Endocytosis is a fundamental process occurring in all eukaryotic cells. Live cell imaging of endocytosis has helped to decipher many of its mechanisms and regulations. With the pulsed-pH (ppH) protocol, one can detect the formation of individual endocytic vesicles (EVs) with an unmatched temporal resolution of 2 s. The ppH protocol makes use of cargo protein (e.g., the transferrin receptor) coupled to a pH-sensitive fluorescent protein, such as superecliptic pHluorin (SEP), which is brightly fluorescent at pH 7.4 but not fluorescent at pH <6.0. If the SEP moiety is at the surface, its fluorescence will decrease when cells are exposed to a low pH (5.5) buffer. If the SEP moiety has been internalized, SEP will remain fluorescent even during application of the low pH buffer. Fast perfusion enables the complete exchange of low and high pH extracellular solutions every 2 s, defining the temporal resolution of the technique. Unlike other imaging-based endocytosis assays, the ppH protocol detects EVs without a priori hypotheses on the dynamics of vesicle formation. Here, we explain how the ppH protocol quantifies the endocytic activity of living cells and the recruitment of associated proteins in real time. We provide a step-by-step procedure for expression of the reporter proteins with transient transfection, live cell image acquisition with synchronized pH changes and automated analysis. The whole protocol can be performed in 2 d to provide quantitative information on the endocytic process being studied.
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The MATLAB programs that are used to analyze the ppH data are written for MATLAB2018a with the following MATLAB toolboxes: Image Processing, Wavelet, Statistics and Machine Learning; the programs are formatted as a toolbox, scission_analysis, available at MATLAB Central File Exchange as 72744-scission_analysis (https://fr.mathworks.com/matlabcentral/fileexchange/72744-scission_analysis).
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This article is dedicated to Christien James Merrifield (1972–2017), who was instrumental in the development of the ppH protocol. The authors thank Arnaud Rodriguez (Bordeaux Neurocampus) for taking photographs of the imaging and perfusion setup. This work was supported by the Centre National de la Recherche Scientifique (Interface program), the Fondation Recherche Médicale (FRM ING20101221208) and the Agence Nationale pour la Recherche (CaPeBlE ANR-12-BSV5-005 and LocalEndoProbes ANR-17-CE16-0012) to D.P., the FRM, a pre-doctoral fellowship from the University of Bordeaux to M.R. and Labex BRAIN fellowships to M.R. and L.C.
The authors declare no competing interests.
Peer review information Nature Protocols thanks Emanuele Cocucci, Derek Toomre and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key references using this protocol
Taylor, M. J., Perrais, D. & Merrifield, C. J. PloS Biol. 9, e1000604 (2011): https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1000604
Rosendale, M., Jullié, D., Choquet, D. & Perrais, D. Cell Rep. 18, 1840–1847 (2017): https://www.sciencedirect.com/science/article/pii/S2211124717301663
Rosendale, M. et al. Nat. Comm. 10, 4462 (2019): https://www.nature.com/articles/s41467-019-12434-9
a. General view of a setup equipped with local perfusion for the ppH protocol. One can see (1) the peristaltic pump for perfusion of the chamber with a controlled flow, (2) the syringes containing HBS (pH 7.4) and MBS (pH 5.5) solutions (two syringes in the front). The 5 µm pore size filters and stopcocks are visible under the syringes. The two syringes in the back are optional: they may contain a compound to apply to cells. (3) motorized micromanipulator for positioning the application pipette. (4) XY stage of the microscope (Olympus IX71). b. Close up view on the application pipette and open chamber. (1) three way electrovalves (2) pipette holder (3) glass pipette dipped in the solution (open chamber) (4) heated holder with two blue heating elements and (5) in line solution heater for warming up the solution, and (6) suction needle for evacuation of excess solution. c. Diagram of the perfusion setup. For application of HBS/MBS, the stopcocks of the first two syringes are open while the ones of the other two (HBS/MBS+compound, blue) are closed. To apply the compound, the first two stopcocks are closed and the other two are open.
Extended Data Fig. 2 Effects of ill positioned application pipette on the imaging at pH 7.4 and 5.5 during the ppH protocol.
Images of a COS7 cell transfected with TfR-SEP during the ppH protocol under the perfusion of HBS at pH 7.4 (top images) or MBS at pH 5.5 (bottom images). All images are shown with the same scale. In (a), the positioning is optimal. Note the even fluorescence at pH 7.4 which reflects equilibrium (compare with images in transition from one solution to the other, Figure 2b). At pH 5.5, no homogenous fluorescence is visible, only some dots (corresponding to vesicles) are visible. In (b-f), the application pipette was moved in the directions indicated by the bottom diagrams on the three axes. Values are indicated in µm. For these positions, the exchange is not correct. Either the fluorescence of the ‘pH 7.4’ image is too low (d,e) or the fluorescence of the ‘pH 5.5’ image is too high (b,c,f). Scale bar 10 µm.
User Manual for scission_analysis, the MATLAB toolbox described for analysis of ppH experiments and Supplementary Table 1, listing the files generated by scission_analysis and included in the Supplementary Dataset.
Fast imaging (10 Hz) of a HeLa cell transfected with TfR-SEP with exchange of solutions at pH 7.4 and 5.5, played at real time. Scale bar: 10 µm.
TetraSpeck beads imaged at 50 Hz with TIRF illumination (473-nm laser with a predicted penetration depth of 80 nm with a 150×, 1.45 NA Olympus objective). Note that the immobile beads attached to the coverslip are brightly fluorescent. They appear saturated to better see the moving beads in solution. The moving beads appear very transiently as they enter the evanescent field and disappear as they leave it.
092-1_TfR5.stk generated as described in Step 27, accelerated 40 times. Scale bar: 10 µm.
092-1_TfR7.stk generated as described in Step 27, accelerated 40 times. Scale bar: 10 µm.
092-1_dyn5.stk generated as described in Step 27, accelerated 40 times. Scale bar: 10 µm.
092-1_dyn7.stk generated as described in Step 27, accelerated 40 times. Scale bar: 10 µm.
Merged 092-1_TfR5.stk (green) and 092-1_TfR7.stk (magenta) showing the sites of scission that are located at CCS, and therefore appear white.
Raw imaging files and analysis files generated by scission_analysis. The list of files generated and a quick description are provided in Supplementary Table 1.
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Sposini, S., Rosendale, M., Claverie, L. et al. Imaging endocytic vesicle formation at high spatial and temporal resolutions with the pulsed-pH protocol. Nat Protoc 15, 3088–3104 (2020). https://doi.org/10.1038/s41596-020-0371-z