Quantitative evaluation of the cell penetrating properties of an iodinated Tyr-L-maurocalcine analog. - DyCTim : Dynamique Cellulaire / Tissulaire et Microscopie fonctionnelle Accéder directement au contenu
Article Dans Une Revue BBA - Biochimica et Biophysica Acta Année : 2014

Quantitative evaluation of the cell penetrating properties of an iodinated Tyr-L-maurocalcine analog.

Résumé

L-Maurocalcine (L-MCa) is the first reported animal cell-penetrating toxin. Characterizing its cell penetration properties is crucial considering its potential as a vector for the intracellular delivery of drugs. Radiolabeling is a sensitive and quantitative method to follow the cell accumulation of a molecule of interest. An L-MCa analog containing an additional N-terminal tyrosine residue (Tyr-L-MCa) was synthesized, shown to fold and oxidize properly, and successfully radioiodinated to (125)I-Tyr-L-MCa. Using various microscopy techniques, the average volume of the rat line F98 glioma cells was evaluated at 8.9 to 18.9×10(-7)μl. (125)I-Tyr-L-MCa accumulates within cells with a dose-dependency similar to the one previously published using 5,6-carboxyfluorescein-L-MCa. According to subcellular fractionation of F98 cells, plasma membranes keep less than 3% of the peptide, regardless of the extracellular concentration, while the nucleus accumulates over 75% and the cytosol around 20% of the radioactive material. Taking into account both nuclear and cytosolic fractions, cells accumulate intracellular concentrations of the peptide that are equal to the extracellular concentrations. Estimation of (125)I-Tyr-L-MCa cell entry kinetics indicate a first rapid phase with a 5min time constant for the plasma membrane followed by slower processes for the cytoplasm and the nucleus. Once inside cells, the labeled material no longer escapes from the intracellular environment since 90% of the radioactivity remains 24h after washout. Dead cells were found to have a lower uptake than live ones. The quantitative information gained herein will be useful for better framing the use of L-MCa in biotechnological applications. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

Domaines

Imagerie

Dates et versions

hal-01022916 , version 1 (11-07-2014)

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Citer

Céline Tisseyre, Mitra Ahmadi, Sandrine Bacot, Lucie Dardevet, Pascale Perret, et al.. Quantitative evaluation of the cell penetrating properties of an iodinated Tyr-L-maurocalcine analog.. BBA - Biochimica et Biophysica Acta, 2014, 1843 (10), pp.2356-64. ⟨10.1016/j.bbamcr.2014.03.017⟩. ⟨hal-01022916⟩
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