Multivalent cross-linking of actin filaments and microtubules through the microtubule-associated protein Tau

Abstract

Microtubule-associated proteins regulate microtubule dynamics, bundle actin filaments, and cross-link actin filaments with microtubules. In addition, aberrant interaction of the microtubule-associated protein Tau with filamentous actin is connected to synaptic impairment in Alzheimer's disease. Here we provide insight into the nature of interaction between Tau and actin filaments. We show that Tau uses several short helical segments to bind in a dynamic, multivalent process to the hydrophobic pocket between subdomains 1 and 3 of actin. Although a single Tau helix is sufficient to bind to filamentous actin, at least two, flexibly linked helices are required for actin bundling. In agreement with a structural model of Tau repeat sequences in complex with actin filaments, phosphorylation at serine 262 attenuates binding of Tau to filamentous actin. Taken together the data demonstrate that bundling of filamentous actin and cross-linking of the cellular cytoskeleton depend on the metamorphic and multivalent nature of microtubule-associated proteins.

Fig. 1

Tau interacts with and bundles filamentous actin. a Schematic representation of the importance of MAPs for the cellular cytoskeleton. MAPs (black) regulate microtubule dynamics (blue), bundle actin filaments (yellow), and cross-link actin filaments and microtubules. Aberrant interaction of Tau with F-actin is associated with synaptic dysfunction in Alzheimer’s disease. b Electron micrograph of actin bundles induced by Tau. c Differential centrifugation in combination with a 4–20% gradient gel shows that Tau is associated with actin bundles. Lanes correspond to supernatant (SN), single filaments (PF), and bundles (PB). Open and filled arrowheads mark Tau and actin bands, respectively. d Affinity of the Tau/F-actin interaction measured by fluorescence using NBD-labeled F-actin. Error bars represent s.d. from three experiments. e Selected region of two-dimensional ¹H-¹⁵N HSQC spectra of 441-residue Tau in the absence (gray) and presence of a two-fold excess of F-actin (blue). f Residue-specific changes in ¹H-¹⁵N HSQC signal intensities of Tau upon addition of F-actin (shown in e). I _free and I _bound are intensities in the absence and presence of a two-fold excess of F-actin

Fig. 2

Tau binds to the hydrophobic pocket of actin. a 3D structure of F-actin (pink) in complex with cofilin (light blue) (PDB id: 3J0S). b Influence of a 10-fold excess of cofilin on ¹H-¹⁵N cross-peak intensities of Tau in presence of F-actin (Tau/F-actin molar ratio of 1:1.5). Normalized signal intensities in the absence (black) and presence of cofilin (blue) are shown. c Centrifugation-based sedimentation of F-actin, F-actin + Tau (1.5:1) and F-actin + Tau+cofilin (molar ratio 1.5:1:10). Supernatants (SN) and pellets (PF) were loaded in different lanes of a 4–20% SDS gradient gel. Arrowheads from top to the bottom represent Tau, actin and cofilin bands, respectively. d Cys374 of actin is solvent exposed and can be labeled with the spin label MTSSL^–. e Sequence-specific paramagnetic broadening induced in Tau by MTSSL-tagged F-actin (gray line). I _para and I _dia are signal intensities observed for individual cross-peaks in two-dimensional ¹H-¹⁵N HSQCs of Tau in the presence of a two-fold excess of paramagnetic and diagmagnetic F-actin, respectively. For comparison, the attenuation of Tau signals by diamagnetic F-actin is shown (black bars)

Fig. 3

Tau(254–290) adopts helical structure upon binding to filamentous actin. a Electron micrograph demonstrating bundling of F-actin in presence of Tau(254–290). b Amide–amide region from 2D NOE spectra of Tau(254–290) in the presence (blue) and absence (red) of F-actin. c Ten lowest-energy conformers of F-actin bound Tau(254–290). Structures were aligned from N255 to H268. Hydrophobic residues are colored orange. In addition, K259 (blue) and S262 (light green) are highlighted. d Ten lowest-energy conformers of F-actin bound Tau(254–290) aligned from residue Q276 to V287. e Crystal structure of cofilin (blue) in complex with F-actin (pink; PDB id: 3J0S), superimposed onto the F-actin bound form of Tau(254–290) as derived from protein–protein docking (black). Side chains of selected residues are shown. f Sequence alignment of cofilin with residues 268–254 of Tau

Fig. 4

MARK2 phosphorylation decreases Tau’s affinity for F-actin. a ¹H-¹⁵N HSQC of MARK2-phosphorylated Tau. Phosphorylated serine residues are labeled. b Selected region from a ¹H-¹⁵N HSQC of MARK2-phosphorylated Tau in the absence (gray) and presence of F-actin (orange; Tau/F-actin molar ratio of 1:1.5). c Selected region from a ¹H-¹⁵N HSQC of non-phosphorylated Tau in the absence (gray) and presence of F-actin (blue; Tau/F-actin molar ratio of 1:1.5). d NMR signal broadening induced by F-actin in non-phosphorylated (blue bars) and MARK2-phosphorylated Tau (orange line). Tau residues phosphorylated by MARK2 are highlighted. e Effect of MARK2-phosphorylation on the ability of Tau to bundle actin filaments. f Phosphorylation of S262 decreases the ability of Tau(254–284) to bundle F-actin

Fig. 5

Model of Tau-driven actin bundling F-actin/microtubule network formation. a Schematic representation of cross-linking of actin filaments by helices of Tau, which are separated by flexible linkers. Each helix (shown in red) is bound to a hydrophobic actin pocket in a neighboring filament (gray and light blue). b Sequence alignment of the repeat domains of MAP2c and Tau. Tau residues, which bind to F-actin, are marked by red open bars, experimentally proven F-actin interacting regions by red filled bars, and microtubule-binding sites in orange. KXGS motifs are highlighted. c Because Tau contains multiple interaction sites for both F-actin (this work) and microtubules, Tau can cross-link the cellular cytoskeleton. While short segments in the repeat domain fold into helical structure (red) in complex with F-actin, Tau residues in complex with microtubules form a microtubule-specific hairpin structure (orange)