Eva’s methods
6.1 Molecular Biology
Constructs were generated according to standard molecular procedures (Ausubel, 2003).
Gene amplification via PCR was carried out using the KOD Hotstart DNA polymerase
Kit (Calbiochem/Novabiochem/Novagen) following manufacturer’s instructions.
Restriction digests were performed with restriction enzymes from New England Biolabs.
The ligation (Rapid DNA Ligation Kit from Roche) was transformed into chemically
competent E. coli (Subcloning Efficiency DH5, Invitrogen) according to the protocol
provided. DNA was isolated using Quiagen Miniprep Kits and sequence was confirmed
by the LMB Geneservice Ltd.
6.1.1 Vector list

pGex 4T1, 2 and 3 (Pharmacia Biotech), for generation of GST tagged proteins,
contain a thrombin cleavage site

pET15b and 28c (Novagen), for generation of 6xHis tagged proteins

pGADT7 (Clontech), used for Yeast Two-Hybrid experiments, generates fusion
proteins with the GAL4 activation domain

pGBKT7 (Clontech), used for Yeast Two-Hybrid experiments, generates fusion
proteins with the GAL4 DNA binding domain

pEGFPC2 and N2 (Clontech), used for over expression studies (A. Benmerah)
6.1.2 List of constructs
vector
construct
remarks
-appendage
1
pGex4T2
m--appendage (-earL 695–939)
pGADT7
m--appendage+hinge (-earL653–938)
pGex4T2
m--appendage-F740D
pGex4T2
m--appendage-W840A
pGex4T2
m--appendage-F740D+W840A
pGex4T2
m--appendage-E718A
pGex4T2
m--appendage-E729A
pGex4T2
m--appendage-G742D
pGex4T2
m--appendage-Q784D
pGex4T2
m--appendage-G725E+G742D
-appendage
pGex4T1
h-2-appendage (700–937)
pGex4T1
h-2-appendage-K759E
pGex4T1
h-2-appendage-K808E
pGex4T1
h-2-appendage-Q756A
pGex4T1
h-2-appendage-Y815A
pGex4T1
h-2-appendage-K719E
pGex4T1
h-2-appendage-Q851A
pGex4T1
h-2-appendage-Y888V
pGex4T1
h-2-appendage-R879A
pGex4T1
h-2-appendage-K842E
pGex4T1
h-2-appendage-W841A
pGex4T1
h-2-appendage-Y815A-Y888V
pET15b
h-2-appendage (700–937)
pGex4T2
h2-appendage+hinge (616–937)
2
pGex4T2
h2-appendage+hinge-Y815A
pGex4T2
h2-appendage+hinge-Y888V
pGex4T2
h2-appendage+hinge-Y815A-Y888V
pGADT7
h-2-appendage+hinge
pGex4T2
m-1-appendage (707–943)
pGex4T2
m-1-appendage+hinge (512–943)
pGex5
h-3-appendage (853–1094)
kind gift from
pGex5
h-3-appendage+hinge (810–1094)
Richard
Lundmark and
pGex5
h-4-appendage (570–739)
pGex4T1
h-4-appendage+hinge (535–739)
Sven Carlsson
-appendage
pGex4T2
m--appendage (E3, 704–822)
accessory proteins
pET28C
h-eps15-MD (530-791)
pEGFPC2
h-eps15-MD (620-739)
pGex4T2
r-epsin1 MD (249-401)
pGex4T2
r-AP180 MD (516-915)
pGex4T2
r-Amph1 MD (1-390)
pGex4T3
m-Syj170-MD (1303-1567)
pGex4T3
m-Syj170-MD DPF to DPD mutant
Gex4T2
b--arrestin2-FL
pGex5.1
h--arrestin2 C1 (C-terminal tail fragment, 317–
pGex5.1
410)
kind
h--arrestin2-C1- F389A
gift
3
pGex5.1
h--arrestin2-C1- F392A
from
pGex5.1
h--arrestin2-C1- R396A
Alexandre
pEGFPN1
h--arrestin2-FL
Benmerah
pGBKT7
r-eps15R 1-566
pGBKT7
h-CVAK90
pGBKT7
h-CVAK104
Clathrin
pGex
b-clathrin TD (terminal domain residues 1–363)
The clones were made by Harvey McMahon, Ian Mills or myself unless otherwise stated.
