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 DH5a, 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
	a-appendage
pGex4T2	m-a-appendage (a-earL 695–939)
pGADT7	m-a-appendage+hinge (a-earL653–938)
pGex4T2	m-a-appendage-F740D
pGex4T2	m-a-appendage-W840A
pGex4T2	m-a-appendage-F740D+W840A
pGex4T2	m-a-appendage-E718A
pGex4T2	m-a-appendage-E729A
pGex4T2	m-a-appendage-G742D
pGex4T2	m-a-appendage-Q784D
pGex4T2	m-a-appendage-G725E+G742D
	b-appendage
pGex4T1	h-b2-appendage (700–937)
pGex4T1	h-b2-appendage-K759E
pGex4T1	h-b2-appendage-K808E
pGex4T1	h-b2-appendage-Q756A
pGex4T1	h-b2-appendage-Y815A
pGex4T1	h-b2-appendage-K719E
pGex4T1	h-b2-appendage-Q851A
pGex4T1	h-b2-appendage-Y888V
pGex4T1	h-b2-appendage-R879A
pGex4T1	h-b2-appendage-K842E
pGex4T1	h-b2-appendage-W841A
pGex4T1	h-b2-appendage-Y815A-Y888V
pET15b	h-b2-appendage (700–937)
pGex4T2	hb2-appendage+hinge (616–937)
pGex4T2	hb2-appendage+hinge-Y815A
pGex4T2	hb2-appendage+hinge-Y888V
pGex4T2	hb2-appendage+hinge-Y815A-Y888V
pGADT7	h-b2-appendage+hinge
pGex4T2	m-b1-appendage (707–943)
pGex4T2	m-b1-appendage+hinge (512–943)
pGex5	h-b3-appendage (853–1094)	kind gift from
pGex5	h-b3-appendage+hinge (810–1094)	Richard Lundmark and
pGex5	h-b4-appendage (570–739)	Sven Carlsson
pGex4T1	h-b4-appendage+hinge (535–739)
	g-appendage
pGex4T2	m-g-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-b-arrestin2-FL
pGex5.1	h-b-arrestin2 C1 (C-terminal tail fragment, 317–410)	kind
pGex5.1	h-b-arrestin2-C1- F389A	gift
pGex5.1	h-b-arrestin2-C1- F392A	from
pGex5.1	h-b-arrestin2-C1- R396A	Alexandre
pEGFPN1	h-b-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 mM, 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 b-mercaptoethanol was used instead), 2 mM EDTA (only for GST tagged proteins), 1/1000 protease inhibitor cocktail (set III, Calbiochem) 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-pull-down 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 b-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.

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.

 

6.3.3 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 ml lysate were used per ~50 mg fusion protein on 50 ml glutathione sepharose beads (resulting in 1 mg protein / ml 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 ml 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.

 

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:

 

*          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

 

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 ml/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.

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 mg 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 ml 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 a- and b-appendages were investigated by isothermal titration calorimetry (ITC), using a VP-ITC (MicroCal Inc., USA). All experiments were 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 ml 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 DH and entropy DS. 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 b-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	Peptide sequence	Derived from protein
Syj-P1	LDGFKDSFDLQG	m-synaptojanin
Syj-P3	NPKGWVTFEEEE	m-synaptojanin
b-arrestin P-long	DDDIVFEDFARQRLKGMKDD	h-bArrestin2
b-arrestin P-short	DDIVFEDFARQR	h-bArrestin2
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-a, or GST-b 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 ml/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.

 

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 g adaptin	DDB0214928	ap1g1	adpA
AP1 b adaptin	DDB0204689	ap1b1	adpB
AP1 m adaptin	DDB0191102	apm1,ap1m1	adpC
AP1 s adaptin	DDB0232337	ap1s1	adpD
AP2 a adaptin	DDB0217164	ap2a1	adpE
AP2 m adaptin	DDB0191267	apm2,ap2m1	adpF
AP2 s adaptin	DDB0234236	ap2s1	adpG
AP3 d adaptin	DDB0234240	ap3d1	adpH
AP3 b adaptin	DDB0217414	ap3b1	adpI
AP3 m adaptin	DDB0214930	apm1	adpJ
AP3 s adaptin	DDB0234244	ap3s1	adpK
AP4 e adaptin	DDB0205193 (putative)		adpL
AP4 b adaptin	DDB0234245	ap4b1	adpM
AP4 m adaptin	DDB0219948	apm4	adpN
AP4 s adaptin	DDB0232330	ap4s1	adpO

 

Knockouts derived in this study:

adpE1 (a adaptin-appendage knockout)

adpB1 (b adaptin-appendage knockout)

adpE1,adpB1 (a adaptin-appendage, b adaptin-appendage double knockout)

 

A.8.2 Protein expression and GST-pull-downs

Dictyostelium a- and b-appendages (amino acids 744-989 and 701-942 respectively) as well as a- and b-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. 100ml cells were transferred to 1mm cuvettes (BioRAD) and 20mg of linearised replacement plasmid or circular reporter plasmid added per cuvette. Following a 5 minute incubation on ice, two consecutive 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 mF 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 22^oC. Following overnight incubation, blasticidin (10mg/ml) was added for selection. Medium was changed every 2-3 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 (20mg/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 22^oC in axenic medium with tetracycline (10 mg/ml) and streptomycin (200 mg/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 mg/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 22^oC.

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 10 minutes and then transferred at -80^oC inside a Cryolite Preservation Module (Stratagene) to ensure controlled cooling at a rate of 0.4-0.6^oC per minute. Vials were then transferred to liquid nitrogen (-185^oC) 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 500ml 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 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 100mM 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.

 

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 LC-MS/MS.