Major translocation of calcium upon epidermal barrier insult: imaging and quantification via FLIM/Fourier vector analysis
Major translocation of calcium upon epidermal barrier insult: imaging and quantification via FLIM/Fourier vector analysis
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DOI：
10.1007/s00403-010-1113-9
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发表时间：
2011-03-01
影响因子：
3
通讯作者：
Gratton, Enrico
中科院分区：
文献类型：
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作者：
Behne, Martin J.;Sanchez, Susana;Gratton, Enrico
钙控制着角化细胞和表皮的一系列关键事件：因此，在观察表皮屏障稳态和修复、新生儿屏障建立、分化、信号传导、细胞粘附和各种病理状态时，Ca2+浓度的局部变化及其调控对评估尤为重要。然而，生理和病变状态下的组织和细胞Ca2+浓度仅部分已知，且难以测量。先前对皮肤中Ca2+分布的观察是基于Ca2+沉淀，然后是电子显微镜或质子诱导的x射线发射。细胞和/或亚细胞定位都不能通过这些方法确定。在体外细胞中，荧光染料已广泛用于静态和动态Ca2+浓度的比例测量，也评估细胞器Ca2+浓度。由于缺乏更好的方法，这些发现共同建立了Ca2+在表皮中的作用的当前观点的基础，尽管它们的局限性。在这里，我们报告了一种使用钙绿5N作为钙传感器和相量图方法分离原始寿命成分的方法。因此，荧光寿命成像（FLIM）使我们能够定量评估和可视化Ca2+的动态变化在光显微分辨率在离体活检的非固定表皮，在接近体内条件。比较未受干扰的表皮和屏障损伤后的表皮揭示了主要的变化，更重要的是，屏障破坏后不久，细胞内储存的大量Ca2+被动员起来。这些结果部分地与传统观点相矛盾，其中屏障侮辱废除了Ca2+梯度向地层颗粒。Ca2+ FLIM克服了先前在观察表皮Ca2+动力学方面的局限性，并将进一步深入了解基本的表皮生理学。
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.