Multiphoton FLIM Analyses of Native and UVA-Modified Synthetic Melanins.

To better understand the impact of solar light exposure on human skin, the chemical characterization of native melanins and their structural photo-modifications is of central interest. As the methods used today are invasive, we investigated the possibility of using multiphoton fluorescence lifetime (FLIM) imaging, along with phasor and bi-exponential fitting analyses, as a non-invasive alternative method for the chemical analysis of native and UVA-exposed melanins. We demonstrated that multiphoton FLIM allows the discrimination between native DHI, DHICA, Dopa eumelanins, pheomelanin, and mixed eu-/pheo-melanin polymers. We exposed melanin samples to high UVA doses to maximize their structural modifications. The UVA-induced oxidative, photo-degradation, and crosslinking changes were evidenced via an increase in fluorescence lifetimes along with a decrease in their relative contributions. Moreover, we introduced a new phasor parameter of a relative fraction of a UVA-modified species and provided evidence for its sensitivity in assessing the UVA effects. Globally, the fluorescence lifetime properties were modulated in a melanin-dependent and UVA dose-dependent manner, with the strongest modifications being observed for DHICA eumelanin and the weakest for pheomelanin. Multiphoton FLIM phasor and bi-exponential analyses hold promising perspectives for in vivo human skin mixed melanins characterization under UVA or other sunlight exposure conditions.

A mechanistic view on the aging human skin through ex vivo layer-by-layer analysis of mechanics and microstructure of facial and mammary dermis.

Age-related changes in skin mechanics have a major impact on the aesthetic perception of skin. The link between skin microstructure and mechanics is crucial for therapeutic and cosmetic applications as it bridges the micro- and the macro-scale. While our perception is governed by visual and tactile changes at the macroscopic scale, it is the microscopic scale (molecular assemblies, cells) that is targeted by topical treatments including active compounds and energies. We report here a large dataset on freshly excised human skin, and in particular facial skin highly relevant for cosmetics and aesthetic procedures. Detailed layer-by-layer mechanical analysis revealed significant age-dependent decrease in stiffness and elastic recoil of full-thickness skin from two different anatomical areas. In mammary skin, we found that the onset of mechanical degradation was earlier in the superficial papillary layer than in the deeper, reticular dermis. These mechanical data are linked with microstructural alterations observed in the collagen and elastic networks using staining and advanced imaging approaches. Our data suggest that with ageing, the earliest microstructural and mechanical changes occur in the top-most layers of dermis/skin and then propagate deeper, providing an opportunity for preventive topical treatments acting at the level of papillary dermis.

Simultaneous NAD(P)H and FAD fluorescence lifetime microscopy of long UVA-induced metabolic stress in reconstructed human skin.

Solar ultraviolet longwave UVA1 exposure of human skin has short-term consequences at cellular and molecular level, leading at long-term to photoaging. Following exposure, reactive oxygen species (ROS) are generated, inducing oxidative stress that might impair cellular metabolic activity. However, the dynamic of UVA1 impact on cellular metabolism remains unknown because of lacking adequate live imaging techniques. Here we assess the UVA1-induced metabolic stress response in reconstructed human skin with multicolor two-photon fluorescence lifetime microscopy (FLIM). Simultaneous imaging of nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD) by wavelength mixing allows quantifying cellular metabolism in function of NAD(P)+/NAD(P)H and FAD/FADH2 redox ratios. After UVA1 exposure, we observe an increase of fraction of bound NAD(P)H and decrease of fraction of bound FAD indicating a metabolic switch from glycolysis to oxidative phosphorylation or oxidative stress possibly correlated to ROS generation. NAD(P)H and FAD biomarkers have unique temporal dynamic and sensitivity to skin cell types and UVA1 dose. While the FAD biomarker is UVA1 dose-dependent in keratinocytes, the NAD(P)H biomarker shows no dose dependence in keratinocytes, but is directly affected after exposure in fibroblasts, thus reflecting different skin cells sensitivities to oxidative stress. Finally, we show that a sunscreen including a UVA1 filter prevents UVA1 metabolic stress response from occurring.

Imaging and quantifying drug delivery in skin - Part 2: Fluorescence andvibrational spectroscopic imaging methods.

