Molecular organization and in situ assembly of the human skin barrier

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. LIST OF SCIENTIFIC PAPERS I. The human skin barrier is organized as stacked bilayers of fully extended ceramides with cholesterol molecules associated with the ceramide sphingoid moiety. Iwai I, Han HM*, den Hollander L*, Svensson S, Öfverstedt LG, Anwar J, Brewer J, Bloksgaard M, Laloeuf A, Nosek D, Masich S, Bagatolli LA, Skoglund U, Norlén L. (2012) J Invest Dermatol 132: 2215-25. * These authors contributed equally II. Skin Lamellar Bodies are not Discrete Vesicles but Part of a Tubuloreticular Network. den Hollander L, Han HM, de Winter M, Svensson L, Masich S, Daneholt B, Norlén L. (2016) Acta Derm Venereol 96: 303-8. III. The stepwise formation of the human skin barrier examined in situ on the molecular level. den Hollander L*, Narangifard A*, Iwai I, Han HM, Wennberg C, Lundborg M, Masich S, Daneholt B, Norlén L. * These authors contributed equally