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Assays Measuring Membrane Transport in the Endocytic Pathway
|Assays Measuring Membrane Transport
in the Endocytic Pathway
Significant progress has been made in understanding
mechanisms regulating endocytic membrane
traffic using cell-free assays (Braell, 1987; Davey et al.
1985; Diaz et al.
, 1988; Gruenberg and Howell, 1986;
Woodman and Warren, 1988) (see Fig. 1). Both early
and late endosomes exhibit homotypic fusion properties in vitro
, as in vivo
, yet they do not fuse with each
other (Aniento et al.
, 1993). Transport from early to late
endosomes is achieved by multivesicular intermediates
termed endosomal carrier vesicles (ECV/MVB),
which are presumably translocated on microtubules
between the two compartments (Aniento et al.
Bomsel et al.
, 1990; Gruenberg et al.
, 1989). The vectorial
or heterotypic interactions of ECV/MVBs with late
endosomes have also been reconstituted in vitro
, as has
the involvement of microtubules and motor proteins
in this process (Aniento et al.
, 1993; Bomsel et al.
By reducing the components in these in vitro
a cytosol source, an ATP-regenerating system, salts,
and the purified endosomal membranes, the specificity
of endosomal fusion events has been addressed, and
the molecules and mechanisms involved have been
studied. In fact, a number of conserved molecules, as
well as molecules specific for different steps of the
endocytic pathway, have been identified and/or characterized
using cell-free assays such as those described
in this protocol (for review, see Gruenberg and
|FIGURE 1 Membrane trafficking in the endocytic pathway. The
reconstituted steps of the endocytic pathway described in this protocol
are the (a) fusion of early endosomes with each other, (b) fusion
of ECV/MVBs with late endosomes, and (c) fusion of late endosomes
with each other. An in vitro budding assay for the formation
of ECV/MVBs from early endosomes, which are competent to fuse
with late endosomes, is described in Aniento et al. (1996). As shown,
ECV/MVBs are transported along microtubules from early to late
endosomes. If microtubules are depolymerized in vivo, prior to the
loading of cells with an endocytic tracer, this tracer will accumulate
in ECW/MVBs. These vesicles will then fuse with late endosomes,
loaded with a different marker, in vitro.
This assay for endocytic vesicle fusion is based on
the formation of a complex resulting from a reaction
between two products present in separate populations
of endosomes: avidin and biotinylated horseradish
peroxidase (bHRP). These reaction products can be
internalized into endosomes by fluid phase or
receptor-mediated endocytosis in vivo
. Avidin and a
biotinylated compound are used to provide a fusionspecific
reaction because of the high binding affinity
and low dissociation constant of avidin for biotin. Following
internalization, cells are homogenized and
purified endosomal fractions are prepared, which are
combined in the assay together with cytosol and ATP.
If fusion occurs, a complex is formed between
avidin and bHRP. At the end of the assay, the reaction
mixture is extracted in detergents in the presence of
excess biotinylated insulin as a quenching agent. The
avidin-bHRP complex is then detected by immunoprecipitation
with antiavidin antibodies, and the enzymatic
activity of the bHRP associated with the
immunoprecipitate is quantified. This article describes
techniques for the preparation and partial purification
of three different loaded endosomal fractions from
BHK cells: early endosomes, endosomal carrier vesicles,
and late endosomes. In addition, this article
describes the preparation of the cytosol source used,
as well as the techniques for the fusion assays
II. MATERIAL AND
Standard laboratory rockers for washing cells and a
large 37°C water bath, which can fit a metal plate of
dimensions of 20 × 33 cm, are used. Large rectangular
ice buckets (Cat. No. 1-6030), from NeoLab GmbH, can
also accommodate metal plates of the same dimensions.
Cell scrapers (flexible rubber policemen) with a
silicone rubber piece of about 2cm, cut at a sharp angle, and attached to a metal bar, are made. A standard
low-speed cell centrifuge and Beckman ultracentrifuges
and rotors are used. The refractometer (Cat.
