Tau protein diffuses along the microtubule lattice - PubMed (original) (raw)

Tau protein diffuses along the microtubule lattice

Maike H Hinrichs et al. J Biol Chem. 2012.

Abstract

Current models for the intracellular transport of Tau protein suggest motor protein-dependent co-transport with microtubule fragments and diffusion of Tau in the cytoplasm, whereas Tau is believed to be stationary while bound to microtubules and in equilibrium with free diffusion in the cytosol. Observations that members of the microtubule-dependent kinesin family show Brownian motion along microtubules led us to hypothesize that diffusion along microtubules could also be relevant in the case of Tau. We used single-molecule total internal reflection fluorescence microscopy to probe for diffusion of individual fluorescently labeled Tau molecules along microtubules. This allowed us to avoid the problem that microtubule-dependent diffusion could be masked by excess of labeled Tau in solution that might occur in in vivo overexpression experiments. We found that approximately half of the individually detected Tau molecules moved bidirectionally along microtubules over distances up to several micrometers. Diffusion parameters such as diffusion coefficient, interaction time, and scanned microtubule length did not change with Tau concentration. Tau binding and diffusion along the microtubule lattice, however, were sensitive to ionic strength and pH and drastically reduced upon enzymatic removal of the negatively charged C termini of tubulin. We propose one-dimensional Tau diffusion guided by the microtubule lattice as one possible additional mechanism for Tau distribution. By such one-dimensional microtubule lattice diffusion, Tau could be guided to both microtubule ends, i.e. the sites where Tau is needed during microtubule polymerization, independently of directed motor-dependent transport. This could be important in conditions where active transport along microtubules might be compromised.

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Figures

FIGURE 1.

FIGURE 1.

Tau molecules diffuse along MTs. A, TIRF microscopy images of a Cy5-labeled MT (red) and TMR-labeled hTau40 molecules (green) at a Tau:tubulin ratio of 100 p

m

:50 n

m

. hTau40-TMR shows clear co-localization and enrichment on Cy5-labeled MTs. B, sequential frames of a TMR-labeled hTau40 molecule (green) moving along an immobilized Cy5-labeled MT (red) in the absence of ATP. The Tau:tubulin ratio was 100 p

m

:50 n

m

. Only 5% of the Tau molecules were TMR-labeled. Time intervals were as indicated. C, kymograph of the movement of the hTau40-TMR molecule shown in B. Horizontal dashed lines indicate start and end of diffusive interaction, respectively. The extreme positions along the MT reached during this diffusive encounter of ∼15 s are depicted as vertical dashed lines. D, mean squared displacement <x_2> of the same hTau40-TMR molecule (black squares) plotted against time increment, i.e. different time intervals over which displacement was determined. Data fitted by a linear regression (black line) to the equation <_x_2> = 2_Dt yielded a diffusion coefficient D of 0.292 μm2/s. As an example for directed motion the gray circles depict data derived from a single kinesin-1 molecule moving linearly along an MT with a speed of 0.481 μm/s in the presence of 1 m

m

ATP. Error bars represent the S.E. of the squared displacement values.

FIGURE 2.

FIGURE 2.

The diffusive behavior of Tau molecules is independent of the total Tau concentration. A, diffusion coefficients of individual hTau40-TMR molecules at different Tau concentrations. Concentration of tubulin was 50 n

m

. B, distribution of diffusion coefficients of 170 hTau40-TMR molecules at various total Tau concentrations. A Gaussian fit (black curve) yielded a mean value for the diffusion coefficient D of 0.153 ± 0.019 μm2/s. C, interaction times between individual hTau40-TMR molecules and Cy5-labeled MTs at different Tau concentrations in the presence of 50 n

m

tubulin. D, distribution of interaction durations of 170 hTau40-TMR molecules at various total Tau concentrations. An exponential decay (black curve) fitted to the data yielded, when corrected for photobleaching (

supplemental Fig. S5_D_

), a diffusion time constant of 24.41 ± 1.78 s. E, distances on MTs scanned by individual hTau40-TMR molecules during their interaction times at different Tau concentrations in the presence of 50 n

m

tubulin. F, distribution of observed distances on MTs scanned by individual hTau40-TMR molecules (n = 170) at various total Tau concentrations.

FIGURE 3.

FIGURE 3.

The C terminus of tubulin facilitates Tau binding to MTs and Tau diffusion. A, TIRF microscopy images of three (1–3) Cy5-labeled subtilisin-digested MTs (red) immobilized together with unlabeled undigested MTs. TMR-labeled hTau40 molecules (green) bound preferentially to undigested but not to subtilisin-digested MTs, making the unlabeled undigested MTs visible (indicated as dashed white lines). B, 60-s kymographs of few but mainly stationary hTau40-TMR molecules on Cy5-labeled subtilisin-digested MTs (1–3, from A). More hTau40-TMR molecules bound to two unlabeled undigested MTs († and ‡, from A) and showed clear movement. Scale bars, 2.5 μm. C, Coomassie Brilliant Blue-stained SDS-PAGE of undigested unlabeled MTs (Ø-MT) and partially Cy5-labeled subtilisin-digested MTs (s-MT, 200 μg/ml subtilisin for 20 min at 35 °C), marker: 50, 60 kDa.

FIGURE 4.

FIGURE 4.

The diffusion of Tau molecules along MTs is sensitive to ionic strength and pH. A, diffusion coefficient D of TMR-labeled hTau40 molecules diffusing along MTs versus potassium acetate concentration added to buffer BRB12 resulting in final ionic strength of ∼40, 80, 120, and 140 m

m

, respectively. Error bars represent error margins of the Gaussian fits. B, diffusion time, corrected for photobleaching, of TMR-labeled hTau40 molecules versus potassium acetate added to buffer BRB12. Error bars represent error margins of the exponential decay fits to the observed duration and the TMR fluorescence decay. C, MT sections scanned by hTau40-TMR molecules (calculated from the diffusions constants and the respective corrected interaction times) versus potassium acetate concentration. Error margins were calculated from the error margins in A and B. D, diffusion coefficient D of hTau40-TMR molecules diffusing along MTs versus pH of buffer BRB12. Error bars represent error margins of the Gaussian fits. E, photobleaching corrected diffusion time of hTau40-TMR molecules versus pH of buffer BRB12. Error bars represent error margins of the exponential decay fits to the observed duration and the TMR fluorescence decay. F, MT sections scanned by hTau40-TMR molecules versus pH of buffer BRB12 (calculated from the respective diffusions constants and interaction times). Error margins were calculated from the error margins in E and F.

FIGURE 5.

FIGURE 5.

Diffusion of an IgG antibody along immobilized MTs. A, 60-s kymographs of rhodamine-labeled anti-dynein IgG antibodies (40 ng/ml) moving bidirectionally along two different immobilized Cy5-labeled MTs. B, mean squared displacement <x_2> (black squares) of a rhodamine-labeled antibody (bright mobile molecule in the leftmost kymograph in A) plotted against the time increment. The data were fitted by a linear regression (black line) to the equation <_x_2> = 2_Dt yielding the diffusion coefficient D (0.0497 μm2/s). Error bars represent the S.E. of the squared displacement values. For comparison, the dashed line represents data derived from the diffusing hTau40-TMR molecule shown in Fig. 1_D_.

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