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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 exhibits kiss-and-hop behavior also in axons of primary neurons. (A) Triple fluorescence micrographs showing expression of Halo-tau (red) in lentivirally infected wild-type mouse cortical neurons. Staining against tubulin (DM1A, green) and nuclei (4′,6-diamidino-2-phenylindole [DAPI], blue) is shown. Right, Western blot indicating expression of exogenous Halo-tau (arrow) in cultures from wild-type (wt) or TAU−/− mice (KO). The presence of endogenous tau in wt mice is indicated by arrowheads. Bottom, staining for actin as a loading control. Scale bar, 20 μm. (B) Triple fluorescence micrographs after a fixation-extraction protocol, which reveals cytoskeletal association. Cortical neurons from TAU−/− mice were lentivirally infected to express Halo-tau (red). Top, overview; bottom, individual growth cone region. Note the enrichment of Halo-tau at the transition between the axon shaft and the growth cone as indicated by arrows. Staining against tubulin (DM1A, green) and nuclei (DAPI, blue) is shown. Scale bar, 20 μm (top), 10 μm (bottom). (C) Localization of a single Halo-tau molecule in an axon of a TAU−/− neuron over time. For comparison, thickness and density of axonal MTs are schematically indicated by the green bars. Top, enlargement of the indicated segment. Note that the MT–MT distance in axons of cortical neurons is much smaller than in neurites of PC12 cells. (D) Dwell times of Halo-tau in neurites of PC12 cells (39 ± 4 ms; n = 7) and axons of primary cultures (36 ± 5 ms; n = 4). Data represent mean ± SEM.
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Figure 6: Tau exhibits kiss-and-hop behavior also in axons of primary neurons. (A) Triple fluorescence micrographs showing expression of Halo-tau (red) in lentivirally infected wild-type mouse cortical neurons. Staining against tubulin (DM1A, green) and nuclei (4′,6-diamidino-2-phenylindole [DAPI], blue) is shown. Right, Western blot indicating expression of exogenous Halo-tau (arrow) in cultures from wild-type (wt) or TAU−/− mice (KO). The presence of endogenous tau in wt mice is indicated by arrowheads. Bottom, staining for actin as a loading control. Scale bar, 20 μm. (B) Triple fluorescence micrographs after a fixation-extraction protocol, which reveals cytoskeletal association. Cortical neurons from TAU−/− mice were lentivirally infected to express Halo-tau (red). Top, overview; bottom, individual growth cone region. Note the enrichment of Halo-tau at the transition between the axon shaft and the growth cone as indicated by arrows. Staining against tubulin (DM1A, green) and nuclei (DAPI, blue) is shown. Scale bar, 20 μm (top), 10 μm (bottom). (C) Localization of a single Halo-tau molecule in an axon of a TAU−/− neuron over time. For comparison, thickness and density of axonal MTs are schematically indicated by the green bars. Top, enlargement of the indicated segment. Note that the MT–MT distance in axons of cortical neurons is much smaller than in neurites of PC12 cells. (D) Dwell times of Halo-tau in neurites of PC12 cells (39 ± 4 ms; n = 7) and axons of primary cultures (36 ± 5 ms; n = 4). Data represent mean ± SEM.

Mentions: PC12 cells are a well-characterized neuronal model with a rather homogeneous cell population and well-defined microtubule distribution in processes. They do not, however, develop axonal-somatodendritic polarity. To examine the behavior of tau in axons, we prepared primary mouse cortical cultures and introduced Halo-tagged tau by lentiviral gene transfer (Bakota et al., 2012; Figure 6A, left). To avoid potential interference of endogenous mouse tau, we prepared cultures from TAU−/− animals. A Western blot confirmed 1) the absence of endogenous mouse tau in the TAU−/− cultures (arrowheads, Figure 6A, right) and 2) the expression of Halo-tau at a level comparable to that of the endogenous one (arrow, Figure 6A, right).


