Abstract
Pulse shape analysis (PulSA) is a flow cytometry-based method that involves the measurement of the pulse width and height of a fluorescently labeled molecule simultaneously, enabling a multidimensional analysis of protein localization in a cell at high speed and throughput. We have used the method to detect morphological changes in organelles such as Golgi fragmentation, track protein trafficking from the cell surface, and also discriminate cells with different target protein localizations such as the Golgi, lyso-endosomal network, and the plasma membrane. Here, we describe the basic experimental setup and analytical methods for performing PulSA to examine membrane trafficking processes. We illustrate in particular the application of PulSA for monitoring the trafficking of the membrane-bound enzyme furin and morphological changes to the Golgi caused by Brefeldin A.
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References
Abraham VC, Taylor DL, Haskins JR (2004) High content screening applied to large-scale cell biology. Trends Biotechnol 22:15–22
Wersto RP, Chrest FJ, Leary JF, Morris C, Stetler-Stevenson MA, Gabrielson E (2001) Doublet discrimination in DNA cell-cycle analysis. Cytometry 46:296–306
Kang K, Lee SB, Yoo JH, Nho CW (2010) Flow cytometric fluorescence pulse width analysis of etoposide-induced nuclear enlargement in HCT116 cells. Biotechnol Lett 32:1045–1052
Ramdzan YM, Polling S, Chia CP, Ng IH, Ormsby AR, Croft NP, Purcell AW, Bogoyevitch MA, Ng DC, Gleeson PA, Hatters DM (2012) Tracking protein aggregation and mislocalization in cells with flow cytometry. Nat Methods 9:467–470
Chia PZ, Ramdzan YM, Houghton FJ, Hatters DM, Gleeson PA (2014) High-throughput quantitation of intracellular trafficking and organelle disruption by flow cytometry. Traffic 15:572–582
Thomas G (2002) Furin at the cutting edge: from protein traffic to embryogenesis and disease. Nat Rev Mol Cell Biol 3:753–766
Chia PZ, Gasnereau I, Lieu ZZ, Gleeson PA (2011) Rab9-dependent retrograde transport and endosomal sorting of the endopeptidase furin. J Cell Sci 124:2401–2413
Klausner RD, Donaldson JG, Lippincott-Schwartz J (1992) Brefeldin A: insights into the control of membrane traffic and organelle structure. J Cell Biol 116:1071–1080
Ramdzan YM, Wood R, Hatters DM (2013) Pulse shape analysis (PulSA) to track protein translocalization in cells by flow cytometry: applications for polyglutamine aggregation. Methods Mol Biol 1017:85–93
Acknowledgments
This work was supported by funding from the National Health and Medical Research Council of Australia and the Australian Research Council.
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Toh, W.H., Houghton, F.J., Chia, P.Z.C., Ramdzan, Y.M., Hatters, D.M., Gleeson, P.A. (2015). Application of Flow Cytometry to Analyze Intracellular Location and Trafficking of Cargo in Cell Populations. In: Tang, B. (eds) Membrane Trafficking. Methods in Molecular Biology, vol 1270. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2309-0_17
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DOI: https://doi.org/10.1007/978-1-4939-2309-0_17
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