Employing fluorescence spectroscopy and the membrane-embedded dye Laurdan we experimentally show broad order-disorder-transitions in membranes of various cell lines and red blood cell ghosts. A custom-made setup allows for the determination of fluorescence spectra and the determination of the generalized polarization (GP) as a measure for membrane order in the temperature range of -40°C to +90°C of µl-volumes of cell suspension. While artificial lipid membranes like phosphatidylcholine show sharp transitions as known from calorimetry measurements, living cells in a physiological temperature range do only show linear changes in generalized polarization. However, extending the temperature range shows the existence of broad transitions and their sensitivity to cholesterol content, pH and anaesthetic. Moreover, adaptation to culture conditions like decreased temperature and morphological changes like detachment of adherent cells or dendrite growth are accompanied by changes in membrane order as well. The observed GP changes are equivalent to temperature changes dT in the range of -12K < dT <+6K.

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Employing fluorescence microscopy, we measure the uptake of various fluorescent dyes into adherent HeLa cells and determine simultaneously the degree of membrane lipid chain order on a single cell level. The latter is measured by spectral analysis of the membrane-embedded dye Laurdan with different filter sets and quantified by determining the generalized polarization GP. First, we find that the mean GP value of single cells varies within a cell population in a range that is equivalent to a temperature variation of 9 K. We exploit this natural variety of lipid membrane chain order to examine the uptake of fluorescent dyes as a function of GP at constant temperature. Using this approach, we experimentally show that transport across the cell membrane quantified by uptake of fluorescent dyes correlates with the membrane phase state. Specifically, we observed higher membrane transport with increasing lipid chain order. As a result, HeLa cells that adapted to lower culture temperatures by reducing lipid membrane chain order show less rhodamine B transport than the 37°C culture. Aside from the cell intrinsic membrane phase state environmental factors have impact on the transport across the cell membrane as well. While temperature reduces lipid order, we found that locally high cell densities increase lipid order of cell membranes, and in turn leading to increased dye uptake. To demonstrate the physiological relevance of this concept, we analyzed cell phase state and transport at different stages of an in vitro wound healing process. While the uptake within a confluent cell layer is high due to the high degree of lipid chain order it decreases from the wound edge towards the wound center where the membrane lipid chain order is lowest.

The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adbi.202200282.

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