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Single-molecule tracking of tau reveals fast kiss-and-hop interaction with microtubules in living neurons.

Janning D, Igaev M, Sündermann F, Brühmann J, Beutel O, Heinisch JJ, Bakota L, Piehler J, Junge W, Brandt R - Mol. Biol. Cell (2014)

Bottom Line: Furthermore, we observed by quantitative imaging using fluorescence decay after photoactivation recordings of photoactivatable GFP-tagged tubulin that, despite this rapid dynamics, tau is capable of regulating the tubulin-microtubule balance.Our data imply a novel kiss-and-hop mechanism by which tau promotes neuronal microtubule assembly.The rapid kiss-and-hop interaction explains why tau, although binding to microtubules, does not interfere with axonal transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Osnabrück, D-49076 Osnabrück, Germany.

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Tau promotes microtubule assembly in neurites of living cells. (A) Live-cell imaging of a PC12 cell coexpressing mCherry-tagged tau (left, inset) and PAGFP-tagged α-tubulin (PAGFP-tub) before and after fluorescence photoactivation. The position of activation is indicated by a violet box. Fluorescence decay of PAGFP-tub in the activated region is indicated by the arrows. Scale bar, 10 μm. (B) Determination of the axes of symmetry (left) and the plot of normalized intensity distributions (right) as they were used to define the ROI. (C) FDAP curves of PAGFP-tub in the ROI. The presence of mCherry-tau decreases fluorescence decay of PAGFP-tub (left), whereas a control protein (3×mCherry) has no effect (right). For comparison, FDAP of a soluble protein (3×PAGFP) is shown. Values are expressed as mean ± SEM (n = 29–36). Fractions of polymerized PAGFP-tub were estimated based on fitting of the respective FDAP curves to a reaction-diffusion model (bottom).
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Figure 5: Tau promotes microtubule assembly in neurites of living cells. (A) Live-cell imaging of a PC12 cell coexpressing mCherry-tagged tau (left, inset) and PAGFP-tagged α-tubulin (PAGFP-tub) before and after fluorescence photoactivation. The position of activation is indicated by a violet box. Fluorescence decay of PAGFP-tub in the activated region is indicated by the arrows. Scale bar, 10 μm. (B) Determination of the axes of symmetry (left) and the plot of normalized intensity distributions (right) as they were used to define the ROI. (C) FDAP curves of PAGFP-tub in the ROI. The presence of mCherry-tau decreases fluorescence decay of PAGFP-tub (left), whereas a control protein (3×mCherry) has no effect (right). For comparison, FDAP of a soluble protein (3×PAGFP) is shown. Values are expressed as mean ± SEM (n = 29–36). Fractions of polymerized PAGFP-tub were estimated based on fitting of the respective FDAP curves to a reaction-diffusion model (bottom).

Mentions: The data show that tau only briefly resides on one particular MT until it hops to a neighboring one. We asked whether this short-lived interaction is compatible with an effect of tau on microtubule polymerization in living cells. To address this question, we coexpressed mCherry-tagged tau or, as a control, 3×mCherry together with PAGFP-tagged α-tubulin in neuronally differentiated PC12 cells. Tau-induced changes in MT dynamics were then monitored by FDAP measurements from cells in which the fluorescence of PAGFP had been focally activated in the middle of a process (Gauthier-Kemper et al., 2012; Figure 5, A and B). The normalized intensity of the control (3×PAGFP) showed a considerably faster decay than that of α-tubulin (Figure 5C), indicating reaction-dependent diffusion of tubulin versus free diffusion of 3×PAGFP. The presence of mCherry-tau significantly slowed the decay of α-tubulin, whereas 3×mCherry did not have any effect (Figure 5C, left vs. right). To determine the fractions of free and bound tubulin, we fitted the FDAP curves to a reaction-diffusion model (see Materials and Methods). In the absence of exogenous tau, about two-thirds of PAGFP-tubulin was polymerized in the cell processes. The exogenous mCherry-tau increased polymerization by ∼10% (Figure 5C, bottom). This was consistent with previous results from in vitro experiments, in which tau was shown to induce microtubule polymerization in a concentration-dependent manner (Brandt and Lee, 1993a). Taken together, the data indicate that tau is capable of promoting MT assembly in neurites despite its rapid kiss-and-hop dynamics. However, it should be taken into account that the experiment uses an overexpression system in which tubulin and tau are expressed on top of the respective endogenous proteins. It remains to be shown whether it is also the physiological function of tau to promote microtubule assembly in neurites.


