Parallelized TCSPC for dynamic intravital fluorescence lifetime imaging: quantifying neuronal dysfunction in neuroinflammation.

Two-photon laser-scanning microscopy has revolutionized our view on vital processes by revealing motility and interaction patterns of various cell subsets in hardly accessible organs (e.g. brain) in living animals.

Major translocation of calcium upon epidermal barrier insult: imaging and quantification via FLIM/Fourier vector analysis.

Calcium controls an array of key events in keratinocytes and epidermis: localized changes in Ca(2+) concentrations and their regulation are therefore especially important to assess when observing epidermal barrier homeostasis and repair, neonatal barrier establishment, in differentiation, signaling, cell adhesion, and in various pathological states. Yet, tissue- and cellular Ca(2+) concentrations in physiologic and diseased states are only partially known, and difficult to measure. Prior observations on the Ca(2+) distribution in skin were based on Ca(2+) precipitation followed by electron microscopy, or proton-induced X-ray emission.

CD44 regulates tight-junction assembly and barrier function.

Upon barrier disturbance, adult CD44 knockout (KO) mice show delayed recovery of epidermal barrier function. This correlates with the loss of apical polarization of lamellar body (LB) secretion. As tight junctions (TJs) are crucial for barrier function and regulate polarized targeting of vesicles, we hypothesized that CD44 regulates TJs and associated cell polarity complexes, which in turn contributes to altered skin barrier function in CD44 KO mice.

Alteration of tight junction proteins is an early event in psoriasis: putative involvement of proinflammatory cytokines.

Psoriasis is an inflammatory skin disease characterized by hyperproliferation of keratinocytes, impaired barrier function, and pronounced infiltration of inflammatory cells. Tight junctions (TJs) are cell-cell junctions that form paracellular barriers for solutes and inflammatory cells. Altered localization of TJ proteins in the epidermis was described in plaque-type psoriasis.

Regulation of epidermal tight-junctions (TJ) during infection with exfoliative toxin-negative Staphylococcus strains.

Tight Junction (TJ) proteins have been shown to exert a barrier function within the skin. Here, we study the fate of TJ proteins during the challenge of the skin by bacterial colonization and infection. We investigated the influence of various exfoliative toxin-negative Staphylococcus strains on TJ, adherens junction (AJ), desmosomal proteins, and actin in a human keratinocyte infection culture and in a porcine skin infection model.

Involucrin expression is decreased in Hailey-Hailey keratinocytes owing to increased involucrin mRNA degradation.

Hailey-Hailey disease (HHD) (MIM 16960) is an autosomal-dominant blistering skin disease caused by a mutation in the Ca2+-ATPase ATP2C1 (protein SPCA1), responsible for controlling Ca2+ concentrations in the cytoplasm and Golgi in human keratinocytes. Cytosolic Ca2+ concentrations, in turn, play a major role in the regulation of keratinocyte differentiation. To study how ATP2C1 function impacts keratinocyte differentiation, we assessed involucrin expression in HHD keratinocytes.

Targeted disruption of the protein kinase SGK3/CISK impairs postnatal hair follicle development.

Members of the serum- and glucocorticoid-regulated kinase (SGK) family are important mediators of growth factor and hormone signaling that, like their close relatives in the Akt family, are regulated by lipid products of phosphatidylinositol-3-kinase. SGK3 has been implicated in the control of cell survival and regulation of ion channel activity in cultured cells. To begin to dissect the in vivo functions of SGK3, we generated and characterized Sgk3 null mice.

Stratum corneum acidification in neonatal skin: secretory phospholipase A2 and the sodium/hydrogen antiporter-1 acidify neonatal rat stratum corneum.

At birth, human stratum corneum (SC) displays a near-neutral surface pH, which declines over several days to weeks to months to an acidic pH, comparable to that of adults. Recent studies suggest that an acidic pH is required for normal permeability barrier homeostasis and SC integrity/cohesion. We assessed here the basis for postnatal acidification in the neonatal rat, where SC pH, as measured with a flat surface electrode, declines progressively from near-neutral levels (pH 6.