All constructs were sequenced.
Abbreviations: (m) mouse, (r) rat, (b) bovine, (h) human, (MD) motif domain
6.2 Protein purification
6.2.1 Growth and lysis of BL21 (DE3) E.coli
DNA was transformed into chemically competent BL21 (DE3) pLysS cells (Stratagene)
according to the provided protocol. A single colony was inoculated into 50 ml LB media
supplemented with 0.050 mg/ml ampicillin (or for pET vectors 0.01 mg/ml kanamycin
instead) and 0.034 mg/ml chloramphenicol and grown for ~5 hours at 37 C until OD600
~0.6. 25 ml culture were added to 1 l LB in 2 l flasks supplemented with the appropriate
antibiotics and incubated for ~2 hours at 37 C until log phase. IPTG was added to a
concentration of 40 M, the temperature decreased to 18 C and incubated over night.
The bacteria were harvested by centrifugation at 4 000 rpm in the Sorvall RC-3B plus
centrifuge. They were resuspended in 150 mM NaCl, 20 mM HEPES pH 7.4, 2 mM DTT
(for 6xHis tagged proteins 5mM -mercaptoethanol was used instead), 2 mM EDTA
(only for GST tagged proteins), 1/1000 protease inhibitor cocktail (set III, Calbiochem)
4
and 1/1000 of DNAseI (1 mg/ml in 1M MgCl) and snap-frozen in liquid nitrogen. Lysis
was achieved by thawing the bacteria at room temperature and the lysate was clarified by
spinning at 40 000 rpm for 40 min in the Beckman-Coulter Optima L-80 XP
ultracentrifuge.
Comments: for good lysis no Mg should be present during lysis and lysate should be
mixed on room temperature for 30 min. Then Mg and DNAseI should be added and
incubated again for approx. 20 min on room temperature.
Alternatively: BL21 (DE3) cells can be used and french pressed instead.
6.2.2 Affinity purification for GST fusion proteins
The soluble extract of bacterial lysate was incubated with glutathione sepharose beads
(GE Healthcare) for 45 min at 4 C. The beads were washed five times with 300 mM
NaCl, 20 mM HEPES pH 7.4, 2 mM DTT and 2mM EDTA, including one 20 min long
wash, followed by two more washes with 150 mM NaCl, 20 mM HEPES (pH 7.4) and 2
mM DTT. Aliquots of GST-tagged protein on beads were frozen and used for GST-pulldown assays.
When removal of the GST tag was required, beads were incubated with thrombin (Serva)
over night at 16 C or at room temperature for 2 hours. For elution of the tagged protein
20mM glutathione was used (repeated incubation for 10 min on room temperature).
6.2.3 Affinity purification for 6xhis tagged proteins
The soluble exrtract of bacterial lysate was incubated with a minimal volume of 15 ml
Ni-NTA agarose (Qiagen) for 45 min at 4 C (10 mM imidazole was added as well). The
beads were washed twice in 50 mM NaCl, 20 mM HEPES pH 7.4, 5mM mercaptoethanol and 20 mM imidazole. Further washing was done on the Akta FPLC
Purifier system (Amersham Biosciences) with a gradient up to 300 mM imidazole. Peak
fractions containing the protein of interest were pooled.
5
6.2.4 Anion-exchange
A Q-sepharose column was used as a second purification step. It was particularly useful
for removal of previously used thrombin. Proteins were applied at a salt concentration of
150 mM NaCl and a gradient to 1 M NaCl was used over five column volumes to effect
elution.