Understanding the delivery and diffusion of topically-applied drugs on human skin is of paramount importance in both pharmaceutical and cosmetics research. This information is critical in early stages of drug development and allows the identification of the most promising ingredients delivered at optimal concentrations to their target skin compartments. Different skin imaging methods, invasive and non-invasive, are available to characterize and quantify the spatiotemporal distribution of a drug within ex vivo and in vivo human skin. The first part of this review detailed invasive imaging methods (autoradiography, MALDI and SIMS). This second part reviews non-invasive imaging methods that can be applied in vivo: i) fluorescence (conventional, confocal, and multiphoton) and second harmonic generation microscopies and ii) vibrational spectroscopic imaging methods (infrared, confocal Raman, and coherent Raman scattering microscopies). Finally, a flow chart for the selection of imaging methods is presented to guide human skin ex vivo and in vivo drug delivery studies.

Imaging and quantifying drug delivery in skin - Part 1: Autoradiography and mass spectrometry imaging.

In this two-part review we present an up-to-date description of different imaging methods available to map the localization of drugs on skin as a complement of established ex-vivo absorption studies. This first part deals with invasive methods which are grouped in two classes according to their underlying principles: i) methods using radioactivity such as autoradiography and ii) mass spectrometry methods such as MALDI and SIMS. For each method, a description of the principle is given along with example applications of imaging and quantifying drug delivery in human skin. Thanks to these techniques a better assessment of the fate of drugs is obtained: its localization on a particular skin structure, its potential accumulation, etc. A critical comparison in terms of capabilities, sensitivity and practical applicability is included that will help the reader to select the most appropriate technique depending on the particular problem to be solved.

Human pollution exposure correlates with accelerated ultrastructural degradation of hair fibers.

Exposure to pollution is a known risk factor for human health. While correlative studies between exposure to pollutants such as polycyclic aromatic hydrocarbons (PAHs) and human health exist, and while in vitro studies help to establish a causative connection, in vivo comparisons of exposed and nonexposed human tissue are scarce. Here, we use human hair as a model matrix to study the correlation of PAH pollution with microstructural changes over time. Two hundred four hair samples from 2 Chinese cities with distinct pollution exposure were collected, and chromatographic-mass spectrometry was used to quantify the PAH-exposure profiles of each individual sample. This allowed us to define a group of less contaminated hair samples as well as a more contaminated group. Using transmission electron microscopy (TEM) together with quantitative image analysis and blind scoring of 82 structural parameters, we find that the speed of naturally occurring hair-cortex degradation and cuticle delamination is increased in fibers with increased PAH concentrations. Treating nondamaged hair fibers with ultraviolet (UV) irradiation leads to a more pronounced cortical damage especially around melanosomes of samples with higher PAH concentrations. Our study shows the detrimental effect of physiological concentrations of PAH together with UV irradiation on the hair microstructure but likely can be applied to other human tissues.

In vivo quantitative molecular absorption of glycerol in human skin using coherent anti-Stokes Raman scattering (CARS) and two-photon auto-fluorescence.

The penetration of small molecules through the human skin is a major issue for both safety and efficacy issues in cosmetics and pharmaceutic domains. To date, the quantification of active molecular compounds in human skin following a topical application uses ex vivo skin samples mounted on Franz cell diffusion set-up together with appropriate analytical methods. Coherent anti-Stokes Raman scattering (CARS) has also been used to perform active molecule quantification on ex vivo skin samples, but no quantification has been described in human skin in vivo. Here we introduce and validate a framework for imaging and quantifying the active molecule penetration into human skin in vivo. Our approach combines nonlinear imaging microscopy modalities, such as two-photon excited auto-fluorescence (TPEF) and coherent anti-Stokes Raman scattering (CARS), together with the use of deuterated active molecules. The imaging framework was exemplified on topically applied glycerol diluted in various vehicles such as water and xanthan gel. In vivo glycerol quantitative percutaneous penetration over time was demonstrated, showing that, contrary to water, the xanthan gel vehicle acts as a film reservoir that releases glycerol continuously over time. More generally, the proposed imaging framework provides an enabling platform for establishing functional activity of topically applied products in vivo.

Whole-GUV patch-clamping.

