The Huntington's disease, HD, is an incurable, neurodegenerative pathology caused by a mutation in gene encoding huntingtin protein. The huntingtin is primarily associated with vesicles and microtubules, and probably involved in microtubule-dependent transport processes. To study the influence of mutant huntingtin on the dynamism of microtubules, we used in vivo visualization of EB3 protein, which regulates the dynamic properties of microtubules by bending and stabilizing the growing plus-ends.
To load fluorescently-labeled EB3 into human skin fibroblasts, we applied plasmid transfection. We used the primary fibroblast culture to obtain from the EB3 patient's skin biopsy for this study. Deliver the biopsy to laboratory within a few hours in DMEM medium, supplemented with 15 microgram per milliliter of penicillin, and 50 units per milliliter of streptomycin.
Place biopsy tissue in a six centimeters Petri dish, together with a small amount of medium. Using a sterile scalpel, cut the biopsy sample into pieces about 0.5 to one millimeters in size. Place one, two, obtained fragments into a 3.5 centimeters Petri dish, and place a sterile cover slip over the biopsy pieces.
Slowly add 1.5 milliliters of a growth medium. Culture fiberblasts in the growth medium in a CO2 incubator, in 5%CO2 that is seven Centigrade, 80%humidity. After four, seven days, first keratinocytes, then fibroblasts begin to migrate from the tissue to the bottom of the dish.
Cell cultivation. For transfection visualization, glass confocal plate dishes, confocal dishes should be used. Cover the dish bottom with autoclaved 0.1%gelatin solution prepared in distillate water.
Incubate for 15 minutes. Prepare the culture medium. Mix thoroughly, store tall at plus 4 Centigrade.
Warm the medium to plus 37 Centigrade, before adding to the cells. Assess the culture under the microscope. Remove the medium, and wash the cells with a sterile DPBS.
Add one milliliter of pre-warmed 0.25%trypsin solution to the cells. Check the cells under the microscope if they detach from the substrate completely. Deactivate trypsin with one milliliter of the culture medium.
Transfer the cell suspension into a 15-milliliter conical tube. Centrifuge the tube at 200 G for five minutes. Remove the supernatant, resuspend the cell palette in one milliliter of the culture medium.
Count the cell number. Calculate the required number of cells. De-seed them with a density of eight to 15, multiplied by 10 to the power of three, passed by a centimeter.
This is panned into milliliters of the culture medium. Remove the gelatin solution from the culture dish prepared for inoculation, and immediately add two milliliters of the cell suspension. Cultivate fibroblasts at plus 37 Centigrade in a CO2 incubator.
Refresh the medium every two, four days. For experiments, use the cells of four, 11 passages. By the time of transfection, the confluence should be no more than 70, 80%Replace the culture medium with a fresh one 24 hours before transfection.
Prepare a DNA-lipid complex, based on the area and density of the cell seeding. Liposomal transfection agent, and the density of the cell seeding of one, multiplied by 10 to the power of four cells, passed by a centimeter was used. Add three microliters of commercial reagent to 125 microliters of Opti-MEM, containing no antibiotic.
Without touching the tube walls, gently resuspend. Dilute plasmid DNA, GFP-EB3, adding one microgram of the plasmid DNA to 125 microliters of OptiMEM. Gently resuspend.
Add diluted plasmid DNA to each tube of diluted transfection reagent, one to one ratio. Incubate for 30 minutes. Add the DNA lipid complex to the six centimeters Petri dish with cells.
Mix with a cruciform swing for 30 seconds. Incubate cells with a transfection agent for 24 hours, then change the medium to fresh one. Analyze the efficiency after inspection in 24 and 48 hours.
Preparing for imaging. Before live imaging of cells, change the culture medium to a medium without PH indicator dye to reduce autofluorescence. Carefully apply mineral oil to the medium cell phase to completely cover the medium, isolating it from the external environment to reduce the O2 penetration, and the medium evaporation.
Use a macro lamp and all image in 60-fold, 100-fold objective lens with high numeral aperture to take image. For in viva observations, the microscope must be equipped with an incubator to maintain the necessary conditions for the cells, including hitting the optic table, and the lens to pass 37 Centigrade, a closed chamber was CO2 supply, and humidity-level support. Place the confocal dish with the cells in the holder of the microscope before imaging.
Ensure that the dish and the camera are securely attached to the holder to avoid drifting while taking images. Setting the imaging parameters. Choose the low exposure values, since slide induces cell-damaging, reacts with oxygen spaces.
To study the dynamics of microtubules in human skin fibroblasts, we selected a 300 millisecond exposure. Focus on the object of interest. For long-time, time-lapse imaging, the automatic focus stabilization system, perfect focus system is necessary, since there may be a shift along the set axis, and the object of interest will thus be out of focus.
Choose the optimal imaging conditions, depending on the cell's photosensitivity, and the rate of the flare chrome fading. Since microtubules are highly dynamic structures, are reasonably short time in turn can be selected, and the frame rate must be sufficiently high. To investigate the microtubule dynamics in skin fibroblasts, we used a frequency of one frame per second, for three, five minutes.
When selecting the next object to get image, move away from the already imaged area, since under the influence of light, there is an elapsable photo-bleeding. Since we used a relatively high imaging frequency, the shutter didn't close between images, and the lamp was late for the entire period of imaging, which is fading increased. Choose the optimal videos for studying the microtubule dynamics visually, taking into account the quality of transfection, the quality of the microtubules'images, optimal signal-to-noise ratio, and the absence of drifting of analyzed cell.
Use the selected videos to study the microtubules plus-ends dynamics, by trussing them in the Fiji program. The most critical step in the protocol is transfection, ensuring sufficient protein expression. Good signal-to-noise ratio, no photobleaching of microtubules, and absence of cell drifting are critical for imaging.
In vivo visualization of microtubule dynamics provides vital information on the properties of mutant huntingtin. Our protocol can be applied to studies of other diseases, whose pathology implicates dynamic properties of cytoskeleton.