6.2.5 Gel Filtration
Most proteins were applied to a size exclusion column as a final purification step. For all
proteins used in ITC the buffer used was 50 mM NaCl, 100 mM HEPES pH 7.4, and 2
mM DTT. Depending on molecular weight of the protein and quantity of the protein a
Sephadex 200 or 75 16/60 or 26/60 (Amersham Biosciences) was used.
6.3 GST-fusion protein co-sedimentation assays (pull-downs)
6.3.1 Preparation of rat brain cytosol
For preparation of rat brain lysate typically one frozen rat brain (Harlan, Sera-Lab Ltd)
was defrosted on ice. In a 15 ml Teflon-glass homogeniser the brain was homogenised in
4 ml of homogenisation buffer (150 mM NaCl, 20 mM HEPES pH 7.4, 2 mM DTT,
1/1000 protease inhibitor cocktail (set III, Calbiochem) and 0.1% Triton X-100, this
buffer was also used as washing buffer). The lysate was centrifuged at 50 000 rpm in the
TLA 100.4 rotor (Beckman-Coulter Optima TL ultracentrifuge).
6.3.2 Preparation of HeLa cytosol
For preparation of HeLa lysate 1x108 cells were trypsinised and washed in 150 mM
NaCl, 20 mM HEPES pH 7.4, 2 mM DTT, 1/1000 protease inhibitor cocktail (set III,
Calbiochem). Cells were solubilised with NP40 (not to disrupt the nuclei) and debris was
pelleted with a spin in a desktop eppendorf centrifuge (13 000 rpm, 30 min 4 C). 0.1%
Triton X-100 was added and incubated on ice for 10 min. The lysate was cleared by
spinning at 50 000 rpm in the TLA 100.4 rotor.
6
6.3.3 Preparation of rat liver cytosol
Preparation of rat liver extract was similar to rat brain lysate, except an initial spinning
step to was used to deplete the lipid content. Two rat livers were homogenised in 4 ml
homogenisation buffer (see above) and centrifuged in SW41 (Beckman) tubes at 30 000
rpm for 30 min using a SW41 swing-out rotor. A clear white lipid layer could be
observed. A hole was pinched in the bottom of the SW41 tube to collect the lower layer
and avoid the upper lipid layer. The lipid free lysate was spun again at 50 000 rpm in the
TLA 100.4 rotor and used for GST-pull-down assays.
6.3.4 GST-pull-downs
500 l lysate were used per ~50 g fusion protein on 50 l glutathione sepharose beads
(resulting in 1 g protein / l beads). The extract was incubated with the beads for 40 min
at 4 C and then rapidly washed three times using the washing buffer described above
(also used as homogenisation buffer). After the last wash, all buffer was removed and 50
l sample buffer were added. The samples were incubated at 95 C for 5 min and beads
were pelleted. The supernatant was analysed via SDS-PAGE (Polyacrylamide Gel
Electrophoresis, Nu-PAGE Gel system from Invitrogen, typically using 4-12% Bis-Tris
10 well gels with provided tank buffers) followed by Coomassie staining and mass
spectrometric analysis or alternatively Western blotting.
6.3.5 Cross-linked GST proteins
In the mass spec analysis it was a major problem that the GST-tagged proteins used to
pull binding partners, masked a large portion of the gel in the region of 50 kDa. In order
to find the binding partners that have this molecular weight, the GST-tagged proteins
were cross-linked to the beads using AffiGel 15 (BioRad) according to manufacturer’s
guidelines.
7
6.3.6 ‘Bead bound versus solution’ GST-pull-downs
To analyse the protein interaction partners of free appendages, GST-appendages were
incubated with brain extract for 40 min and then captured these by centrifugation through
a layer of GSH beads on a filter (spin-X centrifuge tube filters, Fisher Scientific).
Subsequently the beads were washed three times using the washing buffer described
above. This was compared with bead-bound GST-appendages incubated with extract for
40 min before capturing the beads coupled to GST-appendages on a filter.
