Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 85 (3), 2015-27

The Mechanical Properties of Hydrated Intermediate Filaments: Insights From Hagfish Slime Threads

Affiliations

The Mechanical Properties of Hydrated Intermediate Filaments: Insights From Hagfish Slime Threads

Douglas S Fudge et al. Biophys J.

Abstract

Intermediate filaments (IFs) impart mechanical integrity to cells, yet IF mechanics are poorly understood. It is assumed that IFs in cells are as stiff as hard alpha-keratin, F-actin, and microtubules, but the high bending flexibility of IFs and the low stiffness of soft alpha-keratins suggest that hydrated IFs may be quite soft. To test this hypothesis, we measured the tensile mechanics of the keratin-like threads from hagfish slime, which are an ideal model for exploring the mechanics of IF bundles and IFs because they consist of tightly packed and aligned IFs. Tensile tests suggest that hydrated IF bundles possess low initial stiffness (E(i) = 6.4 MPa) and remarkable elasticity (up to strains of 0.34), which we attribute to soft elastomeric IF protein terminal domains in series with stiffer coiled coils. The high tensile strength (180 MPa) and toughness (130 MJ/m(3)) of IF bundles support the notion that IFs lend mechanical integrity to cells. Their long-range elasticity suggests that IFs may also allow cells to recover from large deformations. X-ray diffraction and congo-red staining indicate that post-yield deformation leads to an irreversible alpha-->beta conformational transition in IFs, which leads to plastic deformation, and may be used by cells as a mechanosensory cue.

Figures

FIGURE 1
FIGURE 1
(A) SEM of a mature gland thread cell (without its plasma membrane) from Eptatretus stoutii. These unique cells manufacture a single, continuous bundle of keratin-like IFs that eventually occupies the vast majority of the cell volume. Scale bar = 25 μm. (B) SDS-PAGE of isolated hagfish threads demonstrates that the threads are composed of 67 kDa IF proteins, with very little contamination from other proteins (D. Fudge and J. Gosline, unpublished data).
FIGURE 2
FIGURE 2
Micromechanical testing apparatus used to measure the tensile properties of isolated lengths of hagfish threads in seawater. VDA = video dimension analyzer.
FIGURE 3
FIGURE 3
Stress-strain curves for the eight hagfish threads used to measure the initial tensile stiffness (Ei). The heavy dark line is the average stiffness calculated from these data (Ei = 6.4 ± 0.9 MPa).
FIGURE 4
FIGURE 4
Ultimate tensile behavior of hagfish threads in seawater. (A) Typical stress-strain curve for hagfish threads strained to failure. Inset at top left is detail of stress-strain curve within box at lower left. (B) Plot of the instantaneous stiffness as a function of strain. Roman numerals denote the four distinct regions of the stress-strain curve.
FIGURE 5
FIGURE 5
Recovery behavior of hagfish threads in seawater. (A) Typical load cycle in region I, showing completely reversible deformation. (B) Typical load cycle into region II, showing that deformation past the yield point is mostly plastic. (C) Results from trials in which threads were extended to a given strain, held, and allowed to recover. Note that deformation is elastic up to a strain of 0.35, and plastic thereafter.
FIGURE 6
FIGURE 6
CR staining of hagfish threads after straining in seawater. (A) Unstrained and unstained threads showed considerable birefringence, whereas CR staining of threads extended to ɛ < 0.35 caused them to swell and lose their birefringence and mechanical integrity (not shown). (BF). Threads stained with CR after extension to ɛ > 0.35 retained birefringence and mechanical integrity, and displayed increasing metachromasia as strain increased. Threads appeared orange-yellow when strained to ɛ = 0.35 (B); green when strained to ɛ = 0.50; blue at ɛ = 0.75 (D); blue-violet at ɛ = 1.0 (E); and pale magenta to colorless at ɛ = 1.50. Scale bar = 10 μm.
FIGURE 7
FIGURE 7
X-ray diffraction patterns for hagfish thread bundles strained in seawater. (A) Unstrained threads exhibited a typical “α-pattern,” whereas threads extended to a strain of 1.0 exhibited a typical “β-pattern” (C). Thread extended to a strain of 0.60 exhibited a mixed pattern, suggesting the presence of both α-helix and β-sheet structure (B). Diffraction maxima (dark spots) are labeled according to the molecular spacings (in Angstroms, Å) to which they correspond.
FIGURE 8
FIGURE 8
The effect of subfilament sliding on the persistence length of IFs assuming a Young's modulus of 7.0 MPa (A) and 2 GPa (B). Persistence length was calculated assuming an IF diameter of 10 nm for the case of one subfilament, and holding the total cross-sectional area constant for all other cases. Note that under the assumption that IFs are as stiff as keratins, even the maximum amount of subfilament sliding predicts a persistence length an order of magnitude higher than measured values.
FIGURE 9
FIGURE 9
Proposed mechanical behavior of IF dimers in mechanical region I. Assumptions of the model and flow of logic for the analysis are described in the text. The model suggests that in region I, the vast majority of the deformation occurs in highly extensible and low-stiffness terminal domains in series with stiff-coiled coils.
FIGURE 10
FIGURE 10
Proposed mechanical behavior of IF dimers in regions I–III. In region I, deformation occurs almost exclusively in the terminal domains. In region II, α-helices within the coiled coil motif begin to extend into β-sheets (denoted by the crimped lines). By the end of region III, all of the coiled coil α-helices have been extended to β-sheets. Region IV corresponds to the straining, slippage, and ultimate rupture of β-sheets and β-sheet crystals.

Similar articles

See all similar articles

Cited by 45 PubMed Central articles

See all "Cited by" articles

Publication types

Feedback