TAT-Mediated Aequorin Transduction: An Alternative Approach for Effective Calcium Measurements in Plant Cells

Abstract

Cell-penetrating peptides are short cationic peptides with the property of translocating across the plasma membrane and transferring macromolecules otherwise unable to permeate cell membranes. We investigated the potential ability of the protein transduction domain derived from amino acids 47-57 of the human immunodeficiency virus type 1 (HIV-1) TAT (transactivator of transcription) protein to be used as a nanocarrier for the delivery of aequorin, a Ca(2+)-sensitive photoprotein widely used as a reliable Ca(2+) reporter in cell populations. The TAT peptide, either covalently linked to apoaequorin or ionically bound to plasmids encoding differentially targeted aequorin, was supplied to plant suspension-cultured cells. The TAT-aequorin fusion protein was found to be rapidly and effectively translocated into plant cells. The chimeric molecule was internalized in fully active biological form and at levels suitable to monitor intracellular Ca(2+) concentrations. Plant cells incubated for just 5 min with TAT-aequorin responded to different environmental stimuli with the expected Ca(2+) signatures. On the other hand, TAT-mediated plasmid internalization did not provide the necessary level of transformation efficiency to allow calibration of luminescence signals into Ca(2+) concentration values. These results indicate that TAT-mediated aequorin transduction is a promising alternative to traditional plant transformation methods to monitor intracellular Ca(2+) dynamics rapidly and effectively in plant cells.

Cell-Penetrating Peptides

“…Baumgärtner et al [35] found that the cellular internalization of the serpin protein C inhibitor (PCI) is supported by plasma membrane PE, which enables its rapid targeting to the nucleus both in vitro and in vivo. Therefore, PCI was considered as an excellent candidate for the nuclear supply of cargo.…”

“…CPP-endosomal escape is one major challenge in CPP cargo delivery and several studies have focused on investigating the mechanisms of endosomal escape of CPPs [ 35 ]. In an elegant experiment, the biological endosome can be modeled using the bacteriorhodopsin (BR)-reconstituted LUV system (BR-LUV).…”

“…This single artifact has likely caused decades of confusion about CPP mechanism of action because leakage of entrapped probes due to peptide-induced aggregation and fusion, while a common experimental observation, is likely to be completely unrelated to the biological activity of CPPs. To separate true membrane permeabilization from artifacts caused by aggregation and fusion, vesicle-vesicle interactions can be blocked by the addition of 4 mol % of PEG2000 lipids [35]. The effects of PEG2000 lipids on peptide-induced aggregation of vesicles are shown in Fig.…”

“…The number of cell surface receptors classifi ed as scavenger receptors is continually growing from the fi rst suggestion of classes A-F in 1997 [ 35 ] to now include 19 receptors that divided into class A through I [ 33 ]. The receptors share the ability to bind polyanionic ligands with low specifi city.…”

“…Similar to the case with i3 agonist pepducins, an i3 antagonist pepducin, P2pal-18S, was strongly biased toward blocking PAR2-Gq and PAR-Gi signaling pathways including MAPK activation, but had no effect on triggering PAR2-ligand-activated endocytosis [ 27 ]. As PAR2 endocytosis is dependent on β-arrestin engagement, it was not surprising that P2pal-18S was unable to inhibit β-arrestin interactions with PAR2 [ 35 ].…”