Download Cell delivery mechanism of protein/lipid complexes studied by

Cell delivery mechanism of protein/lipid complexes studied by
correlative microscopy
Laurence Dallet 1,2,3,4*, Marion Decossas 1,2, Pauline Peuziat 3,4, Bruno Pitard 3,4 and
Olivier Lambert1,2
1Univ.
Bordeaux, CBMN UMR 5248, Bordeaux INP, IECB, Pessac
2CNRS, CBMN UMR5248, Pessac
3 Unité INSERM UMR 1087, CNRS UMR 6291, Nantes
4
Université de Nantes, Faculté de médecine, L’institut du Thorax, Nantes
Many studies have investigated the use of cationic lipids for gene delivery or
DNA vaccination more recently, those cationic lipids may also be efficient for cellular
uptake of proteins [1], [2]. Thus, taking advantage of the capability of cationic lipids,
original therapy involving antibody represents a new approach to treat some diseases
such as cystic fibrosis (CF). A mutation (ΔF508) of CFTR (Cystic Fibrosis
Transmembrane conductance Regulator) causing its retention in endoplasmic
reticulum (ER) seem responsible for CF. Recently, it has been shown that keratin 8
(K8), component of intermediate filaments, was involved in this retention by
interacting with the domain of mutated CFTR [3]. The intracellular delivery of
antibodies against K8 may prevent the interaction between mutated CFTR and K8
and then changes the intracellular trafficking of CFTR.
In this context, this study aims at understanding the cellular trafficking of
complexes (cationic lipid/protein) and the mechanisms governing the internalization
process. To ensure that the cells analyzed by electron microscopy to contain
fluorescent anti-K8 antibody, correlative microscopy approach (CLEM: Correlative
Light and Electron Microscopy) has been performed. This technique is based on the
analysis by fluorescence microscopy and electron microscopy of the same cell,
grown on adequate support (MaTtek support).
Preliminary results have shown that the intracellular delivery of anti-K8-FITC
antibody via cationic lipids allowed to partially restore the functionality of the channel
[4]. We have found effective lipid formulation to intracellular delivery of anti-K8
antibody. After having optimized the protein/lipid ratios, the antibody is internalized
efficiently in HeLa cells and is distributed in the cell at filaments. Ultrathin sections
(50 nm) of the cells containing the antibody are observed by electron microscopy.
The results obtained have been able to demonstrate the association of anti-K8
antibody to its target: intermediate filaments. Studies to identify the internalization
and intracellular trafficking pathways are underway to deepen the antibody
mechanism of action.
This methodological work can analyze and better understand therapeutic
protein delivery mechanisms. The methodology will be shifted to HeLa cells
expressing wild-type CFTR and the mutated CFTR to analyze the localization of
CFTR
[1]
[2]
[3]
[4]
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