Use of Brain Cytosolic Extracts for
Studying Actin-Based Motility of Listeria monocytogenes
Cell motility is essential for numerous biological
events. Unicellular organisms, for instance, use
directed movement to find and ingest food. In multicellular
organisms, cell motility is required for the
morphogenetic movements that accompany embryogenesis,
fibroblast migration during wound healing,
and the chemotactic movement of immune cells
during an immune response. Cell motility is characterised
by the formation of cellular extensions that,
depending on their morphology and cellular context,
are called lamellipodia, ruffles, or filopodia. In recent
decades many cell biological, biochemical, and biophysical
studies have established that the formation of
these structures depends on the activity of the actin
cytoskeleton and its associated proteins. More specifically,
the assembly of a network of actin filaments at
the leading edge of motile cells provides the propulsive
force for the extension of these structures (see
Small et al.
, 2002). Despite much effort, however, the
complexity of cell motility has precluded the detailed
analysis of the molecular mechanisms and components
that govern this process.
Since the mid-1980s, much work focused on the
intracellular actin-based motility of the gram-positive
bacterium Listeria monocytogenes
its own uptake by phagocytic and nonphagocytic
cells and, once free in the cytoplasm, recruits host cell
cytoskeletal components, which are then rearranged
into phase-dense actin tails. The assembly of actin
monomers at the actin filament (+) ends abutting
the bacterial surface provides the propulsive force
that allows Listeria
to move within the infected cells
and spread to adjacent cells while avoiding exposure
to the host's humoral immune system. As these
bacteria imitate the protrusive behaviour of
lamellipodial edges, Listeria
motility is considered a
simplified model system for actin filament dynamics
during cell motility (Cossart and Bierne, 2001;
Frischknecht and Way, 2001). As one approach towards
defining the molecular basis of bacterial motility,
we and others have developed simple in vitro
that support actin-based Listeria
motility based on Xenopus
, platelets, and mouse brain extracts (Theriot et al.
, 1994; Marchand et al.
, 1995; Laurent and
Carlier, 1998; Laurent et al.
, 1999; May et al.
These cell-free systems in combination with bacterial
genetics and cell biological studies have
been essential for the characterisation of two key regulators
of actin cytoskeleton dynamics: Ena/VASP
proteins and the Arp2/3 complex (Pistor et al.
2000; Smith et al.
, 1996; Niebuhr et al.
, 1997; May et al.
1999; Skoble et al.
, 2000, 2001; Geese et al.
They also provided the basis for further development
of in vitro
motility systems that culminated in the
reconstruction of bacterial motility using a limited
set of purified proteins (Loisel et al.
, 1999). This
article describes procedures for the preparation
and use of mouse brain extracts for studying Listeria
II. MATERIALS AND
Calcium chloride (Cat. No. 102378), magnesium
chloride hexahydrate (Cat. No. 105832), potassium
chloride (Cat. No. 4936), HEPES (Cat. No. 10110),
sucrose (Cat. No. 1.07654), sodium chloride (Cat. No.
1.06404), disodium hydrogen phosphate dihydrate
(Cat. No. 1.06580), sodium dihydrogen phosphate
hydrate (Cat. No. 6346), and paraffin (Cat. No. 1.07160)
are from Merck. EGTA (Cat. No. E-3889), methylcellulose
(Cat. No. M-0555), ATP (Cat. No. A-2383), erythromycin
(Cat. No. E-6376), and lanoline (Cat. No.
L-7387) are from Sigma. Chymostatin (Cat. No. 17158),
leupeptin (Cat. No. 51867), pepstatin (Cat. No. 52682),
PEFABLOCK (Cat. No. 31682), aprotinin (Cat. No.
13178), and creatine kinase (Cat. No. 127566) are from
Boehringer-Ingelheim. Dithiothreitol (DTT, Cat. No.
43815), Tris-HCL (Cat. No. 93363), and glycerol (Cat.
No. 49770) are from Fluka. Cell-Tak (Cat. No. 354241)
is from BD Biosciences. Creatine phosphate (Cat. No.
