Abstract
Autophagy, a highly regulated homeostatic degradative process, allows cells to reallocate nutrients from less important to more essential processes under extreme conditions of starvation. Autophagy also prevents the buildup of damaged proteins and organelles that cause chronic tissue damage and disease. Although a topic of great interest with involvement of multiple signaling pathways, there are limitations in real-time detection of the autophagic process. EMD Millipore has developed technologies where prepackaged, ready-to-use, high-titer lentiviral particles, “lentiviral biosensors,” encoding GFP- or RFP-tagged proteins provide a convenient and robust solution for fluorescent imaging of cells undergoing autophagy. Compared to nonviral transfection methods, lentiviral transduction, in many cases, offers higher transfection efficiency and more homogeneous protein expression, particularly for traditionally hard-to-transfect primary cell types. Lentiviral biosensors are ideal for use with fixed and live cell fluorescent microscopy, and are nondisruptive towards cellular function. GFP- or RFP-protein localization matches well with antibody-based immunostaining and demonstrates altered patterns of expression upon treatment with modulators of cell function and phenotype. Lentiviral biosensors provide a broadly effective, convenient method for visualization of cell behavior under a variety of physiological and pathological treatment conditions, in both endpoint and real-time imaging modalities. In this study, we focus on lentiviral biosensors containing GFP-LC3 and RFP-LC3 to study the formation of autophagosomes.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Goldman RD, Swedlow JR, Spector DL (2009) Live cell imaging: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Coutu DL, Schroeder T (2013) Probing cellular processes by long-term live imaging—historic problems and current solutions. J Cell Sci 126:3805–3815
Fein MR, Egeblad M (2013) Caught in the act: revealing the metastatic process by live imaging. Dis Model Mech 6:580–593
Protocol Online. Your Lab’s reference book. http://www.protocol-online.org/
Pauwels K, Gijsbers R, Toelen J, Schambach A, Willard-Gallo K, Verheust C, Debyser Z, Herman P et al (2009) State-of-the-art lentiviral vectors for research use: risk assessment and biosafety recommendations. Curr Gene Ther 9:459–474
Merzlyak EM, Goedhart J, Shcherbo D, Bulina ME, Shcheglov AS, Fradkov AF, Gaintzeva A, Lukyanov KA, Lukyanov S, Gadella TW, Chudakov DM (2007) Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat Methods 4:555–557
Subach OM, Gundorov IS, Yoshimura M, Subach FV, Zhang J, Grüenwald D, Souslova EA, Chudakov DM, Verkhusha VV (2008) Conversion of red fluorescent protein into a bright blue probe. Chem Biol 15:1116–1124
Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 21:5720–5728
Klionsky DJ et al (2008) Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4:151–175
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Long, K. et al. (2015). Analysis of Autophagosome Formation Using Lentiviral Biosensors for Live Fluorescent Cellular Imaging. In: Mor, G., Alvero, A. (eds) Apoptosis and Cancer. Methods in Molecular Biology, vol 1219. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1661-0_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1661-0_12
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1660-3
Online ISBN: 978-1-4939-1661-0
eBook Packages: Springer Protocols