No. 79729) is from Carl Zeiss Inc., and the pump for
collecting sucrose gradients (peristaltic pump P-l) is
from Pharmacia Fine Chemicals. A rotating wheel
(such as Snijders Model 34528) with a speed of about
10 rotations per minute should be used. All tissue
culture reagents, including modified Eagle's medium
(MEM), are from either Sigma Chemical Company
or GIBCO-BRL/Life Technologies. Peroxidase from
horseradish (HRP) (Cat. No. P-8250), ATP (disodium
salt, Cat. No. A-5394), and deuterium oxide (D2
No. D-4501) are from Sigma Chemical Company, Ltd.
Biotinyl-ε-aminocaproic acid N
ester (biotin-X-NHS, Cat. No. 203188) is from
Calbiochem. Avidin (egg white, Cat. No. A-887) is
from Molecular Probes. Creatine phosphate (Cat. No.
621714), creatine phosphokinase (Cat. No. 127566), and
precipitate of yeast hexokinase,
1400U/ml, Cat. No. 1426362] are from Boehringer-
Mannheim GmbH. Protein A-Sepharose beads (Cat.
No. CL-4B) are from Pharmacia. Antiavidin antibodies
are generated by injecting purified avidin into rabbits
and are affinity purified prior to use. Antiavidin antibodies
are also available commercially from several
companies. BCA protein assay reagents (Cat. No.
23223) are from Pierce, and Bio-Rad protein assay
reagents (Cat. No. 500-0006) are from Bio-Rad Laboratories
A. Internalization of Endocytic Markers into
Early Endosomes (EE) from BHK Cells
- Internalization media (IM): MEM containing 10
mM HEPES and 5 mM D-glucose, pH 7.4. Filter sterilize
and store at 4°C.
- Phosphate-buffered saline (PBS): 137mM NaCI,
2.7 mM KCL, 1.5 mM KH2PO4, and 6.5 mM Na2HPO4;
should be pH 7.4. Filter sterilize and store at 4°C.
- Biotinylated horseradish peroxidase: Dissolve 20mg
of HRP in 9.5ml of 0.1M NaHCO3/Na2CO3, pH 9.0,
buffer (make fresh and check pH carefully) in a small
glass Erlenmeyer flask. Dissolve 20mg of biotin-XNHS
in 0.5 ml dirnethylformamide. Mix by adding the
biotin dropwise to the HRP mixture while gently stirring
or shaking the Erlenmeyer and incubate at room
temperature with gentle stirring for at least 45min
(a 50:1 molar excess of biotin is important). Quench
unreacted active groups with 1 ml of 0.2M glycine,
pH 8.0 (use KOH to pH), by adding dropwise while
mixing, and mix for an additional 15 min at room temperature.
Transfer to 4°C. Dialyze the mixture extensively
against PBS-or IM at 4°C (at least four changes
of 200 ml each time). The final dialysis should be in IM.
Measure protein concentration (should be about
2ml/ml) and HRP enzymatic activity (should be
unchanged). Aliquot in sterile tubes, freeze in liquid
N2, and store at -20°C until use. Immediately before
use, thaw quickly and warm to 37°C.
- Avidin: Avidin powder dissolved in IM at 3mg/
ml. Make fresh immediately before use and warm to
- PBS/BSA: 5mg/ml BSA in PBS-. Make fresh
before use and cool to 4°C.
B. Internalization of Endocytic Markers into
Endosomal Carrier Vesicles (ECV) and Late
- Cell culture: Maintain monolayers of baby hamster
kidney (BHK-21) cells as described in Gruenberg et al. (1989). For a fusion assay of 5-10 points, eight petri
dishes (10 cm diameter) should be prepared 16 h before
the experiment: four for preparing bHRP-labeled EEs
and four for preparing avidin-labeled EEs.
- Fluid-phase internalization: Wash each 10-cm dish
of cells twice with 5 ml ice-cold PBS-on ice. This and
other washes on ice to follow are performed most
easily by placing four dishes onto a metal plate in a
large ice bucket on a rocker. After the last wash,
remove PBS and place the dish on a metal plate in
a 37°C water bath. Add at least 3ml/dish bHRP or
avidin solution prewarmed to 37°C. Incubate for
- Washes: From now on, all work should be done
at 4°C or on ice. Return the dishes to the metal plate
in the ice bucket. Remove the avidin or bHRP solution
and wash dishes three times for 5 min with 5 ml icecold
PBS/BSA followed by 2 × 5 min with 5 ml ice-cold
- Homogenization and fractionation: Go directly to
- Nocodazole stock: 10mM in dimethyl sulfoxide
(DMSO), aliquoted, and stored at -20°C.