6.3.7 Western blotting and list of antibodies
Proteins were transferred from polyacrylamide gels onto nitrocellulose (Protean) at a
limiting current of 200 mA per gel for 2 hours. After blotting, the filters were blocked
with milk (Marvel powder) for 15 min and incubated with primary antibody in 10% (v/v)
Goats serum (Sigma) in TBST for 1 hour at room temperature or over night at 4 C.
Secondary anti-rabbit or anti-mouse antibodies conjugated to horseradish peroxidase
(Biorad) were used at 1/10 000 dilution and incubated at room temperature for 40 min.
The blots were developed using ECL reagent (Amersham Biociences or SuperSignal
West Femto from Pierce) and the blots were exposed for varying lengths of time to
medical imaging film (Kodak).
Primary antibodies used for Western blots:
detected protein
company
dilution
poly
AAK
Ra 41*
1: 5 000
mono
Amphiphysin1 and
BD Bioscience
1:10 000
monoclonal
or
polyclonal
2
mono
AP180
BD Bioscience
1:10 000
mono
-adaptin
BD Bioscience
1:10 000
mono
Auxilin
Gift from Ernst
1: 1 000
8
Ungewickell (18a6)
mono
-adaptin 1,2
Sigma
1:10 000
mono
-arrestin1
BD Bioscience
1: 5 000
mono
Clathrin
BD Bioscience
1:10 000
mono
Dab2 (Doc2)
BD Bioscience
1:10 000
mono
Dynamin1,2,3
BD Bioscience
1: 5 000
poly
Endophilin1
Ra 37*
poly
Eps15
Santa Cruz (C-20)
1: 5 000
poly
Epsin1
Ra 14*
1: 5 000
mono
HIP1
AbCam
1: 5 000
mono
Hsc70
BD Bioscience
1: 5 000
poly
Intersectin2
kind gift from Tom Sudhoff
1: 3 000
(S750)
poly
NECAP
kind gift from Peter
1: 1 000
McPherson
mono
Numb
BD Bioscience
1: 5 000
poly
Sorting nexin 9
Richard Lundmark
1: 8 000
poly
Stonin
kind gift from Volker
1: 1 000
Haucke
poly
*
Synaptojanin
Ra 59*
1: 5 000
these antibodies were raised by Harlan SeraLab against the following
proteins:
Ra 41: hAAK kinase domain (1-326)
Ra 37: endophilin N-terminus
Ra 14: 6xHis epsin1 DPW domain (MD, 249-401)
Ra 59: alkaline phosphatase treated FL Synaptojanin
9
6.4 Mass Spectrometry analysis (in collaboration with Sew-Yeu
Peak-Chew)
Proteins were separated on PAGE-Gels and Coomassie stained bands were excised.
Peptides of in-gel trypsin digested protein bands were separated by liquid
chromatography on a reverse phase C18 column (150 x 0.075 mm i.d., flow rate 0.15
l/min). The eluate was introduced directly into a Q-STAR hybrid tandem mass
spectrometer (MDS Sciex, Concord, Ontario, Canada). The spectra were searched against
a NCBI non-redundant data-base with MASCOT MS/MS Ions search
(www.matrixscience.com). For protein with a low number of peptides their identity has
been confirmed by searching the PeptideSearch nrdb database using sequence tags from
the data.
10
6.5 Yeast Two-Hybrid
For verification of a small selection of interaction partners identified in mass spec
analysis, yeast two-hybrid experiments were carried out using the MATCHMAKER
Two-Hybrid System 3. The bait (the appendages plus hinge regions) was cloned into the
GAL4 activation domain containing vector pGADT7 and the possible interaction partners
(CVAK 90/104 and eps15R 1-566) were cloned into the GAL4 DNA-binding domain
containing plasmid pGBKT7.