Defective cyclic guanosine monophosphate-gated calcium channels and the pathogenesis of psoriasis.

A positive association between intake of calcium channel blockers and psoriasis has been observed recently. Intake of blockers of voltage-gated calcium ion channels is associated with outbreaks of psoriasis after a latent period in patients with and without a previous family history of psoriasis. This suggests that interfering with calcium influx may trigger psoriasis.

Human keratinocyte ATP2C1 localizes to the Golgi and controls Golgi Ca2+ stores.

Hailey-Hailey disease (MIM16960) is a blistering skin disease caused by mutations in the Ca2+ ATPase ATP2C1. We found that the abnormal Ca2+ signaling seen in Hailey-Hailey disease keratinocytes correlates with decreased protein levels of ATP2C1. Human ATP2C1 protein approximated 115 kDa in size.

Actin reorganization is abnormal and cellular ATP is decreased in Hailey-Hailey keratinocytes.

Actin reorganization and the formation of adherens junctions are necessary for normal cell-to-cell adhesion in keratinocytes. Hailey-Hailey disease (HHD) is blistering skin disease, resulting from mutations in the Ca2+ ATPase ATP2C1, which controls Ca2+ concentrations in the cytoplasm and Golgi of human keratinocytes. Because actin reorganization is among the first responses to raised cytoplasmic Ca2+, we examined Ca2+-induced actin reorganization in normal and HHD keratinocytes.

Neonatal development of the stratum corneum pH gradient: localization and mechanisms leading to emergence of optimal barrier function.

Although basal permeability barrier function is established at birth, the higher risk for infections, dermatitis, and percutaneous absorption of toxic agents may indicate incomplete permeability barrier maturation in the early neonatal period. Since stratum corneum (SC) acidification in adults is required for normal permeability barrier homeostasis, and lipid processing occurs via acidic pH dependent enzymes, we hypothesized that, in parallel with the less acidic surface pH, newborn SC would exhibit signs of incomplete barrier formation. Fluorescence lifetime imaging reveals that neonatal rat SC acidification first becomes evident by postnatal day 3, in extracellular "microdomains" at the SC- stratum granulosum (SG) interface, where pH-sensitive lipid processing is known to occur.

NHE1 regulates the stratum corneum permeability barrier homeostasis. Microenvironment acidification assessed with fluorescence lifetime imaging.

The outermost epidermal layer, the stratum corneum (SC), exhibits an acidic surface pH, whereas the pH at its base approaches neutrality. NHE1 is the only Na(+)/H(+) antiporter isoform in keratinocytes and epidermis, and has been shown to regulate intracellular pH. We now demonstrate a novel function for NHE1, as we find that it also controls acidification of extracellular "microdomains" in the SC that are essential for activation of pH-sensitive enzymes and the formation of the epidermal permeability barrier.

Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient.

Two-photon fluorescence lifetime imaging is used to identify microdomains (1-25 microm) of two distinct pH values within the uppermost layer of the epidermis (stratum corneum). The fluorophore used is 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), whose lifetime tau (pH 4.5, tau = 2.

Glucosylceramide synthesis and synthase expression protect against ceramide-induced stress.

Ceramides (Cers), critical for epidermal barrier function, also can inhibit keratinocyte proliferation, while glucosylceramides (GlcCers) exert pro-mitogenic effects. Since alterations in Cer-to-GlcCer ratios appear to modulate cellular growth versus apoptosis, we assessed whether keratinocytes up-regulate GlcCer synthesis as a protective mechanism against Cer-induced stress. Exogenous sphingomyelinase (SMase) treatment of cultured human keratinocytes (CHK) initially decreased proliferation and cellular sphingomyelin (50-60% decrease; P < 0.