TAT-mediated aequorin transduction: an alternative approach for effective calcium measurements in plant cells.
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.
Dipartimento di Biologia, Università di Padova, Via U. Bassi 58/B, 35131 Padova, Italy.
Elisabetta
E
Zonin
Roberto
R
Moscatiello
Manuela
M
Miuzzo
Nadia
N
Cavallarin
Maria Luisa
ML
Di Paolo
Dorianna
D
Sandonà
Oriano
O
Marin
Marisa
M
Brini
Alessandro
A
Negro
Lorella
L
Navazio
D000331:Aequorin / Q000378:metabolism*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002470:Cell Survival; D004247:DNA / Q000235:genetics; D018552:Daucus carota / Q000166:cytology; D004705:Endocytosis; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D049449:Luminescence; D008856:Microscopy, Fluorescence; D049329:Nanostructures; D059828:Plant Cells / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport; D011523:Protoplasts / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013025:Glycine max / Q000166:cytology / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D000331:Aequorin; D004247:DNA; D002118:Calcium