Several colonies of the yeast strain AH109 were resuspended in 50 ml of YPD (20 g/l
Difco peptone, 10 g/l Yeast extract and 2% Glucose) and incubated at 30 C for 16-18
hours until stationary phase (OD600 ~1.5) was reached. The overnight culture was
diluted into 300 ml YPD to OD600 ~ 0.3 and incubated for 3 hours at 30 C after which
the OD600 had reached ~ 0.6. Cells were harvested, washed twice in distilled water and
resuspended in 1.5 ml freshly prepared 1x TE / 1x LiAc (10 mM Tris-HCl,1 mM EDTA
pH 7.5 / 100 mM LiAc pH 7.5). Sequentially 0.1 g plasmid DNA and 0.1 mg herring
testes carrier DNA (Invitrogen), 0.1 ml yeast competent cells and 0.6 ml sterile 1x TE/ 1x
LiAc with 40% PEG were added and well mixed after each step. After incubation at 30
C for 30 min 70 l DMSO were added, mixed by gentle inversion and heat shocked at
42 C for 15 min. Cells were chilled on ice and pelleted. The pellet was resuspended in
0.5ml 1x TE buffer and plated on double selection plates (-Leu/ -Trp). After 3 to 4 days
colonies appeared which were restreaked on quadruple selection plates (-Leu/ -Trp/ -His/
-Ade) to select for interaction of bait and putative binding partner.
6.6 Biophysical methods
6.6.1 Isothermal Titration Calorimetry (ITC)
Binding of ligands to - and -appendages were investigated by isothermal titration
calorimetry (ITC), using a VP-ITC (MicroCal Inc., USA). All experiments were
11
performed in 100 mM HEPES (pH 7.4), to minimise heat release due to acid-base
interactions, 50 mM NaCl, to bring the ionic strength to near-physiological and 2 mM
DTT at 10 °C and protein concentrations were determined by absorbance at 280 nm.
Ligands were injected into the experimental cell containing 1.36 ml of the binding protein
in 40-50 steps of typically 7 l every 3.5 minutes, until a 3-4 fold molar excess of ligand
in the cell was reached. The concentration of the binding protein in the cell was chosen so
that it was at least 5-fold higher that the dissociation constant (based on previous
experimental estimations). Ligands could be either peptides or purified protein domains.
Peptides were synthesised by Southampton Peptides Ltd with a purity of >95% and were
weighed on an analytical balance. Where possible, concentrations were verified by
absorbance measurements. Ligands were ideally 20 times more concentrated than the
binding protein in the cell.
Data analysis was carried out using the manufacturer’s software, Origin 5 or 7. Affinities
were calculated via fitting titration curves to the data. This resulted in the stoichiometry
N, the binary equilibrium constant Ka, the dissociation constant Kd (which equals 1/Ka),
the enthalpy H and entropy S. The heat of dilution of the ligand into buffer was
measured and subtracted from the data prior to fitting.
Where the concentrations of protein/peptides can be accurately measured, accurate values
for the stoichiometry of interactions can be obtained. Where proteins are not a single
species due to degradation then the stoichiometry may be inaccurate but the affinity can
still be measured if the concentration of the ligand in the syringe is accurate.
All proteins used for ITC were purified via affinity resins, Q-sepharose and gel filtration
before concentrating and freezing in aliquots.
The affinity measurement for both GST tagged and non-GST tagged versions of appendage proteins were identical showing the GST dimerisation was not significant to
the experiments.