- IM/BSA: IM containing 2mg/ml BSA. Make fresh
before use and warm to 37°C.
- All solutions listed in Section III,A.
C. Homogenization and Fractionation of Cells
- Cell culture: For a fusion assay of 5-10 points, 10
dishes (10cm) of BHK cells should be prepared as
described in Section III,A, step 1. For ECV-LE fusion
assays, use 5 dishes for bHRP-labeled ECVs and
5 dishes for avidin-labeled LEs, For LE-LE fusion
assays, use 5 dishes for bHRP-labeled LEs and 5 dishes
for avidin-labeled LEs.
- Nocodazole pretreatment for ECV preparation: Intact
microtubules are required for the delivery of endocytosed
markers to the LE. Therefore, markers accumulate
in transport intermediates (ECVs) in the absence
of microtubules. Whereas stable microtubules are cold
sensitive, dynamic microtubules are depolymerized
easily in the presence of nocodazole (Aniento et al.,
1993; Bomsel et al., 1990). In BHK cells, microtubules
can be depolymerized efficiently in the presence of
nocodazole, whereas cold treatment is without effect.
For ECV preparation, depolymerize the microtubules
immediately before the experiment with 10µM nocodazole
at 37°C for 1-2 h in media used to grow cells in
a 5% CO2 incubator. Following this step, nocodazole
(10µM) should remain present in all solutions up to
the homogenization step. For LE preparation, do not
treat with nocodazole or include nocodazole in any
- Fluid-phase internalization: Wash each 10-cm dish
of cells twice with 5 ml ice-cold PBS+/- 10µM nocodazole
on ice, as in Section III,A, step 2. After the last
wash, remove the PBS and place the dish on a metal
plate in a 37°C water bath. Add at least 3 ml bHRP or
avidin solution for making LEs or bHRP + 10 µM nocodazole
for making ECVs. Incubate for 10min.
- Chase: Remove bHRP or avidin and wash twice
quickly at 37°C with 10ml PBS/BSA+/- 10µM nocodazole,
prewarmed to 37°C. Remove last wash, and
add 8ml IM/BSA+/- 10µM nocodazole, prewarmed
to 37°C. Incubate at 37°C (in water bath or in a 37°C incubator without CO2) for 45 min.
- Washes: Remove IM/BSA, move dishes to ice
bucket, and wash 2 × 5min with 5ml cold PBS/BSA
followed by 5 min with 5 ml cold PBS on ice.
- PBS: See Section IIIA.
- 300mM imidazole stock: Dissolve imidazole in
H2O and adjust pH to 7.4 with NaOH, filter sterilize,
and store at 4°C
- Homogenization buffer (HB): Add imidazole from
300mM stock to H2O and dissolve sucrose such that
the final concentrations are 250 mM sucrose and 3 mM imidazole. Filter sterilize and store at 4°C.
- 62% sucrose solution: For 100ml, add 1 ml of imidazole
from 300mM stock to 15ml H2O. Add 80.4g
sucrose and dissolve by stirring at 37°C. Add H2O and
mix until the refractive index is 1.4464.
- 10 and 16% sucrose solutions in D2O: For 100 ml,
add 1 ml imidazole from 300mM stock to 50ml D2O.
For 10% solution, add 10.4g sucrose, and for 16% solution,
add 17.0g sucrose. Dissolve sucrose, add D2O,
and mix until the refractive index is 1.3479 for the 10%
solution and 1.3573 for the 16% solution.
D. Measurement of Latency and Balance
Sheet for Gradients
- Cell scraping: All of the following steps should be
performed on ice or at 4°C. After the last wash, remove
all PBS. Add 2ml/dish PBS and rock the dish so that
cells do not dry. Using a flexible rubber policeman,
scrape round 10-cm dishes by first scraping in a circular
motion around the outside of the dish, followed by
a downward motion in the middle of the dish. Scrape gently in order to obtain "sheets" of cells. Using a
plastic Pasteur pipette, gently transfer the scraped
"sheets" of cells from four or five dishes into a 15-ml
tube on ice.
