresrial, available at www.jneurosci.org). cence recovers evenly in the entire bleached region rather than starting from the borders of the bleached zone (in contrast to large We used FSM with the CFP-tau conbleached areas in axons; see Fig. 4). b, Time-lapse imaging of cell shown in a in 2 s intervals (black and white; left), color spectrum view (right). Scale bar, 2 ␮m. c, Fluorescence recovery of b at center of bleached zone, reaching ⬃50% of initial value with a struct in Vero cells. The binding of tau to half-time of 20 s. d, Mean recovery rate ⫾ SEM of CFP-tau, YFP-tubulin, and CFP-tau8R. Note that CFP-tau recovers rapidly MTs is more dynamic than that of all (half-time, ⬃3 s), tightly binding tau8R slowly (⬃20 s), and tubulin very slowly (⬃90 s) because it is mostly integrated into the other published MAPs so far but close to MT wall. the t1/2 of ensconsin (MAP2, t1/2 ⫽ 60 s; MAP4, t1/2 ⫽ 44 s; ensconsin, t1/2 ⫽ 4 s) (Olmsted et al., 1989; Bulinski et al., (90 s), because tubulin is trapped within the microtubule lattice. 2001). Because our results were all derived from FRAP experThus the recovery time of tau in Vero cells is extremely fast comiments, we wanted to confirm them with a second indepenpared with MAP2 and MAP4 (15–30 times) (Olmsted et al., 1989; dent method such as FSM (Waterman-Storer et al., 1998). In Samsonov et al., 2004). This result is consistent with the fast this method, a low concentration of CFP-tau should lead to an diffusion seen in axons. uneven distribution of tau on MTs, resulting in a speckled Next we wanted to quantify the rates of tau exchange on miappearance (Bulinski et al., 2001). The dynamics of these crotubules for the special geometry of an axon. Areas of 2– 4 ␮m speckles can be used to study tau dynamics on MTs. Unexpectlength along RGC axons were bleached with a 405 nm laser. In edly, low concentration of CFP-tau in Vero cells showed a such small areas of the axon, the rate limiting step is the release of uniform localization along microtubules (Fig. 7a,c). To visutau from the microtubule, rather than diffusion, resulting in a alize lower tau concentrations, we enhanced the signal intenhalf-time that depends on microtubule binding (see supplemensity of single tau molecules by generating derivatives carrying tal material, available at www.jneurosci.org). The time-lapse up to three EGFP molecules at the N terminus. This revealed a video of the 8R-tau mutant (Fig. 6a) shows the clear borders after speckled distribution of tau along MTs, which was followed bleaching. The half-time of MT rebinding is ⬃15 s, similar to the over time (Fig. 7b,d,e). MT binding and diffusion was still results in Vero cells. CFP-tau (wild type) has a rebinding halfrapid because most of the speckles were visible only over a time of 3.8 s, close to the 4KXGA mutant at 4.2 s (Fig. 6c). This short time period (2–5 s), confirming the results from the confirms the results from the diffusion experiments that under



FRAP measurements. Thus, no movement of dots along microtubules was visible so that evidence for transport could not be obtained in this experiment. Rapid movement of eight-repeat tau through bleached zones of RGC axons in short filament-like structures The bleaching experiments did not reveal a transport mechanism of tau but showed that tau can distribute rapidly by diffusion because of the short-lived MT interaction, although on average, most of tau is bound to MTs. The results argue against the possibility that tau is transported along the axon as part of moving vesicles or protein complexes, because this kind of transport would have been visible in the bleaching experiments. A possible mechanism could be the transport as single proteins or in small accumulations not detectable with our microscopic system. A second possibility is that tau is transported along with a moving particle to which it attaches in a dynamic manner. Because tau has a dynamic interaction with MTs, we considered the possibility that tau binds to moving MT pieces, which have been postulated as the transport units of tubulin in neurons (He et al., 2005). To test Figure 6. FRAP of CFP-tau on MTs in axons of RGC neurons. a, Photobleaching of CFP-tau8R in a 3 ␮m area of an axon. Because this hypothesis, the CFP-8Rtau mutant diffusion over this short distance is fast, the half-time of recovery represents the off-rate of tau from the MTs. b, Mean signal and CFP-tau were analyzed more care- intensities along the axon shown in a before the bleach (black), directly after the bleach (red) and 15.4 s after the bleach (light blue). c, Mean recovery time ⫾ SD of different tau mutants and of tau at different positions along the axon, showing only small fully. If tau were transported on MT variations (3– 4 s). wt, Wild type. d, Comparison of tau distribution along the axon by diffusion and transport (computed on the fragments, the bleached area should be basis of observed and published rates of transport and diffusion; details in supplemental material, available at www. longer visible with CFP-8Rtau because it jneurosci.org). The advance of the “front” of tau (defined as half of the initial tau concentration) is plotted versus time. Top, Long binds to MT more tightly and diffuses time axis (30 d). Bottom, Initial short time axis (16 h). The modeling shows that diffusion can move tau in 4 d by 1 mm along the more slowly. The FRAP experiment of axon (top, solid black line) before it becomes too slow to account for the published transport rate of ⬃0.003 ␮m/s (solid blue line) large regions of the axon (30 – 60 ␮m) (Mercken et al., 1995). At longer distances (beyond the intersection between the black and blue solid curves), diffusion is too was repeated with an Olympus FV1000 inefficient to distribute tau, and active transport is needed for axons longer than ⬃1 mm. The spreading of tau into the axon microscope, which is more sensitive, reported by Utton et al. (2002) (bottom, short pink line, marked by cross) could be fully explained by diffusion alone. generates bleached regions more effiwild-type four-repeat tau can be observed piggybacking on MT ciently, and allows image collection during bleaching. The fragments from the cell body into axons, we applied the protocol sensitivity and laser intensity were chosen so that the signal of Ahmad and Baas (1995). Primary cortical rat neurons were before the bleach was strongly oversaturated to be able to grown for 3 d and transfected with CFP-tau adenovirus, and 24 h detect very weak signals. Under these conditions, it was possilater, tau was visible on the MTs in the cell body (Fig. 9a). The ble to visualize CFP-8Rtau in 2- to 6-␮m-long filament-like neurons were incubated for 6 h with 10 ␮g/ml nocodazole to structures moving through the bleached zone (Fig. 8a,b). destroy the MT cytoskeleton (Fig. 9b). After nocodazole washMovement events were observed at a frequency of 0.2 events out, the cells were incubated for 10 min at 37°C. During this per minute in an axon stretch of 100 ␮m. Instantaneous vetime, the MTs resume growth from the centrosomes and reach locities ranged between 0.2–2 ␮m/s in both directions, with lengths of ⬃5 ␮m (Fig. 9c). The addition of 40 nM vinblastine mean velocities ⬃0.7 ␮m/s and pausing frequencies ⬃2/min stops MT polymerization, and subsequently, it was possible to (Fig. 8c,d). The velocities are typical for motor protein-driven observe MTs with CFP-tau being severed from the centrosome transport. and moving to the periphery (Fig. 9d– h). Because the MTs are limited to the close proximity of the centrosome in this experAnterograde transport of tau on fast moving microtubules in iment, the moving MT fragments have to move to the periphcortical neurons ery of the cell body and into axons in an actin-dependent In bleaching experiments, the tau mutant with a duplicated remanner (Ahmad and Baas, 1995). A fraction of these taupeat domain and enhanced MT affinity (8R-tau) was the only tau containing MT fragments entered the axon (Fig. 9d). These species visible as part of a moving filament-like structure. The MTs are moving at rates up to 1 ␮m/s (Fig. 9d,h). Together results suggest that tau can be transported on small MT fragments these results show that axonal transport of tau occurs at least through the axon, but the images from wild-type tau become blurred because of the short dwell time on MTs. To test whether in part by piggybacking on fast-moving MT fragments.



