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

Abstract

Calcium controls an array of key events in keratinocytes and epidermis: localized changes in Ca2+ 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 Ca2+ concentrations in physiologic and diseased states are only partially known, and difficult to measure. Prior observations on the Ca2+ distribution in skin were based on Ca2+ precipitation followed by electron microscopy, or proton-induced X-ray emission. Neither cellular and/or subcellular localization could be determined through these approaches. In cells in vitro, fluorescent dyes have been used extensively for ratiometric measurements of static and dynamic Ca2+ concentrations, also assessing organelle Ca2+ concentrations. For lack of better methods, these findings together build the basis for the current view of the role of Ca2+ in epidermis, their limitations notwithstanding. Here we report a method using Calcium Green 5N as the calcium sensor and the phasor-plot approach to separate raw lifetime components. Thus, fluorescence lifetime imaging (FLIM) enables us to quantitatively assess and visualize dynamic changes of Ca2+ at light-microscopic resolution in ex vivo biopsies of unfixed epidermis, in close to in vivo conditions. Comparing undisturbed epidermis with epidermis following a barrier insult revealed major shifts, and more importantly, a mobilization of high amounts of Ca2+ shortly following barrier disruption, from intracellular stores. These results partially contradict the conventional view, where barrier insults abrogate a Ca2+ gradient towards the stratum granulosum. Ca2+ FLIM overcomes prior limitations in the observation of epidermal Ca2+ dynamics, and will allow further insights into basic epidermal physiology.

Endoplasmic Reticulum Calcium Regulates Epidermal Barrier Response and Desmosomal Structure

“…Figure 1a and b represent the averaged position of the calcium concentration peaks in each epidermal layer. We observed a dramatic shift of the calcium concentration distribution toward higher calcium levels immediately after barrier perturbation in all epidermal layers (Figure 1a and b, respectively, and Supplementary Figure S1) in agreement with data previously reported in mice (Behne et al, 2011). To monitor calcium fluxes during barrier recovery, we imaged tape-stripped and acetone-treated samples 4 and 24 hours after perturbation.…”

“…While initial studies using fixed and sectioned tissue (Mauro et al, 1998; Menon et al, 1992) showed that after acute barrier perturbation, Ca 2+ drops in the viable layers and then recovers as the barrier recovers, a more recent study (Behne et al, 2011) reported a moderate increase in intracellular and extracellular calcium concentration as soon as 30 minutes after barrier perturbation by acetone lipid extraction in hairless mice. We find that the conflicting results on the Ca 2+ gradient response to barrier perturbation reported by different groups can be attributed to different experimental preparations (see Supplementary Figure S3 online).…”

Parallelized TCSPC for Dynamic Intravital Fluorescence Lifetime Imaging: Quantifying Neuronal Dysfunction in Neuroinflammation

“…Therefore, dynamic intravital fluorescence lifetime imaging (FLIM) is required [52]–[53]. Here we present a novel parallelized TCSPC (p-TCSPC) device featuring a 80 MHz average count rate, enabling us to demonstrate 4D-FLIM in vivo .…”

Lowered Humidity Produces Human Epidermal Equivalents with Enhanced Barrier Properties

“…5,6 In normal skin, there is a characteristic intraepidermal calcium gradient, with peak concentrations in the stratum granulosum and lowest levels in the stratum basale. [7][8][9] Ca 2 + plays an important role in keratinocyte differentiation 10 and in regulation of lamellar body secretion during barrier recovery. 11 Normal skin also has a pH gradient where the SC has pH 4.5-5.5 and the viable epidermis displays neutral pH.…”