List of peptides used in ITC (and crystallography, see below):
Name
Syj-P1
Peptide sequence
LDGFKDSFDLQG
Derived from protein
m-synaptojanin
12
Syj-P3
NPKGWVTFEEEE
m-synaptojanin
-arrestin P-long
DDDIVFEDFARQRLKGMKDD
h-Arrestin2
-arrestin P-short
DDIVFEDFARQR
h-Arrestin2
ARH
LDDGLDEAFSRLAQSRTNPQ
h-ARH
ARH-mut
LDDGLREAFSRLAQSRTNPQ
h-ARH
Amph DNF 12-mer
INFFEDNFVPDI
r-amphiphysin2
Epsin P3
EPDEFSDFDRLR
r-epsin1
EpsinR P1
SADLFGGFADFG
r-epsinR
Eps15 P-long
SATDPFASVFGNESFGDGFADFSTL
h-eps15
Eps15 P-short
SFGDGFADFSTL
h-eps15
FADF-7mer
DDFADFS
FGGF-7mer
DDFGGFS
6.7.2 Surface Plasmon Resonance (SPR)
SPR experiments were performed using a BIA 2000 apparatus (BIAcore). GST, GST-,
or GST- were immobilized via amine coupling (according to manufacturer’s
instructions) on a CM5 (carboxymethyl) chip. Recombinant 6 His h-eps15-MD was
then injected at a concentration of 300 nM to saturate the surface. Running buffer was 20
mM HEPES pH 7.4, 150 mM NaCl. Dissociation was measured over 7,000 sec at a flow
rate of 20 l/min. Nonspecific binding was measured as 6his h-eps15-MD binding to
the GST surface and subtracted from the experimental data. SPR data was analyzed using
BIAevaluation software provided by the manufacturer.
13
Dictyostelium Methods
A.8.1 General materials and nomenclature
The technicians of the Cell Biology division provided media for the growth of
Dictyostelium, together with solutions of KK2, TBE and other salts.
Solution
Composition
Axenic medium
1.43% bactopeptone, 0.715% yeast extract, 1.54%
glucose, 0.05% Na2HPO4 and 0.05% KH2PO4
Cell freezing medium
Horse serum, 7.5% DMSO
H50
20 mM HEPES, 50 mM KCl, 10 mM NaCl, 1 mM
MgSO4, 5 mM NaHCO3, 1 mM NaHPO, pH7.0
NS (New salts)
20 mM KCl, 20 mM NaCl, 1 mM CaCl2x6H20
KK2
16.5 mM KH2PO4, 3.9 mM K2HPO4 and 2 mM MgSO4
HKM
25 mM HEPES pH7.4, 125 mM KAc, 5 mM MgAc
Dictyostelium nomenclature used following Dictybase guidelines:
protein
DDB numbers
old gene name
new gene name
AP1  adaptin
DDB0214928
ap1g1
adpA
AP1  adaptin
DDB0204689
ap1b1
adpB
AP1  adaptin
DDB0191102
apm1,ap1m1
adpC
AP1  adaptin
DDB0232337
ap1s1
adpD
AP2  adaptin
DDB0217164
ap2a1
adpE
AP2  adaptin
DDB0191267
apm2,ap2m1
adpF
AP2  adaptin
DDB0234236
ap2s1
adpG
14
AP3  adaptin
DDB0234240
ap3d1
adpH
AP3  adaptin
DDB0217414
ap3b1
adpI
AP3  adaptin
DDB0214930
apm1
adpJ
AP3  adaptin
DDB0234244
ap3s1
adpK
AP4  adaptin
DDB0205193
adpL
(putative)
AP4  adaptin
DDB0234245
ap4b1
adpM
AP4  adaptin
DDB0219948
apm4
adpN
AP4  adaptin
DDB0232330
ap4s1
adpO
Knockouts derived in this study:
adpE1 ( adaptin-appendage knockout)
adpB1 ( adaptin-appendage knockout)
adpE1,adpB1 ( adaptin-appendage,  adaptin-appendage double knockout)
A.8.2 Protein expression and GST-pull-downs
Dictyostelium - and -appendages (amino acids 744-989 and 701-942 respectively) as
well as - and -appendages plus hinge (amino acids 660-989 and 616-942 respectively)
have been cloned into the pGex4T2 vector and expressed in BL21 pLysS cells. Molecular
cloning, protein expression and pull-downs have been performed as previously described
in Chapter 6. Antibodies were described earlier (Chapter 6).
