Molecular Organization of the Skin Barrier

Abstract

Cryo-electron microscopy of vitreous sections allows for investigation directly in situ of the molecular architecture of skin. Recently, this technique has contributed to the elucidation of the molecular organization of the skin's permeability barrier and its stepwise formation process. The aim of this review is to provide an overview of the procedure for cryo-electron microscopy of vitreous sections, its analysis using atomic detail molecular dynamics modelling and electron microscopy simulation, and its application in the investigation of the barrier structure and formation process of the skin.

Fig. 1

Specimen collection, vitrification, sectioning, and mounting for cryo-electron microscopy of vitreous sections (CEMOVIS). (a) A biological specimen is collected in vivo by surgical excision. (b) The specimen is vitrified by fast immersion in liquid nitrogen under pressure (2 kbar). (c) The vitrified specimen is cut with a diamond knife at cryo-conditions (–140°C) into ultrathin (30-nm) sections. (d) The cryo-sections are transferred to a cryo-EM grid, which is mounted in (e) a cryo-EM sample holder that in turn is inserted into (f) the cryo-electron microscope.

Fig. 2

Collection of a cryo-electron microscopy of vitreous sections (CEMOVIS) defocus image series. (a) Initially, a low-resolution overview image is collected at approximately 1000–5000 times magnification, followed by (b) a medium-resolution orientation image collected at approximately 5000–30,000 times magnification, to select the specimen location (for example red square in (b)) where (c) a high-resolution defocus image series can be collected at approximately 80,000–115,000 times magnification. Electron-dense (black) spots in (a) and (b) correspond to surface ice contamination. Wave-like diagonal patterns in (a) and (b) are due to section compression during cutting. Scale bars in (a) 1.0 μm and in (b) 500 nm. Pixel-size in (c) is 0.188 nm. SG and SC1-3 in (b): stratum granulosum and stratum corneum layer 1–3, respectively. Adapted from Norlén and Al-Amoudi 2004 (25) (a, b) and Iwai et al., 2012 (27) (c), with permission.

Fig. 3

Molecular model building subject to molecular dynamics (MD) simulation. (a) Building an atomic model based on a known molecular composition. (b) The atomic model is equilibrated by MD simulation, and then (c) multiplied in 3 dimensions, before processing for (d) electron microscopy (EM) simulation.

Fig. 4

Electron microscopy (EM) simulation. Simulated cryo-electron microscopy of vitreous sections (CEMOVIS) defocus series images for candidate atomic models are created with a computer program simulating (a) the interaction between the candidate atomic model’s electron scattering potential map and the microscope’s electron beam, (b) the optical transformation effect of the microscope’s lens system, and (c) the image formation on the microscope’s detector.

Fig. 5

Comparison between simulated and original electron micrographs. The simulated electron micrographs are compared with the original cryo-electron micrographs at different microscope defocus levels. If differences are identified, the atomic model is updated, and the simulation renewed. This process is repeated until optimal correspondence is attained between simulated and original electron micrographs.

Fig. 6

Cryo-electron micrograph overview of human stratum corneum. Note the approximately homogeneous corneocyte density, size, and form throughout the stratum corneum, except for the lowermost corneocytes that are generally thicker (lower right corner). Electron-dense spot (black) corresponds to surface ice contamination. Wave-like diagonal pattern in the upper right corner is due to section compression during cutting. SC1-12: stratum corneum cell 1–12. Scale bar: 1.0 μm. Adapted from Norlén and Al-Amoudi 2004 (25), with permission.

Fig. 7

Cryo-electron micrographs of lowermost stratum corneum. SG: stratum granulosum cell. SC1-3: stratum corneum cell 1–3. Scale bar 500 nm. Adapted from Norlén and Al-Amoudi 2004 (25), with permission.

Fig. 8

Cryo-electron micrograph overview of the interface between viable and cornified cell layers. SG: stratum granulosum cell. SC1: first stratum corneum cell. Scale bar: 500 nm. Adapted from Al-Amoudi et al., 2005 (35), with permission.

Fig. 9

Cryo-electron micrograph of the tubuloreticular (lamellar body) system at the interface between viable and cornified epidermis. Multiple apparent active sites of skin barrier formation (black boxes) corresponding to the tubuloreticular system (lamellar bodies) (white asterisk), can be seen. White dotted line marks the interface between viable and cornified epidermis. SC: stratum corneum; SG: stratum granulosum. Scale bar: 500 nm. Adapted from den Hollander et al., 2016 (28), with permission.

