Structure and Dynamics of Single-isoform Recombinant Neuronal Human Tubulin

Abstract

Microtubules are polymers that cycle stochastically between polymerization and depolymerization, i.e. they exhibit "dynamic instability." This behavior is crucial for cell division, motility, and differentiation. Although studies in the last decade have made fundamental breakthroughs in our understanding of how cellular effectors modulate microtubule dynamics, analysis of the relationship between tubulin sequence, structure, and dynamics has been held back by a lack of dynamics measurements with and structural characterization of homogeneous isotypically pure engineered tubulin. Here, we report for the first time the cryo-EM structure and in vitro dynamics parameters of recombinant isotypically pure human tubulin. α1A/βIII is a purely neuronal tubulin isoform. The 4.2-Å structure of post-translationally unmodified human α1A/βIII microtubules shows overall similarity to that of heterogeneous brain microtubules, but it is distinguished by subtle differences at polymerization interfaces, which are hot spots for sequence divergence between tubulin isoforms. In vitro dynamics assays show that, like mosaic brain microtubules, recombinant homogeneous microtubules undergo dynamic instability, but they polymerize slower and have fewer catastrophes. Interestingly, we find that epitaxial growth of α1A/βIII microtubules from heterogeneous brain seeds is inefficient but can be fully rescued by incorporating as little as 5% of brain tubulin into the homogeneous α1A/βIII lattice. Our study establishes a system to examine the structure and dynamics of mammalian microtubules with well defined tubulin species and is a first and necessary step toward uncovering how tubulin genetic and chemical diversity is exploited to modulate intrinsic microtubule dynamics.

Keywords: cryo-EM; cryo-electron microscopy; cytoskeleton; dynamic instability; engineered tubulin; microscopy; microtubule; microtubule dynamics; microtubule structure; post-translational modification; recombinant tubulin; tubulin; tubulin isoform.

FIGURE 1.

Structure of unmodified single-isoform human α1A/βIII microtubules. A, mass spectra and SDS-polyacrylamide gel (inset) of recombinant human α1A/βIII-tubulin purified to >99% homogeneity. The experimentally determined masses for α1A- and βIII-tubulin were 50,477.8 and 51,163.6 Da, respectively. The theoretical masses for α1A- and βIII-tubulin are 50,476.8 and 51,162.4 Da, respectively. B, cryo-EM map (4.2 Å resolution, 2.8 σ contour) and model of GMPCPP recombinant human α1A/βIII microtubules viewed from the lumen (three protofilaments shown). A central protofilament (Pf2) makes lateral contacts with adjacent protofilaments (Pf1 and Pf3); α-tubulin, orange, β-tubulin, red (Pf1, Pf3); α-tubulin, cyan; β-tubulin, purple (Pf2). C, E-site in βIII-tubulin shows clear density for GMPCPP and its three phosphate groups. D, model and map of the βIII-tubulin lateral interface (boxed and colored as in B). βIII-specific residues are in green. E, superposition of the α1A/βIII (colored as in B) and brain (PDB, 3JAT; atomistic models of brain microtubules use the βII isotype sequence because it constitutes ∼50% of these preparations (28, 44); yellow) microtubule structures; residues specific to βIII are in green. F, βIII sequence variability concentrates at the lateral interface. Green spheres denote residues that are different between the βIII and βII isotypes, the most abundant tubulin isoforms in brain tubulin preparations (10).

FIGURE 2.