transport problems (Stamer et al., 2002). Time-lapse studies of the tau particles showed rapid but intermittent bidirectional movements at instantaneous velocities of ⬃0.3 ␮m/s. They largely ceased during treatment with nocodazole, indicating that movements are dominated by MT-dependent transport (Fig. 10c–f ).

Discussion Tau, one of the major MAPs in the brain, is important for axonal outgrowth and for stabilizing microtubules as tracks for axonal transport (Drubin and Kirschner, 1986). Tau is normally sorted into the axonal compartment; however, in disease states such as Alzheimer’s, it becomes missorted into the somatodendritic compartment and forms abnormal aggregates (Lee et al., 2001). In previous studies, we noticed that tau has the potential of inhibiting MT-based transport by interfering with the attachment of motors to MTs. Both kinesin and dynein are inhibited, but the effect is more pronounced with kinesin. This leads to a predominance of retrograde flow and net inhibition of anterograde flow, so that vesicles and organelles tend to accumulate in the cell body. The consequence is the starvation and decay of cell processes (Trinczek et al., 1999; Stamer et al., 2002; Vershinin et al., 2006). Surprisingly, tau itself is able to enter cell processes despite the general inhibition of anterograde transport, suggesting some distinct mode of distribution. This prompted us to investigate the transport of Figure 7. FSM of 3GFP-tau on microtubules. a, CFP-tau in Vero cells stains MTs evenly. b, 3GFP-tau shows a speckled distri- tau in neurons. Previous radiolabeling bution along MTs. c, d, Magnifications of marked areas in a and b. e, Kymograph of a MT from the cell in b. The time lapse consists studies had shown that tau propagates of 20 pictures spaced 1.5 s (30 s total), illustrating the appearance and disappearance of speckles of fluorescent 3GFP-tau. f, In the overall at the rate of slow axonal transport same kymograph, 3 s intervals are separated by white lines. Red arrows indicate the residence time of single 3GFP-tau molecules (⬃0.2– 0.4 mm/d, equivalent to ⬃0.003 on the MT. This is consistent with the half-time of tau-MT binding defined by the FRAP experiment (⬃3 s) (Fig. 5), indicated by the ␮m/s), close to that of tubulin (Mercken et blue vertical arrows on the right (this study and Olmsted et al., 1989; Samsonov et al., 2004). al., 1995). This was confirmed by fluorescence microscopy using GFP-labeled tau transfected into neuronal cultures (Utton Rapid bidirectional movement of tau clusters from the distal et al., 2002). These authors also provided evidence that tau forms end of the axon local accumulations (particles) that move rapidly at instantaAt higher tau concentrations and longer expression times (⬎2 neous rates of ⬃0.3 ␮m/s, close to the rate of fast axonal transd), the pattern of tau localization changes its character in corport, but with extended pauses (Utton et al., 2005). This would tical and RGC neurons. Instead of a distribution with a make tau transport akin to the slow axonal transport described smooth gradient (type-1 distribution), CFP-tau begins to acfor neurofilaments (Wang et al., 2000). cumulate in particles along the axon. In the two crossing RGC We approached the tau transport problem by adenoviral neurons of Figure 10a, one has a low CFP-tau intensity and a transfection of different XFP-labeled tau variants into differentismooth tau distribution, and the other has stronger tau signals ated primary neuronal cultures and observing them by several with a smooth background and additional tau “particles.” To time-resolved microscopic methods, including confocal, deconvisualize complete cortical neurons from the cell body to the volution, FRAP, and FSM. This enabled us to distinguish three distal end, they were imaged in multiple series of z-stacks after modes of tau propagation into axons and their dependence on 2 d of tau expression, revealing a similar accumulation of tau microtubule interactions. The main results are discussed below, into globular structures as in RGC neurons. Interestingly, in and the main observations on diffusion and photobleach recovall neurons, these particles begin to appear near the distal end ery are summarized in Table 1. of the axon, not near the cell body, where tau was first visible The major mechanism determining the tau distribution in the after transfection (Fig. 10b). This indicates that the clustering of tau is a secondary effect that may result from tau-induced short-to-intermediate range is its rapid diffusion. The apparent