Fig. 10

Cryo-electron micrographs of lamellar bodies located at the interface between stratum granulosum and stratum corneum. Note that the locally ordered granular regions (green stars) are closely associated with the broad-lamellar regions (blue stars), seemingly integrating without a clear interface. A–B represents lamellar bodies located within the cytoplasm of the topmost stratum granulosum cell and C–D represent lamellar bodies located intercellularly between the topmost stratum granulosum cell and the lowermost stratum corneum cell. Green stars: locally ordered granular pattern. Blue stars: symmetrical broad-lamellar pattern with 50–55 Å periodicity. Scale bars: 100 nm. Adapted from Narangifard et al., 2018 (30), with permission.

Fig. 11

Cryo-electron micrographs of the intercellular space of the lowermost (1^st–4^th cell layers) stratum corneum. Note that the single-band pattern with 2.0–2.5 nm periodicity (dark-brown stars) occupies the intercellular space completely, seemingly embedding the 2-band pattern with 5.5–6.0 nm periodicity (red stars) that starts to appear pairwise peripherally and centrally (a–h). Occasionally, stacks of the 2-band pattern with 11.0–12.0 nm periodicity (yellow stars in (j)) could also be detected in association with the single-band pattern with 2.0–2.5 nm periodicity (dark-brown stars in (j)) and the 2-band pattern with 5.5–6.0 nm periodicity (red stars in (j)). Right inset in (a): Enlargement of the single-band pattern with 2.0–2.5 nm periodicity; Left inset in (a): Enlargement of the 2-band pattern with 5.5–6.0 nm periodicity. Scale bars (a–d, h, j): 100 nm; Scale bars (left and right insets in (a, e–g, i): 10 nm. Adapted from Narangifard et al., 2021 (32), with permission.

Fig. 12

Cryo-electron micrographs of the 2-band pattern with 11.0–12.0 nm periodicity representing the mature skin barrier lipid structure. (a–f) Cryo-electron micrographs of the intercellular space of the midpart (above the 5^th cell layer) stratum corneum. Yellow stars: 2-band pattern with 11.0–12.0 nm periodicity. Section thicknesses (a–f): 30–50 nm; Scale bars (a–b) 50 nm, (c–f) 25 nm. Adapted from Narangifard et al., 2021 (32), with permission.

Fig. 13

Schematic view of skin. Left column (I): full-thickness skin; Mid column (II): epidermis; Right column (III): interface between viable and cornified epidermis. Green and blue areas: tubolureticular (lamellar body) network of a secretory (stratum granulosum) cell; Dark-brown area: intercellular space between first and second corneal (stratum corneum) cells; Pink area: intercellular space between second and third corneal cells; Yellow area: intercellular spaces above the third corneal cell. Desmosomes connecting the secretory cell to the first corneal cell are indicated by transverse striations (section III, black). Tight junctions in the stratum granulosum cell periphery are indicated by stars (section II, red). Adapted from Norlén et al., 2022 (42), with permission.

Fig. 14

Cryo-electron microscopy (cryo-EM) patterns of the barrier formation process. (a) Granular cryo-EM pattern of the tubolureticular (lamellar body) network of secretory (stratum granulosum) cell (green); (b) lamellar cryo-EM pattern with 50–55 Å periodicity of the tubolureticular network of secretory cell (blue); (c) lamellar cryo-EM pattern with 20–25 Å periodicity of the intercellular space between first and second corneal (stratum corneum) cells (dark-brown); (d) lamellar cryo-EM pattern with 55–60 Å periodicity of the intercellular space between second and third corneal cells (pink); (e) lamellar cryo-EM pattern with 110–120 Å periodicity of the intercellular spaces above the third corneal cell (yellow). Adapted from Norlén et al., 2022 (42), with permission.