A.8.3 Dictyostelium transformation methods
Cells between 1-2x106 cells/ml were washed twice in ice-cold electroporation buffer H50
and resuspended at 4x107 cells/ml in the same buffer. 100l cells were transferred to
1mm cuvettes (BioRAD) and 20g of linearised replacement plasmid or circular reporter
plasmid added per cuvette. Following a 5 minute incubation on ice, two consecutive
15
pulses, with a 5 second recovery between them, were delivered through each cuvette
using a BioRad Gene Pulser set at 0.85 kV and 25 F with no external resistance.
Immediately after electroporation, 0.5ml of axenic medium was added per cuvette and
cells were left to recover on ice for 5 minutes. They were then plated in axenic medium in
tissue culture plates at various dilutions, and incubated at 22oC. Following overnight
incubation, blasticidin (10g/ml) was added for selection. Medium was changed every 23 days after initial selection, and successful transformations were allowed to form clones
and reach confluence or cloned out on bacterial plates (Knecht and Pang, 1995).
For generation of knockout strains constructs were cloned into pLPBLP plasmid and
linearised prior to transformation. The GFP-CLC construct was kindly provided by Terry
O’Halloran and the resistance used was G418 (20g/ml). To remove the blasticidin
cassette the pLPBLP-cre-loxP plasmid was transformed (G418 resistant).
A.8.4 Handling Dictyostelium: growth and storage
Wild-type strain Ax2 and its derivatives were grown at 22oC in axenic medium with
tetracycline (10 g/ml) and streptomycin (200 g/ml), either in tissue culture plates or
shaken at 180 rpm in conical flasks. Reporter construct transformants of Ax2 and its
derivatives were grown in axenic medium supplemented with G418 (20 g/ml). Once
established in axenic medium cells were maintained by sub-culturing back to 105 cells/ml
as they reached stationary phase.
For growth curves cells were counted twice a day until stationary phase was reached.
Cells growing on SM-agar plates with Klebsiella aerogenes were propagated by picking
cells from a growth zone onto the surface of a K.aerogenes covered agar plate. Plates
were stored in an incubator at 22oC.
For experiments cells were harvested during the log phase (1-6 x 106 cells/ml) from
axenically growing cultures. Cells were counted using a haemocytometer (Improved
Neubauer) or a coulter counter (Beckman Coulter, Z1).
Mutants and newly isolated strains were stored frozen in liquid nitrogen. After growth
around 108 cells were collected from plates or shaking suspension directly into 1.5 ml of
cell freezing medium, in a 2 ml cryo-vial (Nunc, Denmark). Cells were chilled on ice for
16
10 minutes and then transferred at -80oC inside a Cryolite Preservation Module
(Stratagene) to ensure controlled cooling at a rate of 0.4-0.6oC per minute. Vials were
then transferred to liquid nitrogen (-185oC) where cells can be preserved indefinitely.
A.8.5 Dextran uptake
Cells were grown to log phase and used at 5x106 cells/ml. The assay was carried out in
axenic media whilst shaking at 180 rpm using a desktop shaker (Gyrotory Shaker Model
G2). 4 mg/ml FITC Dextran (FD70 Sigma) was added to the cells at t0. A 0.4 ml sample
of the shaken suspension was taken before, and every 10 minutes after for one hour. The
samples were placed in 1.5 ml eppendorf tubes containing 1 ml KK2 buffer + 1% BSA
(Sigma) on ice. After the time course cells were spun down in a microfuge to pellet the
cells. The cell pellet was resuspended and washed twice in ice-cold KK2 + 1% BSA.
Cells were then lysed in 500l 0.1M Tris pH 8.6 with 0.2% Triton.
The fluorescence was measured using a fluorimeter (Perkin Elmer LS 50B) with
emission/excitation 520/490nm.
A.8.6 Microscopy
Confocal Microscopy
Live cells were imaged on a BioRad Radiance confocal inverted microscope with a 60x
oil immersion objective. Cells were placed in a chambered coverslip (Lab-Tek, USA) and
immersed in KK2. Fluorescence images were collected using a 488 nm argon laser set at
minimal intensity. Differential interference contrast (DIC) images were collected
simultaneously. The data was collected as Tif files and converted into a movie format
using Quicktime Pro or Image J software.