diffusion constant of CFP-tau (D ⬇ 3 ␮m 2/s) is surprisingly high, only fourfold less than the diffusion constant of the free cytosolic protein and ensures that tau can distribute evenly throughout the cytosolic volume. This means, for example, that tau can move significantly into axons (millimeter range) without active transport (Fig. 6d). In fact, the rates of spreading by diffusion agree well with the rates considered as active transport by Utton et al. (2002). Because only a low tau/tubulin ratio is required for MT stabilization, diffusion of tau would suffice, at least for short segments of axons. At first glance, the high mobility of tau appears contradictory to the view of tau as a microtubule-associated protein. Indeed, the majority of tau is concentrated on the microtubule surface, but because the exchange rates are high (t1/2 ⬇ 4 s), this does not prevent tau from redistributing along the microtubule or between different microtubules. The mechanism of diffusion can only explain some aspects of tau distribution, and active transport makes the major con- Figure 8. Bidirectional axonal transport of tau in small filamentous structures in RGC neurons. a, Time lapse of a FRAP expertribution over long distances (as part of iment showing anterograde CFP-tau movement in a 5 ␮m filamentous structure at a velocity of 0.4 ␮m/s. The image sequence slow axonal transport at rates of 0.2– 0.4 starts before the bleach event. The recovery by diffusion is visible by the appearance of the CFP signal along the axon and the mm/d) (Mercken et al., 1995). Axonal mi- anterograde entry of a tau containing structure into the bleached region is indicated by arrows. In the right column the same crotubules are mostly stationary (Sabry et time-lapse series was strongly contrast enhanced and blurred, and in every image, the fluorescence was subtracted by the signal al., 1995), but short microtubule frag- of a later image (3 positions) to visualize tau movement without the background of recovery. b, Analogous time-lapse series but ments or tubulin oligomers moving along with a retrograde movement event of tau. The tau-containing structure is visible as a 4-␮m-long filament moving rapidly (0.8 the axon would provide such a mechanism ␮m/s) along the axon. c, Anterograde displacement of tau structure along the axon with time and instantaneous velocities (for review, see Baas and Buster, 2004). (average speed, ⬃0.6 ␮m/s; instantaneous velocities range, ⬃0.2–1.3 ␮m/s). d, Retrograde displacement of a tau structure and instantaneous velocity (average speed, ⬃0.4 ␮m/s; instantaneous velocities, ⬃0.2– 0.9 ␮m/s). The movement of such MT fragments has been observed by tubulin fluorescence of well localized fluorophores so that their movement becomes (Ahmad and Baas, 1995; Wang and Brown, 2002), but the movevisible. ment of tau has been difficult to detect. This can be explained by Tau phosphorylation and tau mutations are important issues the loose coupling between MT and tau, which tends to blur the in Alzheimer’s disease and frontotemporal dementias. As menfluorescence of labeled tau. It requires tightly binding tau variants tioned above, tau pseudophosphorylated in the repeat domain (such as 8R-tau) to visualize the movement of the tau component (KXGE mutant) behaves essentially as a free molecule, consistent in such oligomeric complexes. In this context, we note that there with the detachment of tau from MTs by this type of phosare conflicting interpretations regarding the nature of the short phorylation (Drewes et al., 1997). In contrast, the nonphostubulin-containing structures. They have been regarded either as phorylatable protein (KXGA mutant) behaves identically with MT fragments/tubulin oligomers (Wang and Brown, 2002; He et wild-type tau in terms of distribution and diffusion, arguing al., 2005), or alternatively as tubulin-containing vesicles (Ma et that phosphorylation affects only a small fraction of the exal., 2004). The binding of tau to these structures provides strong pressed tau in these cells. Thus the highly dynamic behavior of support for the first option, because tubulin contained in a vesicle tau can be explained without invoking phosphorylation, in would not be readily accessible to cytosolic tau. contrast to previous proposals (Samsonov et al., 2004). The The relationship between MT affinity, diffusion, and visualtwo FTDP-17 mutants tested, ⌬K280 and P301L, are very simization is well illustrated by different tau variants: when critical ilar to wild-type tau with respect to diffusion and dwell time sites within the repeat domain (KXGS motifs) are pseudophoson MT. This means that the mutations in the tau gene do not phorylated (to KXGE), the MT affinity is strongly reduced. These affect MT interactions as such but operate on other levels, variants are no longer “MT-centered” but “volume-centered,” possibly isoform distribution, aggregation, phosphorylation, because they diffuse almost freely (D ⬇ 11 ␮m 2/s) and thus fill or degradation. the available space, regardless of MTs. However, they fail to reach Regarding the mechanism of long-range transport of tau, aca high concentration at far distances, where diffusion becomes cording to the work of Utton et al. (2005), we expected to observe slow because they are not tied to moving carriers (MT fragtau particles moving along axonal MTs in a rapid stop-and-go ments). Conversely, tightly binding variants such as 8R-tau manner, consistent with the accepted view of slow axonal transspend nearly all their time on a microtubule surface (t1/2 ⬇ 15 s), port (Wang et al., 2000). We indeed observed inhomogeneous hence their diffusion is much slower (D ⬇ 1 ␮m 2/s). Thus, comtau accumulations with typical sizes in the 1–10 ␮m range. Howplexes of MT fragments with 8R-tau contain a sufficient number



Figure 9. Anterograde transport of tau on small MT filaments. a, Cortical mouse neurons transfected with CFP-htau40 and imaged after 24 h. The scheme (top row) shows MTs in red and the nucleus in blue; the images in the second row show CFP-tau. Images were processed with NIH Image (contrast, smooth, and shadow) to enhance contrast. b– d, The shape of the cell is indicated by a dashed line in orange color. b, After 24 h microtubules were depolymerized by nocodazole for 6 h. c, Image 10 min after nocodazole washout. Tau accumulates on small MTs growing out of the centrosome (arrow). d, After blocking MT dynamics by adding 40 nM vinblastine, tau localizes on small anterogradely moving MT fragments (length, ⬃2– 8 ␮m) detached from the centrosome. The boxed area was enlarged and signal enhanced in the top right corner. The orange arrow points to an MT moving into an axon, and white arrows point to moving MTs. e, Time-lapse series of tau cotransported with anterogradely moving MTs. f, Small MTs (white arrows) build up the usual MT array for neuronal cell bodies lying along the plasma membrane instead of forming an aster. Orange arrows indicate MTs already moved far away from the centrosome. g, Overlay of pictures 1 (red), 3 (green), and 4 (blue) of time-lapse series (e). Arrows indicate the movement of 3 MTs. h, Distances traveled by the three MTs in g versus time. Scale bar, 10 ␮m.

phosphorylation level (Drubin and Kirschner, 1986; Garcia and Cleveland, 2001). Several mechanisms of axonal sorting have been envisaged, e.g., preferential routing of tau mRNA to the axon (Hirokawa et al., 1996; Aronov et al., 2002), preferential stabilization in the axon (e.g., by MT interactions and/or phosphorylation), and preferential loss of MT binding and degradation in the dendrites (Kanai and Hirokawa, 1995). In fact, when tau protein is microinjected, it rapidly diffuses into both axons and dendrites, but subsequently it persists only in axons (Hirokawa et al., 1996). Our data show that a fraction of tau moves actively on MT fragments. This takes place even under conditions in which tau also fills cell bodies and dendrites, similar to the distribution in AD. The anterograde traffic of MT fragments is sustained by the motor protein dynein (50%) and actin filaments (50%) (He et al., 2005). This feature, combined with the rapid diffusion, would explain why tau transport works even if the kinesin-dependent transport of vesicles and organelles is already inhibited. In this view, the initial distribution of expressed CFP-tau can mostly be accounted for by diffusion, whereas the long-range distribution requires an active transport component. Conversely, the pathological somatodendritic distribution of tau in AD would not be caused by missorting in the strict sense but rather to a higher effective diffusion rate of hyperphosphorylated tau because it is largely detached from microtubules, combined with a failure of tau degradation.