- Centrifuge at 1200rpm for 5min at 4°C. Gently
- Add 1 ml HB to pellet, using a plastic Pasteur
pipette, gently pipette up and down one time and
add an excess of HB (4-5ml) to change buffer. Centrifuge
again at 2500rpm for 10min at 4°C. Remove
- Homogenization: It is important that cells are
homogenized under conditions where endosomes are
released from cells, yet where latency is high so that
the endosomes are not broken and retain their internalized
marker. First add 0.5 ml HB to the cell pellet.
Using a 1-ml pipetman, gently pipette up and down
until the pellet is resuspended and particles can no
longer be seen by eye. Do not introduce air bubbles.
Using a 22-gauge needle connected to a narrow 1-ml
Tubercutine syringe, prewet the needle and syringe
with HB so that no air is introduced. Insert the needle
into the cell homogenate, slowly pull up on the syringe
until most of the cell homogenate is in the syringe, and
gently expel without bubbles. Repeat this procedure
until plasma membranes are broken, yet nuclear membranes
are not. Monitor homogenization as follows.
Take 3µl of homogenate and place in a 50-µl drop of
HB on a glass slide. Mix and cover with a glass coverslip.
Observe by phase-contrast microscopy, using a
20× objective. Homogenize until unbroken cells are no
longer observed, yet nuclei, which appear as dark
round or oblong structures, are not broken. Usually
between 3 and 10 up-and-down strokes through
the needle are necessary. Centrifuge homogenate at
2000rpm for 10min at 4°C and carefully collect the
postnuclear supernatant (PNS) and nuclear pellet.
- Save a 50-µl aliquot of each PNS fraction for
measuring latency and for calculating the balance
sheet as described in Section III,D. Adjust the sucrose
concentration of the remaining PNS to 40.6% by
adding about 1.1 volume of 62% sucrose solution per
volume of PNS. Mix gently but thoroughly, without
bubbles. Check sucrose concentration using a
- Place adjusted PNS in the bottom of a SW60 centrifuge
tube. On top of the PNS, layer 1.5 ml of 16%
sucrose solution in D2O, followed by 1 ml of 10%
sucrose solution in D2O, and fill tube with HB. Steps
should be layered so that interfaces are clearly seen
and not disturbed. See Gruenberg and Gorvel (1992)
for diagram of gradients.
- Centrifuge gradients in SW60 rotor at 35,000rpm
for 1 h at 4°C.
- Carefully remove the interfaces from the gradients
after centrifugation by first placing gradients in a
test tube rack with a black backdrop. The interfaces
should appear white. The layer of white lipids on top
of the gradient should be removed carefully. Collect
fractions at 4°C using a peristaltic pump at speed 2,
with capillary tubes connected to each end. Place the
outgoing end into a collection tube and collect the top
interface carefully (10%/HB interface = LE + ECV fraction)
first. Collect by holding the capillary tube directly
in the middle of the wide interface and slowly move
in a circular motion until most of the white interface is
collected into the smallest possible volume. Wash the
pump tubing with water and then collect the EE
(16/10%) interface into another tube. Fractions can be
frozen and stored in liquid N2 until use in fusion assays
if they are carefully frozen quickly in liquid N2 and
thawed quickly at 37°C immediately before use.
- HRP stocks: 1-10ng HRP in 0.1ml HB, for
- HB: See Section IIIC.
- HRP reagent: 0.342mM o-dianisidine and 0.003%
H2O2 in 0.05M Na-phosphate buffer, pH 5.0, containing
0.3% Triton X-100. To prepare, use very clean glassware
or plasticware (as in for tissue culture) and mix
12 ml of 0.5M Na-phosphate buffer, pH 5.0 (filter sterilized),
and 6ml of 2% Triton X-100 (filter sterilized)
with 111 ml sterile H2O. Add 13mg o-dianisidine, dissolve
gently, and add 1.2ml 0.3% H2O2 (filter sterilized).