Fig. 15

The 5 stages identified in the barrier formation process. (a) highly folded and highly hydrated glucosyl-ceramide based conventional lipid bilayer (green rectangle); (b) stacked monolayer with mixed hairpin- and splayed ceramides (blue rectangle); (c) stacked monolayer with splayed ceramides and chain interdigitation (dark-brown rectangle); (d) stacked bilayer with splayed ceramides and chain interdigitation (pink rectangle); (e) stacked bilayer with splayed ceramides without chain interdigitation (yellow rectangle). Molecular colour codes: glucosyl-ceramide and ceramide molecules (green carbon atoms), cholesterol molecules (yellow carbon atoms), free fatty acid molecules (orange carbon atoms). Oxygen (red atoms), hydrogen (white atoms), and nitrogen (dark-blue atoms) are coloured the same in all lipid molecules and water. Adapted from Norlén et al., 2022 (42), with permission.

Fig. 16

Reorganization steps of the barrier formation process. Lower row: cleavage of the sugar groups of glucosyl-ceramides (a–b), followed by dehydration (b, c), resulting in a collapse into stacks of tightly packed flat lipid bilayers. (c, d) Ceramide chain flipping resulting in a reorganization of the ceramides from a hairpin (folded) into a splayed (extended) conformation. Sliding along the molecular length axes resulting in (i) interdigitation of the lipid chains (d–e) followed by (ii) separation of the ceramides’ fatty acid- and sphingoid chains, as well as of the cholesterols and the free fatty acids, into different bands of the lamellar structure (e, f), and finally (iii) un-interdigitation of the lipid chains (f, g), yielding the mature skin barrier lipid structure (g). (a) Highly folded and highly hydrated glucosyl-ceramide based conventional lipid bilayer (left green rectangle); (b) flattened and highly hydrated stacked ceramide-based conventional lipid bilayer (right green rectangle); (c) stacked lowly hydrated bilayer with hairpin ceramides (left blue rectangle); (d) stacked monolayer with mixed hairpin- and splayed ceramides (right blue rectangle); e) stacked monolayer with splayed ceramides and chain interdigitation (dark-brown rectangle); (f) stacked bilayer with splayed ceramides and chain interdigitation (pink rectangle); (g) stacked bilayer with splayed ceramides without chain interdigitation (yellow rectangle). Molecular colour codes: glucosyl-ceramide and ceramide molecules (green carbon atoms), cholesterol molecules (yellow carbon atoms), free fatty acid molecules (orange carbon atoms). Oxygen (red atoms), hydrogen (white atoms), and nitrogen (dark-blue atoms) are coloured the same in all lipid molecules and water. Adapted from Norlén et al., 2022 (42), with permission.

Fig. 17

Architecture of the mature barrier structure. (a) Drawing of epidermis. (b) Atomistic molecular dynamics (MD) model of the mature skin barrier structure. (c) MD simulation box of the model in (b). (d) Schematic drawing of the basic molecular arrangement in (c): a lipid bilayer of fully stretched (splayed) ceramides with cholesterol (yellow molecules) largely associated with the ceramides’ shorter sphingoid side and with free fatty acids (orange molecules) associated with the ceramides’ longer fatty acid side. Acyl-ceramides (ceramide EOS) (light-blue molecules) protrude their ester-bound lignoceric acid ends into the interface between opposing ceramide fatty acid tail ends. (e–g) Cryo-EM defocus series of the skin’s barrier structure. (h–j) Simulated EM defocus series of the model system in B) ((e, h) defocus –1.3 mm; (f, i) –2.7 mm; (g, j) –4.0 mm). White boxes in (h–j) represent the position of the simulation box visualized in (c). (k) Electron density pattern of the stratum corneum intercellular space in plastic embedded skin stained with ruthenium tetroxide (RuO₄), graciously obtained from Dr Philip Wertz and adapted from Madison et al. 1987 (43). (l) Enlarged view of the area marked by a white box in (k). (m) Van der Waals representation of oxygens and nitrogens (the most electronegative atoms in the system) of the model system in (b). (m) Rough estimate of what the electron density pattern of the model system in (b) would look like after RuO₄ staining. (e–m) Adapted from Lundborg et al., 2018a (29). Molecular colour codes: ceramide molecules (green (ceramide), and light-blue (acyl-ceramide), carbon atoms), cholesterol molecules (yellow carbon atoms), free fatty acid molecules (orange carbon atoms). Oxygen (red atoms), hydrogen (white atoms), and nitrogen (dark-blue atoms) are coloured the same in all lipid molecules and water. Adapted from Norlén et al., 2022 (42), with permission.