Live imaging of alterations in cellular morphology and organelles during cornification using an epidermal equivalent model

Abstract

the stratum corneum plays a crucial role in epidermal barrier function. Various changes occur in granular cells at the uppermost stratum granulosum during cornification. To understand the temporal details of this process, we visualized the cell shape and organelles of cornifying keratinocytes in a living human epidermal equivalent model. three-dimensional time-lapse imaging with a two-photon microscope revealed that the granular cells did not simply flatten but first temporarily expanded in thickness just before flattening during cornification. Moreover, before expansion, intracellular vesicles abruptly stopped moving, and mitochondria were depolarized. When mitochondrial morphology and quantity were assessed, granular cells with fewer, mostly punctate mitochondria tended to transition to corneocytes. Several minutes after flattening, DNA leakage from the nucleus was visualized. We also observed extension of the cell-flattening time induced by the suppression of filaggrin expression. Overall, we successfully visualized the time-course of cornification, which describes temporal relationships between alterations in the transition from granular cells to corneocytes. The human epidermis is a heterogeneous, multilayered structure constructed from keratinocytes 1. All keratinocytes are originally produced at the lowest layer of the epidermis, the stratum basale (SB), following which they move towards the skin surface while differentiating through the stratum spinosum (SS) and stratum granulosum (SG) and finally transform into corneocytes at the border between the SG and the most outer layer, the stratum corneum (SC). Due to the robust hydrophobic features of the SC and tight junctions that form at the second layer of the SG, these factors play a crucial role in protection against physical damage and harmful factors outside the body as well as the prevention of water loss from inside the body 2-4. The terminal differentiation of the uppermost granular cells to corneocytes, the cells of the SC, is called cornification, which is a kind of programmed cell death. During cornification, a variety of phenomena occur in granular cells 3. One of the most obvious changes is in their thickness. Corneocytes are very thin with a thickness of approximately 0.2-0.5 μm 5. Furthermore, corneocytes do not contain nuclei and other organelles but rather are filled with densely packed keratin filaments throughout their cytoplasm 3,6. The condensation of keratin filaments is induced by interaction with filaggrin monomers 7,8. At the cell periphery, the cornified envelope, a structure consisting of various cross-linked intracellular proteins such as involucrin and loricrin, is formed 9. Adjacent corneocytes are interconnected by corneodesmosomes, which are modified desmosomes, and their intercellular space is filled with lipids 2. Several recent reports have elucidated that autophagy is involved in the elimination of nuclei and mitochondria during terminal differentiation 10,11. It has also been reported that DNA is degraded by both DNase1L2 ...

NIX initiates mitochondrial fragmentation via DRP1 to drive epidermal differentiation

“…Mitochondria undergo cyclic fission and fusion ( Chan, 2020 ) and serve as versatile regulators of metabolism, oxidative stress, and cell death, including in keratinocytes ( Bhaduri et al, 2015 ; Hamanaka and Chandel, 2013 ). Although epidermal mitochondria have been known to fragment late during keratinocyte differentiation ( Ipponjima et al, 2020 ; Mellem et al, 2017 ), the importance of this process in epidermal morphogenesis remains untested, and the mechanisms driving mitochondrial breakdown in keratinocytes have not been elucidated.…”

Water‐in‐oil microemulsions composed of monoolein enhanced the transdermal delivery of nicotinamide

“…As can be seen in the confocal fluorescence images (Figure 6a), red signals from rhodamine B were observed at the surface of the corneocytes and around wrinkles in the skin treated with microemulsions for 1 and 2 hours. After 4 hours, red signals with a polygon shape were observed, which is typical for the stratum granulosum [39, 40]. Rhodamine B penetrated mainly along the wrinkles until 2 hours, then penetrated through the intercellular spaces in the epidermal cells during the steady‐state penetration (4 hours~).…”

Hyperspectral Three-Dimensional Fluorescence Imaging Using Snapshot Optical Tomography

“…In applications currently using fluorescent imaging, the additional spatial information may lead to more accurate classification or even the discovery of a new correlation. In order to fully investigate cellular morphology during cornification, a recent study [39] used eight fluorophores in separate trials, while only capturing 2D images. Since cellular structures naturally form in 3D, it is difficult to fully observe biological phenomena with standard 2D microscopy methods.…”