Supplementary material for Boldogh et al. (2001) Proc. Natl. Acad. Sci. USA 98 (6), 3162–3167. (10.1073/pnas.051494698)
Materials and Methods
Actin Affinity Chromatography and Sedimentation Assay for mABP Activity.
F-actin affinity columns were prepared as described previously (1). Affi-Gel 10 (0.5 ml; Bio-Rad) and 0.5 ml of Sepharose CL-6B (Amersham Pharmacia) were mixed in a plastic syringe with 0.5 ml of F-actin (2 mg/ml) in 50 mM Hepes (pH 7.4) containing 0.1 M KCl, 0.2 mM CaCl2, 0.2 mM ATP, 5 mM MgCl2, 10% glycerol, and 10 µg/ml phalloidin and kept overnight at 4°C. After washing, 0.5–0.7 mg of F-actin remained on the column. For affinity chromatography, the F-actin column was equilibrated with 5 vol of column buffer (0.6 M sorbitol/50 mM Hepes, pH 7.4/0.2 M KCl/1 mM EGTA/3 mM MgCl2/10% glycerol/1 mM DTT/1 mM PMSF and protease inhibitor cocktail). The protease inhibitor cocktail is described in ref. 2.Mitochondria were isolated as described previously (2). To release salt-sensitive peripheral proteins from the outer membrane, mitochondria (30 mg/ml) were incubated in SM1 buffer (0.6 M sorbitol/20 mM Hepes-KOH, pH 7.4/1 M KCl/2 mM MgCl2/1 mM PMSF and protease inhibitor cocktail) for 15 min on ice. Thereafter, the mixture was subjected to centrifugation (10,000 ´ g for 5 min at 4°C). Salt-extractable peripheral proteins (SE) were recovered in the supernatant. Salt extract from 50 mg Nycodenz-purified mitochondria was diluted with column buffer until the final concentration of KCl was 0.2 M and applied to the column. To remove nonspecifically bound proteins, the column was washed twice with 2 vol of column buffer. Elution of ATP sensitive actin-binding proteins was carried out in two steps by applying 2 vol of the column buffer containing first 0.1 mM ATP, then 1 mM ATP. The flow rate during chromatography was maintained at 0.05 ml/min. The fractions from each step were collected.
To test the ability of the fractions to restore actin-binding activity of salt-washed mitochondria, 5% (vol/vol) of the salt extract and the column flow through, and 12% (vol/vol) of the eluates from the second wash, the 0.1-mM ATP wash, and the 1-mM ATP wash were incubated with salt-washed mitochondria. Mitochondria were then separated from the mixture by centrifugation at 10,000 ´ g at 4°C, suspended in RM buffer, and used in the sedimentation assay (2) using F-actin prepared from blocks of baker's yeast (3). In short, mitochondria (2.5 mg/ml) were incubated with phalloidin-stabilized F-actin (4.4 µM) for 10 min at 30°C in 80 µl of RM buffer (0.6 M sorbitol/20 mM Hepes-KOH, pH 7.4/100 mM KCl/2 mM MgCl2/1 mM DTT/1 mg/ml fatty acid-free BSA/1 mM PMSF and protease inhibitor cocktail). To deplete the system of ATP, apyrase (type VI, Sigma) was added to a final concentration of 12.5 units/ml (4). Mitochondria were separated from unbound actin filaments by centrifugation through a sucrose cushion (25% sucrose/20 mM Hepes-KOH, pH 7.4/1 mM PMSF, and protease inhibitor cocktail) at 12,500 ´ g for 10 min. Proteins recovered in the mitochondrial pellet were separated by SDS/PAGE (5) and identified by Western blot analysis (6) using polyclonal antibodies raised against the mitochondrial marker protein porin (gift from G. Schatz, Biozentrum, University of Basel, Switzerland) and monoclonal antibody raised against actin (c4d6; ref. 7).
Peptide Sequencing.
Proteins in the fractions from the actin affinity column were separated by SDS/PAGE and silver-stained (8). Silver-stained proteins with potential ATP-sensitive actin binding activity were excised and subjected to in-gel trypsin digestion (9). For microelectrospray high performance liquid chromatography, columns were constructed from 360 micron o.d. ´ 100 micron i.d. fused silica capillary with the column tip tapered to a 5- to10-micron opening. The columns were packed with PerSeptive Biosystems (Framingham, MA) POROS 10 R2, a 10-mm reversed-phase packing material, to a length of 10-12 cm. The flow from the HPLC pumps (typically 150 ml/min) was split precolumn to achieve a flow rate of 500 nl/min. The mobile phase used for gradient elution consisted of (solution A) 0.5% acetic acid and (solution B) acetonitrile/water 80:20 (vol/vol) containing 0.5% acetic acid. The gradient was linear from 0-40% B in 50 min followed by 40-80% B in 10 min or 0-60 B% in 30 min. Mass spectra were recorded on an LCQ ion trap mass spectrometer (Finnigan MAT, San Jose, CA) equipped with a microelectrospray ionization source (10). Electrospray was performed at a voltage of 1.6 kV. Tandem mass spectra were acquired automatically during the entire gradient run as previously described (11).An S. cerevisiae protein sequence database was searched directly with the tandem mass spectra using the computer program sequest (12). This database was obtained from the Saccharomyces Genome Database (Stanford University) and represents the complete genome sequence. Sequences of potential contaminants such as human keratin and bovine trypsin were added to the database. Each high scoring amino acid sequence matched to a tandem mass spectrum was verified by manually inspecting the fit of the amino acid sequence to the corresponding tandem mass spectrum. Protein identifications were considered conclusive when two or more tandem mass spectra matched to the same protein.
