Scanning-fluctuation correlation spectroscopy was used to detect subresolution organizational fluctuations in the lipid liquid-crystalline phase for single lipid model systems. We used the fluorescent probe Laurdan which is sensitive to the amount of water in the membrane to show that there is a spatial heterogeneity on the scale of few pixels (the size of the pixel is 50 nm). We calculated the pixel variance of the GP function and we found that the variance has a peak at the phase transition for 3 different samples made of pure lipids. The pixel variance has an abrupt change at the phase transition of the membrane and then it slowly decreases at higher temperature. The relatively large variance of the GP indicates that the liquid phase of the membrane is quite heterogeneous even several degrees higher than the phase transition temperature. We interpreted this result as evidence of an underlying microscale structure of the membrane in which water is not uniformly distributed at the micron scale. Imaging of these microstructures shows that the pixels with different GP tend to concentrate in specific domains in the membrane. In the case of single lipid membrane, the statistical and fluctuation analysis of the GP data shows that even such simple lipid systems are capable of generating and maintaining stable structural and organizational heterogeneities.

We used a combination of imaging and fluctuation techniques to investigate the temporal evolution of gel phase domains at the onset of phase separation, as well as the correlation between domain topology and local lipid ordering in GUVs composed of a binary mixture of DPPC/DLPC 1:1. The data acquired at temperatures immediately above the transition temperature of the two lipids suggest fluctuations in the lipid organization with a lifetime <0.1 s and a characteristic length of 1.2 microm. As the temperature is decreased below the transition temperature of one of the lipids, coupling between the two leaflets of the bilayer is observed to begin within the first five minutes after the onset of phase separation. However, domains confined to only one leaflet can be found during the first 45-50 min after the onset of phase separation. Our analysis using a two-state model (liquid and gel) indicates that for the first 45-50 min from the onset of phase separation the two lipid phases do not strongly influence the phase behavior of each other on the micron-length scale. At longer times, behavior that deviates from the two-state model is observed and appears to be correlated to domain morphology.

Ca(2+) fluxes direct keratinocyte differentiation, cell-to-cell adhesion, migration, and epidermal barrier homeostasis. We previously showed that intracellular Ca(2+) stores constitute a major portion of the calcium gradient especially in the stratum granulosum. Loss of the calcium gradient triggers epidermal barrier homeostatic responses. In this report, using unfixed ex vivo epidermis and human epidermal equivalents we show that endoplasmic reticulum (ER) Ca(2+) is released in response to barrier perturbation, and that this release constitutes the major shift in epidermal Ca(2+) seen after barrier perturbation. We find that ER Ca(2+) release correlates with a transient increase in extracellular Ca(2+). Lastly, we show that ER calcium release resulting from barrier perturbation triggers transient desmosomal remodeling, seen as an increase in extracellular space and a loss of the desmosomal intercellular midline. Topical application of thapsigargin, which inhibits the ER Ca(2+) ATPase activity without compromising barrier integrity, also leads to desmosomal remodeling and loss of the midline structure. These experiments establish the ER Ca(2+) store as a master regulator of the Ca(2+) gradient response to epidermal barrier perturbation, and suggest that ER Ca(2+) homeostasis also modulates normal desmosomal reorganization, both at rest and after acute barrier perturbation.

Multiphoton fluorescence microscopy has now become a relatively common tool among biophysicists and biologists. The intrinsic sectioning achievable by multiphoton excitation provides a simple means to excite a small volume inside cells and tissues. Multiphoton microscopes have a simplified optical path in the emission side due to the lack of an emission pinhole, which is necessary with normal confocal microscopes. This article illustrates examples in which this advantage in the simplified optics is exploited to achieve a new type of measurements. First, dual-emission wavelength measurements are used to identify regions of different phase domains in giant vesicles and to perform fluctuation experiments at specific locations in the membrane. Second, we show how dual-wavelength measurements are used in conjunction with scanning fluctuation analysis to measure the changes in the geometry of the domains and the incipient formation of gel domains when the temperature of the giant vesicles is gradually lowered.

We used a technique that allows us to visualize local and morphological changes of the membrane of more component giant unilamellar vesicles due to high pressure perturbation. Under these conditions, thermally induced processes are largely suppressed, and the bending rigidity and line tension are influenced by pressure-induced changes in lipid molecular packing and shape only. We studied the effect of pressure on the lateral organization and morphology of the model raft system DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)/sphingomyelin/cholesterol as well as of the fluid mixture POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)/DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine) by two-photon excitation fluorescence microscopy. The pressure-dependent experiments were carried out using a sample cell made from a thin fused silica capillary. The use of Laurdan as fluorescence label allowed us to also follow the lipid phase state by calculating the generalized polarization (GP) values of the vesicles and extracting their average value. During the compression cycle, a reduction in the volume of the vesicles is observed, accompanied by an increase of the average GP value, indicating an increasingly tighter packing of the lipids. Interestingly, the two systems studied show phenomena of budding and fission, and these at surprisingly low pressures of 200-300 bar. Moreover, these budding processes are not directly related to phase transitions to an overall ordered conformational state of the lipid membrane, which occur at much higher pressures. The topological changes of the lipid vesicles are irreversible and exhibit a different behavior depending on whether the pressure is increased or decreased. The results are discussed in light of the various contributions to the free energy functional of lipid vesicles. Finally, the biological relevance of these studies is highlighted.