TIR-FM
4x105 cells were plated on glass bottom dishes (WillCo-dish) submerged in KK2 buffer.
The cells were imaged using an Olympus IX70 microscope (Southhall, UK) and Argon
laser (Melles Griot, Carlsbad CA). Fluorescence was excited with an exponentially
declining, so-called evanescent field generated by total internal reflection of a 488-nm
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argon laser at the coverslip-cell interphase. Cells were viewed with a 60X oil immersion
objective lens with a numerical aperture of 1.45 (Olympus). Images were acquired at 1
Hz, captured with a Princeton intstruments (Trenton NJ) cooled I-PentaMAX camera,
analysed and processed with Metamorph (Universal Imaging). QuickTime movies were
generated.
A.8.7 Plaque development on bacterial plates
Cells growing in axenically were harvested, washed in KK2 buffer and counted. Five to
20 cells were co-plated with K. aeorogenes on SM agar plates. Developmental stages
were then viewed and photographed under phase optics after 3, 4 and 5 days.
A.8.8 cAMP recognition and movement
Streaming
1x106 Ax2 control or mutant cells were allowed to attach to the surface of plastic dishes
(typically 3.5 cm2 Falcon tissue culture dishes) after being washed in KK2 buffer. Axenic
media was replaced with starving buffer (KK2 + 2 mM MgSO4 +0.1 mM CaCl2) and
incubated for the indicated time at room temperature. Cells were then viewed and
photographed under phase optics.
Movement towards a cAMP releasing needle
1x108 cells were harvested from axenic growth, washed twice with KK2 buffer and
resuspended at 2x107 cells / ml in KK2 + 2 mM MgSO4 +0.1 mM CaCl2. After starvation
for 1 hour in shaking suspension the cells were pulsed every 6 minutes with 60 – 100nM
cAMP for 5 hours using a peristaltic pump (to develop them to a cAMP responsive state).
4x105 cells were immersed in KK2 in microscope dishes (Nunc 2-well) and their
movement towards a needle containing 100M cAMP was imaged on a BioRad Radiance
confocal inverted microscope with a 20x objective. Differential interference contrast
(DIC) images were collected. The data was collected as Tif files and converted into a
movie format using Quicktime Pro or Image J software.
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A.8.9 CCV preps in Dictyostelium
Ax2 and mutant cells were grown in 750 ml suspension culture and harvested shortly
before reaching stationary phase. After washing once in KK2 buffer, cells were
resuspended in 150 mM NaCl, 20 mM HEPES pH 7.4 and 2 mM DTT and freeze thawed
to induce lysis. The lysate was spun at 7 000 rpm for 20 minutes in a SS34 rotor
(Beckman centrifuge) and the supernatant was collected. Following ultracentrifugation of
the supernatant at 45 000 rpm for 40 minutes in a 70Ti rotor (Sorvall centrifuge) the
pellet was collected, resuspended and homogenised (rotor homogeniser, Philip Harris
Scientific) in 10 ml HKM buffer. An equal volume of HKM containing 12.5% Ficoll and
12.5% sucrose was added and mixed by inversion. Following centrifugation in a 70Ti
rotor at 25 000 rpm for 20 minutes the supernatant was diluted 1:5 in HKM buffer and
centrifuged for 60 minutes at 35 000 rpm in a 45Ti ultracentrifuge rotor. The pellet was
resuspended in 15 ml HKM buffer, homogenised and left on ice for 1 hour. Insoluble
material was sedimented at 13 000 rpm for 10 min in a 70 Ti ultracentrifuge rotor and the
supernatant was carefully layered over an 8% (w/v) sucrose cushion made up in
concentrated HKM and D2O. The sample was spun in a swing out rotor (SW40) for 2
hours at 25 000 rpm. The collected pellet (CCVs) was resuspended in sample buffer and
loaded on a SDS Page Gel. Coomassie stained bands were cut out and analysed by LCMS/MS.
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