Visualization of Mitochondria, Arp2/3 Subunits, and the Actin Cytoskeleton.
ARC15 wild-type sister (DNY226-4d) and arc15-GFP insertion mutant (DNY226-4c) strains were grown to mid-log phase overnight in YPR at 30°C. ARP2 parent (YPH499) and arp2-1 mutant (YMW81) cells were grown to mid-log phase in YPD at 22°C and then shifted to 37°C for 3 hr. The actin cytoskeleton was visualized in fixed cells by using rhodamine-phalloidin (Molecular Probes) as described previously (13). To determine the level of F-actin in actin cables in mother cells, mid-log phase yeast were fixed and stained with the fluorescent, F-actin-binding agent, Alexa-594 phalloidin (Molecular Probes). Thereafter, the fluorescence intensity of F-actin in mother cells was measured on a Zeiss Axioplan II microscope (see below) and quantitated using ip lab software (Scanalytics, Fairfax, VA). Analysis was restricted to mother cells that contained polarized actin cables and no actin patches. Latrunculin-A (P. Crews, University of California, Santa Cruz) treatment was carried out as described previously (14).Mitochondria were visualized in living cells using a fusion protein consisting of the mitochondrial signal sequence of citrate synthase 1 fused to green fluorescent protein (GFP; CS1-GFP). CS1-GFP was expressed in DAUL1 cells using a centromere-based plasmid under control of the GAL1-10 promoter (15). Cells were transformed using the lithium acetate method (16). Samples were mounted on microscope slides and visualized by fluorescence microscopy as described below. CS1-GFP labeling of mitochondria is specific and has no detectable effect on mitochondrial morphology or movement under our experimental conditions.
For some experiments, mitochondria were visualized in living cells using the membrane potential sensing dye, 3,3'-dihexyloxacarbocyanine iodide (DiOC6; Molecular Probes). The cell density of mid-log phase samples was adjusted to 1 ´ 107 cells/ml. Thereafter, the sample was incubated in medium containing 20 ng/ml of DiOC6 for 5 min at room temperature. Cells were washed once and resuspended to a final concentration of 2 ´ 108 cells/ml in medium. Samples were mounted on microscope slides and visualized by fluorescence microscopy. At the concentrations used, DiOC6 is specific for mitochondria and has no detectable effect on cell viability (13).
The method used for indirect immunofluorescence is a modification of published methods (17). All samples were fixed by addition of paraformaldehyde and potassium phosphate buffer (pH 6.5) to the cell culture medium to final concentrations of 3.7% and 100 mM, respectively, and incubation for 15 min under growth conditions. Cells were collected by centrifugation and resuspended in a second fixative solution (100 mM KPO4, pH 6.5/2 mM MgCl2/5% paraformaldehyde) for 1 hr at room temperature. Fixative was removed by three washes with NS [20 mM Tris·HCl, pH 7.6/0.25 M sucrose/1 mM EDTA/1 mM MgCl2/0.1 mM ZnCl2/0.1 mM CaCl2/0.8 mM PMSF/0.05% (vol/vol) 2-mercaptoethanol). Cell walls were removed from fixed cells by incubation with zymolyase (18). Fixed spheroplasts were applied to polylysine-coated coverslips and allowed to adhere to the coverslips for 20 min. In some cases, immobilized spheroplasts were subjected to additional fixation by incubation in methanol (- 20°C, 6 min) followed by incubation in acetone (- 20°C, 30 sec).
Immobilized spheroplasts were then gently washed in PBS and incubated in PBT [1´ PBS/0.1% (vol/vol) Triton X-100/0.02% (vol/vol) sodium azide/1% (wt/vol) BSA] for 5 min at room temperature. This procedure was followed by incubation with primary antibody for 2 hr at room temperature. Mitochondria were visualized with a rabbit polyclonal antiserum raised against total mitochondrial outer membrane proteins (18). Arp2p was visualized using a rabbit polyclonal anti-peptide antibody (19) generously provided by B. Winsor (Centre National de la Recherche Scientifique, Strasbourg, France). Myc-tagged Arc15p was visualized using a monoclonal anti-myc antibody (20). Subsequent to incubation with primary antibodies, spheroplasts were washed with PBT and incubated with fluorescently labeled secondary antibody for 30-60 min at room temperature. The secondary antibodies used for these studies, FITC-coupled goat anti-rabbit IgG (Boehringer Mannheim) or rhodamine-coupled goat anti-mouse IgG (Kirkegaard & Perry Laboratories), were reconstituted, stored, and used according to the manufacturer's instructions. The spheroplasts were washed with PBS to remove unbound secondary antibody and mounted on microscope slides using mounting solution (1 mg/ml p-phenylenediamine, 90% (wt/vol) glycerol, and 1´ PBS).