Studying how the membrane modulates ion channel and transporter activity is challenging because cells actively regulate membrane properties, whereas existing in vitro systems have limitations, such as residual solvent and unphysiologically high membrane tension. Cell-sized giant unilamellar vesicles (GUVs) would be ideal for in vitro electrophysiology, but efforts to measure the membrane current of intact GUVs have been unsuccessful. In this work, two challenges for obtaining the "whole-GUV" patch-clamp configuration were identified and resolved. First, unless the patch pipette and GUV pressures are precisely matched in the GUV-attached configuration, breaking the patch membrane also ruptures the GUV. Second, GUVs shrink irreversibly because the membrane/glass adhesion creating the high-resistance seal (>1 GΩ) continuously pulls membrane into the pipette. In contrast, for cell-derived giant plasma membrane vesicles (GPMVs), breaking the patch membrane allows the GPMV contents to passivate the pipette surface, thereby dynamically blocking membrane spreading in the whole-GMPV mode. To mimic this dynamic passivation mechanism, beta-casein was encapsulated into GUVs, yielding a stable, high-resistance, whole-GUV configuration for a range of membrane compositions. Specific membrane capacitance measurements confirmed that the membranes were truly solvent-free and that membrane tension could be controlled over a physiological range. Finally, the potential for ion transport studies was tested using the model ion channel, gramicidin, and voltage-clamp fluorometry measurements were performed with a voltage-dependent fluorophore/quencher pair. Whole-GUV patch-clamping allows ion transport and other voltage-dependent processes to be studied while controlling membrane composition, tension, and shape.

Keratin network modifications lead to the mechanical stiffening of the hair follicle fiber.

The complex mechanical properties of biomaterials such as hair, horn, skin, or bone are determined by the architecture of the underlying fibrous bionetworks. Although much is known about the influence of the cytoskeleton on the mechanics of isolated cells, this has been less studied in tridimensional tissues. We used the hair follicle as a model to link changes in the keratin network composition and architecture to the mechanical properties of the nascent hair. We show using atomic force microscopy that the soft keratinocyte matrix at the base of the follicle stiffens by a factor of ∼360, from 30 kPa to 11 MPa along the first millimeter of the follicle. The early mechanical stiffening is concomitant to an increase in diameter of the keratin macrofibrils, their continuous compaction, and increasingly parallel orientation. The related stiffening of the material follows a power law, typical of the mechanics of nonthermal bending-dominated fiber networks. In addition, we used X-ray diffraction to monitor changes in the (supra)molecular organization within the keratin fibers. At later keratinization stages, the inner mechanical properties of the macrofibrils dominate the stiffening due to the progressive setting up of the cystine network. Our findings corroborate existing models on the sequence of biological and structural events during hair keratinization.

Dynamics of membrane tethers reveal novel aspects of cytoskeleton-membrane interactions in axons.

Mechanical properties of cell membranes are known to be significantly influenced by the underlying cortical cytoskeleton. The technique of pulling membrane tethers from cells is one of the most effective ways of studying the membrane mechanics and the membrane-cortex interaction. In this article, we show that axon membranes make an interesting system to explore as they exhibit both free membrane-like behavior where the tether-membrane junction is movable on the surface of the axons (unlike many other cell membranes) as well as cell-like behavior where there are transient and spontaneous eruptions in the tether force that vanish when F-actin is depolymerized. We analyze the passive and spontaneous responses of axonal membrane tethers and propose theoretical models to explain the observed behavior.

Filopodial retraction force is generated by cortical actin dynamics and controlled by reversible tethering at the tip.

Filopodia are dynamic, finger-like plasma membrane protrusions that sense the mechanical and chemical surroundings of the cell. Here, we show in epithelial cells that the dynamics of filopodial extension and retraction are determined by the difference between the actin polymerization rate at the tip and the retrograde flow at the base of the filopodium. Adhesion of a bead to the filopodial tip locally reduces actin polymerization and leads to retraction via retrograde flow, reminiscent of a process used by pathogens to invade cells. Using optical tweezers, we show that filopodial retraction occurs at a constant speed against counteracting forces up to 50 pN. Our measurements point toward retrograde flow in the cortex together with frictional coupling between the filopodial and cortical actin networks as the main retraction-force generator for filopodia. The force exerted by filopodial retraction, however, is limited by the connection between filopodial actin filaments and the membrane at the tip. Upon mechanical rupture of the tip connection, filopodia exert a passive retraction force of 15 pN via their plasma membrane. Transient reconnection at the tip allows filopodia to continuously probe their surroundings in a load-and-fail manner within a well-defined force range.

How filopodia pull: what we know about the mechanics and dynamics of filopodia.

In recent years, the dynamic, hair-like cell protrusions called filopodia have attracted considerable attention. They have been found in a multitude of different cell types and are often called "sensory organelles," since they seem to sense the mechanical and chemical environment of a cell. Once formed, filopodia can exhibit complex behavior, they can grow and retract, push or pull, and transform into distinct structures. They are often found to make first adhesive contact with the extracellular matrix, pathogens or with adjacent cells, and to subsequently exert pulling forces. Much is known about the cytoskeletal players involved in filopodia formation, but only recently have we started to explore the mechanics of filopodia together with the related cytoskeletal dynamics. This review summarizes current advancements in our understanding of the mechanics and dynamics of filopodia, with a focus on the molecular mechanisms behind filopodial force exertion.

