Membrane transport in cell ensembles is modulated by the membrane state

Abstract

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.

Microfluidic Transfection System and Temperature Strongly Influence the Efficiency of Transient Transfection

“…The phase state of the lipids correlates with a number of observable parameters (including bending stiffness, permeability, membrane viscosity, as well as membrane thickness and area), and it can be influenced by a variety of factors (for example, by cholesterol levels, salt content, or the presence of an electric field). − Moreover, it has been shown that various transport processes for smaller molecules as well as nanoparticles depend on the phase state (or lipid order) of lipid and plasma membranes for both synthetic liposomes , and also biological membranes . One of the most important factors influencing the phase state of lipid membranes is temperature ( T ). ,− While the phase transition of synthetic one or two component liposomes is limited to a T range of just a few degrees, biological cells display a markedly broader phase transition range as well as a noticeable scattering of the phase state between individual cells, even under the same external conditions. , …”

“…17,25−27 While the phase transition of synthetic one or two component liposomes is limited to a T range of just a few degrees, 17 biological cells display a markedly broader phase transition range as well as a noticeable scattering of the phase state between individual cells, even under the same external conditions. 17,28 For endocytotic uptake pathways, both membrane tension as well as the curvature of the membrane required for particle uptake must be taken into consideration. 29 It, therefore, stands to reason that there could also be a link between particle uptake rates and the thermodynamic state of the membrane when it comes to polyplex uptake.…”

“… 16 , 24 − 26 While the phase transition of synthetic one or two component liposomes is limited to a T range of just a few degrees, 16 biological cells display a markedly broader phase transition range as well as a noticeable scattering of the phase state between individual cells, even under the same external conditions. 16 , 27 …”