The overall aim of the following procedures is to generate purified disc. One aggregates able to invade recipient cells. Two different methods to accomplish this goal will be demonstrated with the first method.
The asome forming fluorescent protein disc one fusion protein is over expressed in NLF human neuroblastoma donor cells. The cells are then washed with PBS collected and lyed without the use of detergents. The DNA is digested and agro zones are purified using a sucrose gradient System.
Agrios purified by this method have a low cell invasion efficiency. The alternative approach is to overexpress amino acids 5 98 to 7 85 of the recombinant disc one protein in e coli. The protein fragment is then labeled with DITE 5 94 dialyzed and purified using an NI NTA matrix which removes any remaining unbound dye multimeric disc.
One fragments generated by this method have a high cell invasion efficiency to assess host cell invasion. Either the purified fluorescent protein disc, one risomes or the labeled recombinant disc. One fragment are incubated with the recipient cells of interest.
Trian blue is then added to quench the extracellular fluorescence and invasion is visualized by confocal microscopy. So far, protein aggregation has not been a recognized feature of the chronic mental diseases like schizophrenia. Where the disrupted in schizophrenia one protein is a candidate cell invasiveness of protein aggregates is a hallmark feature for protein confirmational diseases.
Here we show with two methods that disc one protein aggregates are cell invasive and hence that disc one related disorders or disc one neuropathies are protein confirmational disorders. The two methods are firstly the purification of full length disc, one ASOS from neuroblastoma cell lines and secondly, the expression of a C terminal multimeric disc. One fragment in e coli as well as the purification and labeling with the fluorescent dye.
While the first method only leads to a low efficient cell invasion of oh 0.3%of the cells taking up the purified microsomes, the pur recombinant protein fragment infects up to 20%of the exposed cells. The methods described here should be transferrable to other proteins where acrosome formation or multimeric assembly have been convincingly demonstrated. In each of the 10 10 centimeter dishes.
Seed 1.5 times 10 to the six human neuroblastoma and LF cells in supplemented RPMI 1640 medium and grow overnight. The next day cells should be 70 to 80%confluent label five dishes for transfection with PC DNA 3.1 MRFP and EGFP tagged human full-length disc one and five for transfection with EGFP plasmid alone. For each 10 centimeter dish, combine 15 micrograms of plasmid DNA 30 microliters met affecting and 750 microliters.
Opti MEM in a micro centrifuge tube and makes by pipetting up and down, incubate at room temperature for 20 minutes. Then to transfect the cells, add the DNA transfection reagent solution, dropwise to the cells incubate the cells at 37 degrees Celsius with 5%CO 2 48 hours after transfection monitored the expression of MRFP full length disc one and confirmed the formation of agro zones under a fluorescence microscope. Some cells harbor massive fluorescent protein inclusion as shown here.
This protocol combines a method to purify Lewy bodies from brain tissue and the protocol to isolate larger aggregates or agro zones since already existing methods are based on the usage of detergents, the isolation of detergent free protein for the application in cell invasiveness assays is critical. Next, aspirate the medium wash with PBS and add 400 microliters of PBS containing protease inhibitor cocktail. Then scrape the cells and add one milliliter of cell suspension and 10 ceramic beads to a two milliliter lysis tube.
Lys the cells using three 62nd pulses on a pre-cell lyse 24 homogenizer. The mechanical force of the process may increase the temperature within the sample to above 37 degrees Celsius. So care should be taken to chill the sample on ice After the lysis procedure, after cooling the cells on ice, add 10 millimolar magnesium chloride 40 units per milliliter DNAA and incubate for 60 minutes at 37 degrees Celsius with shaking.
Prepare 10 milliliters each of 80%50%and 20%sucrose weight per volume in PBS. Then in a 15 milliliter conical tube, prepare the sucrose gradient starting with two milliliters of 80%sucrose followed by two milliliters of 50%and two milliliters of 20%sucrose. Pour the gradient slowly and be careful not to disturb already poured layers.
Next layer the digested lysate on top of the gradient and centrifuge for 15 minutes at 1000 times G at four degrees Celsius. With the break off, carefully collect the interface between the 50 to 80%sucrose layer with a pipette and mix with five volumes of a 15 milliliter conical tube centrifuge for 15 minutes at 1000 times G at four degrees Celsius. Carefully remove the supernatant and keep the agri zone containing pellet.
Repeat washing two additional times in this interface. Agri zones are fluorescent and can be visualized in the microscope under UV light after the last wash, discard the supernatant and resuspend the pellet in 250 to 500 microliters of PBS. This pellet contains the purified agro zones.
Add 10 microliters of MRFP disc, one asome suspension to each well of a 24 well plate containing SHSY five Y human neuroblastoma cells. Constitutively expressing soluble GFP disc one C terminus on sterile glass cover slips and incubate for 48 to 72 hours following the incubation. Wash the cells three times with PBS and quench extracellular fluorescence with 0.04%trian blue for five minutes.