621714) is from Roche. Vaseline (Cat. No. 16415) is
from Riedel-de Haen. Brain heart infusion (BHI)
culture medium and Bacto agar are from Difco
Laboratories. Rhodamine-labelled actin (Cat. No.
AR05) is from Cytoskeleton. A glass homogeniser
equipped with a Teflon piston, glass slides (76 × 26
mm), glass coverslips (22 × 22mm), forceps, scissors,
and razor blades are from local suppliers. Bacterial
motility is observed using an Axiovert 135TV microscope
(Zeiss) equipped with a Plan-Apochromat
100×/1.4 NA oil immersion objective. Images can be
acquired with a cooled, back-illuminated CCD camera (TE/CCD-1000 TKB; Roper Scientific) driven by IPLab
Spectrum software (Scanalitics).
A. Explantation of Mouse Brains
Phosphate-buffered saline (PBS)
O, and 4mM
O, pH 7.4.
For 1 litre, weigh out 7.65g NaCl, 0.21g
O, and 0.72 g Na2
in 900 ml H2
O, adjust pH to 7.4 with 1N
bring volume to 1 litre. Store at 4°C.
- Kill mice using CO2 or by cervical dislocation.
- Remove skin and fur from the head using thintipped
scissors and discard.
- Cut the skull bone by sliding the scissors along the
sagittal suture (Fig. 1a, dashed red line).
- Cut the frontal, parietal, and interparietal bones
along their sutures and remove them (Fig. 1a,
dashed green line).
- Gently pinch the surface of the brain to lift the
meninges up and gently ease the brain out of the
- Put explanted brains in ice-cold PBS.
- Wash brains three times in ice-cold PBS to remove
tissue debris and blood residues.
- Proceed to Section III,B or flash freeze the brains in
liquid nitrogen. Store frozen brains at -80°C.
|FIGURE 1 Diagram of the explantation of mouse brains. (a) Dorsal view of mouse skull showing the nasal
(1), eye socket (2), nasal process of incisive bone (3), zygomatic process (4), frontal bone (5), parietal
(6), interparietal bone (7), and zygomatic bone (8). (b) Dorsal view of skull after cutting (along the dashed
green and red lines) and removing the frontal, parietal, and interparietal bones. (c) Dorsal view of explanted
B. Preparation of Cytosolic Extracts
- Homogenisation buffer (HB): 20mM HEPES, pH
7.5, 100mm KCl, 1 mM MgCl2·H2O, 1 mM EGTA, and
0.2mM CaCl2. For 1 litre, weigh out 4.76g HEPES,
7.45g KCl, 0.2g MgCl2·H2O, 0.38g EGTA, and 0.02g
MgCl2. Dissolve in 900ml H2O, adjust pH to 7.5
with 1N NaOH, and bring volume to 1 litre. Store at
- Protease inhibitors: 20mg/ml chymostatin, ling/
ml leupeptin, 1mg/ml pepstatin, 167mM Pefabloc,
and 10mg/m! aprotinin. To prepare stock solutions,
dissolve 1mg chymostatin in 50 µl dimethyl sulfoxide,
0.5mg leupeptin in 500µl H2O, 0.5mg pepstatin in
500µl methanol, 20mg Pefabloc in 500µl H2O, and
0.5mg aprotinin in 50µl H2O. Aliquot and store at
- 0.1M ATP: For 50ml, weigh out 2.75g ATP.
Dissolve in H2O, aliquot, and store at -20°C.
- 0.1M DTT: For 50ml, weigh out 0.77g DTT.
Dissolve in H2O, aliquot, and store at -20°C.
- 2M sucrose: For 20ml, weigh out 13.69 g sucrose.
Dissolve in H2O, aliquot, and store at -20°C.
- Homogenisation buffer supplemented with protease
inhibitors (HBI): HB containing 60µg/ml chymostatin,
5 µg/ml leupeptin, 10 µg/ml pepstatin, 4mm Pefabloc,
2µg/ml aprotinin, 0.5mM ATP, and 1mm DTT.
