Human microtubule-associated-protein tau regulates the number of protofilaments in microtubules: a synchrotron x-ray scattering study

Abstract

Microtubules (MTs), a major component of the eukaryotic cytoskeleton, are 25 nm protein nanotubes with walls comprised of assembled protofilaments built from alphabeta heterodimeric tubulin. In neural cells, different isoforms of the microtubule-associated-protein (MAP) tau regulate tubulin assembly and MT stability. Using synchrotron small angle x-ray scattering (SAXS), we have examined the effects of all six naturally occurring central nervous system tau isoforms on the assembly structure of taxol-stabilized MTs. Most notably, we found that tau regulates the distribution of protofilament numbers in MTs as reflected in the observed increase in the average radius R(MT) of MTs with increasing Phi, the tau/tubulin-dimer molar ratio. Within experimental scatter, the change in R(MT) seems to be isoform independent. Significantly, R(MT) was observed to rapidly increase for 0 < Phi < 0.2 and saturate for Phi between 0.2-0.5. Thus, a local shape distortion of the tubulin dimer on tau binding, at coverages much less than a monolayer, is spread collectively over many dimers on the scale of protofilaments. This implies that tau regulates the shape of protofilaments and thus the spontaneous curvature C(o)(MT) of MTs leading to changes in the curvature C(MT) (=1/R(MT)). An important biological implication of these findings is a possible allosteric role for tau where the tau-induced shape changes of the MT surface may effect the MT binding activity of other MAPs present in neurons. Furthermore, the results, which provide insight into the regulation of the elastic properties of MTs by tau, may also impact biomaterials applications requiring radial size-controlled nanotubes.

Figure 1

(A) Schematic of a MT decorated with adsorbed tau isoforms. (B) Six isoforms of human wild-type tau. tau isoforms possess either three or four imperfect repeats (R), which differ by exclusion or inclusion of a 31 amino acids that contain R2 and interrepeat between R1 and R2. Isoforms also differ in the N-terminal region by possessing either zero, one, or two 29 amino acids, thereby generating short (S-), medium (M-), or long (L-) isoforms. The numbers below each isoform refer to the first residue of the isoform, the beginning residues for the proline-rich region, the repeat region, and the C-terminal tail, and the last residue of the isoform.

Figure 2

Assays measuring the distinct binding of tau isoforms to the MT surface. Fraction of tau (A) and tubulin (B) in pellets as a function of the tau/tubulin-dimer molar ratio in the reaction mixture (Φ) for each of the six tau isoforms. (C) Plot of ϕ (tau/tubulin-dimer in pellets) versus Φ. ϕ measures the fraction of tau bound to the MT surface whereas Φ takes into account all tau (attached and in buffer) and all tubulin (in MTs and in buffer).

Figure 3

Light and transmission electron microscopy images of MTs with or without bound tau. Polarized light microscopy images of MTs (A) and 3RS tau-MTs at tau to tubulin-dimer molar ratio Φ = 1/10 (B) in 1.5 mm quartz capillaries show thread-like nematic liquid crystalline texture. Differential interference contrast microscopy images show oriented and uniformly distributed MTs (C) and MTs with bound 3RS tau isoforms at Φ = 1/10 (D) (scale bar, 10 μm). (E) TEM image of an MT with bound 3RS at Φ = 1/6 (scale bar = 100 nm). MT concentrations were as follows: A and C, 4.4 mg/mL; B, 3.8 mg/mL; D, 2.2 mg/mL; and E, 0.1 mg/mL.

Figure 4

(A and B) SAXS data showing that tau regulates the mean radius of MTs, or equivalently, the MT protofilament number (N_pf). SAXS results of MTs with bound 4RS tau as a function of tau to tubulin-dimer molar ratio in the reaction mixture (Φ) before (A) and after (B) background subtraction. With increasing tau, the SAXS profile shifts to lower q implying an increase in the MT radius. Colored lines are results of fits of the data to the one-box model with the MT electron density shown in B (inset) (see Fig. S1 for SAXS results of five other tau isoforms). (C) The three-box electron density model description of MAP tau bound to the surface of a MT for Φ_4RS (4RS-tau/tubulin-dimer) = 1/10. The first box corresponds to the tubulin wall of the MT with an electron density relative to water = 0.07817 e/ų (35–37) and thickness 49 Å. The second box is the region containing bound MAP tau, which consists of 144 residues comprising the repeat domains and the proline region (Fig. 1B). The thickness is taken to be the diameter of an adsorbed hydrated unstructured polypeptide ≈ 10.88 Å; diameter = 6.88 Å plus a 2 Å hydration layer. (The diameter of polypeptide tau was estimated from the volume of tau (= weight of tau/mass density of tau (=1.41 g/cc)) and using a contour length of 3.5 Å × 383 residues.) The third box contains the N-terminus projection domain (165 residues) and the thickness of this region is assumed to be 2R_g, where R_g = 1.927N^0.6 = 41.2 Å (38). The electron densities (relative to water) for the second and third boxes (with bound and projection regions of tau) were taken to be 0.005 e/ų and 0.0009 e/ų, respectively. They were calculated by estimating the fraction of tau in each region and using ρ (electron density) = tau-volume-fraction × ρ_tau + water-volume-fraction × ρ_water. The electron density of tau (ρ_tau) was taken to be 0.462 e/ų. The tau-volume-fractions were first calculated for tau/tubulin dimer = 1 (where each tubulin dimer is assumed to have one tau attached to its surface area 50 Å × 80 Å) and multiplied by ϕ (the actual tau/tubulin-dimer ratio). (D) The solid line, which does not fit the data, is the result of a calculation using the three-box model at a constant 〈R_in^MT〉 = 77.9 Å (or equivalently protofilament number 〈N_pf〉 = 13) described in C for Φ_4RS (4RS-tau/tubulin-dimer) = 1/10.

Figure 5

(A) Schematic showing cross-sections of two MTs showing that with increasing tau binding the distribution of protofilaments in MTs shifts toward MTs with larger N_pf, which leads to increases in 〈R_in^MT〉. (B–G) The inner radius 〈R_in^MT〉 for six tau isoforms plotted versus Φ, the tau/tubulin-dimer molar ratio in the reaction mixture (B–D), and ϕ, the tau/tubulin-dimer in pellets, which measures only the fraction of tau bound to the MT surface (E–G). For all six tau isoforms, the radial size of MTs increases as a function of increasing tau.

Figure 6

Salt dependence of the interactions between tau and MTs. (A) The salt dependence of 〈R_in^MT〉 for 4RS tau obtained from SAXS data. (B–D) Assays measuring the fraction of tau and tubulin in pellets (B and C) and the decrease in ϕ (tau/tubulin-dimer in pellets) with increasing KCl showing the nonspecific electrostatic nature of tau interactions with MTs (D). (E) Plot of 〈R_in^MT〉 for 4RS tau versus ϕ, where changes in ϕ (∝tau coverage) result from preparations at fixed Φ = 1/2 (open black circles) and Φ = 1/40 (open green circles) with different amounts of added salt. The blue squares are 〈R_in^MT〉 for 4RS tau, in the absence of salt, plotted versus ϕ due to changes in Φ (data from Fig. 5E). For clarity only two typical error bars are plotted for data with open black and green circles.