References ever, we believe that this is not the normal physiological mechanism but rather a sign of degenerating axons, akin to incipient axonal swellings. The reasons are that (1) tau particles occur only at high levels of tau expression and (2) more importantly, they originate not in the cell body or proximal axon, but in the distal regions of axons. It is known that tau induces defects in axonal traffic because it interferes with motor–microtubule interactions (Mandelkow et al., 2004). This causes gradual axonal degeneration, starting at the extremities and accompanied by the accumulations of proteins, vesicles, and organelles. We consider the tau particles to be a sign of this process. In contrast, the physiological tau carriers (MT fragments) are barely visible (see above); they represent a small fraction and originate at the cell body or proximal axon. Given the rapid diffusion of tau, one may ask why tau is confined to axons in differentiated neurons or, conversely, why the appearance in the somatodendritic compartment is a sign of a disease state. The sorting mechanism for tau and other MAPs becomes effective only toward the end of the fetal stage, concomitant with the expression of adult tau isoforms and a reduction of

Ahmad FJ, Baas PW (1995) Microtubules released from the neuronal centrosome are transported into the axon. J Cell Sci 108:2761–2769. Andreadis A (2005) Tau gene alternative splicing: expression patterns, regulation and modulation of function in normal brain and neurodegenerative diseases. Biochim Biophys Acta 1739:91–103. Aronov S, Aranda G, Behar L, Ginzburg I (2002) Visualization of translated tau protein in the axons of neuronal P19 cells and characterization of tau RNP granules. J Cell Sci 115:3817–3827. Baas PW, Buster DW (2004) Slow axonal transport and the genesis of neuronal morphology. J Neurobiol 58:3–17. Banker G, Goslin K (1988) Developments in neuronal cell culture. Nature 336:185–186. Barghorn S, Zheng-Fischhofer Q, Ackmann M, Biernat J, von Bergen M, Mandelkow EM, Mandelkow E (2000) Structure, microtubule interactions, and paired helical filament aggregation by tau mutants of frontotemporal dementias. Biochemistry 39:11714 –11721. Biernat J, Gustke N, Drewes G, Mandelkow EM, Mandelkow E (1993) Phosphorylation of Ser262 strongly reduces binding of tau to microtubules: distinction between PHF-like immunoreactivity and microtubule binding. Neuron 11:153–163. Bulinski JC, Odde DJ, Howell BJ, Salmon TD, Waterman-Storer CM (2001) Rapid dynamics of the microtubule binding of ensconsin in vivo. J Cell Sci 114:3885–3897.



Butner KA, Kirschner MW (1991) Tau protein binds to microtubules through a flexible array of distributed weak sites. J Cell Biol 115:717–730. Chang S, Svitkina TM, Borisy GG, Popov SV (1999) Speckle microscopic evaluation of microtubule transport in growing nerve processes. Nat Cell Biol 1:399 – 403. Chen J, Kanai Y, Cowan NJ, Hirokawa N (1992) Projection domains of MAP2 and tau determine spacings between microtubules in dendrites and axons. Nature 360:674 – 677. Drewes G, Ebneth A, Preuss U, Mandelkow EM, Mandelkow E (1997) MARK, a novel family of protein kinases that phosphorylate microtubule-associated proteins and trigger microtubule disruption. Cell 89:297–308. Drubin DG, Kirschner MW (1986) Tau protein function in living cells. J Cell Biol 103:2739 –2746. Garcia ML, Cleveland DW (2001) Going new places using an old MAP: tau, microtubules and human neurodegenerative disease. Curr Opin Cell Biol 13:41– 48. Goedert M, Jakes R (2005) Mutations causing neurodegenerative tauopathies. Biochim Biophys Acta 1739:240 –250. Figure 10. Tau forms dynamic axonal clusters when highly overexpressed. a, c, d, RGC neurons 2 d after transfection Gustke N, Trinczek B, Biernat J, Mandelkow EM, with CFP-htau40. Two axons are shown in a: black and white (left) and color spectrum image (right) to enhance the Mandelkow E (1994) Domains of tau protein resolution of the signal intensities. Axon 1 has a low expression of CFP-htau40 and therefore a smooth distribution. Axon and interactions with microtubules. Biochem- 2 has a threefold higher CFP-htau40 concentration, leading to an uneven tau distribution. The boxed area of axon 1 is istry 33:9511–9522. signal enhanced to verify that the tau distribution is uniform. b, Cortical neurons 2 d after transfection with CFP-tau. The He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, top shows an axon with an even tau distribution in the proximal part and a clustered tau distribution in the distal part. Note Vogelstein B (1998) A simplified system for that after 2 d of expression the tau gradient is no longer visible in the 1-mm-long axon. The magnified image (inset; boxed) generating recombinant adenoviruses. Proc shows that the transition between smooth and granular tau distribution is quite abrupt. The axon in the bottom shows a Natl Acad Sci USA 95:2509 –2514. clustered tau distribution along the whole visible part of the axon at a higher magnification. c, d, Time-lapse studies of He Y, Francis F, Myers KA, Yu W, Black MM, Baas CFP-tau clusters in axons of RGC neurons. Top left, Frame time in seconds; arrows mark moving particles. Left frames show PW (2005) Role of cytoplasmic dynein in the anterograde movement (v ⫽ 0.4 ␮m/s), and right frames show retrograde (0.2 ␮m/s). d, Moving tau clusters in the same axonal transport of microtubules and neuroconditions but after treatment with 10 ␮m nocodazole for 6 h to destroy all MTs (v ⫽ 0.1 ␮m/s). e, Life history of moving filaments. J Cell Biol 168:697–703. Hirokawa N, Takemura R (2005) Molecular mo- tau cluster (position vs time). Red, pink, and white in bar indicate phases of anterograde or retrograde movements, and tors and mechanisms of directional transport pausing. Note that the movement is intermittent and bidirectional. Fast phases in both direction have speeds of ⬃0.5 ␮m/s, but the net movement during the observation period (180 s) is much slower (0.02 ␮m/s anterogradely). f, in neurons. Nat Rev Neurosci 6:201–214. Hirokawa N, Funakoshi T, Sato-Harada R, Ka- Histograms depict mean (left) and instantaneous (right) velocity distributions of moving tau clusters. nai Y (1996) Selective stabilization of tau in axons and microtubule-associated protein 2C in cell bodies and dendrites contributes to polarized localization of cytoskeletal proteins in mature neurons. J Cell Biol 132:667– 679. MARK/PAR1 kinase is a regulator of microtubule-dependent transport in Hollenbeck PJ, Saxton W (2005) The axonal transport of mitochondria. axons. J Cell Biol 167:99 –110. J Cell Sci 118:5411–5419. Mercken M, Fischer I, Kosik KS, Nixon RA (1995) Three distinct axonal Hong M, Zhukareva V, Vogelsberg-Ragaglia V, Wszolek Z, Reed L, Miller transport rates for tau, tubulin, and other microtubule-associated proBI, Geschwind DH, Bird TD, McKeel D, Goate A, Morris JC, Wilhelmteins: evidence for dynamic interactions of tau with microtubules in vivo. sen KC, Schellenberg GD, Trojanowski JQ, Lee VM (1998) J Neurosci 15:8259 – 8267. Mutation-specific functional impairments in distinct tau isoforms of Miller KE, Sheetz MP (2006) Direct evidence for coherent low velocity axhereditary FTDP-17. Science 282:1914 –1917. onal transport of mitochondria. J Cell Biol 173:373–381. Kanai Y, Hirokawa N (1995) Sorting mechanisms of tau and MAP2 in Olmsted JB, Stemple DL, Saxton W, Neighbors B, McIntosh JR (1989) Cell neurons: suppressed axonal transit of MAP2 and locally regulated cycle-dependent changes in the dynamics of MAP 2 and MAP 4 in culmicrotubule binding. Neuron 14:421– 432. tured cells. J Cell Biol 109:211–223. Kosik KS, Finch EA (1987) MAP2 and tau segregate into dendritic and axRoy S, Coffee P, Smith G, Liem RK, Brady ST, Black MM (2000) Neurofilaonal domains after the elaboration of morphologically distinct neurites: ments are transported rapidly but intermittently in axons: implications an immunocytochemical study of cultured rat cerebrum. J Neurosci for slow axonal transport. J Neurosci 20:6849 – 6861. 7:3142–3153. Sabry J, O’Connor TP, Kirschner MW (1995) Axonal transport of tubulin in Lee VM, Goedert M, Trojanowski JQ (2001) Neurodegenerative tauopaTi1 pioneer neurons in situ. Neuron 14:1247–1256. thies. Annu Rev Neurosci 24:1121–1159. Samsonov A, Yu JZ, Rasenick M, Popov SV (2004) Tau interaction with Lim SS, Sammak PJ, Borisy GG (1989) Progressive and spatially differentimicrotubules in vivo. J Cell Sci 117:6129 – 6141. ated stability of microtubules in developing neuronal cells. J Cell Biol Sontag E, Nunbhakdi-Craig V, Lee G, Brandt R, Kamibayashi C, Kuret J, 109:253–263. White III CL, Mumby MC, Bloom GS (1999) Molecular interactions Lu M, Kosik KS (2001) Competition for microtubule-binding with dual among protein phosphatase 2A, tau, and microtubules. Implications for expression of tau missense and splice isoforms. Mol Biol Cell 12:171–184. the regulation of tau phosphorylation and the development of tauopaMa Y, Shakiryanova D, Vardya I, Popov SV (2004) Quantitative analysis of thies. J Biol Chem 274:25490 –25498. microtubule transport in growing nerve processes. Curr Biol 14:725–730. Mandelkow EM, Thies E, Trinczek B, Biernat J, Mandelkow E (2004) Stamer K, Vogel R, Thies E, Mandelkow E, Mandelkow EM (2002) Tau

