体外荧光法检测核内体早期动力学-丁香实验

A fluorescence-based in vitro assay for investigating early endosome dynamics

Sina V Barysch^{1 ,} ² , Reinhard Jahn¹ & Silvio O Rizzoli²

ABSTRACT

Early endosomes receive material from the plasma membrane by fusion with endocytotic vesicles. This material is sorted within endosomes and directed to subdomains at which carrier vesicles bud. These vesicles are then transported toward different cellular destinations. In this article, we describe a protocol for the cell-free reconstitution of endosome docking/fusion and sorting/budding, which is based on labeling of endosomes by endocytotic uptake with fluorescent cargoes. The protocol includes (i) the preparation of fluorescently labeled endosomes, (ii) assays for docking/fusion and for sorting/budding in vitro and (iii) imaging of the reaction mix by fluorescence microscopy to quantify docking, fusion, cargo sorting and budding using counting of single organelles. Production of endosome stocks requires approximately 1 d. The in vitro reactions can then be performed separately (~1 d) and are conveniently carried out with multiple samples in parallel. The assay can be adapted for studying the dynamics of organelles other than endosomes.

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Procedure

Overview

Steps 1 - 8 Preparation of rat brain cytosol
Steps 9 - 10 Culture of PC12 cells
Steps 11 - 30 Preparation of postnuclear supernatants
Steps 31 - 39 Preparation of docking/fusion or sorting/budding reactions
Steps 40 - 43 Imaging of the samples
Steps 44 - 53 Data analysis

