Paclitaxel was purchased from Sigma. Anti-gamma-synuclein antibodies were purchased from Santa Cruz Inc., anti-beta tubulin antibodies from Cytoskeleton Inc, and anti-beta actin antibodies were from Sigma. Alexa568-anti-goat secondary antibody was purchased from Invitrogen. Human recombinant gamma-synuclein was purchased from Alpha Diagnostic International. Human recombinant MAP2 and kinesin heavy chain motor domain protein were purchased from Cytoskeleton Inc. MitoTracker Red was purchased from Invitrogen.
Breast cancer cell line T47D and GFP-tubulin HeLa cell line (kindly provide by Dr. Tim Yen at Fox Chase Cancer) that stably expressing GFP tagged tubulin were cultured in DMEM supplemented with 10% FBS, L-glutamine and P/S. A2780 parental cells and A2780 cells stably overexpressing human gamma-synuclein were established as previously described [15
] and maintained in RPMI supplemented with 10% FBS, L-glutamine, P/S and insulin.
The method for stably transfecting human gamma-synuclein gene into A2780 cells was described previously [15
]. For transient transfection of human gamma-synuclein or empty control vector into GFP-tubulin HeLa cells, Lipofectamine2000 (Invitrogen) was used and the protocols provided by the company were followed. Briefly, GFP-tubulin HeLa cells were cultured to approximately 80~90% confluency before transfection. pCDNA3-gamma-synuclein and empty control vector were mixed with lipid in Opti-MEM at recommended ratios and incubated for 30 min at room temperature to form DNA-lipid complex. DNA-lipid complexes were added to cell culture and incubated overnight. The transfection efficiency was examined either by immunofluorescence staining or western blot analysis.
Immunofluorescence staining and microscopy
To visually assess the co-localization between GFP microtubules and gamma-synuclein, GFP-tubulin HeLa cells with transient gamma-synuclein expression were cultured on two-well or four-well chamber slides at approximately 60~70% confluency. To preserve the microtubule structure, cells were treated with microtubule stabilization buffer (MTSB, 4 M glycerol, 100 mM PIPES pH 6.9, 1 mM EGTA) for 1 min followed by 2 min in MTSB + 0.5% TritonX-100 and immediate fixation in 3.7% paraformaldehyde for 5-7 min. For gamma-synuclein staining, the slides were washed with PBS three times and blocked with 3% BSA. The slides were then washed with PBS and incubated at room temperature with goat-anti-human-gamma-synuclein antibody diluted in 3% BSA (1:100). 1 hr later the slides were washed with PBS three times again and incubated with Alexa568-anti-goat secondary antibody for 1 hr. The slides was washed three times with PBS and mounted with Vectashield containing DAPI. For mitochondria staining, A2780 cells seeded in two-well or four-well chamber slides were incubated with MitoTracker for 15 min at room temperature. Then slides were washed with PBS and fixed with methanol for 5 min. The slides were washed again and subjected to mounting and sealing. For Immunofluorescence microscopy, image was captured (600×) either using Nikon ECLIPSE TE300 microscope or Nikon E800 upright microscope with BioRad Radiance 2000 confocal scanhead. LaserSharp2000 software was used for confocal image acquisition and procession.
Tubulin polymerization assay
Tubulin polymerization assay was performed using Tubulin Polymerization Assay Kit (Cytoskeleton, Inc.) and followed the protocols provided by the manufacturer. Briefly, 200 μl of highly purified tubulin from bovine brain at 20 μg/μl concentration was mixed with 420 μl ice cold tubulin polymerization buffer to give a final concentration of 3 mg/ml tubulin in 8 nM PIPES pH6.9, 2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP and 10.2% glycerol. 100 μl of the diluted tubulin added with DAPI was immediately loaded into each of 6 wells of prewarmed (37°C) 96-well plate that has been pre-added with two wells of 10 μl PBS as negative control, two wells of 10 μl of paclitaxel as positive control (final concentration 3 μM) and two wells of 10 μl of recombinant human gamma-synuclein (final concentration 25 μM). The plate was immediately subjected for kinetic reading at 37°C by excitation at 360 nm and emission at 420 nm for 1hr. Polymerized microtubules in the presence of gamma-synuclein were centrifuged for 90 min at 22,500 × g. Supernatants and pellets were separated and subjected to SDS-PAGE analysis. For scanning electron microscopy, microtubules from tubulin polymerization assay were centrifuged at 22,500 × g for 20 min. The pellet was fixed with 2% glutaraldehyde and 1% tannic acid and subjected for microscopy analysis.