Then wash with PBS to remove the trian blue. Next, place the dish on ice and add 4%PFA and PBS for 10 minutes to fix the cells. Then wash once with sterile water and mount the cover slips on glass slides with prolonged gold with DPI mounting medium.
Confirm the uptake in invasion of exogenously. Applied aggro zones by colocalization with recruited proteins expressed by recipient cells in Zack images. This Zack encompasses approximately six micrometers in depth.
24 pictures shot in steps of 200 nanometers. The fluorescence of the MRFP disc one agri zone shown in red recruits the cytosolic GFPC terminal disc. One fragment shown in green colocalization presents as yellow and indicates that the agri zones have entered the recipient cytosol and recruited formerly soluble disc one protein.
In this part of the protocol human disc one will be labeled with DITE 5 94. This D allows the direct monitoring of the recombinant protein without the need of additional immuno staining. Begin by freeing protein prepared as described in the accompanying document from beam mercaptoethanol by dialyzing it three times in two liters of PBS at a dilution of one to 2000.
Next to label one milligram disc one with DITE 5 94 IDE dissolve one milligram per milliliter protein in PBS and add five millimolar TCEP to recover free TH groups add 20 microliters of the DMF dissolved dye to the reaction and incubate for two hours at room temperature using a SLI dialysis cassette with a molecular weight cutoff of 10, 000. Daltons dialyze the protein three times in PBS at a ratio of one to 2000 for two hours each time. Next to further increase the purity of the labeled protein.
A hiss tag-based affinity purification on a NI NTA column is performed. Wash one milliliter NI NTA matrix with 10 milliliters. PBS then apply the labeled dialyze protein to the column and let it run over the column.
Slowly the solution will lose a part of its blue color due to the binding of the protein to the NTA matrix unlabeled free dye will remain in the flow through. Once the protein solution is entered. The column, wash the column three times with 20 milliliters PBS to elute the protein add two milliliters of PBS containing 500 millimolar ole to the column.
Set the valve to slow speed and elute dropwise while monitoring the colored band on the N NI NTA column. To be alluded, this colored fraction of the eluate contains a labeled recombinant protein transfer the EIT to a SLI dialysis cassette and dialyze the EIT three times to 10 millimolar NAPI at a dilution of one to 2000 for two hours each in two liters after dialysis. Push the EIT through a sterile 0.45 micrometer syringe filter, then aliquot in five times 100 microliter samples and snap freeze in liquid nitrogen.
The total amount of protein should be 0.5 to one microgram per milliliter in each 100 microliter aliquot. To assess the invasiveness of the protein add medium containing five to 10 micrograms per milliliter labeled recombinant disc one protein to each well of a 24 well plate containing recipient SH SY five Y human neuroblastoma cells. Constituently expressing the soluble GFP tag disc one C terminus on sterile glass cover slips incubate for 48 to 72 hours.
Quench extracellular fluorescence fix and mount the cells as done for the agri zone treated cells confirm the invasion of exogenously applied recombinant protein by Zack Imaging. When the labeled protein is taken up, it is detected within the perimeter of recipient cells in images generated by confocal microscopy. This Zack encompasses approximately five micrometers of depth at 24 steps of 220 nanometers.
Here the recombinant disc one fragment 5 98 to 7 85 shown in red is taken up by recipient cells expressing the soluble C terminal GFP tagged disc one shown in green to determine the invasiveness of disc. One protein aggregates agro zones consisting of MRFP tagged full length disc one were purified from NLF cells and applied to recipient SH SY five Y cells as described in this photo as shown here. A-M-R-F-P tagged disc one acrosome invaded a human neuroblastoma SH SY five Y cell expressing soluble GFP tagged C terminal disc one.
Furthermore, the MRFP tagged disc one asome was able to recruit soluble GFP tagged disc one into insoluble aggregates to determine the invasiveness of DIA light labeled recombinant disc one was applied to SH SY five Y neuroblastoma cells expressing soluble GFP disc one as shown here. Recombinant disc one protein malters shown in red invaded the recipient cell yellow dots indicate a recruitment of soluble GFP tagged C terminal disc one into aggregates labeled recombinant disc one expressed and purified from e coli was also cell invasive to neurons in vivo when the multimeric protein was stereotactically injected into the medial prefrontal cortex of a rat. Zack imaging confirms the presence of recombinant disc one protein in rat cortical neuron stained with an antibody against neuronal nuclei.
We have just shown you two methods for generating disc one aggregates for studying cell invasion. Using either of these methods, we can investigate the cellular effects of disc one aggregates uptake without the potentially confounding variables of recombinant gene expression by transfection of foreign DNA. These techniques can be applied to investigate the biological effects of disc one protein pathology in vitro in cells, and in vivo by intracerebral inoculation or other administration in animal models.
We are confident that these techniques may also be transferred to other protein aggregates eventually with slight modifications to the protocol adopted to each particular protein. When doing this procedure, be sure to dedicate time for diligent analysis of confocal microscopy images.