Points from here (point 1) up to and including point 8 are related to Preparation of rat brain cytosolTiming : 4�5 h Prepare sucrose buffer with protease inhibitors (1 μg ml⁻¹ pepstatin A (1:1,000 vol/vol of pepstatin A stock solution) and 0.2-mM PMSF (1:1000 vol/vol of PMSF stock solution)) for about 5 ml per rat brain.
Critical step Stir the buffer thoroughly while slowly adding PMSF stock solution, as PMSF might precipitate otherwise.
Kill 10�40 rats and remove their brains into a beaker with sucrose buffer (without protease inhibitors, kept on ice).
Caution Follow national and institutional guidelines for animal handling.
Wash brains 2�3 times with ice-cold sucrose buffer (without protease inhibitors) to remove most of the blood remaining in the solution.
Add 10 brains into the Glass/Teflon Potter Elvehjem Tissue Grinder, add about 5 ml ice-cold sucrose buffer (with protease inhibitors, from Step 1) per brain and homogenize brains by 10 strokes (one stroke consists of one up and down movement) at 900 r.p.m. with an overhead stirrer.
Caution Do not apply too much pressure during the first stroke, as the first homogenization of brains is difficult and should be performed slowly; the glass homogenizer might break otherwise.
Centrifuge homogenate at 3,000g (5,000 r.p.m. in a Sorvall SS-34 rotor) for 10 min at 4 °C. Transfer the supernatant into a fresh tube and discard the pellet.
Centrifuge the supernatant at 32,000g (16,500 r.p.m. in a Sorvall SS-34 rotor) for 15 min at 4 °C. Transfer the supernatant into a fresh tube and discard the pellet.
Centrifuge the supernatant at minimum 100,000g (we use 330,000g (90,000 r.p.m. in a Beckman 100.3 rotor)) for 30 min at 4 °C to obtain the cytosol in the supernatant.
Transfer the cytosol-containing supernatant into a fresh tube and divide into small aliquots (0.3�1 ml), determine the protein concentration using a standard assay¹⁶ , snap-freeze the aliquots in liquid nitrogen and store them at −80 °C. The protein concentration of the cytosol concentrations should be in the range between 6 and 10 mg ml⁻¹ .Pause Point If stored at −80 °C, rat brain cytosol can be used for at least 12 months.
Points from here (point 9) up to and including point 10 are related to Culture of PC12 cellsTiming : 30 min Split PC12 cells 1:6 into 6�30 plates (∅ 15 cm) using standard procedures for passaging PC12 cells (or any other cell line)¹⁷ .
Critical step Dissociate the cells thoroughly, as otherwise clumps may remain that may lead to inefficient cargo uptake. We resuspend the cell pellet in 5 ml fresh PC12 culture medium and break cell aggregates by passing the cells 20 times though a 200-μl pipette tip attached to a 10-ml plastic pipette.
Allow cells to grow for several days until they reach 80% confluence.
Critical step Cells should reach a high degree of confluence; otherwise, it is difficult to detach them from the plates.
Points from here (point 11) up to and including point 30 are related to Preparation of postnuclear supernatantsTiming : 3�5 h Prepare PBS-BSA (freshly, 30 ml for each labeled PNS) and store on ice, together with thawed aliquots of internalization medium (15 ml per PNS), homogenization buffer (50 ml per PNS) and PBS (10 ml per PNS).
Prewarm trypsin/EDTA and PBS (37 °C), but store PC12 culture medium in the refrigerator.
Remove old medium from 6�12 plates of cells and wash each plate with 5 ml PBS.
Add 2 ml trypsin/EDTA per plate and incubate at room temperature with strong shaking.
Critical step Cells should be exposed to trypsin/EDTA only until they start detaching from the plates. Longer exposure to trypsin/EDTA might cause the degradation of receptors that are required for the uptake of fluorescent cargoes.
Stop the reaction with 5 ml cold PC12 medium per plate, collect the cells from the plates (using a 10-ml plastic pipette) and transfer them into 50-ml plastic tubes.
Critical step Cells need to be detached, which may require mechanical force such as repetitively squirting of the medium onto the plate. This procedure is easier when longer trypsin/EDTA incubations have been used (Step 14); it is more efficient in highly confluent cultures (Step 10).
Leave tubes on ice and repeat Steps 13�15 for the next 6�12 plates of cells.
When all cells are collected, centrifuge all tubes at 250g for 5 min at 4 °C.
Discard the supernatant and resuspend the cell pellet in 10 ml of ice-cold PBS and transfer to a 15-ml plastic tube. Centrifuge again at 250g for 5 min at 4 °C.
Discard the supernatant and resuspend the cell pellet in 10 ml ice-cold internalization medium and centrifuge the cells as before (250g , 5 min, 4 °C). If more than one PNS label is needed (e.g., for docking/fusion assay) and two differently labeled PNS fractions are required, resuspend the cell pellet in 20 ml ice-cold internalization medium and divide into equal amounts in two 15-ml plastic tubes before centrifugation (250g , 5 min, 4 °C), for the separate PNSs.
Estimate the volume of the resulting cell pellet(s).
Critical step This is just an approximation but is necessary for calculating the amounts of fluorescent cargoes to be added. The volume should be in the range of 0.5�3 ml.
Remove supernatants and warm the pellets at 37 °C for 5 min in a water bath.
Add fluorescent cargoes in uptake medium to the cell pellets, as described in the options below, and incubate for 5 min at 37 °C. The medium should have the same volume as the estimated cell pellet(s) and should thus show cargo concentrations twice as high as finally desired (e.g., to label 1 ml cell pellet with 50 μg ml⁻¹ fluorescent transferrin, 1 ml uptake medium with a concentration of 100 μg ml⁻¹ transferrin is required). The cargo composition is dependent on whether the docking/fusion assay (A) or the sorting/budding assay (B) should be used as follows:
1. Docking/fusion assay