Single-molecule tracking of tau reveals fast kiss-and-hop interaction with microtubules in living neurons.

Janning D, Igaev M, Sündermann F, Brühmann J, Beutel O, Heinisch JJ, Bakota L, Piehler J, Junge W, Brandt R - Mol. Biol. Cell (2014)

Tau promotes microtubule assembly in neurites of living cells. (A) Live-cell imaging of a PC12 cell coexpressing mCherry-tagged tau (left, inset) and PAGFP-tagged α-tubulin (PAGFP-tub) before and after fluorescence photoactivation. The position of activation is indicated by a violet box. Fluorescence decay of PAGFP-tub in the activated region is indicated by the arrows. Scale bar, 10 μm. (B) Determination of the axes of symmetry (left) and the plot of normalized intensity distributions (right) as they were used to define the ROI. (C) FDAP curves of PAGFP-tub in the ROI. The presence of mCherry-tau decreases fluorescence decay of PAGFP-tub (left), whereas a control protein (3×mCherry) has no effect (right). For comparison, FDAP of a soluble protein (3×PAGFP) is shown. Values are expressed as mean ± SEM (n = 29–36). Fractions of polymerized PAGFP-tub were estimated based on fitting of the respective FDAP curves to a reaction-diffusion model (bottom).
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Figure 5: Tau promotes microtubule assembly in neurites of living cells. (A) Live-cell imaging of a PC12 cell coexpressing mCherry-tagged tau (left, inset) and PAGFP-tagged α-tubulin (PAGFP-tub) before and after fluorescence photoactivation. The position of activation is indicated by a violet box. Fluorescence decay of PAGFP-tub in the activated region is indicated by the arrows. Scale bar, 10 μm. (B) Determination of the axes of symmetry (left) and the plot of normalized intensity distributions (right) as they were used to define the ROI. (C) FDAP curves of PAGFP-tub in the ROI. The presence of mCherry-tau decreases fluorescence decay of PAGFP-tub (left), whereas a control protein (3×mCherry) has no effect (right). For comparison, FDAP of a soluble protein (3×PAGFP) is shown. Values are expressed as mean ± SEM (n = 29–36). Fractions of polymerized PAGFP-tub were estimated based on fitting of the respective FDAP curves to a reaction-diffusion model (bottom).
Mentions: The data show that tau only briefly resides on one particular MT until it hops to a neighboring one. We asked whether this short-lived interaction is compatible with an effect of tau on microtubule polymerization in living cells. To address this question, we coexpressed mCherry-tagged tau or, as a control, 3×mCherry together with PAGFP-tagged α-tubulin in neuronally differentiated PC12 cells. Tau-induced changes in MT dynamics were then monitored by FDAP measurements from cells in which the fluorescence of PAGFP had been focally activated in the middle of a process (Gauthier-Kemper et al., 2012; Figure 5, A and B). The normalized intensity of the control (3×PAGFP) showed a considerably faster decay than that of α-tubulin (Figure 5C), indicating reaction-dependent diffusion of tubulin versus free diffusion of 3×PAGFP. The presence of mCherry-tau significantly slowed the decay of α-tubulin, whereas 3×mCherry did not have any effect (Figure 5C, left vs. right). To determine the fractions of free and bound tubulin, we fitted the FDAP curves to a reaction-diffusion model (see Materials and Methods). In the absence of exogenous tau, about two-thirds of PAGFP-tubulin was polymerized in the cell processes. The exogenous mCherry-tau increased polymerization by ∼10% (Figure 5C, bottom). This was consistent with previous results from in vitro experiments, in which tau was shown to induce microtubule polymerization in a concentration-dependent manner (Brandt and Lee, 1993a). Taken together, the data indicate that tau is capable of promoting MT assembly in neurites despite its rapid kiss-and-hop dynamics. However, it should be taken into account that the experiment uses an overexpression system in which tubulin and tau are expressed on top of the respective endogenous proteins. It remains to be shown whether it is also the physiological function of tau to promote microtubule assembly in neurites.

Bottom Line: Furthermore, we observed by quantitative imaging using fluorescence decay after photoactivation recordings of photoactivatable GFP-tagged tubulin that, despite this rapid dynamics, tau is capable of regulating the tubulin-microtubule balance.Our data imply a novel kiss-and-hop mechanism by which tau promotes neuronal microtubule assembly.The rapid kiss-and-hop interaction explains why tau, although binding to microtubules, does not interfere with axonal transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Osnabrück, D-49076 Osnabrück, Germany.

Show MeSH
Related in: MedlinePlus