The sense is in the fingertips: The distal end controls filopodial mechanics and dynamics in response to external stimuli.

Small hair-like cell protrusions, called filopodia, often establish adhesive contacts with the cellular surroundings with a subsequent build up of retraction force. This process seems to be important for cell migration, embryonic development, wound healing, and pathogenic infection pathways. We have shown that filopodial tips are able to sense adhesive contact and, as a consequence, locally reduce actin polymerization speed. This induces filopodial retraction via forces generated by the cell membrane tension and by the filopodial actin shaft that is constantly pulled rearwards via the retrograde flow of actin at the base. The tip is also the weakest point of actin-based force transduction. Forces higher than 15 pN can disconnect the actin shaft from the membrane, which increases actin polymerization at the tip. Together, this points toward the tip as a mechano-chemical sensing and steering unit for filopodia, and it calls for a better understanding of the molecular mechanisms involved.

Filopodium retraction is controlled by adhesion to its tip.

Filopodia are thin cell extensions sensing the environment. They play an essential role during cell migration, cell-cell or cell-matrix adhesion, by initiating contacts and conveying signals to the cell cortex. Pathogenic microorganisms can hijack filopodia to invade cells by inducing their retraction towards the cell body. Because their dynamics depend on a discrete number of actin filaments, filopodia provide a model of choice to study elementary events linked to adhesion and downstream signalling. However, the determinants controlling filopodial sensing are not well characterized. In this study, we used beads functionalized with different ligands that triggered filopodial retraction when in contact with filopodia of epithelial cells. With optical tweezers, we were able to measure forces stalling the retraction of a single filopodium. We found that the filopodial stall force depends on the coating of the bead. Stall forces reached 8 pN for beads coated with the ß1 integrin ligand Yersinia Invasin, whereas retraction was stopped with a higher force of 15 pN when beads were functionalized with carboxyl groups. In all cases, stall forces increased in relation to the density of ligands contacting filopodial tips and were independent of the optical trap stiffness. Unexpectedly, a discrete and small number of Shigella type three secretion systems induced stall forces of 10 pN. These results suggest that the number of receptor-ligand interactions at the filopodial tip determines the maximal retraction force exerted by filopodia but a discrete number of clustered receptors is sufficient to induce high retraction stall forces.

Single-molecule protein unfolding and refolding using atomic force microscopy.

Over the past few years, atomic force microscopy (AFM) became a prominent tool to study the mechanical properties of proteins and protein interactions on a single-molecule level. AFM together with other mechanical, single-molecule manipulating techniques (Bustamante et al., Nat Rev Mol Cell Biol 1:130-136, 2000) made it possible to probe biological molecules in a way that is complementary to single-molecule methods using chemicals or temperature as a denaturant (Borgia et al., Annu Rev Biochem 77:101-125, 2008). For example, AFM offered new insights into the process of protein folding and unfolding by probing single proteins with mechanical forces. Since many proteins fulfill mechanical function or are exerted to mechanical forces in their natural environment, AFM allows to target physiologically relevant questions. Although the number of proteins unfolded with AFM continually increases (Linke and Grutzner, Pflugers Arch 456:101-115, 2008; Zhuang and Rief, Curr Opin Struct Biol 13:88-97, 2003; Clausen-Schaumann et al., Curr Opin Chem Biol 4:524-530, 2000; Rounsevell et al., Methods 34:100-111, 2004), the total number of proteins studied so far is still relatively small (Oberhauser and Carrion-Vazquez, J Biol Chem 283:6617-6621, 2008). This chapter aims at giving protocol-like instructions for people who are actually getting started using AFM to study mechanical protein unfolding or refolding. The instruction includes different approaches to produce polyproteins or modular protein chains which are commonly used to screen for true single-molecule AFM data traces. Also, the basic principles for data collection with AFM and the basic data analysis methods are explained. For people who want to study proteins that unfold at small forces or for people who want to study protein folding which also occurs typically at small forces (<30 pN), an averaging technique is explained, allowing to increase the force resolution in this regime. For topics that would go beyond the scope of this chapter - as, for example, the details about different cantilever calibration methods - references are provided.

Full distance-resolved folding energy landscape of one single protein molecule.