Konzack et al. • Mobility of Tau in Neurons blocks traffic of organelles, neurofilaments, and APP vesicles in neurons and enhances oxidative stress. J Cell Biol 156:1051–1063. Stoothoff WH, Johnson GV (2005) Tau phosphorylation: physiological and pathological consequences. Biochim Biophys Acta 1739:280 –297. Trinczek B, Ebneth A, Mandelkow EM, Mandelkow E (1999) Tau regulates the attachment/detachment but not the speed of motors in microtubuledependent transport of single vesicles and organelles. J Cell Sci 112:2355–2367. Utton MA, Connell J, Asuni AA, van Slegtenhorst M, Hutton M, de Silva R, Lees AJ, Miller CC, Anderton BH (2002) The slow axonal transport of the microtubule-associated protein tau and the transport rates of different isoforms and mutants in cultured neurons. J Neurosci 22:6394 – 6400. Utton MA, Noble WJ, Hill JE, Anderton BH, Hanger DP (2005) Molecular

J. Neurosci., September 12, 2007 • 27(37):9916 –9927 • 9927 motors implicated in the axonal transport of tau and alpha-synuclein. J Cell Sci 118:4645– 4654. Vershinin M, Carter B, Razafsky D, King SJ, Gross SP (2006) Multiplemotor based transport and its regulation by Tau. Proc Natl Acad Sci USA 104:87–92. Wang L, Brown A (2002) Rapid movement of microtubules in axons. Curr Biol 12:1496 –1501. Wang L, Ho CL, Sun D, Liem RK, Brown A (2000) Rapid movement of axonal neurofilaments interrupted by prolonged pauses. Nat Cell Biol 2:137–141. Waterman-Storer CM, Desai A, Bulinski JC, Salmon ED (1998) Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells. Curr Biol 8:1227–1230.

1

Swimming against the Tide: Mobility of the Microtubule Associated Protein Tau in Neurons Sven Konzack, Edda Thies, Alexander Marx, Eva-Maria Mandelkow, Eckhard Mandelkow Max-Planck-Unit for Structural Molecular Biology Notkestrasse 85, 22607 Hamburg, Germany Tel. +49-40-89982810, Fax +49-40-89716810 E-mail: [email protected], [email protected] *Corresponding author

Supplement Mathematical Methods: Estimation of diffusion constants from hydrodynamic properties: We used the StokesEinstein equation, D = kBT/6πηRH (where kB is the Boltzmann constant, T absolute temperature, η the viscosity of the medium, and RH the hydrodynamic radius) to estimate the diffusion coefficients D of GFP, tau, and the GFP-tau fusion proteins. The viscosity of the axonal cytosol was assumed to be 3.4 cP, 5 times larger than that of water at 37°C [Popov & Poo, 1992]. The hydrodynamic radius for GFP (RH = 2.4 nm, corresponding to D = 28 µm2/s) was calculated from the formula for globular proteins, RH/nm = 0.595 (MW/kDa)0.427 (MW = molecular weight), in agreement with experimental values (Luby-Phelps, 2000). The hydrodynamic radius of tau protein (RH =5.6 nm; corresponding to D = 12 µm2/s ) was taken from (Cleveland et al., 1977). This is larger than the value expected for globular proteins of equivalent mass because tau is a natively unfolded protein (Schweers et al., 1994). The empirical formula RH/nm = 0.221 N 0.57 (N= number of amino acids) for highly denatured proteins (Wilkins et al., 1999) predicts RH = 7.1 nm (corresponding to D = 9.4 µm2/s) for a protein of the same number of amino acids as tau (N = 441), indicating that tau protein is not completely unfolded. The same formula can be used to calculate an upper limit for the hydrodynamic radius of the GFP-tau fusion protein (N = 679), assuming a completely unfolded protein of the same number of amino acid residues: RH,max = 9.1 nm (corr. to Dmin = 7.4 µm2/s). Considering that the GFP moiety of the fusion protein is compactly folded, and that the tau moiety also retains some residual structure, the true value is problably closer to RH,min = 5.6 nm (the value of tau protein alone) than to RH,max. This is consistent with the diffusion constant measured for GFP-tau (D = 11 µm2/s), which corresponds to RH = 6.1 nm. Alternatively, the combined RH of a fusion protein with two globular domains can be estimated from (M1+M2)Rtot2 = M1R12 + M2R22 + M1M2 d2/(M1+M2). Since the values for RH and MW of tau (5.6 nm, 48 kDa) are much larger than for GFP (2.4 nm, 28 kDa), the presence of GFP in the fusion protein makes only a minor contribution to the combined RH (6.06 nm, +8%) Modelling of free diffusion and diffusion with binding to microtubules: Experimental values for the diffusion coefficients were determined by FRAP from the half time of recovery, t1/2, at the centre of an extended bleached region within the axon. To define the relation between t1/2 and D, the axon was modeled as a cylinder (tube of constant cross section) and the diffusion of tau was described by solving the one-dimensional diffusion equation for free