Shortly before use add to 20ml of HB 60µl of 20mg/ml
chymostatin, 100µl of 1mg/ml leupeptin, 200µl of
1mg/ml pepstatin, 476µl of 167mM Pefabloc, 4µl of
10mg/ml aprotinin, 200µl of 0.1M DTT, and 50µl
of 0.1M ATP.
C. Preparation of Bacteria
- After the last wash in PBS (see Section III,A, step 7)
remove PBS and weigh the brains.
- Cut the brains into small pieces using a razor blade
(keep brains on ice).
- Add 0.75ml of HBI per gram of wet tissue (keep
brain suspension on ice).
- Transfer brain suspension into a glass homogeniser
- Grind brain tissue for 20 passages of the pestle on
- Centrifuge crude extract at 15,000g for 1h at 4°C.
- Recover clarified supernatant (cytosolic brain
extract) and supplement it with 150mM sucrose,
50mg/ml creatine kinase, 30mM creatine phosphate,
and 0.5 mM ATP.
- Aliquot and flash freeze in liquid nitrogen. Store
frozen aliquots at -80°C.
- Brain heart infusion (BHI) broth: Prepare liquid
medium according to the manufacturer's instruction,
autoclave, filter, and store at 4°C. For agar
plates, add Bacto-agar (15g/liter of BHI broth),
autoclave, and pour 30ml in a 10-cm petri dish.
Store plates at 4°C.
- Erythromycin stock solution: Dissolve 50mg of erythromycin
in 10ml of pure ethanol. Store at 4°C.
D. Listeria Motility Assay
- Streak the bacteria onto BHI agar plates. Incubate
at 37°C for 24h.
- Put 5ml of BHI (supplemented with 50µg/ml
erythromycin) in a 15-ml sterile Falcon tube. Scrape a
few colonies off the BHI plate using a sterile pipette tip
or a flamed bacteriological loop. Inoculate the broth
and grow bacteria overnight at 37°C with vigorous
- Transfer bacterial culture to a centrifuge tube and
pellet the bacteria at 10,000g for 3 min.
- Wash bacterial pellet three times in homogenisation
buffer. After the final washing step, resuspend
pellet in a final volume of homogenisation buffer corresponding
to the initial volume of bacterial culture.
- Alternatively, supplement the overnight culture
with 20% glycerol, aliquot, and store at -80°C.
- 2% methycellulose in homogenisation buffer (stock
solution): Heat 100ml of HB to 60°C and then add 2g
of methylcellulose. Stir vigorously until the methycellulose
dissolves. Cool down and store at room
- 0.5% methycellulose in homogenisation buffer
(working solution): To make 10 ml, mix 7.5 ml of HB with
2.5 ml of 2% methycellulose. Store at 4°C.
- VALAP: Mix vaseline, lanoline, and paraffin in a
1:1:1 ratio (w/w/w) and homogenise at 75°C. Store
at room temperature.
- G buffer: 5mM Tris-HCl, pH 7.6, 0.5mM ATP,
0.1 mM CaCl2, and 0.5 mM DTT. For 1 litre, weigh out
0.8 g Tris-HCl, 0.01 g CaCl2, and then add 5 µl of 0.1M ATP and 5µl of 0.1M DTT. Dissolve in 900ml H2O, adjust pH to 7.6 with 1N NaOH, and bring volume to
1 litre. Store at 4°C.
- Rhodamine-labelled actin: Add 6µl of G buffer to
one aliquot of rhodamine-labelled actin. Mix gently
and store on ice.
- Wash bacteria three times in homogenisation
buffer. Resuspend pellet in 20µl homogenisation
- In a small Eppendorf tube, mix 4 µl brain extract,
4 µl of 0.5% methycellulose, 0.5 µl bacteria suspension,
and 0.2µl rhodamine-labelled actin. Mix by pipetting
up and down gently. Do not vortex. Incubate mixture
at room temperature for 10min.