  1. Incubate one set of cells with 500 μg ml⁻¹ dextran Alexa 488, 10 kDa (prepared at 1 mg ml⁻¹ in internalization medium).
  2. Incubate a second set of cells with 500 μg ml⁻¹ dextran Alexa 594, 10 kDa (prepared at 1 mg ml⁻¹ in internalization medium).
2. Sorting/budding assay
  1. For dual labeling with transferrin and LDL: simultaneously incubate cells with 50 μg ml⁻¹ transferrin-Alexa 488 and 3 μg ml⁻¹ LDL-DiI (prepared at 100 μg ml⁻¹ transferrin and 6 μg ml⁻¹ LDL in internalization medium).
    Critical step LDL easily oxidizes, which affects the uptake efficiency. The exact concentration may need to be adjusted, with concentrations of up to 10 μg ml⁻¹ being needed after several weeks of storage. To delay the oxidation process, it is recommended to keep the LDL-DiI stock solution (Invitrogen) always sealed with parafilm.
  2. For triple labeling with transferrin, LDL and cholera toxin, incubate cells with 50 μg ml⁻¹ transferrin-Alexa 488, 3 μg ml⁻¹ LDL (both as above) and 3 μg ml⁻¹ cholera toxin subunit B-Alexa 647 (i.e., a concentration of 6 μg ml⁻¹ for the uptake medium). Note that any other combinations of labeled endocytotic cargoes can be used.
Stop the uptake reaction by chilling the cells on ice, centrifuge them at 250g for 5 min at 4 °C and remove the uptake medium.
Wash the cell pellet(s) three times with 10 ml ice-cold PBS-BSA; centrifuge after each wash at 250g for 5 min at 4 °C.
Prepare 30�50 ml homogenization buffer with protease inhibitors by adding PMSF, pepstatin A, leupeptin and aprotinin (1:1,000 (vol/vol) of the respective stock solutions).
Wash the cell pellet(s) once with ice-cold homogenization buffer and resuspend the pellet(s) in ice-cold homogenization buffer with protease inhibitors (Step 25).
Critical step Use no more than 4× the volume of the cell pellet or the PNSs will be too dilute.
Crack the cells with a ball homogenizer on ice. Use 1-ml disposable syringes for passing 1 ml of resuspended cells 10�20 times through the precooled ball homogenizer. Repeat the procedure, depending on the volume of cells.
Critical step Before cell cracking, wash the assembled ball homogenizer thoroughly with homogenization buffer (with protease inhibitors, Step 25) to remove air bubbles that would cause damage to the endosomes.
Transfer the cell homogenate into 1.5-ml tubes and centrifuge at 1,200g for 15 min at 4 °C. This will lead to a separation of the homogenate into two layers: the bottom fraction is the nuclear pellet and the upper fraction is the PNS.
Critical step It is sometimes difficult to see the separation of the two layers properly. Use a bright light source (lamp or bright window) to better illuminate the samples. If no band is visible, further dilute the samples with ice-cold homogenization buffer (with protease inhibitors, Step 25) and repeat the centrifugation process.
Remove the supernatant fractions into a new tube and determine the protein concentration of the PNS using a standard method¹⁶ . The PNS concentration is usually between 10 and 16 mg ml⁻¹ .Troubleshooting
Prepare small aliquots (100�400 μl), snap-freeze in liquid nitrogen and store at −80 °C.Pause Point If stored at −80 °C, PNS fractions can be used for at least 12 months.
Points from here (point 31) up to and including point 39 are related to Preparation of docking/fusion or sorting/budding reactionsTiming : 2�4 h Thaw aliquots of PNS (from Step 30), cytosol (from Step 8), DHM buffer, KAc buffer, ATP solution, CP solution, CK solution and homogenization buffer.
Critical step Be careful to thaw PNS and cytosol slowly on ice to avoid unwanted protein degradation or breaking of organelles. This might require up to 30 min, depending on the size of the aliquots.
Critical step Store aliquots of PNS in the dark to avoid bleaching of the fluorescent cargoes.
Sonicate multifluorescent TetraSpeck beads for 2�5 min in the bath sonicator and prepare a 1:1,000 (vol/vol) bead stock solution in PBS (2-μl beads in 2 ml PBS). This can be kept at 4 °C for up to 2 weeks.
Wash 18-mm glass coverslips in 100% ethanol and place them into 12-well tissue culture plates.
Dilute bead stock solution (from Step 32) 1:100 (vol/vol) in PBS to obtain the final bead solution and pipette 1 ml of it into each well. Remove the air bubbles that might be present between the plate and the coverslip to avoid coverslips floating on centrifugation.
Centrifuge plates at 5,880g for 45 min at 4 °C (we use 5,460 r.p.m. in a Heraeus Multifuge); this fixes the beads onto the coverslips. Do not remove the solution after centrifugation.
Prepare cytosol mix in the following order on ice, depending on whether an ATP-regenerating system (A) or an ATP-depleting system (B) is required, according to whether a positive control (in presence of ATP) or a negative control (in absence of ATP) is desired:
1. ATP-regenerating system
  1. Prepare cytosol mix (volumes correspond to one reaction of 50 μl, scale up accordingly): 100 μg cytosol (usually 10�17 μl, depending on the concentration), 0.9 μl DHM buffer, 2.25 μl KAc buffer, 1.67 μl ATP solution, 1.67 μl CP solution and 1.67 μl CK solution.
    The final concentration of the reaction corresponds therefore to 100 μg cytosol, 11.25 mM HEPES, 1.35 mM magnesium acetate, 0.18 mM DTT, 45 mM potassium acetate, 3.3 mM ATP, 26.7 mM creatine phosphate and 6.7 μg creatine kinase (=5.3 U).
2. ATP-depleting system
  1. Centrifuge 5 μl hexokinase (1,500 U ml⁻¹ , supplied as ethanol precipitate) at 16,000g for 2 min at 4 °C.
  2. Remove the supernatant and resuspend the hexokinase pellet in 5 μl glucose solution (250 mM).
  3. Cytosol mix (volumes correspond to one reaction of 50 μl; scale up accordingly): 100 μg cytosol (usually 10�17 μl, depending on the concentration), 0.9 μl DHM buffer, 2.25 μl KAc buffer and 5 μl hexokinase-glucose buffer (see above).
    
