TMA-DPH as Specific Plasma Membrane Fluidity Probe for Intact Cells and its Limitation

Abstract

Fluorescence anisotropy measurements to determine membrane fluidity have been generally performed with diphenylhexatriene (DPH) on purified membrane samples. This probe could not be used satisfactorily with intact cells, because it did not present any specificity for a particular type of membrane and labelled all the hydrophobic regions of the cell. We have recently focussed our attention on the cationic derivative of DPH, trimethylamino-diphenylhexatriene (TMA-DPH). This molecule displays excellent photophysical properties in hydrophobic media: lifetime 6 ns (DPH 8 ns), quantum yield 0.55 (DPH 0.75), and is not fluorescent in water. 1,2 In comparison with DPH and the other fluorescent probes previously used in membrane fluidity determinations it was given evidence 3 ,4 by fluorescence micrography and by fluorescence intensity measurements, to have two particularly favorable features : (i) it is incorporated in membranes very rapidly (ii) when it is interacted with intact cells, it is retained specifically in the plasma membranes for periods of typically 30 min. before being internalized.These properties made TMA-DPH a relevant fluorescent probe for fluidity measurements of the plasma membranes of intact cells. As such it has been used satisfactorily in monitoring fluidity changes in : mast cells upon histamine release,s erythrocytes upon parasitemia by plasmodium,6 purified mitochondria upon treatment by succinate. 7 On the other hand, the evidence of a partition equilibrium of TMA-DPH between membranes and water 4 suggested an original method to monitor exocytosis phenomena by simple fluorescence intensity measurements. 8 LIMITATIONS TO ITS USE Internalization featuresThe kinetics of the internalization of TMA-DPH after its stay in the cell plasma membranes depend actually on the cell type. In fibroblasts, lymphocytes and mast cells the process started after 30 min. at 37°C.

An ω-3, but Not an ω-6 Polyunsaturated Fatty Acid Decreases Membrane Dipole Potential and Stimulates Endo-Lysosomal Escape of Penetratin

“…Although the steady-state fluorescence anisotropy of TMA-DPH might be influenced by several factors related to dynamic and static aspects of fluidity, and therefore linking it to membrane order or fluidity is problematic, depolarization of TMA-DPH fluorescence does indeed correlate with membrane disorder (Engel and Prendergast, 1981;do Canto et al, 2016). Furthermore, TMA-DPH shows relatively slow internalization (Kubina et al, 1986), therefore its fluorescence originates almost exclusively from the cell membrane, similarly to F66 that we demonstrated previously to remain in the plasma membrane of cells under our experimental conditions (Kovacs et al, 2017). On the other hand, significant positive correlations were found between PY3174 generalized polarization and F66 emission ratios (Figure 5), which must be due to the proposed strong relationship between the organization of membraneassociated water molecules and the magnitude of DP (Simon et al, 1992;Smondyrev and Berkowitz, 2001;O'Shea, 2003O'Shea, , 2005Starke-Peterkovic et al, 2006;Wang, 2012;Zakany et al, 2020).…”