2 diffusion or by numerical integration of the reaction-diffusion equations for diffusion with binding to microtubules. Free diffusion: The one-dimensional diffusion equation (1)

!c( x, t ) ! 2 c( x, t ) =D !t !x 2

with initial conditions (2)

c ( x , t = 0) = c 0

for x
L/2

L/2 < x < L/2

(outside the bleached zone) (inside the bleached zone)

is solved by using the well-known solution of (1) for initial conditions corresponding to a step function (e.g. Segel, 1980). Using the notation (3)

erf ( x ) =

2 #

!

x 0

2

e " s ds

( error function )

the solution of equation (1) with initial conditions (2) is given by

(4)

c c( x, t ) = 0 2

& ,L ) ,L )# * +x' * - x '! $ 2 ' - erf * 2 '! . $2 - erf * 2 Dt 2 Dt * ' * '! $ * ' * ' $% + ( + (!"

At the center of the bleached region ( x = 0 ) this equation simplifies to (5)

& & L ## c( x = 0, t ) = c0 $$1 ' erf $ ! !! % 4 Dt " " %

which leads to (6)

D = 0.275

L2 . t1 / 2

Diffusion with binding to microtubules: Equations (1) and (6) should apply to freely diffusing molecules like GFP and the 4KXGE-tau mutant. For wildtype tau and the 8R-tau mutant, reversible binding to MTs must be taken into account. Instead of a simple diffusion equation for a single species, two equations for the bound and for the detached molecules (concentrations cbound and cfree) are required to describe diffusional motion and MT binding kinetics. Assuming that the total concentration of tau (ctotal = cbound + cfree) is far below saturation of MTs everywhere in the axon, association and dissociation can both be described by (pseudo) first order rate equations (rate constants kon and koff), leading to: (7a) (7b)

!c free ! 2 c free = + koff cbound " kon c free + D , !t !x 2 "cbound = !k off cbound + k on c free . "t

The system of reaction-diffusion equations (7a, b) with initial conditions (2) does not have an analytical solution, but two limiting cases can be discussed easily. In the case L 0 (small bleached zone) the diffusion is "fast", relative to MT binding, and therefore the experiment yields information on MT binding. Conversely, in the case L ∞ the diffusion is "slow" compared to MT binding so that the experiment yields information on diffusion. Specifically, if L is sufficiently small, such that diffusion of the free molecules can be considered fast

3 compared to the MT binding kinetics, then diffusional equilibration of cfree is virtually instantaneous over all relevant distances. Thus cfree is the same at every point, and equations (7a, b) with initial conditions (2) reduce to a set of three ordinary differential equations for the concentrations cfree, cbound,1, and cbound,2 where indices 1 and 2 refer to the bleached and the unbleached region: (8a) (8b) (8c)

dc free = + k off cbound ,1 ! k on c free dt dcbound ,1 = !k off cbound ,1 + k on c free , dt dcbound , 2 = !k off cbound , 2 + k on c free , dt

cbound ,1 (t = 0) = 0 , cbound , 2 (t = 0) = c0 .

Equations (8a) and (8b) (or (8a) and (8c)) are the rate equations describing reversible association and dissociation of tau and MTs. At equilibrium ( dc free / dt = dcbound / dt = 0 ) the concentrations obey the relationship (9)

c eqfree c

eq bound

=

k off k on

=

! free . ! bound

!1 !1 Here, " free = k on , " bound = k off denote the mean time periods the molecules spend in the free

or the bound state before binding or unbinding, respectively. Equations (8a,b,c) can be solved by first considering a cylinder of finite length > L , then increasing its length to infinity. The result for the bleached region is (10a)

c free (t ) = c eqfree,0 = const,

(10b)

eq , 0 cbound ,1 (t ) = cbound 1! e

(

! koff t

)

,0 eq , 0 eq , 0 eq , 0 where c eq free and cbound are the equilibrium concentrations before bleach, c free + cbound = c0 .

By combination of (10a) and (10b), the total concentration at the bleached region is: (10c)

(

eq , 0 ctotal ,1 (t ) = c eqfree,0 + cbound 1' e

' koff t

)= c &$$1 ' k 0

%

k on 'k t # e off ! . ! off + k on "

Thus, recovery after bleaching of a small region within the axon follows an exponential time dependence with rate constant koff. Equation (10b) can be obtained more easily by considering the bleached instead of the unbleached molecules, i.e. by solving reactiondiffusion equations (7a, b) for initial conditions complementary to (2), that is c( x, t = 0) = c0 for ! L 2 < x < L 2 , and zero elsewhere. Since the diffusion is assumed to be fast, c free in this case drops immediately to zero, c free = 0 for all x and for all t > 0. Thus, t =0 cbound (t ) = cbound e

! koff t

eq , 0 = cbound e

! koff t

. If we assume that bleaching does not change the

fundamental properties of the molecules (but only their visibility), and if the system was at equilibrium before the bleach, then it will stay at equilibrium all the time, and any change in the concentration of the bleached molecules must be compensated by redistribution of the unbleached molecules. Thus the solutions for the bleached and the unbleached molecules follow the same (complementary) time dependence. Since this is a general argument that

4 does not rely on the special geometry of the system, the result in equation (10c) is valid for any geometry and can be applied, in principle, to the axon (one-dimension case) or the cell body (three-dimension case) as long as the bleached region is sufficiently small. If the bleached region is large, and if we disregard local details at the boundaries between the bleached and the unbleached regions, but only consider the long-term behavior at large scales (characteristic time t c " L2 D >> ! = ! free + ! bound , where ! is the mean time a molecule needs to cycle through the bound and the unbound state), then the reactiondiffusion equation (7a) can be simplified: If t c >> ! , each molecule attaches and detaches many times during all relevant time intervals; thus, the long-term molecular motions are the same as those of freely diffusing molecules, if only the fraction of time the molecules are freely diffusing is taken into account. Hence, the behavior of molecules interacting with MTs can be described with a simple diffusion equation by substituting the real time, t, by the effective time, t diff , i.e. the time during which the molecules are actually diffusing: (11)

!c ! 2c =D 2 !t diff !x

with

t diff =

! free k off t= t , ! bound + ! free k on + k off

or (11')

k off !c ! 2c = D 2. !t k on + k off !x

!1 !1 + k off Thus, at very long time scales ( t >> k on ) molecules that bind to MTs reversibly behave

like freely diffusing molecules with an apparent diffusion coefficient (12)

Dapp =

k off k on + k off

D .

