In vitro incubation of a reaction mix that contains labeled endosomes, cytosol and an ATP-regenerating system at physiological temperature leads to both docking and fusion, or sorting and budding of the endosomes (Steps 31–39). Depending on the combination of labeled endosomes used, either docking/fusion or sorting/budding is preferentially visualized. The purpose of the assay is to test the function of different molecules in the docking/fusion or sorting/budding processes. Therefore, inhibitors or activators of different molecules should be tested (see Step 37). Various components can be used, from ion chelators (such as calcium- or magnesium-chelating compounds), chemical inhibitors for different processes (such as actin depolymerizing reagents), antibodies for different receptors or effectors (such as clathrin antibodies) to purified effector proteins (e.g., Rab proteins).
To quantify docking/fusion or sorting/budding, we centrifuged the endosomes onto glass coverslips in which they adsorb and can be imaged by epifluorescence microscopy in different color channels (Steps 40–43). Alternatively, it is possible to attach endosomes to coverslips using other approaches such as dilution in low-melt agarose, which is then allowed to harden on the coverslips. For the docking/fusion assay, double-labeled ('yellow') endosomes are visible after incubation. In contrast, sorting/budding leads to the separation of the two labels, resulting in a decrease of double-labeled endosomes.
To distinguish truly double-labeled (fused) from closely apposed (docked) endosomes, we use the following data analysis procedure (Steps 44–53): for each labeled particle a center of intensity is determined in the respective color channel, thus providing information about its precise localization. The distance between the closest neighboring spots (endosomes) in different colors serves as a readout for docking, fusion, sorting and budding. Note that the ability to determine the distance between the two organelles in two channels (i.e., the ability to determine the precise position of each organelle in its respective color channel) is fully independent of diffraction, unlike the situation in which both organelles are similarly labeled. Therefore, this procedure allows information to be obtained beyond the diffraction limit. To correct for drifts between the images acquired in the different channels, we use multicolored fluorescent beads for alignment. The analysis can be performed using various image analysis programs such as Metamorph (Universal Imaging Corporation), ImageJ (NIH), Matlab (The Mathworks Inc.) or Excel (Microsoft Corporation).
As an important control, the procedure should also be performed in conditions in which only multicolor fluorescent beads are added. This allows the determination of the minimal distance between the centers of intensity of perfectly colocalizing objects—a parameter that may vary between different microscopy setups (see Step 50).
Adult Wistar rats
Caution Experiments involving animals must be carried out in accordance with all appropriate national and institutional regulations.
PC12 cells (clone 251), a similar PC12 line can be obtained commercially from the American Type Culture Collection (ATCC, cat. no. CRL-1721.1)
Dulbecco's modified Eagles's medium (DMEM; Lonza, cat. no. 12-733F)
Glutamine (Lonza, cat. no. 17-605E)
Penicillin-streptomycin (Lonza, cat. no. 17-602E)
Fetal calf serum (FCS Gold; PAA Laboratories, cat. no. A15-649)
Horse serum (Biochrom, cat. no. S9135)
Trypsin/EDTA (Lonza, cat. no. 17-161E)
TetraSpeck microspheres, 0.2 μm (Invitrogen, cat. no. T7280)
Fluorescent cargo (all Invitrogen): transferrin-Alexa 488 (T13342), LDL-DiI (L3482), cholera toxin subunit B-Alexa 647 (C34778), dextran-Alexa 488 (D22910) and dextran-Alexa 594 (D22913)
Protease inhibitors: leupeptin hemisulfate (AppliChem, cat. no. A2183), aprotinin (AppliChem, cat. no. A2132), pepstatin A (Peptide Institute, cat. no. 4039), PMSF (Roth, cat. no. 6367.3), alternatively, Complete EDTA-Free protease inhibitor tablets (Roche, cat. no. 1873580)
OptiMEM (Invitrogen, cat. no. 31985)
Creatine phosphate (Roche, cat. no. 621722)
Creatine kinase (Roche, cat. no. 127566)
ATP (AppliChem, cat. no. A1348)
HEPES (Gerbu Biotechnik, cat. no. 1009)
Magnesium acetate (Merck, cat. no. 1.05819)
1,4-DTT (Roth, cat. no. 6908.2)
Potassium acetate (Merck, cat. no. 1.04936)
Hexokinase (Roche, cat. no. 1426362)
Sodium chloride, NaCl (Merck, cat. no. 1.06404)
Disodium hydrogen phosphate, Na2HPO4 (Merck, cat. no. 1.06580)
Sucrose (Merck, cat. no. 1.07687)
Imidazole (AppliChem, cat. no. A10730)
Bovine serum albumin, BSA (AppliChem, cat. no. A1391)
D-Glucose (Merck, cat. no. 1.08342)
Ethanol (Merck, cat. no. 1.00983)
Sodium hydroxide, NaOH (Merck, cat. no. 1.06498)
Potassium hydroxide, KOH (Merck, cat. no. 1.05033)
Hydrochloric acid, HCl (Merck, cat. no. 1.09057)
Dissection tools
Standard cell culture equipment (laminar flow hoods, incubators, cooling centrifuges, water baths, refrigerator and freezers, cell culture plates and flasks)
Plastic flasks (15-ml and 50-ml tubes and flasks of different sizes) and plastic pipettes (5, 10, 25 and 50 ml)
Critical All steps must be performed in the absence of detergent traces, i.e., using plasticware or using specially cleaned glassware.
Centrifuge, Sorvall RC5B Plus, with a SS-34 rotor and polycarbonate flanged tubes (Sorvall, cat. no. 03146)
Critical Tubes must be free of detergent traces.
Ultracentrifuge, Beckman TL-100, with a TLA-100.3 rotor and polycarbonate tubes (Beckman, cat. no. 349622)
Critical Tubes must be free of detergent traces.
Glass/Teflon Potter Elvehjem Tissue Grinder, 55 ml (OMNI International, cat. no. 07-358054)
Overhead stirrer (Kika Labortechnik, cat. no. RW20 DZM.n)
Ball homogenizer (Isobiotech, cat. no. 202 09 547.9, see EQUIPMENT SETUP)
Disposable syringe, NORM-JECT (1 ml Ins/TBC; Henke-Sass & Wolf)
Critical This should be free of latex and silicon oils.
Refrigerated table-top centrifuge (Eppendorf, cat. no. 5415 R)
Reaction tubes, polycarbonate, 0.2 ml (Beckman, cat. no. 343775)
Lid for reaction tubes: rubber sealing from 1-ml syringes (Dispomed, cat. no. 22009)
12-well plates (Corning Costar, 3513)
18-mm glass coverslips (Marienfeld Superior, cat. no. 0111580)
Plate centrifuge (Heraeus Multifuge 4 KR) with a rotor and inlays for plates (Heraeus HIGHplate windshielded rotors)
Open imaging chamber for 18-mm coverslips (see EQUIPMENT SETUP)
Epifluorescence microscopy setup (see EQUIPMENT SETUP)
Bath sonicator (Bandelin Sonorex RK 100)
Dark (covered) water bath with an agitation device (GFL 1086)
Forceps
Pasteur pipettes (plastic)
Zeiss Axiovert 200M fluorescence microscope (see EQUIPMENT SETUP)
METAMORPH (Universal Imaging Corporation)
Matlab (The Mathworks Inc.)