Cyclic stretch reveals a mechanical role for intermediate filaments in a desminopathic cell model.

Mechanics is now recognized as crucial in cell function. To date, the mechanical properties of cells have been inferred from experiments which investigate the roles of actin and microtubules ignoring the intermediate filaments (IFs) contribution. Here, we analyse myoblasts behaviour in the context of myofibrillar myopathy resulting from p.D399Y desmin mutation which disorganizes the desmin IF network in muscle cells. We compare the response of myoblasts expressing either mutated or wild-type desmin to cyclic stretch. Cells are cultivated on supports submitted to periodic uniaxial stretch of 20% elongation amplitude and 0.3 Hz frequency. We show that during stretching cycles, cells expressing mutated desmin reduce their mean amplitude both for the elongation and spreading area compared to those expressing wild-type desmin. Even more unexpected, the reorientation angles are altered in the presence of p.D399Y desmin. Yet, at rest, the whole set of those parameters are similar for the two cell populations. Thus, we demonstrate that IFs affect the mechanical properties and the dynamics of cell reorientation. Since these processes are known due to actin cytoskeleton, these results suggest the IFs implication in mechanics signal transduction. Further studies may lead to better understanding of their contribution to this process.

New aspects of the alpha-helix to beta-sheet transition in stretched hard alpha-keratin fibers.

The putative transformation of alpha-helices into beta-sheets has been studied for more than 50 years in the case of hard alpha-keratin. In a previous study of stretched keratin fibers, we specified the conditions for beta-sheet appearance within horsehair: the formation of beta-sheets requires at least 30% relative humidity. However, this phenomenon was observed in the whole tissue. Then there was no clear chemical identification of the beta-sheets (keratin or matrix proteins) and the exact location of the beta-sheets across the fiber could not be specified. In this study, using wide-angle x-ray scattering and high spatial resolution infrared microspectroscopy, we could determine and characterize the structural elements across hair sections stretched in water, which provides new information about the aforementioned transition. Our results show that the process can be split into three steps: 1), unraveling of the alpha-helical coiled-coil domains, which starts at roughly 5% macroscopic strain; 2), further transformation of the unraveled coiled-coils into beta-sheet structures, which occurs above roughly 20% macroscopic strain; and 3), spatial expanding of the beta-structured zones from the sample center to its periphery.

Evidence for various calcium sites in human hair shaft revealed by sub-micrometer X-ray fluorescence.

New information about calcium status in human scalp hair shaft, deduced from X-ray micro-fluorescence imaging, including its distribution over the hair section, the existence of one or several binding-types and its variation between people, is presented. The existence of two different calcium types is inferred. The first one corresponds to atoms (or ions) easily removable by hydrochloric acid, located in the cortex (granules), in the cuticle zone and also in the core of the medulla, which can reasonably be identified as calcium soaps. The second type consists of non-easily removable calcium atoms (or ions) that are located in the medulla wall, probably also in the cuticle, and rather uniformly in the cortex; these calcium atoms may be involved in Ca(2+)-binding proteins, and their concentration is fairly constant from one subject to another. In addition to its nonuniform distribution across the hair section, the second striking feature of the first type calcium content is its high variability from one subject to another, by up to a factor 10. We expect this information to be useful for analyzing in more detail the relationship between hair calcium and environmental and medical factors.

A new deformation model of hard alpha-keratin fibers at the nanometer scale: implications for hard alpha-keratin intermediate filament mechanical properties.

The mechanical behavior of human hair fibers is determined by the interactions between keratin proteins structured into microfibrils (hard alpha-keratin intermediate filaments), a protein sulfur-rich matrix (intermediate filaments associated proteins), and water molecules. The structure of the microfibril-matrix assembly has already been fully characterized using electron microscopy and small-angle x-ray scattering on unstressed fibers. However, these results give only a static image of this assembly. To observe and characterize the deformation of the microfibrils and of the matrix, we have carried out time-resolved small-angle x-ray microdiffraction experiments on human hair fibers stretched at 45% relative humidity and in water. Three structural parameters were monitored and quantified: the 6.7-nm meridian arc, which is related to an axial separation between groups of molecules along the microfibrils, the microfibril's radius, and the packing distance between microfibrils. Using a surface lattice model of the microfibril, we have described its deformation as a combination of a sliding process and a molecular stretching process. The radial contraction of the matrix is also emphasized, reinforcing the hydrophilic gel nature hypothesis.

