The Skin’s Barrier: A Cryo-EM Based Overview of its Architecture and Stepwise Formation

A major role of the skin is to serve as a barrier toward the environment. The skin’s permeability barrier consists of a lipid structure positioned in the stratum corneum. Recent progress in high-resolution cryo-electron microscopy (cryo-EM) has allowed for eluci-dation of the architecture of the skin’s barrier and its stepwise formation process representing the ﬁnal stage of epidermal differentiation. In this review, we present an overview of the skin’s barrier structure and its formation process, as evidenced by cryo-EM.

Human skin serves to uphold homeostasis by preventing water loss from the body as well as by preventing the permeation of exogenous substances into the body. The major physical barrier property is located in the skin's topmost epidermal layer-the stratum corneum. It consists of a lipid structure positioned intercellularly between cornified dead cells.
The formation of the skin's barrier represents the final stage of epidermal differentiation. The epidermal cells move from the basal layer, through several layers of cells evolving into secretory cells, to the stratum corneum with cornified cells. Barrier formation is initiated in the topmost secretory cell layers-the stratum granulosum (SG) (Figure 1, right column). The barrier lipids are synthesized in the endoplasmic reticulum and Golgi apparatus. They subsequently appear as a partly granular (green pattern) and partly lamellar (blue pattern) material inside an extensive tubuloreticular (lamellar body) membrane system (green pattern) (den Hollander et al., 2016;Elias et al., 1998;Yamanishi et al., 2019). The lipids are finally discharged into the intercellular space at the interface between SG and stratum corneum. Once in the stratum corneum intercellular space, the secreted lipids transform into laterally extended, stacked sheets (brown, pink, and yellow patterns), a process completed at the third to fifth stratum corneum layer. The stacked sheets are then observed in the intercellular space throughout the stratum corneum.
Discharge of lamellar lipid precursors from the apical side of SG2 cells has so far not been observed in human skin with cryo-electron microscopy (cryo-EM).
Electron microscopy (EM) visualizations of the stacked sheets came with Breathnach et al. (1973) and Elias and Friend (1975). Madison et al. (1987) further pioneered EM visualization by the introduction of ruthenium tetroxide (RuO 4 ) staining. In this way, the stacked sheets' characteristic broad-narrow-broad electron-lucent band staining pattern was discovered (Hou et al., 1991;Madison et al., 1987).
In our laboratory, we have applied cryo-EM of vitreous sections (CEMOVIS) to elucidate in situ the molecular organization and the formation process of the skin's barrier structure (den Hollander et al., 2016;Iwai et al., 2012;Lundborg et al., 2018a;Narangifard et al., 2021Narangifard et al., , 2018Wennberg et al., 2018). In cryo-EM, the tissue is preserved down to the molecular level at near-native conditions (Al-Amoudi et al., 2004;Dubochet et al., 1988). In the analysis of the cryo-EM images, atomic-detail molecular models subject to molecular dynamics (MD) simulation followed by EM simulation is employed. The image analysis procedure comprises the following three steps: (i) construction of candidate molecular models, (ii) generation of simulated electron micrographs on the basis of these models, and (iii) finally a comparison of the observed cryo-electron micrographs with the simulated ones.