The authors developed a technique to apply high hydrostatic pressure to giant unilamellar vesicles and to directly observe the consequent structural changes with two-photon fluorescence microscopy imaging using high numerical aperture oil-immersion objectives. The data demonstrate that high pressure has a dramatic effect on the shape of the vesicles, and both fluidity and homogeneity of the membrane.

Extracellular Ca²⁺ (Ca²⁺_o) is a crucial regulator of epidermal homeostasis and its receptor, the Ca²⁺-sensing receptor (CaSR), conveys the Ca²⁺_o signals to promote keratinocyte adhesion, differentiation, and survival via activation of intracellular Ca²⁺ (Ca²⁺_i) and E-cadherin-mediated signaling. Here, we took genetic loss-of-function approaches to delineate the functions of CaSR in wound re-epithelialization. Cutaneous injury triggered a robust CaSR expression and a surge of Ca²⁺_i in epidermis. CaSR and E-cadherin were co-expressed at the cell-cell membrane between migratory keratinocytes in the nascent epithelial tongues. Blocking the expression of CaSR or E-cadherin in cultured keratinocytes markedly inhibited the wound-induced Ca²⁺_i propagation and their ability to migrate collectively. Depleting CaSR also suppressed keratinocyte proliferation by downregulating the E-cadherin/epidermal growth factor receptor/mitogen-activated protein kinase signaling axis. Blunted epidermal Ca²⁺_i response to wounding and retarded wound healing were observed in the keratinocyte-specific CaSR knockout (^EpidCasr^-/-) mice, whose shortened neo-epithelia exhibited declined E-cadherin expression and diminished keratinocyte proliferation and differentiation. Conversely, stimulating endogenous CaSR with calcimimetic NPS-R568 accelerated wound re-epithelialization through enhancing the epidermal Ca²⁺_i signals and E-cadherin membrane expression. These findings demonstrated a critical role for the CaSR in epidermal regeneration and its therapeutic potential for improving skin wound repair.

Ca(2+) fluxes direct keratinocyte differentiation, cell-to-cell adhesion, migration, and epidermal barrier homeostasis. We previously showed that intracellular Ca(2+) stores constitute a major portion of the calcium gradient especially in the stratum granulosum. Loss of the calcium gradient triggers epidermal barrier homeostatic responses. In this report, using unfixed ex vivo epidermis and human epidermal equivalents we show that endoplasmic reticulum (ER) Ca(2+) is released in response to barrier perturbation, and that this release constitutes the major shift in epidermal Ca(2+) seen after barrier perturbation. We find that ER Ca(2+) release correlates with a transient increase in extracellular Ca(2+). Lastly, we show that ER calcium release resulting from barrier perturbation triggers transient desmosomal remodeling, seen as an increase in extracellular space and a loss of the desmosomal intercellular midline. Topical application of thapsigargin, which inhibits the ER Ca(2+) ATPase activity without compromising barrier integrity, also leads to desmosomal remodeling and loss of the midline structure. These experiments establish the ER Ca(2+) store as a master regulator of the Ca(2+) gradient response to epidermal barrier perturbation, and suggest that ER Ca(2+) homeostasis also modulates normal desmosomal reorganization, both at rest and after acute barrier perturbation.

Differentiation of normal human keratinocytes (NHK) grown in vitro as a monolayer to confluency can be triggered with an acute increase in concentration of extracellular Ca⁺⁺ . Over several days, induced by Ca⁺⁺ , the cells form pseudostratified sheets that somewhat resemble the basic organization of the intact skin. This experimental system is widely used in studies of keratinocyte biology and skin pathology. However, expression pattern of the genes considered as markers for cells in specific layers of epidermis in vivo does not always match the specific pattern observed in vitro and might lead to misinterpretation of data. Here, we demonstrate that among 18 markers of terminally differentiated keratinocytes of stratum granulosum (SG) and stratum corneum (SC) in vivo, only four (CDSN, KPRP, LCE1C and SPRR4) have reproduced their expression pattern in vitro. Our data suggest that findings based on two-dimensional (2D) Ca⁺⁺ -induced terminal differentiation of NHK in vitro should be subjected to additional scrutiny before conclusions could be made and, if possible, verified in other experimental system that might more faithfully represent the in vivo microenvironment.

Objective

With rapid development of nanotechnology, there is increasing interest in nanoparticle (NP) application and its safety and efficacy on human skin. In this study, we utilized confocal laser scanning microscopy to estimate NP skin penetration.

Methods

Three different-sized polystyrene NPs marked with red fluorescence were applied to human skin, and Calcium Green 5N was used as a counterstain. Dimethyl sulfoxide (DMSO) and ethanol were used as alternative vehicles for NPs. Tape stripping was utilized as a barrier-damaged skin model. Skin biopsies dosed with NPs were incubated at 4°C or 37°C for 24 hours and imaged using confocal laser scanning microscopy.

Results

NPs were localized in the stratum corneum (SC) and hair follicles without penetrating the epidermis/dermis. Barrier alteration with tape stripping and change in incubation temperature did not induce deeper penetration. DMSO enhanced NP SC penetration but ethanol did not.

Conclusion

Except with DMSO vehicle, these hydrolyzed polystyrene NPs did not penetrate intact or barrier-damaged human "viable" epidermis. For further clinical relevance, in vivo human skin studies and more sensitive analytic chemical methodology are suggested.