Internalization of the lipophilic fluorescent probe trimethylamino-diphenylhexatriene follows the endocytosis and recycling of the plasma membrane in cells.

The lipophilic fluorescent probe trimethylamino-diphenylhexatriene (TMA-DPH) has been shown previously to behave as a marker of plasma membrane in living cell systems, and it has therefore been widely used in membrane fluidity studies via fluorescence anisotropy measurements. However, progressive internalization of this probe in cells could lead to unsuitable interferences, when long incubations times were required. The mechanism of this internalization had not yet been elucidated. We present here fluorescence-intensity kinetic results and fluorescence micrographic data on L929 cells and on mouse bone-marrow macrophages, which allow us to identify the mechanism as fluid-phase pinocytosis: the probe remains associated with the plasma membrane throughout its internalization-recycling flow and it is finally concentrated in lysosomes. The study was facilitated by the partition equilibrium property of TMA-DPH between plasma membranes and the external aqueous medium, which allowed to immediately distinguish the internalized fraction of the probe from the peripheral labelling, by simply washing cells. This conclusion is confirmed by the features of the influence of temperature on TMA-DPH internalization.

The plasma-membrane internalization and recycling is enhanced in macrophages upon activation with gamma-interferon and lipopolysaccharide; a study using the fluorescent probe trimethylaminodiphenylhexatriene.

The lipophilic fluorescent probe trimethylaminodiphenylhexatriene (TMA-DPH), previously used as a plasma membrane marker in membrane fluidity and exocytosis studies, was shown, to monitor the plasma-membrane internalization-recycling shuttle movement in cells. Using this approach we present here kinetic and dose-response data, which give evidence that the plasma membrane flow is enhanced in bone marrow macrophages from various mouse strains, upon in vitro activation with gamma interferon (IFN-gamma) or bacterial lipopolysaccharide (LPS), within physiological dose ranges. The effect studied evolved in line with the usual development kinetics of macrophage activation. Complementary assays on membrane fluidity, surface charge density and membrane surface indicated no related changes. From these experiments it is concluded that the observed enhancement of the plasma membrane traffic does not originate from specific limited membrane modifications, but is merely a particular feature of the overall macrophage activation.