Antibody crosslinking assay
To prepare preformed paclitaxel stabilized microtubules, 8 μg of unlabeled tubulin with trace amount of Rhodamine–labeled tubulin (mass ratio = 120:1) was mixed with 5 μl of General Tubulin Buffer (80 nM Piperazine-N,N′-bis [2-ethanesulfonic acid] sequisodium salt; 2.0 mM MgCl2; 0.5 mM Ethylene glycol-bis (b-amino-ethyl ether) N,N,N′,N′-tetra-acetic acid, pH 6.9) and 2 μl of cushion buffer (80 nM Piperazine-N,N′-bis [2-ethanesulfonic acid] sequisodium salt; 2.0 mM MgCl2; 0.5 mM Ethylene glycol-bis (b-amino-ethyl ether) N,N,N′,N′-tetra-acetic acid, 60% v/v glycerol, pH 6.9]. Both General Tubulin Buffer and cushion buffer were purchased from Cytoskeleton, Inc. The mixture was incubated for 5 min at 37°C and immediately added with 100 μl of paclitaxel at 200 nM. The integrity of the preformed microtubules was checked using fluorescence microscope. To perform antibody crosslinking assay, 50 μl of preformed microtubules was mixed with 200 ng of either anti-tubulin or 200 ng anti-BSA antibodies or with 200 ng or 600 ng anti-gamma-synuclein antibodies. The bundle formation was examined 5 min later under fluorescence microscope (200×).
Microtubule bundling by gamma-synuclein and MAP2
10 μg tubulin was mixed with 4 μl the General Tubulin Buffer, 2 μl cushion buffer and (1) 2 μl or 4 μl of recombinant human gamma-synuclein (1 μg/μl), (2) 2 μl of recombinant MAP2 or BSA (1 μg/μl), (3) 2 μl of gamma-synuclein (1 μg/μl) plus 2 μl MAP2 (1 μg/μl), (4) 4 μl of gamma-synuclein (1 μg/μl) plus 2 μl MAP2 (1 μg/μl), or (5) 2 μl of BSA (1 μg/μl) plus 2 μl MAP2 (1 μg/μl). After the mixture was incubated at room temperature for 15 min, the sample was examined by fluorescence microscopy (200×).
Chamber perfusion assay
This assay was modified from kinesin motility assay protocol used for Kinesin Motility Assay Biochem Kit (Cytoskeleton Inc). All the working solutions including General Tubulin Buffer, blocking buffer and wash buffer was provided by the kit. Briefly, 4 μg of BSA (negative control), recombinant human gamma-synuclein, or recombinant human kinesin (positive control) were dissolved in 11 μl of General Tubulin Buffer. To coat the chamber, solutions were perfused into 3 individual perfusion chambers and incubated at room temperature for 5 minutes. After coating, 11 μl of blocking solution was perfused through perfusion chamber for 5 min to prevent non-specific binding. To test the binding activity, 10 μl of preformed fluorescent microtubules were perfused through perfusion chamber and incubated for 5 min, followed by two times of washing with 20 μl of wash buffer to remove unbounded microtubules. The chambers were then subjected to examination under fluorescence microscope (200×).
Cytoskeleton was isolated using Microtubules/Tubulin In Vitro Assay Kit (Cytoskeleton Inc.). Briefly, T47D cells grown on 10 cm cell culture plate were covered with warmed (37°C) Lysis and Microtubule Stabilization Buffer (component of the kit) along with ATP, GTP and phosphatase inhibitors, collected, homogenized with 25 G syringe and incubated for 10 min at 37°C. 95% cell rupture was achieved based on microscopy examination. The homogenate was centrifuged at 50,000 × g for 1 hr at 37°C. The supernatant and the pellet were subjected to western blot analysis.
In vivo microtubule bundle measurement
GFP-tubulin HeLa cells were transfected with gamma-synuclein or empty vector as control. Three days later both cultures were treated with paclitaxel (10 μM) for 5 hr to induce microtubule bundle formation. 10 fields were randomly chosen and subjected for fluorescence microscopy imaging (600×). All the images were taken using the same exposure time. The experiments were repeated twice. The intensity and the area of the microtubule bundle were measured using ImageJ software. The results were plotted as the percentage of microtubule bundle reduction mediated by gamma-synuclein overexpression versus vector controls. The P value was calculated using a two-tailed t test.