Light Microscopy.
Images were collected with a Zeiss Axioplan II microscope using a Plan-Apochromat ´ 100, 1.4 numerical aperture objective lens, and one of two cooled charge-coupled device (CCD) cameras (Star-1, Photometrics, Tucson, AZ; or Orca-100, Hamamatsu Photonics, Bridgewater, NJ). Illumination with a 100-W mercury arc lamp was controlled with a shutter (Uniblitz D122, Vincent Associates, Rochester, NY). Image enhancement and analysis were performed on a Macintosh Quadra 800 computer using the public domain program nih image version 1.60.Quantitation of Mitochondrial Movement in Vivo.
Mitochondria were defined as motile if they displayed linear movement for three consecutive still frames. In all cases, the only portion of the organelle that was evaluated for movement was the tip of the organelle. Moreover, for any given cell, movement was evaluated only in a single optical plane. The velocities of motile mitochondria were determined by measuring the change in position of the tip of each moving mitochondrion as a function of time in time-lapse series recorded at 20-sec intervals over 10 min of real time. In wild-type cells ( ARP2 and ARC15 ), only velocities of organelles undergoing linear movement for at least three consecutive frames (1 min of real time) were measured. In the arp2-1 and arc15-GFP mutants, linear movements were not observed; therefore, the velocities of non-linear movements were measured. For all velocity measurements, nih image version 1.60 was used to determine the change in position (x-y coordinates) of mitochondria per unit time, and these were averaged to give a mean velocity. Polarized movement is defined as that which achieves a net displacement toward the bud in budding cells and is expressed as the percentage of all motile organelles exhibiting polarized movement over the time-lapse course.To determine the extent of mitochondrial movement, a single imaging plane within a cell was scored positive for movement if it contained motile mitochondria. In wild-type cells, approximately 10% of cell sections imaged showed movement. This is an underestimate of the level of movement, because it did not account for movement (i) in the z axis, (ii) in multiple focal planes within a given cell, (iii) in the bud, or (iv) at regions of the organelle other than the tips.
Preparation of Anti-Arp2 Antibody
. Preparation and affinity purification of anti-Arp2p antibody was carried out as described previously (19). Briefly, the peptide 40-RAEERASVATPLKDI-54 was synthesized, conjugated to ovalbumin, and used for polyclonal antibody production in rabbits (Research Genetics, Huntsville, AL). Individual antisera with highest titer against the peptide were affinity purified against the peptide antigen-bound Epoxy-activated Sepharose 6B column ( Amersham Pharmacia) according to the manufacturer’s instructions. After affinity purification, the anti-Arp2p antibody recognizes a protein band from yeast cell extracts that (i) has the apparent molecular weight expected of Arp2p, and (ii) is absent in cells bearing a deletion in the ARP2 gene (data not shown). Isolated mitochondria were solubilized as described in ref. 21. In short, mitochondria were solubilized to 1 mg/ml in a buffer containing 0.5% digitonin, 50 mM NaCl, 30 mM Hepes, pH 7.4, 1 mM PMSF, and protease inhibitor cocktail (see above), incubated for 45 min at 4°C with gentle agitation, and centrifuged at 12,500 ´ g for 10 min at 4°C. Supernatant from 750 µg mitochondria was mixed with 15µl of protein G-Sepharose beads (Amersham Pharmacia) coupled to anti-myc antibody and incubated for 2 hr at 4°C with gentle rotation. Thereafter, the beads were washed twice with solubilization buffer and once with the solubilization buffer without digitonin. Proteins bound to protein G-Sepharose beads were eluted with 1´ SDS/PAGE sample buffer. After separation by SDS/PAGE, proteins were immunoblotted with a monoclonal antibody to the myc epitope (see above) and polyclonal antibodies raised against Arp2p (see above) and the mitochondrial marker protein, cytochrome b2 (gift from G. Schatz, Biozentrum, University of Basel, Switzerland).Other Methods.
To detect actin, Arp2p, and Arc15p-myc levels in the DNY262 strain, whole cell extract was prepared by vortexing mid-log phase yeast cells with 0.5-mm glass beads in a solution consisting of 10% glycerol, 10 mM EGTA, 1% Triton X-100, 50 mM Tris·HCl, pH 7.5, 150 mM NaCl, 2 mM PMSF, and protease inhibitor cocktail. Protein concentration of the cell lysate was determined using the BCA assay following the vendor's protocol (Pierce). Gel electrophoresis, immunoblots, and antibodies used for Western blot analysis were described above.1. Miller, K. G., Field, C. M., Alberts, B. M. & Kellogg, D. R. (1991) Methods Enzymol. 196, 303- 319.
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