Tau is an abundant microtubule-associated protein in neurons. Tau aggregation into insoluble fibrils is a hallmark of Alzheimer’s disease and other types of dementia1, yet the physiological state of tau molecules within cells remains unclear. Using single-molecule imaging, we directly observe that the microtubule lattice regulates reversible tau self-association, leading to localized, dynamic condensation of tau molecules on the microtubule surface. Tau condensates form selectively permissible barriers, spatially regulating the activity of microtubule-severing enzymes and the movement of molecular motors through their boundaries. We propose that reversible self-association of tau molecules, gated by the microtubule lattice, is an important mechanism of the biological functions of tau, and that oligomerization of tau is a common property shared between the physiological and disease-associated forms of the molecule.
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Source data for all statistical analyses can been found on Supplementary Table 1. All other data that support the findings of this study are available from the corresponding authors on reasonable request.
The custom analysis code used in this study is available from the corresponding authors on reasonable request.
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We thank all of the members of the McKenney and Ori-McKenney laboratories for feedback and B. Monroy for initial tau observations; J. Al-Bassam for feedback and M. Braun and S. Diez for sharing unpublished data. R.J.M. is supported by grants from NINDS (R00NS089428) and from NIGMS (R35GM124889). K.M.O.-M. is supported by grants from NICHD (R00HD080981), and from the Pew Charitable Trusts (A19-0406). S.S. is supported by grant number R01NS109176. M.V. is supported by a grant from the NSF (ENG-1563280).
The authors declare no competing interests.
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Integrated supplementary information
a, SDS-PAGE gel showing proteins used in this study. Protein constructs are listed to the right. N = 1 blot. b, Kymograph and images of the same MT as GFP-2N4R tau is added and subsequently washed out from the chamber. Scale bars: 2 μm (vertical), 2 min (horizontal). (left) and 10 min. (right). N = 2 chambers. c, Pixel intensity distribution of tau along MTs at various concentrations and MT types. WT (N) = 43, 57, 40, and 50. GMP-CPP (N) = 44, 96, and 82. Subtilisin (N) = 67, 52, 43, and 47 MTs, respectively. d, Distribution frequency of condensate lengths. N = 4, 52, 133, 233, 276, and 330 MTs for increasing tau concentrations respectively. e, Top: Intensity distribution of single TMR-SNAP-2N4R tau molecules. Fit to a Gaussian distribution is shown in red. N = 428 molecules from two independent trials. Bottom: Distribution of single- and two-step photobleaching observed for TMR-SNAP-2N4R tau molecules. Examples of single- and two-step photobleaching traces from the analysis. No molecules were observed to bleach in more than two steps. N = 49 molecules from 4 independent trials.
a, Images of mouse hippocampal neurons at different days cultured in vitro. Neurons were immunostained with two different pan-tau antibodies, GTX49353 (Genetex) and T46 (Thermo Fisher). Scale bar: 25 μm. N = 4 preparations of neurons. b, Quantification of tau puncta frequency in neuron processes. Data represented the mean ± SD. N = 18, 26, and 23 images (from ≥ 3 different neuron preps) for DIV3, DIV4, and DIV7, respectively.
Supplementary Figure 3 The C-terminal Pseudo-Repeat Region of Tau Licenses the Rest of the Molecule Into Tau Condensates.
a, Schematic of tau isoforms and constructs. Orange boxes: alternatively spliced N-term. inserts. Blue: proline-rich domain. Green: MT binding repeats. Yellow: pseudo-repeat domain. b, Top: Images of tau isoforms incorporating into full-length tau (2N4R) condensates. Middle: intensity plots of both channels. Bottom: tau isoform intensities on the lattice and within 2N4R condensates. Scale bar: 2 μm. N = 176 and 211 continuous MT segments (3 chambers each) for 2N4R. N = 127 and 143 segments (3 chambers) for 2N3R. 309 and 158 segments (3 chambers) for 0N3R. c, Top: images of constructs incorporating in 2N4R-tau condensates. Middle: intensity plot of both channels. Bottom: isoform intensities on the lattice and in condensates. Scale bar: 2 μm. N = 152 and 119 segments (3 chambers) for MTBD, 189 and 208 segments (4 chambers) for Proj., 182 and 149 segments (4 chambers) for C-Term, 337 and 296 segments (6 chambers) for bonsai. d, Top: images of Mini-Tau and pseudo-repeat truncations incorporating into 2N4R-tau condensates. Middle: intensity plot of both channels. Bottom: tau isoform intensities on the lattice and in condensates. Scale bar: 2 μm. N = 144 and 239 segments (3 chambers) for Mini-Tau, 145 and 122 segments (3 chambers) for Mini-TauΔ9, 120 and 75 segments (4 chambers) for Mini-TauΔ28. Data represented the mean ± SD. For b, c, and d Student’s T-test (two sided). e, Sequence alignments showing identity conservation (top) and hydrophobicity (bottom) of the tau pseudo-repeat region. The pseudo-repeat is highlighted by green box above.
a, Cumulative frequency graph and table of pause times for DDX molecules. N = 7, 8, 4, and 10 chambers for DDB, DDH, DDF, and DDB-Lis1, respectively. b, Summary graph of all the behaviors for DDX complexes upon encountering tau condensates. Statistical significance calculated by both Pearson’s chi-squared test and Fisher’s exact test. c, Kymograph of a DDB molecule undergoing bidirectional movement when encountering a tau patch. Scale bar: 2 μm, 15 sec. N = 7 chambers. d, Model of MT-binding footprints of dynein, kinesin and tau. a, Left: Kinesin motor domain (KIF5B, pdb: 4HNA; green) footprint at the interface of the tubulin dimer and overlapping tau MT-binding repeats (R2x4, pdb: 6CVN; orange). Arrow highlights steric clash between kinesin motor domain and tau. Right: Dynein motor domain (DYNC1H1, pdb: 3JLT; yellow). e, End-on view of 4 tubulin dimers, shown from the minus (-) end. Arrow highlights steric clash between kinesin motor domain and tau. f, MT-lattice view. Arrow highlights steric clash between kinesin and tau.
Supplementary Figs. 1–4, title and legend for Supplementary Table 1, titles and legends for Supplementary Videos 1–5.
Statistics source data.
Tau forms high density condensates on MTs.
Single-molecule behaviour of tau outside and within condensates.
Tau condensates form at specific locations on the MT lattice.
Behaviour of Dynein-Dynactin-BicD2 (DDB) complexes at tau condensates.
Tau condensates exclude spastin and protect MTs from severing.
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Tan, R., Lam, A.J., Tan, T. et al. Microtubules gate tau condensation to spatially regulate microtubule functions. Nat Cell Biol 21, 1078–1085 (2019) doi:10.1038/s41556-019-0375-5
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