Avoid magnetic stirring. Solution should be
clear. Store at 4°C in the dark.
- 1 mM KCN in H2O
- Protein assay system (such as the BCA protein
assay reagent or the Bio-Rad protein assay system)
E. Preparation of BHK Cell Cytosol
- Load a 20-µl aliquot of bHRP PNS into an airfuge
tube or a small tabletop ultracentrifuge tube of the
Beckman TL-100 type and fill the tube with a known
volme of HB. Mix thoroughly by pipetting without air
bubbles. Centrifuge at 4°C for 20min at 20psi in an
airfuge or at 200,000g for 20min in a tabletop ultracentrifuge
rotor (such as Beckman TLA-100.1). Transfer
the supernatant to another tube. Resuspend the
pellet in 50 µl HB.
- To measure the latency, adjust samples, blanks,
and standards with HB so that the final volume of each
is 0.1 ml. Assay both the pellet and the supernatant of the latency measurement. If the supernatant volume is
over 0.1ml, assay only 0.1ml. Add 0.9ml of HRP
reagent to each tube, mix quickly, and record the time
with a stop clock. Allow color to develop in the dark,
as this reagent is light sensitive. When a brown color
has begun to develop, read the absorbance at 455 nm
and record the time (results expressed as OD units/
min or ng HRP/min). Stop the reaction with 10µl of
1.0 mM KCN if necessary.
- Calculate latency by first adding the value
(OD/min) for HRP in the pellet to that of HRP in the
supernatant (OD/min after correcting for total supernatant
volume). The value for the pellet divided by the
total value is the percentage latency. Latency should be
over 70% in order to measure endosome fusion.
- The amount of HRP in each gradient fraction collected
from the bHRP gradient can be measured by
assaying an aliquot (about 50µl) of each fraction as
described in step 2.
- Measure the amount of protein in each gradient
fraction using a standard protein assay system, as
described in the manual.
- Calculate percentage yield (percentage of HRP in
each fraction compared to total amount of HRP in
PNS), specific activity (SA) (HRP activity per unit
protein), and relative specific activity (RSA) (divide
specific activity of each fraction by the specific activity
of the PNS). See Gruenberg and Gorvel (1992) for an
example of a typical balance sheet.
- PBS-: See Section IliA.
- HB: See Section IIIC.
- HB + protease inhibitors: HB with the following
protease inhibitors added immediately before use:
10µM leupeptin, 1 µM pepstatin A, 10ng/ml aprotinin,
and, if needed, 1 µm phenylmethylsulfonyl
Two possible cytosol sources for all of the assays
described are BHK and rat liver cytosol. For rat liver
cytosol preparation, refer to Aniento et al.
F. Preparation of Antiavidin Beads for the in
Vitro Fusion Assay Described in Section IIIG
- BHK cells, maintained as described in Section
IIIA, should be plated approximately 16h before the
experiment. Large (245 x 245 x 25 mm) square dishes
are convenient for large cytosol preparations.
- All steps should be performed on ice or at 4°C. Wash dishes four times with excess PBS (50 ml per dish
for large square dishes).
- Remove PBS from the last wash, add 12ml
PBS per dish, and rock the dish so that cells do not
dry. Scrape cells with a rubber policeman using firm,
downward motions, going from top to bottom while
holding the plate at an angle, as described in Section
IIIC, step 1.
- Collect scraped cells into 15-ml tubes (one tube
per dish). Centrifuge at 1200rpm for 5 min at 4°C.
- Remove supernatant and gently add 5ml HB
with a plastic Pasteur pipette and pipette up and down
- Centrifuge at 2500 rpm for 10 min at 4°C. Remove
supernatant and resuspend pellet in 1.2ml HB +
protease inhibitors. Separate into two tubes (about
0.7ml/tube) for homogenization and homogenize
as described in step 4 of Section IIIC.
- Centrifuge at 2500rpm for 15min at 4°C. Add
supernatant (PNS) to a centrifuge tube for the TLS-55
rotor (for the Beckman TL-100 tabletop ultracentrifuge)
and centrifuge in TLS-55 for 45 min at 55,000 rpm
at 4°C. Remove fat from the top using an aspirator.