Barrier formation
Cryo-EM patterns of the barrier formation process ( Figure 2).
During epidermal differentiation, the skin's barrier structure undergoes five apparent maturation stages Analysis of the five cryo-EM patterns using MD model building combined with EM simulation suggests that the granular pattern represents a highly folded and highly hydrated conventional skin lipid bilayer ( Figure 3a) , that the lamellar pattern with 50e55 Å periodicity represents a stacked skin lipid monolayer with mixed hairpin and splayed ceramides ( Figure 3b) , that the lamellar pattern with 20e25 Å periodicity represents a stacked skin lipid monolayer with splayed ceramides and chain interdigitation ( Figure 3c) (Narangifard et al., 2021), that the lamellar pattern with 55e60 Å periodicity represents a stacked skin lipid bilayer with splayed ceramides and chain interdigitation ( Figure 3d) (Narangifard et al., 2021), and that the lamellar pattern with 110e120 Å periodicity represents a stacked skin lipid bilayer with splayed ceramides without chain interdigitation (Figure 3e) (Iwai et al., 2012;Lundborg et al., 2018a).
Reorganization steps of the barrier formation process (Figure 4).
Cryo-EM analysis of the skin's barrier formation process suggests that the formation of the skin's barrier structure starts with the secretion of a highly folded and highly hydrated lipid bilayer into the stratum corneum Once shielded from water, the lipid bilayers' ceramide molecules can now start to stretch out into their preferred splayed conformation with their two hydrocarbon tails pointing in opposite directions (Figure 4d). When all ceramides have become stretched out (Figure 4e), the system can further relax itself and become more tightly packed by letting the ceramide molecules' short and long hydrocarbon tails as well as the cholesterol and free fatty acid molecules separate into different bands within the layered structure. This internal molecular rearrangement is Cryo-EM analysis of the skin's mature barrier structure suggests that it is organized as stacked lipid bilayers of fully stretched (splayed) ceramides with cholesterol largely associated with the ceramides' shorter sphingoid tail and with free fatty acids associated with the ceramides' longer fatty acid tail (Figure 5c and d). Acyl-ceramides (ceramide EOS) (light blue) protrude their ester-bound lignoceric acid ends into the interface between opposing free fatty acid and ceramide fatty acid tail ends. The lignoceric acid ends are mobile and can extend into the opposing layer or fold back into the interface Figure 4. Reorganization steps of the barrier formation process. The lower row indicates (a⇢ ⇢ b) the cleavage of the sugar groups of glucosyl-ceramides followed by (b⇢ ⇢ c) dehydration, 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 (d⇢ ⇢ e) interdigitation of the lipid chains followed by (e⇢ ⇢ f) 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, and finally, (f⇢ ⇢ g) uninterdigitation of the lipid chains, yielding (g) the mature skin barrier lipid structure. (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); and (g) stacked bilayer with splayed ceramides without chain interdigitation (yellow rectangle). Molecular color codes: green carbon atoms for glucosyl-ceramide and ceramide molecules, yellow carbon atoms for cholesterol molecules, and orange carbon atoms for free fatty acid molecules. Oxygen (red atoms), hydrogen (white atoms), and nitrogen (dark blue atoms) are colored the same in all lipid molecules and water.
L Norlén et al.
The Skin's Barrier: Architecture and Formation  Lundborg et al. (2018a). Molecular color codes: green carbon atoms for ceramide molecules, light-blue carbon atoms for acyl-ceramide molecules, yellow carbon atoms for cholesterol molecules, and orange carbon atoms for free fatty acid molecules. Oxygen (red atoms), hydrogen (white atoms), and nitrogen (dark blue atoms) are colored the same in all lipid molecules and water. cryo-EM, cryo-electron microscopy; MD, molecular dynamics; RuO 4 , ruthenium tetroxide.
L Norlén et al.
The Skin's Barrier: Architecture and Formation between the layers. Another feature is that the acyl-ceramides' ester groups make the interface between the ceramides' fatty acid tail ends more polar than the interface between the ceramides' sphingoid tail ends (Figure 5c and d). This arrangement offers a tight and robust barrier structure and is compatible both with cryo-EM ( Figure 5eej) and with the broad-narrow-broad electron-lucent RuO 4 staining band pattern observed using conventional EM (Hou et al., 1991;Madison et al., 1987) (Figure 5kem).

Future perspective
Having access to a near-native reference from healthy skin, it may now be possible to investigate the deviations in lipid structure that could underlie barrier dysfunction in skin diseases such as atopic dermatitis, psoriasis, and the ichthyoses.
The knowledge on the structure of the skin's permeability barrier may facilitate physics-based skin permeability calculations using MD simulation (Lundborg et al., 2018b). This may aid in predicting the properties of drugs interacting with the skin and optimizing them for topical and percutaneous drug delivery. In addition, it may be used for skin toxicity assessment.
The experimental approach exemplified in this paper, consisting in obtaining in situ a near-native structural reference of a biomolecular complex using cryo-EM (CEMOVIS) combined with MD modeling and EM simulation, may be followed by more detailed analysis ex situ and in silico using, for example, X-ray crystallography, single-particle cryo-EM, nuclear magnetic resonance spectroscopy, or MD simulation. It may thus be useful for examining the molecular-level structure/function relationships of biostructures inside any cell or tissue within the constraints of a near-native reference.