Electroendocytosis: stimulation of adsorptive and fluid-phase uptake by pulsed low electric fields.
We present a novel approach for stimulating uptake via endocytic pathways by exposing cells to a train of pulsed low electric fields (LEF) in the range of 2.5-20 V/cm. Electric field treatment of COS 5-7 and HaCaT cells in the presence of BSA-FITC augments the adsorption of the probe to plasma membranes with subsequent enhanced internalization. The uptake of BSA-FITC is maximal when the cells are exposed to LEF in the presence of the probe while uptake of a fluid-phase marker, propidium iodide (PI), is more effective when the probe is added immediately after termination of a 1-min exposure. LEF-stimulated uptake decays with a half-life of about 3 and 1 min for and BSA-FITC and PI, respectively. The uptake is inefficient at 4 degrees C but increases with temperature. The uptake proceeds via cell membrane vesiculation, showing a high extent of colocalization of BSA-FITC with plasma membrane vesicles labeled with a phospholipid fluorescent analogue. Unlike constitutive endocytosis where the BSA-FITC is exposed to acidic pH, in LEF-induced uptake the probe is exposed to the more alkaline pH of the cytosol. The staining kinetics of nuclear targets by PI reflects the release of the probe from the LEF-induced vesicles into the cytosol 1-3 h after exposure. The LEF-induced adsorptive pathway was approximately 2.5 more effective than the LEF-induced fluid-phase one. The observed 5- to 6-fold increase of BSA-FITC uptake induced by LEF may be partially attributed to a clathrin-dependent route (up to 25%), whereas the rest of the uptake may be assigned to macropinocytotic and clathrin/caveolin independent pathways or to a novel, yet unidentified, route driven by LEF. This study provides a basis for a general approach towards the efficient incorporation of a variety of molecules such as antibodies, enzymes or genes into cells.
Antov
Yulia
Y
Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, 69978 Tel-Aviv, Israel.
Barbul
Alexander
A
Korenstein
Rafi
R
D000327:Adsorption; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D002522:Chlorocebus aethiops; D003911:Dextrans / Q000737:chemistry; D004558:Electric Stimulation; D004705:Endocytosis; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry / Q000493:pharmacokinetics*; D006367:HeLa Cells; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D018613:Microscopy, Confocal; D010873:Pinocytosis; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000493:pharmacokinetics*
D003911:Dextrans; D012710:Serum Albumin, Bovine; D016650:Fluorescein-5-isothiocyanate