Published: September 16th, 2016
We describe a method for depletion-rescue experiments that preserves cellular integrity and protein homeostasis. Adenofection enables functional analyses of proteins within biological processes that rely on finely tuned actin-based dynamics, such as mitotic cell division and myogenesis, at the single-cell level.
Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell cytoskeletal structures. This involves the assembly-disassembly of higher-order macromolecular structures at a given time and location, a process that is particularly sensitive to perturbations caused by overexpression of proteins. Methods that can preserve protein homeostasis and maintain near-to-normal cellular morphology are highly desirable to determine the functional contribution of a protein of interest in a wide range of cellular processes. Transient depletion-rescue experiments based on RNA interference are powerful approaches to analyze protein functions and structural requirements. However, reintroduction of the target protein with minimum deviation from its physiological level is a real challenge. Here we describe a method termed adenofection that was developed to study the role of molecular chaperones and partners in the normal operation of dividing cells and the relationship with actin remodeling. HeLa cells were depleted of BAG3 with siRNA duplexes targeting the 3'UTR region. GFP-tagged BAG3 proteins were reintroduced simultaneously into >75% of the cells using recombinant adenoviruses coupled to transfection reagents. Adenofection enabled to express BAG3-GFP proteins at near physiological levels in HeLa cells depleted of BAG3, in the absence of a stress response. No effect was observed on the levels of endogenous Heat Shock Protein chaperones, the main stress-inducible regulators of protein homeostasis. Furthermore, by adding baculoviruses driving the expression of fluorescent markers at the time of cell transduction-transfection, we could dissect mitotic cell dynamics by time-lapse microscopic analyses with minimum perturbation of normal mitotic progression. Adenofection is applicable also to hard-to-infect mouse cells, and suitable for functional analyses of myoblast differentiation into myotubes. Thus adenofection provides a versatile method to perform structure-function analyses of proteins involved in sensitive biological processes that rely on higher-order cytoskeletal dynamics.
Functional inactivation of gene expression in mammalian cells is the gold standard to dissect protein functions. Newly developed technologies of genome editing based on the use of site-specific nucleases such as Zinc-finger nucleases and clustered regularly interspaced short palindromic repeats (CRISPR)/CAS9 now allow the generation of cell lines with targeted gene deletion and mutation1,2. These novel approaches should revolutionize the way we are studying protein function and our understanding of the genetics of human diseases. In some instances, however, long-term or complete gene knockout is not desirable and may provoke secondary cell compensation mech....
1. Preparation of Medium and Solutions (all sterile filtered)
Transfection of BAG3-GFP plasmid DNA using cationic lipids was associated with heterogeneous expression in HeLa cells, some cells showing barely detectable levels of the protein and others bearing very high BAG3 levels (Figure 2A). In these cells, loss of protein homeostasis was evidenced by accumulation of BAG3-GFP into perinuclear aggregates (Figure 2A, arrows). In contrast, cell transduction with adenoviruses carrying BAG3-GFP exhibited more homogenous.......
Here, we described a method enabling depletion-rescue experiments to be performed, which is applicable to functional analyses of cell biological processes that are particularly sensitive to overexpression of proteins affecting the stoichiometry and dynamics of protein complexes and macromolecular structures. Mitotic cell division is an extreme example of finely tuned cell morphodynamics that involves the most dramatic and spectacular changes in the overall structure of a cell. Using adenofection combined with commerciall.......
|C2C12 Mouse Myoblasts
|Adenovirus custom design
|CellLight® Actin-GFP, BacMam 2.0
|CellLight® Tubulin-RFP, BacMam 2.0
|Dulbecco’s modified Eagle’s medium (DMEM), High Glucose
|Fetal Bovine Serum (FBS)
|Glass bottom dishes, 35mm
|Kind gift of Dr Sabine Elowe, Québec, Canada
|Klebig C et al. 2009
|Horse Serum, New Zealand
|Lipofectamine® RNAiMAX Transfection Reagent
|Minimal Essential Medium (MEM) Alpha
|Minimal Essential Medium (MEM) Alpha without Desoxyribonuleosides/Ribonucleosides
|Minimal Essential Medium (MEM) Alpha without Phenol Red
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