    The final concentration of the reaction therefore corresponds to 100 μg cytosol, 11.25 mM HEPES, 1.35 mM magnesium acetate, 0.18 mM DTT, 45 mM potassium acetate, 7.5 U hexokinase and 25 mM glucose.
Prepare docking/fusion (A) or sorting/budding (B) reactions in 200 μl polycarbonate centrifuge tubes on ice.
Critical step Pipette quickly to avoid warming of the solutions. Certain processes (e.g., docking or aggregation) might take place even during short pipetting times.
1. Docking/fusion reaction
  1. Add cytosol mix (from Step 36, usually between 18 and 25 μl).
  2. Add chemicals, inhibitory/modulating proteins, peptides, antibodies (optional; see Experimental design for further information).
  3. Add 100 μg PNS containing dextran-Alexa 488 and 100 μg PNS containing dextran-Alexa 594 (usually between 5 and 8 μl each).
  4. Adjust to 50 μl with homogenization buffer.
2. Sorting/budding reaction
  1. Add cytosol mix (from Step 36, usually between 18 and 25 μl).
  2. Add chemicals, inhibitory/modulating proteins, peptides, antibodies (optional; see Experimental design for further information).
  3. Add 200 μg PNS containing transferrin-Alexa 488 and LDL-DiI (or other double- or triple-labeled PNS) (usually between 10 and 15 μl).
  4. Adjust to 50 μl with homogenization buffer.
Close reaction tubes with a lid and incubate reactions for 45 min at 37 °C with a slow agitation (speed: 20 shakes per minute in the GFL 1086 water bath). Leave controls on ice for 45 min.
Critical step Samples should be kept in the dark to avoid bleaching the fluorescent dyes.
Place the samples on ice for some minutes to cool, pipette 7 μl of each reaction onto the bead-containing solution on the coverslips (from Step 35) and centrifuge the 12-well plates at 5,880g (5,460 r.p.m. in a Heraeus Multifuge) for 45 min at 4°C.
Points from here (point 40) up to and including point 43 are related to Imaging of the samplesTiming : 0.5�6 h Place one coverslip into the open imaging chamber for 18-mm coverslips, cover it with PBS and focus the samples.
Acquire images in the channels of the respective label (Alexa 488, DiI, Alexa 594, Alexa 647, depending on the PNS fractions that are used) and in one additional channel in which only fluorescent beads can be visualized (e.g., blue) with an epifluorescence microscope (see EQUIPMENT SETUP ).
Critical step Be careful with bleedthrough between the different channels and adjust the exposure times to minimize it. Troubleshooting </, , body <>

体外荧光法检测核内体早期动力学

A fluorescence-based in vitro assay for investigating early endosome dynamics

ABSTRACT

Introduction

Introduction

Experimental design

Figure 1: Experimental design of the in vitro endosome docking/fusion and sorting/budding assay.

Materials

REAGENTS

EQUIPMENT

REAGENT SETUP

PC12 culture medium

Sucrose buffer

Homogenization buffer

PMSF stock solution

Pepstatin A stock solution

Leupeptin stock solution

Aprotinin stock solution

PBS

PBS-BSA

DHM buffer

KAc buffer

ATP solution

CP solution

CK solution

Glucose solution

Internalization medium

EQUIPMENT SETUP

Epifluorescence microscopy setup

Ball homogenizer

Open imaging chamber for 18-mm coverslips

Procedure

Overview