Synchrotron X-ray Diffraction Study of Microtubules Buckling and Bundling under Osmotic Stress: A Probe of Interprotofilament Interactions

Self Cite

Microtubules (MTs) are hollow cytoskeletal filaments assembled from αβ-tubulin heterodimers. Tau, an unstructured protein found in neuronal axons, binds to MTs and regulates their dynamics. Aberrant Tau behavior is associated with neurodegenerative dementias, including Alzheimer's. Here, we report on a direct force measurement between paclitaxel-stabilized MTs coated with distinct Tau isoforms by synchrotron small-angle X-ray scattering (SAXS) of MT-Tau mixtures under osmotic pressure (P). In going from bare MTs to MTs with Tau coverage near the physiological submonolayer regime (Tau/tubulin-dimer molar ratio; Φ Tau = 1/ 10), isoforms with longer N-terminal tails (NTTs) sterically stabilized MTs, preventing bundling up to P B ∼ 10,000-20,000 Pa, an order of magnitude larger than bare MTs. Tau with short NTTs showed little additional effect in suppressing the bundling pressure (P B ∼ 1,000-2,000 Pa) over the same range. Remarkably, the abrupt increase in P B observed for longer isoforms suggests a mushroom to brush transition occurring at 1/13 < Φ Tau < 1/10, which corresponds to MT-bound Tau with NTTs that are considerably more extended than SAXS data for Tau in solution indicate. Modeling of Tau-mediated MT-MT interactions supports the hypothesis that longer NTTs transition to a polyelectrolyte brush at higher coverages. Higher pressures resulted in isoform-independent irreversible bundling because the polyampholytic nature of Tau leads to short-range attractions. These findings suggest an isoform-dependent biological role for regulation by Tau, with longer isoforms conferring MT steric stabilization against aggregation either with other biomacromolecules or into tight bundles, preventing loss of function in the crowded axon environment.Tau | intrinsically disordered proteins | microtubule | SAXS | force measurement

Section: Resultssupporting

confidence: 80%

Section: Resultssupporting

confidence: 77%

Direct force measurements reveal that protein Tau confers short-range attractions and isoform-dependent steric stabilization to microtubules

Chung

Choi

Miller

et al. 2015

Self Cite

“…If this were true, the mechanical properties and response that we obtain would not be those of a capsid that was indented in an equilibrium fashion. It could be argued, for example, that frequency dependence of the elastic constants accounts for the very large difference between the Young's moduli of tubelin microtubules determined from AFM measurements (7,26) and those obtained from osmotic compression measurements (27), which are four orders of magnitude smaller. On the other hand, estimates of E made from an analysis of thermal fluctuations of a microtubule, an equilibrium measurement, are in good accord with the AFM studies (28), and recent high-indentation measurements on 29 show that the capsids regain their initial heights within milliseconds.…”

Section: Discussionmentioning

confidence: 99%

Nanoindentation studies of full and empty viral capsids and the effects of capsid protein mutations on elasticity and strength

Michel

Ivanovska

Gibbons³

et al. 2006

280

305

The elastic properties of capsids of the cowpea chlorotic mottle virus have been examined at pH 4.8 by nanoindentation measurements with an atomic force microscope. Studies have been carried out on WT capsids, both empty and containing the RNA genome, and on full capsids of a salt-stable mutant and empty capsids of the subE mutant. Full capsids resisted indentation more than empty capsids, but all of the capsids were highly elastic. There was an initial reversible linear regime that persisted up to indentations varying between 20% and 30% of the diameter and applied forces of 0.6 -1.0 nN; it was followed by a steep drop in force that is associated with irreversible deformation. A single point mutation in the capsid protein increased the capsid stiffness. The experiments are compared with calculations by finite element analysis of the deformation of a homogeneous elastic thick shell. These calculations capture the features of the reversible indentation region and allow Young's moduli and relative strengths to be estimated for the empty capsids.atomic force microscopy ͉ cowpea chlorotic mottle virus ͉ finite element analysis ͉ biomechanics

“…results in k long ϭ 4 N͞m (43), which is 40 times larger than the elastic constant measured in a transverse compression by an AFM tip, k trans ϭ 0.1 N͞m (7). Thus, a large amount of experimental data, including the assembly͞disassembly dynamics of MTs, electron crystallography maps (41,42), cryoelectron microscopy (19,38,44), and small-angle x-ray diffraction (21,45), suggest a pronounced mechanical anisotropy of MTs.…”

Section: Discussionmentioning

confidence: 99%

“…where x , y , and z are the stresses along the x, y, and z axes and x , y , and z are the corresponding strains; E 1 and E 2 are the longitudinal and transverse Young moduli, respectively, and 12 , 21 , and 23 are the Poisson ratios. ‡ ‡ The shear stress-strain relationships are described by the following matrix:…”

Section: Linear Elasticity Theory For Composite Structuresmentioning

confidence: 99%

Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length

Pampaloni

Lattanzi²,

Jonáš

et al. 2006

328

376

Microtubules are hollow cylindrical structures that constitute one of the three major classes of cytoskeletal filaments. On the mesoscopic length scale of a cell, their material properties are characterized by a single stiffness parameter, the persistence length ഞp. Its value, in general, depends on the microscopic interactions between the constituent tubulin dimers and the architecture of the microtubule. Here, we use single-particle tracking methods combined with a fluctuation analysis to systematically study the dependence of ഞ p on the total filament length L. Microtubules are grafted to a substrate with one end free to fluctuate in three dimensions. A fluorescent bead is attached proximally to the free tip and is used to record the thermal fluctuations of the microtubule's end. The position distribution functions obtained with this assay allow the precise measurement of ഞ p for microtubules of different contour length L. Upon varying L between 2.6 and 47.5 m, we find a systematic increase of ഞp from 110 to 5,035 m. At the same time we verify that, for a given filament length, the persistence length is constant over the filament within the experimental accuracy. We interpret this length dependence as a consequence of a nonnegligible shear deflection determined by subnanometer relative displacement of adjacent protofilaments. Our results may shine new light on the function of microtubules as sophisticated nanometer-sized molecular machines and give a unified explanation of seemingly uncorrelated spreading of microtubules' stiffness previously reported in literature.nanomechanics ͉ protofilaments ͉ single-particle tracking ͉ thermal fluctuation analysis T he mechanics of living cells is largely determined by the cytoskeleton, a self-organizing and highly dynamic network of filamentous proteins of different lengths and stiffnesses (1). Understanding the elastic response of purified cytoskeletal filaments is fundamental for the elucidation of the rheological behavior of the cytoskeleton. Microtubules (MTs) are hollow cylindrical filaments formed by, on average, 13 tubulin protofilaments (PFs) assembled in parallel. The MT outer and inner diameters are Ϸ25 and 15 nm, respectively. In cells, MTs are generally 1-10 m long, whereas in axons their length can be 50-100 m (2).The tubular structure of MTs implies a minimal crosssectional area, hence a high strength and stiffness combined with low density. In recent years, the mechanical properties of MTs have been investigated by several experimental approaches, such as thermal fluctuations (3-6), atomic force microscopy (AFM) (7-10), and optical tweezers (11-13).The standard reference model to describe the mechanical properties of a biopolymer on length scales much larger than any microscopic scale (the tube diameter for MTs) is the worm-like chain model (14,15). It is characterized in terms of a flexural rigidity (neglecting torsional rigidity). The combined effect of flexural rigidity and thermal fluctuations on the conformation of the filament is given by the ratio ...