- Remove 1.7 µl of motility mixture and spot it onto
a glass slide. Gently place a 22 x 22-mm glass coverslip
over the drop and press down until the drop
spreads to the edges. Seal the coverslip edges with
- Observe slide with an upright or inverted microscope.
Actin comet tails can be observed easily by
phase contrast or epifluorescence using a Plan-
Apochromat 100x/1.4NA oil immersion objective
(Figs 2 and 3).
|FIGURE 2 Immunofluorescence microscopy showing Listeria
actin tails formed in mouse brain extracts. Bacteria were incubated
in mouse brain extract for 30min at room temperature and then 5µl
of motility mixture was applied onto Cell Tak-coated cover slips (the
coating procedure was done according to the manufacturer's
instructions) and incubated for 5min on ice. Afterwards, bacteria
were fixed with 4% PFA for 20min at room temperature. Immunolabelling
was done according to Geese et al. (2002) using the
affinity-purified polyclonal antibody K52 to label bacterial surface,
the monoclonal antibody 84H1 to label EVL, and Texas redlabelled
phalloidin to label the actin tails. Primary antibodies were
detected using Alexa 488-conjugated secondary antibodies. Scale
bar: 5 µm.
|FIGURE 3 Dynamics of Listeria motility in mouse brain extracts.
Bacteria were mixed with 4 µl brain extract, 4 µl of 0.5% methycellulose,
and 0.2 µl rhodamine-labelled actin and were incubated at room
temperature for 10min. Thereafter, 1.7µl of motility mixture was
spotted onto a glass slide and a 22 × 22-mm glass coverslip overlaid
on it. Bacterial motility was observed using a Axiovert 135 TV
inverted microscopy equipped with phase-contrast and epifluorescence
optics using a Plan-Apochromat 100×/1.4NA oil immersion
objective. Images were acquired using a cooled, back-illuminated
CCD camera (TE/CCD-1000 TKB; Roper Scientific) driven by IPLab
Spectrum software (Scanalitics). (Top) Rhodamine-labelled actin and
(bottom) the corresponding phase-contrast images.
Scale bar: 10µm.
IV. COMMENTS AND PITFALLS
Although cell-free systems based on egg extracts
of Xenopus laevis
or human platelet extracts provide
excellent in vitro
systems for supporting actin-based
bacterial motility, their ability to do so can be affected
negatively by various factors, such as health of eggs
or quality and age of platelet preparations. In this
context, mouse brain extracts offer a more "robust" in
vitro system that does not seem to be influenced by
external factors such as mouse strain and age.
The reconstitution of Listeria
motility using mouse
brain extracts can have a variety of uses. For instance,
it can be used to study the role of actin cytoskeletal
components involved in Listeria motility by interfering
with their function using specific inhibitors or antibodies,
as described in May et al.
(1999). Moreover, the
procedure described here may be further developed
and adapted to obtain cell-free extracts from normal
cultured cells or cells that lack or express mutated versions of the actin cytoskeletal proteins of interest, thus
widening the spectrum of in vitro
systems available for
studying bacterial motility.
Three main parameters have to be considered to
achieve optimal Listeria
motility with mouse brain
extracts. First, the protein ActA must be expressed at
high levels on the bacterial surface. As most wild-type
strains express low levels of this protein under standard
culture conditions, a Listeria
strain (see Lingnau et al.
, 1996) that constitutively expresses high levels of
this protein must be used in this assay. The second critical
parameter is the total protein concentration of the
brain extract, which must be at least 10mg/ml. The
total protein concentration can be increased by reducing
the amount of homogenisation buffer per gram of
wet tissue. Moreover, mouse brain extracts should not
be diluted more than fourfold as further dilution leads
to loss of activity in the motility assay. Finally, the high
amount of actin present in these extracts induces its
spontaneous polymerisation, characterised by the formation
of short actin bundles. As this actin network
may affect bacterial motility, mouse brain extracts
must be kept on ice to reduce the tendency of actin to
I thank David A. Monner and Jürgen Wehland (GBF,
Department of Cell Biology) for helpful discussions
and valuable support.
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