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- Hollander, Lianne den
(author)
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Molecular organization and in situ assembly of the human skin barrier
- 2016
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Doctoral thesis (other academic/artistic)abstract
- A deficient skin barrier function is a characteristic feature of skin diseases such as eczema, psoriasis and the ichtyoses. A malformation of the lipid matrix might be a major factor in barrier deficient skin disease. To better understand barrier function impairments in the future we studied the in situ assembly and molecular organization of the healthy skin barrier. Presently, two different models for the lipid secretion system, lamellar body system, have been proposed – the Landmann model and the membrane-folding model. The Landmann model proposes lipid secretion into the extracellular space between viable and cornified epidermis via a diffusion/fusion based process of multiple discrete lipid containing vesicles, while the membrane folding model proposes lipid secretion via a single and coherent continuous tuboloreticular system associating the cytoplasmic transGolgi with the extracellular space. In this thesis the continuity versus discreteness of the lamellar body system was addressed with three complementary 3D electron microscopy methodologies - tomography of vitreous sections (TOVIS), freeze-substitution serial section electron tomography (FS-SET) and focused ion beam scanning electron microscopy (FIB-SEM) tomography, using cryo-electron microscopy of vitreous sections (CEMOVIS) as a high-resolution 2D reference. We show that lamellar bodies are not discrete vesicles but part of a tubuloreticular membrane network filling out a large portion of the cytoplasm and being continuous with the plasma membrane of stratum granulosum cells. The next step in skin barrier formation is the lipid molecular assembly. We found five different characteristic high-resolution CEMOVIS patterns that correspond to five different steps in the lipid reorganization process. Using a novel approach combining very-high magnification cryo-electron microscopy of vitreous skin section (CEMOVIS) defocus-series, molecular modelling and electron microscopy simulation we determined the molecular organisation of the lipid matrix in each of the five steps. We conclude that the human skin barrier is formed by a cubic-to-lamellar lipid bilayer phase transition followed by a rearrangement of the ceramide hydrocarbon chains from a hairpin-like to a fully splayed conformation with a concomitant displacement of cholesterol from symmetric to asymmetric distribution between lipid layers. Finally we show with the same novel approach that when the skin barrier formation is complete, the lipid matrix is organized as asymmetrical stacked bilayers of fully extended ceramides with cholesterol associated to the ceramide sphingoid moiety. This rationalizes the skin’s low permeability towards both water and towards hydrophilic and lipophilic substances, as well as the skin barrier’s robustness towards hydration and dehydration, environmental temperature and pressure changes, stretching, compression, bending and shearing. The near-native high resolution molecular description of skin barrier and its formation presented in this thesis opens the door for molecular dynamics modelling as well as in-vitro modelling, of barrier function and its deficiency in normal and diseased skin.
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- Narangifard, Ali, et al.
(author)
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Molecular Reorganization during the Formation of the Human Skin Barrier Studied In Situ
- 2021
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In: Journal of Investigative Dermatology. - : Elsevier BV. - 0022-202X .- 1523-1747. ; 141:5, s. 1243-1253
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Journal article (peer-reviewed)abstract
- In vertebrates, skin upholds homeostasis by preventing body water loss. The skin's permeability barrier is located intercellularly in the stratum corneum and consists of stacked lipid lamellae composed of ceramides, cholesterol, and free fatty acids. We have combined cryo-electron microscopy with molecular dynamics modeling and electron microscopy simulation in our analysis of the lamellae's formation, a maturation process beginning in stratum granulosum and ending in stratum corneum. Previously, we have revealed the lipid lamellae's initial- and end-stage molecular organizations. In this study, we reveal two cryo-electron microscopy patterns representing intermediate stages in the lamellae's maturation process: a single-band pattern with 2.0-2.5 nm periodicity and a two-band pattern with 5.5-6.0 nm periodicity, which may be derived from lamellar lipid structures with 4.0-5.0 nm and 5.5-6.0 nm periodicity, respectively. On the basis of the analysis of the data now available on the four maturation stages identified, we can present a tentative molecular model for the complete skin barrier formation process.
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- Svensson, Lennart, et al.
(author)
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ProViz : a tool for explorative 3-D visualization and template matching in electron tomograms
- 2017
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In: Computer Methods in Biomechanics and Biomedical Engineering: Imaging & Visualization. - : Taylor & Francis. - 2168-1163 .- 2168-1171. ; 5:6, s. 446-454
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Journal article (peer-reviewed)abstract
- Visual understanding is a key aspect when studying electron tomography data-sets, aside quantitative assessments such as registration of high-resolution structures. We here present the free software tool ProViz (Protein Visualization) for visualisation and template matching in electron tomograms of biological samples. The ProViz software contains methods and tools which we have developed, adapted and computationally optimised for easy and intuitive visualisation and analysis of electron tomograms with low signal-to-noise ratio. ProViz complements existing software in the application field and serves as an easy and convenient tool for a first assessment and screening of the tomograms. It provides enhancements in three areas: (1) improved visualisation that makes connections as well as intensity differences between and within objects or structures easier to see and interpret, (2) interactive transfer function editing with direct visual result feedback using both piecewise linear functions and Gaussian function elements, (3) computationally optimised template matching and tools to visually assess and interactively explore the correlation results. The visualisation capabilities and features of ProViz are demonstrated on various biological volume data-sets: bacterial filament structures in vitro, a desmosome and the transmembrane cadherin connections therein in situ, and liposomes filled with doxorubicin in solution. The explorative template matching is demonstrated on a synthetic IgG data-set.
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