Transfer supernatant (cytosol fraction) to a new tube
without disturbing the pellet. Determine the protein
concentration of supernatant. Cytosol should be at
least 15 mg/ml to give a good signal for fusion assays.
Aliquot on ice, freeze quickly, and store in liquid N2 until use.
- PBS/BSA: Dissolve 5 mg/ml BSA in PBS. Filter sterilize
and store at 4°C.
- Sterile PBS: PBS as described in Section III,A, filter
sterilize or autoclave, and store at 4°C.
- Antiavidin antibody: Affinity purify and store
aliquoted in 50% glycerol/PBS at -20°C.
To determine how many antiavidin beads to prepare,
first determine the number of fusion assay
points. From a typical gradient (see Gruenberg and
Gorvel, 1992) about 150µg of EE and 70µg of ECV or
LE are obtained. Optimal amounts of endosomes to
use for fusion assays are 20µg of each EE fraction and
10µg of each ECV or LE fraction. Therefore, a typical
experiment (one gradient each of avidin and bHRPlabeled
fractions) will provide enough endosomes for
about seven fusion assay points.
G. in vitro Assay of Endocytic
- Swell 1.5 g of protein A-Sepharose beads in 10 ml
sterile H2O at room temperature overnight.
- Wash beads three times in 10ml sterile PBS
by centrifuging beads in 15-ml tubes at 3000rpm for
2 min, resuspending in PBS each time.
- After the final wash, resuspend beads in an equal
volume of sterile PBS per volume of packed beads.
Store beads this way up to several months at 4°C.
- One hundred microliters of this 1:1 slurry is
required per fusion assay point. Therefore, for 10 assay
points, block 1 ml of beads by washing 3× in 10ml
PBS/BSA, as described in step 2.
- After final wash, resuspend beads in 10 ml PBS/
BSA. For 10 assay points, add 50 µg of antiavidin antibody
(5µg per 100-µl beads). Rotate tube for at least
5 h at 4°C.
- Wash beads four times in PBS/BSA. After the last
wash, for 10 assay points, resuspend beads in 10ml
- Aliquot 1 ml to each of 10 labeled Eppendorf
tubes. Centrifuge in Eppendorf centrifuge at maximum
speed for 2 min. Remove supernatant. Beads are
now ready for the immunoprecipitation step of the
fusion assay (Section III,G, step 10).
- 50× salts: 0.625M HEPES, 75mM Mg-acetate,
50mM dithiothreitol, pH 7, with KOH. Filter sterilize,
aliquot, and store at -20°C.
- K-acetate (KOAc stock): 1M in H2O. Filter sterilize,
aliquot, and store at -20°C. Note: Depending on the
counterion requirement of the experiment, KOAc must
be replaced by KCl (see Aniento et al., 1993).
- Biotinylated insulin: 1 mg/ml in H2O. Store at 4°C.
- AT?-regenerating system (ATP-RS): Mix 1:1:1
volumes of the following immediately before use.
- 100 mM ATT: Dissolve in ice-cold H2O, titrate
to pH 7.0 with 1M NaOH, filter sterilize,
aliquot on ice, and store at -20°C.
- 800 mM creatine phosphate: Dissolve in ice-cold
H2O, filter sterilize, aliquot on ice, and store at
- 4 mg/ml creatine phosphokinase: To make 4 ml,
add 80 p\ of 0.5M NaHPO4 buffer, pH 7.0, to
1.6 ml H2O on ice. When cool, add 16 mg creatine
phosphokinase. Vortex until dissolved.
Add 2.3 ml ice-cold 87% glycerol. Vortex until
well mixed. Aliquot on ice and store at -20°C.
- Hexokinase: Vortex the suspension, pipette the
desired amount (e.g., 10µg-0.1mg for one assay
point), centrifuge for 2 min in Eppendorf at maximum
speed, and aspirate supernatant. Dissolve pellet in the same volume of 0.25M D-glucose. Prepare immediately
- TxlOO stock: 10% stock of Triton X-100 in H2O
- PBS/BSA and sterile PBS: See Section IIIE
- HEP reagent: See Section IIID.