Again, this conclusion holds for any geometry. By numerical integration of the reaction-diffusion equations (7a, b) for a range of parameters we found that our bleaching experiments can by analyzed in terms of the limiting cases discussed above (see Fig. S1). For the long-range bleaching experiments, approximation by an apparent diffusion coefficient is excellent. Interpretation of the experiments with small bleach regions to determine k off by using equation (10c) is more critical. At very short and very long times, the approximation inevitably breaks down. At medium time scales, the difference to the limiting exponential curve stays nearly constant (Fig. S1b). As a consequence, k off values determined by exponential fitting of the data in the medium time range are within a factor of 2 close to the true value as long as bleaching zone does not exceed ~4 µm (see Fig. S1b for details). Diffusion with active transport by microtubules: To account for the contribution of active transport of tau by attachment to moving fragments of microtubules, equations (7a, b) were expanded into three equations for c free , cmobile = m ! cbound , and c stat = (1 " m ) ! cbound , where m is the fraction of the bound molecules transported by moving fragments of MTs, and an advection term " v

!cmobile was added to the equation of the mobile fraction (v, velocity of !x

moving MT fragments). (13a)

!c free ! 2 c free = + k off ( cmobile + cstat ) " k on c free + D , !t !x 2

5

(13b) (13c)

!cmobile !c = "k off cmobile + m # k on c free " v mobile , !t !x #c stat = "k off c stat + (1 " m) ! k on c free . #t

For the simulation of tau dispersal in axons by diffusion and active transport (see Fig 6d), equations (13a,b,c) were numerically integrated with parameters m = 0.004 and v = 1 µm/s, corresponding to slow axonal transport with an average velocity of 0.003 µm/s. Model calculations of Fig. 6d: The figure shows the long-term (30 days, top) and short term (16 hours, bottom) distribution of tau with different conditions. The blue solid line represents the linear movement of a pulse of labeled tau observed experimentally in slow axonal transport (~0.003 µm/s or ~0.2-0.4 mm/d, Mercken et al., 1995). The long-term movement of this tau is determined by its piggy-backing on MT fragments which move slowly by dynein dependent sliding along other stationary microtubules or microfilaments (Wang & Brown, 2002; He et al., 2005), but otherwise are almost non-diffusible. If diffusion is taken into account (red solid line, top), the front would arrive ~1-2 days earlier at a particular point along the axon, but the slopes (= mean velocities) are nearly the same because long term behavior is dominated by active transport of tau on microtubule fragments. However, up to ~10 days or ~3 mm diffusion is more efficient, as seen from the dashed curve (top) for the KXGE-tau mutant (which does not bind to MTs). In the case of reversible binding of wildtype tau to MTs (black solid line, bottom), the spreading is slowed down because tau molecules spend part of their time on slowly-moving MTs, but even here diffusion exceeds slow transport up to ~3 days or ~1 mm down the axon (intersection of blue and black lines, top). By contrast, the tightly binding construct 8R-tau spends most of its time on the immobile MT cytoskeleton and therefore has a severely restricted diffusion, but even here it advances faster than the MT fragments during the first day. The thick short line (green, bottom) represents the spreading of the labeled tau front reported by Utton et al. (2004) in cortical neurons. From these experimental data one cannot determine the relative contributions of diffusion vs. movement by sliding. Ratio of cytoplasmic to MT-bound tau: To be sure that tau and its mutants are really bound to MTs in the imaged axons the ratio of MT-bound to cytoplasmic tau was determined. CFP-4KXGE-tau and mRFP-tau were co-transfected. CFP-4KXGE-tau has a very low affinity to microtubules because it is pseudo-phosphorylated and therefore and fills the entire volume of the axon, including regions of axonal protrusions where microtubules are not present. mRFP-tau (wildtype) has a high affinity for microtubules, it is thus found mainly in the microtubule rich areas and does not show strong additional signals in axonal protrusions (Fig. 4e). The ratio of the signal intensities of the two tau mutants (mRFP:CFP) in areas without MT (Fig. 4e, arrows) was used to normalize the intensities. The ratio of soluble to MT-bound tau can then be calculated because the intensity of CFP-4KXGE is proportional to cytosolic unbound mutant tau and the mRFP-tau signal reflects the sum of microtubule and cytoplasmic wildtype tau. The microtubule-bound fraction was calculated by subtracting the CFP-4KXGE-tau from mRFP-tau. In 9 separate experiments the ratio of microtubule-bound to unbound cytosolic tau was found to be 3:1. The ratio is consistent with the Kd of Tau (~1 µM).

6 Supplement figure legends Fig. S1: Simulation of the fluorescence recovery after photobleaching by diffusion of tau in axonal segments. The curves in panel a and b show the time dependence of ccen, the concentration of total unbleached tau at the center of a bleached region (relative to the initial concentration before bleach, co) for various lengths L of the bleach zone (L = 1, 2, 4, 8, 16, 32 µm). The curves were obtained by numerical integration of the reaction-diffusion !1 equations (7a, b) with D = 10 µm²/s, ! free = 1 s, " bound = t1 / 2 ln 2 = k off = 3 s. Since the relevant time scales vary over three orders of magnitude (increasing quadratically with L), the over-all recovery in panel (a) is plotted with an L-dependent time scale, 2

t * = (32ìm L ) ! t (e.g. t* = t for L = 32 µm, t* = 1024 t for L = 1 µm, i.e. the end point of the x-axis corresponds to shorter times as L decreases). For comparison, panel (b) shows only the initial phase of the recovery, using the same fixed time scale for all curves (from 0 to 20 seconds). Panel a: The dashed curve corresponds to the analytical solution for infinitely fast binding and unbinding of tau (equation 5 with Dapp = 0.25D). It represents the limiting curve that is approached in the case of very long bleached segments (where binding/unbinding is rapid compared with diffusion along the axon). This curve is almost indistinguishable from the L = 32 µm curve when one uses the above realistic rates for binding/unbinding. For L = 32 µm, it takes 600 s or 10 min to recover to 78% ( t * = t , in this case). The dotted line represents the exponential recovery expected for an infinitely small bleach area (small L limit, equation 10c) over the time range of the 1 µm curve (0 to ~0.6 s, t * = 32 2 ! t = 1024 ! t ). Only the initial, almost linear part of the exponential recovery is visible. The curve starts with a finite value, ! free ! bound + ! free = 0.25 , which corresponds to the concentration of free molecules, since