Investigation of human hair cuticle structure by microdiffraction: direct observation of cell membrane complex swelling.

The cuticle of mammalian hair fibres protects the core of the fibre against physical and chemical stress. The structure and some of the properties of the cuticle have been extensively studied by electron microscopy. However, there is still a need for a less invasive structural probe. For this purpose, microdiffraction experiments have been carried out on human hair samples showing a characteristic small-angle X-ray scattering pattern for the cuticle. This pattern has been assigned to the cell membrane complex (CMC) between each cuticle scale. Using a simple model of the electron density within the CMC, values have been derived for the average thickness of the beta- and delta-layers which are close to those obtained by electron microscopy. In order to illustrate the potentialities of microdiffraction in studying the properties of the cuticle, the effect of water sorption has been monitored. Using the intensity modelling described above, a 10% swelling of the delta-layer's thickness has been observed. This study shows that structural modifications of the CMC by physical or chemical stress can be followed directly on the cuticle of human hair fibres by microdiffraction analysis.

Unraveling double stranded alpha-helical coiled coils: an x-ray diffraction study on hard alpha-keratin fibers.

Transformations of proteins secondary and tertiary structures are generally studied in globular proteins in solution. In fibrous proteins, such as hard alpha-keratin, that contain long and well-defined double stranded alpha-helical coiled coil domains, such study can be directly done on the native fibrous tissue. In order to assess the structural behavior of the coiled coil domains under an axial mechanical stress, wide angle x-ray scattering and small angle x-ray scattering experiments have been carried out on stretched horse hair fibers at relative humidity around 30%. Our observations of the three major axial spacings as a function of the applied macroscopic strain have shown two rates. Up to 4% macroscopic strain the coiled coils were slightly distorted but retained their overall conformation. Above 4% the proportion of coiled coil domains progressively decreased. The main and new result of our study is the observation of the transition from alpha-helical coiled coils to disordered chains instead of the alpha-helical coiled coil to beta-sheet transition that occurs in wet fibers.

Profiling lipids across Caucasian and Afro-American hair transverse cuts, using synchrotron infrared microspectrometry.

Synchrotron-based infrared microscopic measurements have been performed on various hair transverse sections, sampled either from the heads of Caucasian or Afro-American subjects. Lipid content of various virgin hair transverse sections was established, with an unprecedented resolution. The variations in shape and intensity of the CH(2), CH(3), amide I and amide II bands, before and after lipid removal by solvent extraction, were profiled, showing clearly that Caucasian hair often contains lipids localized inside the medulla and to a lesser extent inside the cuticle. This statement does not hold for the Afro-American hair analysed. For this, the FT-IR spectra do not change within the hair section and are insensitive to solvent extraction. The importance of the origin of hair on its physical and chemical properties has to be taken into account in future investigations.

Exploring a biological tissue from atomic to macroscopic scale using synchrotron radiation: example of hair.

A combined approach, using synchrotron radiation-based diffraction and infrared microspectrometry, has been used to study the structure and molecular composition of hair samples. These methods allowed us to get an insight at different structural scales into the composition and structure of hair. Firstly, information about the configuration of amino-acid residues was obtained at atomic scale, secondly, a model was presented for the geometry and the packing of the microfibrils at medium scale and finally different structural zones were evidenced by microdiffraction at macroscopic scale. We also showed that the two main components of hair--proteins and lipids--are not evenly distributed within the fiber. In addition, these two components exhibit different structure, depending upon their location. Moreover, diffraction and microdiffraction data indicate that the cuticle zone is mainly composed of lipid granules, whereas the cortex and the medulla zones are composed primarily of alpha-keratin. Infrared microspectroscopy, using an enhanced lateral resolution thanks to synchrotron radiation, indicates, on one hand, that the protein structure between the cuticle and cortex are different, and on the other hand, that the concentration of lipids, inside the medulla, is much higher than everywhere else. This work emphasizes the complementarity between both techniques, and highlights the potentialities they can offer in the case of various other studies in biology.

Side-chains configurations in coiled coils revealed by the 5.15-A meridional reflection on hard alpha-keratin X-ray diffraction patterns.