Data processing, map quality, and resolution determination for cryo-EM reconstruction of recombinant human α1A/βIII microtubules. A, local resolution estimates calculated using the Bsoft program blocres (51) were used to color the unfiltered whole reconstruction density. Red density corresponds to 3.5 Å resolution, with a continuum of colors indicating the resolution gradient, ending with blue at 5.5 Å resolution. Tubulin is at a higher resolution, ranging from ∼3.5 Å in central regions to ∼4.5 Å in a more flexible peripheral surface-exposed region. Although used for the initial alignment, kinesin-3 is less ordered (resolution of ∼5.5 Å) and is excluded from display items. B, Fourier shell correlation (FSC) curves. The gold standard noise-substitution test (26) on the whole microtubule + kinesin-3 map indicates no overfitting at high resolution and an overall resolution of 4.2 Å (FSCtrue at 0.143 cutoff). C, R_measure (52) fitted curves give the same resolution estimate. Global alignment of whole movie frames improved resolution dramatically, whereas local alignment using an optical flow technique (21) yielded further improvements, especially for frames from early dosing of the data most susceptible to beam-induced motion. D, higher resolution (<4 Å) in the tubulin dimer core is supported by clear density for the backbone and most side chains (see also E). E, representative density for a β-strand in β-tubulin (top) and an α-helix in α-tubulin (bottom). F, reconstructions from the first 12 e⁻/Ų dose data (yellow) showed improved density for some side chains when compared with the 25 e⁻/Ų dose data (gray), regardless of whether they were acidic. The highly negatively charged helix H12 of α-tubulin is shown. Arrowheads indicate acidic side chains that are notable for their different appearance in 12 and 25 e⁻/Ų maps.

FIGURE 3.

Comparison between α1A/βIII and mosaic brain 14 protofilament microtubule structures. A, left panel, dimer displacement compared with the structure of mosaic brain microtubules PDB 3JAT (28) as viewed from the microtubule lumen. The boxed α1A-tubulin protomer from the α1A/βIII structure (orange Cα trace) was superimposed on the α-tubulin protomer from the brain microtubule structure (gray Cα trace). Arrows indicate the gradual increase in displacement of the α1A/βIII heterodimers as one advances toward the plus-end of the protofilament. The GTP and GMPCPP in the N-site of α-tubulin and the E-site of β-tubulin are shown as ball-and-stick. Middle panel, zoomed in view of regions highlighted by boxes in the left panel showing details of the displacement between the dimers from the recombinant α1A/βIII and brain microtubule structures; Right panel, three α1A/βIII heterodimers within one protofilament colored according to main chain displacement from the brain microtubule structure. B, left panel, percentage of seeds that nucleate microtubules at 6 μm tubulin. Brain, α1A/βIII, α1A/βIII + 5% brain tubulin elongated from brain seeds and α1A/βIII-tubulin elongated from α1A/βIII seeds. More than 100 seeds across multiple chambers were counted for these measurements. Right panel, kymograph of microtubule growth for recombinant α1A/βIII at 5.7 μm supplemented with 5% Hilyte 488 brain tubulin (0.3 μm) from brain GMPCPP seeds showing incorporation of the brain tubulin into the α1A/βIII lattice. Horizontal and vertical scale bar, 5 μm and 2 min, respectively.

FIGURE 4.

Dynamic parameters of recombinant human α1A/βIII microtubules. A, schematic of assay design (see under “Experimental Procedures”). B, micrographs of representative dynamic α1A/βIII microtubule ends. Scale bar, 20 nm. C, kymographs showing typical microtubule growth for brain and recombinant α1A/βIII-tubulin at 9 μm. Blue marks the GMPCPP seed. Horizontal and vertical scale bars, 5 μm and 5 min, respectively. D, left panel, kymographs showing a typical depolymerization event for brain and α1A/βIII microtubules. Horizontal and vertical scale bar, 5 μm and 2 s, respectively. Right panel, Tukey plot showing plus-end depolymerization rates at 9 μm tubulin; n = 55 and 58 events for brain and α1A/βIII microtubules, respectively. E, plus-end dynamics of brain and α1A/βIII-tubulin at 9 μm tubulin. Left panel, box-whisker plot (whiskers indicate minimum and maximum) showing growth rates; n = 255 and 504 events for brain and α1A/βIII-tubulin, respectively. Right panel, catastrophe frequencies; n = 48 and 167 microtubules for brain and α1A/βIII-tubulin, respectively. F, minus-end dynamics of brain and α1A/βIII-tubulin at 9 μm tubulin. Left panel, box-whisker plot (whiskers indicate minimum and maximum) showing growth rates; n = 32 and 25 events for brain and α1A/βIII-tubulin, respectively. Right panel, catastrophe frequencies; n = 7 and 16 microtubules for brain and α1A/βIII-tubulin, respectively. Error bars represent S.E. ** and ****, p values < 0.01 and < 0.0001, respectively determined by unpaired t test.