(

)

bleached molecules in solution are instantaneoulsy replaced by unbleached molecules in the limit of zero bleach length. Approach of the 1 µm curve to the dotted line is extremely slow (see also panel b). Theoretically, 100% recovery at t * " ! is expected only for infinitely long cylinders (axons). For finite geometries, the maximum recovery is always smaller than 1 (unless new molecules are created) assuming that the absolute number of fluorescent molecules is irreversibly reduced by bleaching. Note that the gap between the 1 µm curve and the dotted line that remains even at longer times (panel b) is not due to the finite size effect; the boundary conditions used for numerical simulations avoid this effect. Panel b: Curves corresponding to those of panel a are shown within the same time range (0 to 20 s) for all curves. The exponential curve representing the limiting case of an infinitely short bleach zone (dotted line, "0 µm") recovers to virtually 100% over the time of 20 s (time constant of recovery: " bound = k off

!1

= 3 s). For very long bleach length L, recovery starts

slowly and follows a pure diffusion curve with apparent diffusion constant Dapp, reflecting the fact that diffusion is so slow that free and bound molecules are always close to equilibrium. For short segments (L < ~4 µm) recovery after photobleach displays three phases. During a short initial phase, from t = 0 to a fraction of a second, the concentration of unbleached molecules increases rapidly, since diffusion over short distances is fast and the concentration gradient of the diffusing molecules is high at the beginning. Once the concentration in the bleach zone has almost reached the equilibrium concentration of free molecules everywhere in solution, the recovery curve switches over to the second phase. This is governed by the kinetics of unbinding of bleached molecules and their substitution by unbleached molecules. In this phase, recovery follows a curve similar in shape to the limiting exponential curve. However, the recovery curve does not reach the same level as the exponential curve at realistic time scales accessible in experiments. Even for bleaching zones as short as 1 µm, approach to full recovery is extremely slow compared to the exponential curve, due to the fact that with increasing time the relevant distance to be covered by diffusion increases (i.e. the partially depleted region is always expanding, and unbleached molecules have to be supplied from more and more distant regions); thus, the assumption of fast diffusion becomes more and more inadequate. Nevertheless, approximation of the second phase by

7 an exponential function is excellent at least for bleach lengths of 4 µm and below. The apparent time constants, however, differ fom the true value (3 s). The numerical simulations can be used to calculate correction factors for the apparent ! values obtained under the simplifying assumption of an exponential curve. Example: fitting the curves in the entire range from 0 to 20 s with a single exponential function leads to apparent ! values of 3.45 s (L = 1 µm), 3.98 s (2 µm), and 5.15 s (4 µm). Since the bleached spot in the "short L" experiments was between 2 - 4 µm, the correction factors for the k off rates range between 1.3 and 1.8 . It should be mentioned that these correction factors apply for diffusion in a quasi 1dimensional geometry (like a segment of a thin cylinder). For FRAP experiments in a really 3dimensional environement (e.g. small area in the middle of a cell body), approximation of the limiting exponential curve is expected to be much better (thus, correction factors should be closer to 1), since the depletion zone is filled up more efficiently with unbleached molecules arriving from all directions. The curves in panel a may be interpreted in an alternative way: They also represent the different time dependencies expected for varying time constants ! free and ! bound = 3! free at a fixed bleach length, e.g. L = 32 µm. The curves labeled 16, 8, 4, 2, and 1 µm would then correspond to time constants ! free = 4, 16, 64, 256, and 1024 s, respectively. Thus, even in the case of the strongly binding 8R-tau mutant ( ! free of the order of 4 s, corresponding to the curve labeled "16 µm"), the large L limit is a fairly good approximation for the experimental condition we used. Figure S2: RGC neuron expressing CFP-KXGE and mRFP-tau. Cotransfected RGC neurons with mRFP-tau (red) and CFP-4KXGE-tau (green) were used to calculate the ratio of cytoplasmic tau to microtubule-bound tau. The CFP-KXGE-tau mutant does not bind to MTs and can be used as a volume marker because it just shows cytoplasmic localization. Arrows indicate axonal protrusions containing soluble tau but no microtubules. The signals of the two fluorophores were normalized by the signal intensities in these MT free areas. After that procedure the KXGE-tau intensities reflect the intensities of the cytoplasmic portion of mRFP-tau. The signal of the 4KXGE-tau mutant was subtracted from the signal of total tau leading to an image showing only MT-bound tau. The fraction of free tau was calculated by subtracting the signal of MT-bound tau from that of total tau. Arrowheads point to regions with MT-bound and free tau. The procedure is described by the following equations (I = signal intensity, x = factor needed to normalize the signal intensities in the protrusions containing only tau): (1a) x = ICFP-KXGE, protrusion / ImRFP-tau,protrusion (1b) ImRFP-tau,axon, MT-bound = ImRFP-tau, axon – (ICFP-KXGE, axon / x) (1c) ImRFP-tau,axon, unbound = ImRFP-tau, axon – ImRFP-tau, axon, MT-bound Example: ImRFP-tau, axon = 230, ImRFP-tau,protrusion = 30, ICFP-KXGE, axon = 105, ICFP-KXGE, protrusion = 75, yielding (1a) x = 75 / 30 = 2.5 (1b) ImRFP-tau,axon,MT-bound = 230 – (105 / 2.5) = 188 (1c) ImRFP-tau,axon,unbound = 42 Thus, ~80% of the total tau is bound to microtubules, ~20% is unbound cytosolic tau.

Supplement References Cleveland, D.W., S. Y. Hwo, and M.W. Kirschner. 1977. Physical and chemical properties of purified tau factor and the role of tau in microtubule assembly. J. Mol. Biol. 116: 227-247. Luby-Phelps, K. 2000. Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area. Int Rev Cytol. 192:189-221. Popov, S., and M.M. Poo. 1992. Diffusional transport of macromolecules in developing nerve processes. J Neurosci. 12:77-85.

8 Schweers, O., E. Schonbrunn, A. Marx, and E. Mandelkow, 1994. Structural studies of tau protein and Alzheimer paired helical filaments show no evidence for beta structure. J. Biol. Chem. 269: 24290-24297. Segel, L.A. (ed.) (1980). Mathematical models in molecular and cellular biology, Cambridge University Press, Cambridge. Wilkins, D.K., S.B. Grimshaw, V. Receveur, C.M. Dobson, J.A. Jones, and L.J. Smith. 1999. Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques. Biochemistry. 38:16424-16431.