The origin of the 5.15-A meridional reflection on hard alpha-keratin X-ray diffraction patterns is discussed in terms of side-chains conformations. We show it to reveal specific configurations of the side chains which are common to all two-stranded alpha-helical coiled coils. Combining literature data on crystallised coiled coil pieces and molecular dynamics results with our X-ray diffraction pattern simulations, we propose rules for the attribution of chi1 torsion angles for coiled coils involved in fibres whose structure cannot be resolved at atomic resolution: in a (a b c d e f g) heptad repeat, a and d residues, respectively, adopt mean t and g+ configurations, whereas statistical rules are given for the other residues.

Organization of microfibrils in keratin fibers studied by X-ray scattering modelling using the paracrystal concept.

Low-angle X-ray scattering patterns of hard alpha-keratin fibers have been studied for more than 50 years but a completely convincing modelling has never been presented. The models which have been proposed so far are specific to the sample and cannot be adapted to others, mainly because they do not use a parametric analytical expression of the distribution function describing the relative positions of the microfibrils. Our new approach is based on a paracrystal distribution function. In addition, a huge background originating from a non-ordered matrix is taken into account. Various hard alpha-keratins from different origins have been studied using our approach. From the rather good modellings obtained, it appears that the diameter of the microfibril is not origin dependent (7.4 nm) whereas the distances between microfibrils and their electron density profiles are. Hair microfibrils can be reasonably approximated by a solid cylinder but a core and an outer ring are necessary for porcupine. Our method is of course not limited to keratin microfibrils; it can be used for modelling equatorial X-ray scattering profiles of all types of hexagonal fibrillar assemblies, which are in fact widely found in biological tissues.

Rigid-body motions of sub-units in DNA: a correlation analysis of a 200 ps molecular dynamics simulation.

A 200 ps molecular dynamics simulation of the B-form double stranded self-complementary octanucleotide d(CTGATCAG) is analyzed in terms of correlated motions using the canonical analysis approach. Each nucleotide is decomposed in three sub-units corresponding to the base, the sugar ring and the backbone respectively. The correlation between the full dynamics of two sub-units was found to decrease as their mutual distance increases. The interpretation of the full dynamics of sub-units as the superimposition of rigid-body motions (translation and orientation) and deformation shows that the main source of correlation is rigid-body motions. Correlation between sub-units deformation is weak and practically vanishes for sub-units belonging to non-adjacent nucleotides. It is also shown that the correlation is much more important for sub-units of the same strand than of opposite strands. We conclude that the internal dynamics of the octanucleotide may be well described by rigid-body motions, the sub-units deformation having only local influence whereas sub-units translation and rotation have repercussion to long distances. The results presented in this study suggest how the number of degrees of freedom may be reduced for simulating long-time dynamics of oligonucleotides.

Canonical analysis of correlated atomic motions in DNA from molecular dynamics simulation.

We report a method for analyzing atomic correlated motions in biopolymers from trajectories obtained by molecular dynamics simulation. A correlation coefficient based on the canonical analysis of data is defined which is independent on the relative orientation of atomic displacement. To illustrate the method we studied correlation between positional fluctuations of protons in the double-stranded self complementary oligonucleotide d(CTGAT-CAG), deduced from a 200 ps molecular dynamics simulation in the presence of explicit water molecules and counterions. It is found that on this time scale the motions of protons belonging to different residues are poorly coupled while the motion of a base proton is correlated to the motion of the sugar ring protons of the same nucleotide. Such a method may be generalized to study correlated motions of two distinct domains of a macro-molecule.

Molecular dynamics study of the base pair opening process in the self-complementary octanucleotide d(CTGATCAG).

We report an analysis of a 200 ps Molecular Dynamics simulation of the double stranded oligonucleotide d(CTGATCAG) in the presence of 1534 water molecules and 14 Na+ ions. We focus on the opening process of Thymine 5, by analyzing in detail the glycosidic bond rotational motion about the helix axis. The present analysis is mainly based on autocorrelation functions and on mean square displacements. We show that the opening of the base has a Brownian character and we find a rotational diffusion coefficient of 4.7 rad2s-1. Furthermore we estimate the DNA torsional constant to be about 0.5 10(-18) J.rad-2 and the RMS of the angular displacement to be 8.3 degrees. All these values are in fair agreement with those determined experimentally by fluorescence polarization of DNA-Ethidium bromide complexes. This shows that the rotational motions of the bases detected in the range 10(-9)-10(-7) s. by fluorescence techniques are the same as those analyzed in the present study (10(-12)-2 10(-10) s).