Kinetic bulk and single molecule folding experiments characterize barrier properties but the shape of folding landscapes between barrier top and native state is difficult to access. Here, we directly extract the full free energy landscape of a single molecule of the GCN4 leucine zipper using dual beam optical tweezers. To this end, we use deconvolution force spectroscopy to follow an individual molecule's trajectory with high temporal and spatial resolution. We find a heterogeneous energy landscape of the GCN4 leucine zipper domain. The energy profile is divided into two stable C-terminal heptad repeats and two less stable repeats at the N-terminus. Energies and transition barrier positions were confirmed by single molecule kinetic analysis. We anticipate that deconvolution sampling is a powerful tool for the model-free investigation of protein energy landscapes.

Designing the folding mechanics of coiled coils.

Naturally occurring coiled coils are often not homogeneous throughout their entire structure but rather interrupted by sequence discontinuities and non-coiled-coil-forming subsegments. We apply atomic force microscopy to locally probe the mechanical folding/unfolding process of a well-understood model coiled coil when unstructured subsegments with different sizes are added. We find that the refolding force decreases from 7.8 pN with increasing size of the added unstructured subsegment, while the unfolding properties of the model coiled coil remain unchanged. We show that this behavior results from the increased size of the nucleation seed which has to form before further coiled-coil folding can proceed. Since the nucleation seed size is linked to the width of the energetic folding barrier, we are able to directly measure the dependence of folding forces on the barrier width. Our results allow the design of coiled coils with designated refolding forces by simply adjusting the nucleation seed size.

Single molecule mechanics of the kinesin neck.

Structural integrity as well as mechanical stability of the parts of a molecular motor are crucial for its function. In this study, we used high-resolution force spectroscopy by atomic force microscopy to investigate the force-dependent opening kinetics of the neck coiled coil of Kinesin-1 from Drosophila melanogaster. We find that even though the overall thermodynamic stability of the neck is low, the average opening force of the coiled coil is >11 pN when stretched with pulling velocities >150 nm/s. These high unzipping forces ensure structural integrity during motor motion. The high mechanical stability is achieved through a very narrow N-terminal unfolding barrier if compared with a conventional leucine zipper. The experimentally mapped mechanical unzipping profile allows direct assignment of distinct mechanical stabilities to the different coiled-coil subunits. The coiled-coil sequence seems to be tuned in an optimal way to ensure both mechanical stability as well as motor regulation through charged residues.

Tightening the knot in phytochrome by single-molecule atomic force microscopy.

A growing number of proteins have been shown to adopt knotted folds. Yet the biological roles and biophysical properties of these knots remain poorly understood. We used protein engineering and atomic force microscopy to explore the single-molecule mechanics of the figure-eight knot in the chromophore-binding domain of the red/far-red photoreceptor, phytochrome. Under load, apo phytochrome unfolds at forces of approximately 47 pN, whereas phytochrome carrying its covalently bound tetrapyrrole chromophore unfolds at approximately 73 pN. These forces are not unusual in mechanical protein unfolding, and thus the presence of the knot does not automatically indicate a superstable protein. Our experiments reveal a stable intermediate along the mechanical unfolding pathway, reflecting the sequential unfolding of two distinct subdomains in phytochrome, potentially the GAF and PAS domains. For the first time (to the best of our knowledge), our experiments allow a direct determination of knot size under load. In the unfolded chain, the tightened knot is reduced to 17 amino acids, resulting in apparent shortening of the polypeptide chain by 6.2 nm. Steered molecular-dynamics simulations corroborate this number. Finally, we find that covalent phytochrome dimers created for these experiments retain characteristic photoreversibility, unexpectedly arguing against a dramatic rearrangement of the native GAF dimer interface upon photoconversion.

Single-molecule dynamics of mechanical coiled-coil unzipping.

We use atomic force microscopy (AFM) to mechanically unzip and rezip a double-stranded coiled-coil structure at varying pulling velocities. We find that force-extension traces exhibit hysteresis that grows with increasing pulling velocity. This shows that coiled-coil unzipping and rezipping do not occur in thermal equilibrium on our experimental time scale. We present a nonequilibrium simulation that fully reproduces the hysteresis effects, giving detailed insight into dynamics of coiled-coil folding. Using this model, we find that seed formation is responsible for the hysteresis. The seed consists of four alpha-helical turns on both strands of the coiled coil. To obtain equilibrium information from our nonequilibrium experiments, we used the Crooks fluctuation theorem (CFT) to calculate the equilibrium free energy of folding for all of the different pulling velocities. The paper presented here lays the groundwork for the study of self-assembly properties of many physiologically relevant coiled-coil structures at the single-molecule level.