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Abstract

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Materials

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Biology

Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments

Published: September 16th, 2016

DOI:

10.3791/54557

1Département de biologie moléculaire, biochimie médicale et pathologie, Faculté de médecine, Centre de recherche sur le cancer de l'Université Laval, 2Oncology, Centre de recherche du CHU de Québec, Université Laval, 3Laboratoire d'études moléculaires des valvulopathies (LEMV), Groupe de recherche en valvulopathies (GRV), Quebec Heart and Lung Institute/Research Center, 4Department of Surgery, Université Laval

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....

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1. Preparation of Medium and Solutions (all sterile filtered)

  1. C2C12 mouse muscle cells (differentiation studies)
    1. Prepare 500 ml of Growth Medium for C2C12 cell culture maintenance: DMEM High Glucose supplemented with 10 % FBS and 2mM L-Glutamine.
    2. Prepare 100 ml Differentiation Medium (DM) for C2C12 differentiation: DMEM High Glucose supplemented with 2% Horse Serum.
  2. HeLa cells (studies in mitotic cells)
    1. Prepare 500 ml.......

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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.......

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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.......

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This work was supported by the Canadian Institutes of Health Research (Grant no 7077), and by the Bellini Foundation and Roby Fondazione.

....

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Name Company Catalog Number Comments
C2C12 Mouse Myoblasts ATCC CRL-1772
Adenovirus custom design Welgen Custom design
Calcium Chloride Fisher Scientific C79-500
CellLight® Actin-GFP, BacMam 2.0 Thermo Fisher C10582
CellLight® Tubulin-RFP, BacMam 2.0 Thermo Fisher C10614
Dulbecco’s modified Eagle’s medium (DMEM), High Glucose Thermo Fisher 11965-092
EDTA Sigma E5134
Fetal Bovine Serum (FBS) Thermo Fisher 12483-020
Fibronectin Sigma F1141
Glass bottom dishes, 35mm MatTek Corperation P35G-1.5-20-C Case
HeLa-RFP-H2B Kind gift of Dr Sabine Elowe, Québec, Canada Klebig C et al. 2009
HEPES Fisher Scientific BP310-1
Horse Serum, New Zealand Thermo Fisher 16050-122
KCl Fisher Scientific BP366-500
L-Glutamine Thermo Fisher 25030081
Lipofectamine® RNAiMAX Transfection Reagent Thermo Fisher 13778-150
Minimal Essential Medium  (MEM) Alpha  Wisent 310-101-CL
Minimal Essential Medium  (MEM) Alpha without Desoxyribonuleosides/Ribonucleosides Thermo Fisher 12000-022
Minimal Essential Medium  (MEM) Alpha without Phenol Red Thermo Fisher 41061-029
Na2HPO4 Biobasic S0404
NaCl Fisher Scientific BP358-10
OptiMEM Thermo Fisher 11058-021
rAVCMV-LifeAct-TagGFP2 IBIDI 60121
siRNA duplexes Dharmacon Custom design
Thymidine Sigma T9250
Trypsine 2.5% Thermo Fisher 15090-046

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