Abstract
The transactivator of transcription (TAT) protein transduction domain is an 11-amino acid positively charged peptide that has been shown to pull diverse molecules across cell membranes in vitro and in vivo. Fusion proteins constructed with TAT rapidly enter and exit cells and have been shown to cross intracellular membranes as well. Electrostatic interactions between TAT and the cell membrane have been implicated as a part of the mechanism of transduction. Here, we report that TAT transduction causes membrane phospholipid rearrangement as evidenced by detection of phosphatidylserine on the outer surface of the cell membrane. Furthermore, these rearrangements can be blocked by positively charged polylysine, further implicating electrostatic interactions as a part of the mechanism. Neither apoptosis nor necrosis is induced in these cells after exposure to TAT. We conclude that the process of TAT.GFP transduction causes phosphatidylserine to translocate from the inner to the outer leaflet of the plasma membrane. These results provide insight into the mechanism of TAT protein transduction domain transduction.
Highlights
Protein transduction domains (PTDs)1 have been shown to transfer a wide range of cargos across the cell membrane, including large proteins [1], polyanionic oligonucleotides [2], liposomes [3, 4], and even metallic beads [5, 6]
Annexin is specific for PS and only comes into contact with PS when it translocates to the outer surface during apoptosis [22, 23]
Consistent with prior studies, we found that 300 M H2O2 reproducibly kills ϳ40% of the cells via apoptosis, which was evident by a significant increase in caspase-3 activation over controls (3.03 Ϯ 0.082 mean fluorescence units versus 1.12 Ϯ 0.006 for controls; Fig. 1A)
Summary
Materials—Rat tail collagen, nerve growth factor, poly-DL-lysine hydrobromide, hydrogen peroxide, neutral red, Sephadex-G25, and Triton X-100 were obtained from Sigma. Lab-Tek II chambered 1.5 cover glass dishes, and polycarbonate membranes were from Fisher. Dialysis membrane was from Spectrum Laboratories Inc. Caspase-3 assay kit, Alexafluor-568 Annexin-V, and calcein were from Molecular Probes. TAT1⁄7GFP was purified as previously described [19]. General Cell Culture Care—PC12 cells were grown in RMPI 160 medium supplemented with 10% heat-inactivated horse serum and 5% fetal calf serum in 75-cm treated cell culture flasks that were pretreated with 50 g/ml rat tail collagen. Cells were differentiated by plating them with RMPI 1640 medium containing 1% horse serum, supplemented with 100 ng/ml nerve growth factor for a minimum of 48 h.
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