- PBS/BSA/TxlOO: PBS/BSA containing 0.2%
Triton X-100, make immediately before use
- For each fusion assay, at least three points should
be included:mATP, +ATP, and the total. To determine
fusion efficiency, determine the total (maximal possible
fusion value) by mixing 50 //I of each endosomal
fraction in an Eppendorf tube on ice. Add 25 µl Txl00
stock and vortex well. Leave on ice at least 30min and
add PBS/BSA and continue as described in step 9.
- For all other fusion assay points, 3 µl of 50× salts,
8 µl of biotinylated insulin, and 11 µl of KOAc stock are
needed for each point. Make a mixture of these three
components by multiplying the number of assay
points by 3, 8, and 11 and mix the respective amounts
of each component together in one tube. Number
Eppendorf tubes for the appropriate number of assay
points and put them on ice. Add 22µl of the aforementioned
mixture to each tube.
- Add 50µl (750µg-1 mg) of cytosol to each tube
- Add either 5 µl of ATP-RS or 10 µl of hexokinase
to each tube, as appropriate.
- Add 50 µl (7-25 µg) of bHRP-labeled endosomes
and 50µl (7-25µg) of avidin-labeled endosomes to
each tube. Endosomal fractions from the gradients can
be diluted in HB prior to this step, if desired. Mix
gently; avoid introducing air bubbles. Leave tubes on
ice for 3 min.
- Transfer tubes to 37°C for 45 min. Avoid agitation
during this time.
- Return tubes to ice. Add 5µl of biotinylated
insulin to each tube and mix.
- Add 25 µl of Txl00 stock and vortex well. Leave
tubes on ice for 30min.
- Add 1 ml of sterile PBS/BSA to each tube and
- Centrifuge for 2min at maximum speed in an
Eppendorf centrifuge. Transfer supernatants to numbered
tubes containing antiavidin beads, prepared as
in Section IIIE
- Rotate beads for at least 5 h at 4°C.
- Centrifuge in Eppendorf centrifuge at maximum
speed for 2min. Remove supernatant and wash
four times with PBS/BSA/Txl00. Wash once with
- Remove final supernatant and add 900 //I of
HRP reagent to each tube. Allow color to develop in the dark at room temperature. Vortex periodically for
2-3 h or put tubes on rotating wheel in the dark at
room temperature while color develops.
- Centrifuge tubes for 2min in Eppendorf centrifuge.
Measure the absorbance of the supernatants at
Refer to Gruenberg and Gorvel (1992) for an
example of a typical balance sheet for the sucrose gradient
fractionation step. Typical results for fusion
assays are shown in Fig. 2.
Highly purified loaded endosomes can be prepared
by immunoisolation as described in Howell et al.
(1989). Immunoisolated endosomes can then be used
in the fusion assays described in Section IIIG. See
Gruenberg and Gorvel (1992) and Howell et al.
ECV-LE fusion is stimulated by the addition of
polymerized microtubules to the fusion assay. Endogenous
microtubules can be polymerized by adding
taxol to the fusion assay. The preparation of
microtubules is described in the article by Ashford and
Hyman. See Fig. 2 and Aniento et al.
(1993) for more
details on the effects of microtubules and MAPs on
|FIGURE 2 Typical fusion assay results. Fusion efficiency is
expressed as a percentage of total fusion between each set of endosomal
membranes. Total, or maximal, endosome fusion is measured
by mixing bHRP and avidin-containing endosomal fractions together
in the presence of detergent, followed by immunoprecipitation
with antiavidin antibodies and HRP determination. Typical
"total" values (measured as absorbance at 455 nm) are in the range
of 0.6-1.0 ∧455 units for EE fusion assays and 0.3-0.7 ∧455 units for
ECV and LE fusion assays. As a control for nonspecific reactions, the
assay is typically carried out without ATP (-ATP). As shown, the
polymerization of endogenous tubulin present in the cytosol in
the presence of taxol is sufficient to facilitate interactions between
ECVs and LEs (see Aniento et al., 1993).
For ECV and LE preparations, cells should be
homogenized until vesicles are no longer seen around
the periphery of nuclei. If nuclei begin to aggregate
during homogenization, however, this is a sign that
some are broken as free DNA causes aggregation.
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