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 exhibits kiss-and-hop behavior also in axons of primary neurons. (A) Triple fluorescence micrographs showing expression of Halo-tau (red) in lentivirally infected wild-type mouse cortical neurons. Staining against tubulin (DM1A, green) and nuclei (4′,6-diamidino-2-phenylindole [DAPI], blue) is shown. Right, Western blot indicating expression of exogenous Halo-tau (arrow) in cultures from wild-type (wt) or TAU−/− mice (KO). The presence of endogenous tau in wt mice is indicated by arrowheads. Bottom, staining for actin as a loading control. Scale bar, 20 μm. (B) Triple fluorescence micrographs after a fixation-extraction protocol, which reveals cytoskeletal association. Cortical neurons from TAU−/− mice were lentivirally infected to express Halo-tau (red). Top, overview; bottom, individual growth cone region. Note the enrichment of Halo-tau at the transition between the axon shaft and the growth cone as indicated by arrows. Staining against tubulin (DM1A, green) and nuclei (DAPI, blue) is shown. Scale bar, 20 μm (top), 10 μm (bottom). (C) Localization of a single Halo-tau molecule in an axon of a TAU−/− neuron over time. For comparison, thickness and density of axonal MTs are schematically indicated by the green bars. Top, enlargement of the indicated segment. Note that the MT–MT distance in axons of cortical neurons is much smaller than in neurites of PC12 cells. (D) Dwell times of Halo-tau in neurites of PC12 cells (39 ± 4 ms; n = 7) and axons of primary cultures (36 ± 5 ms; n = 4). Data represent mean ± SEM.
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Figure 6: Tau exhibits kiss-and-hop behavior also in axons of primary neurons. (A) Triple fluorescence micrographs showing expression of Halo-tau (red) in lentivirally infected wild-type mouse cortical neurons. Staining against tubulin (DM1A, green) and nuclei (4′,6-diamidino-2-phenylindole [DAPI], blue) is shown. Right, Western blot indicating expression of exogenous Halo-tau (arrow) in cultures from wild-type (wt) or TAU−/− mice (KO). The presence of endogenous tau in wt mice is indicated by arrowheads. Bottom, staining for actin as a loading control. Scale bar, 20 μm. (B) Triple fluorescence micrographs after a fixation-extraction protocol, which reveals cytoskeletal association. Cortical neurons from TAU−/− mice were lentivirally infected to express Halo-tau (red). Top, overview; bottom, individual growth cone region. Note the enrichment of Halo-tau at the transition between the axon shaft and the growth cone as indicated by arrows. Staining against tubulin (DM1A, green) and nuclei (DAPI, blue) is shown. Scale bar, 20 μm (top), 10 μm (bottom). (C) Localization of a single Halo-tau molecule in an axon of a TAU−/− neuron over time. For comparison, thickness and density of axonal MTs are schematically indicated by the green bars. Top, enlargement of the indicated segment. Note that the MT–MT distance in axons of cortical neurons is much smaller than in neurites of PC12 cells. (D) Dwell times of Halo-tau in neurites of PC12 cells (39 ± 4 ms; n = 7) and axons of primary cultures (36 ± 5 ms; n = 4). Data represent mean ± SEM.
Mentions: PC12 cells are a well-characterized neuronal model with a rather homogeneous cell population and well-defined microtubule distribution in processes. They do not, however, develop axonal-somatodendritic polarity. To examine the behavior of tau in axons, we prepared primary mouse cortical cultures and introduced Halo-tagged tau by lentiviral gene transfer (Bakota et al., 2012; Figure 6A, left). To avoid potential interference of endogenous mouse tau, we prepared cultures from TAU−/− animals. A Western blot confirmed 1) the absence of endogenous mouse tau in the TAU−/− cultures (arrowheads, Figure 6A, right) and 2) the expression of Halo-tau at a level comparable to that of the endogenous one (arrow, Figure 6A, right).

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