The overall goal of the following procedure is to determine the submitochondrial localization of a normally nuclear cell cycle kinase and then to analyze how its mitochondrial localization affects the progression of the cell cycle. This method can help answer key questions in the mitochondrial research field, such as how nucleus communicates with the mitochondria during the cell cycle. By tagging proteins with a mitochondria leading sequence, we can take advantage of specifically over expressions in protein in the mitochondria, so we can study their mitochondria-specific functions.
Though this method can provide insight into mitochondria targeting of certain proteins, it can be also be applied to the other organelles, such as the nucleus, ER, golgi, and a lysosome. After culturing, homogenizing, and pelleting cells according to the text protocol, transfer the supernatant into a new tube. And centrifuge the sample at 7, 000 g and 4 degrees Celsius for 10 minutes.
Then use 200 microliters of ice-cold IBC buffer to resuspend the pellet, and divide the homogenate into two aliquots. Centrifuge the samples again at 7, 000 g and 4 degrees Celsius for 10 minutes. And repeat the wash.
After discarding the supernatant, add 30 microliters of cell lysis buffer to one of the pellets, and store the lysate at 80 degrees Celsius for immunoblotting. To carry out sodium carbonate extraction with the second pellet to separate soluble and membrane-bound proteins, add 250 microliters of 0.1 molar sodium carbonate pH 11.0, and incubate on ice for 30 minutes. Centrifuge at 100, 000 g for 20 minutes.
Then collect the supernatant and add an equal volume of freshly made, 20%trichloroacetic acid to precipitate the proteins. Keep on ice for 30 minutes. In the meantime, add 30 microliters of cell lysis buffer to the pellet and sonicate according to the text protocol before storing at 80 degrees Celsius for immunoblotting.
After the 30-minute TCA incubation, centrifuge the reaction at 15, 000 g for 10 minutes. Discard the supernatant, then use 80 microliters of cell lysis buffer to resuspend the pellet. After isolating mitochondrial fractions from cells as described in the text protocol, separate the mitochondrial fractions into 10 equal portions.
Pellet the samples at 7, 000 g and 4 degrees Celsius for 10 minutes. Use 30 microliters of a range of concentrations of hypotonic sucrose buffers with or without trypsin, to dissolve each pellet. And incubate on ice for 30 minutes.
Add 3 microliters of 10 millimolar PMSF to the trypsin-containing vials to stop the trypsin digestion. And incubate on ice for 10 minutes. Centrifuge the samples at 14, 000 g and 4 degrees Celsius for 10 minutes.
And transfer the supernatant into a new tube. To lyse the pellet, add 30 microliters of cell lysis buffer. Sonicate the samples as described in the text protocol and store at 80 degrees Celsius.
To construct mitochondria targeted GFP/RFP-tagged cyclinB-1 Cdk-1 vectors, clone the mitochondria targeting sequence from the precursor of human cytochrome-c oxidase subunit 8A, and frame with the n-terminus of GFP or RFP at the Nhe1 and bAmH1 sites, of pEGFP-N1 or pERFP-N1, using standard molecular cloning techniques. Using the primers outlined in the text protocol, amplify the Cdk1 and cyclinB1 genes following standard techniques. Then use BamH1 to digest the PCR products.
Run the reactions on a 1%agarose gel, before using a razor blade to cut out the correct size DNA fragments. Then use a gel extraction kit to purify the DNA. Next, digest one microgram of MTS-pEGFP-N1 and MTS-pERFP-N1 plasmids with 1 microliter of BamH1 at 37 degrees Celsius for 2 hours.
Then add 1 microliter of calf intestinal alkaline phosphatase and incubate at 37 degrees Celsius for 30 minutes. After running the digestion products on a 1%agarose gel and purifying the DNA as just described, set up a ligation reaction using the reagents listed in the text protocol. Then incubate the reaction at 4 degrees Celsius overnight.
Transform e. coli dH5-alpha competent cells with 10 microliters of the ligation mixture. And grow the bacteria on plates of LB agar plus 10 milligrams per milliliter of kanamycin at 37 degrees Celsius overnight.
The following day, use a sterile pipette tip to pick a colony from a plate. And insert the tip into a tube containing 5 milliliters of LB kanamycin. Incubate the culture overnight and the following morning, use a mini prep kit according to the text protocol to isolate the plasmid.
To tranfect exponentially growing MCF-10A cells, use Cdk1 or cyclinB-1 plasmids to prepare plasmid transfection reagent at a ratio of 1:2 in 100 microliters of serum and antibiotic-free medium. Transfect the cells and incubate at 37 degrees Celsius for 48 hours. Stain and visualize the mitochondria according to the text protocol.
To carry out cell sorting, tranfect 2 times 10 to the 5th cells with the desired vectors in a 6-well plate using a 1:2 ratio of DNA to tranfection reagent prepared in 2.5 milliliters of serum and antibiotic-free medium. After incubating the transfection for 48 hours, use flow cytometry to live-sort the cells stably expressing the GFP-tagged Cdk-1 and RFP-tagged clyclinB1 proteins according to the text protocol. To measure cell cycle length using the EdU labeling flow cytometry assay, seed sorted cells in 6-well plates at a density of 2.5 times 10 to the 5th cells per well.
After an overnight incubation, add EdU to the culture medium at a final concentration of 25 micromolar and incubate for an additional hour. Then at two hour intervals, use 1%BSA in 500 microliters of PBS to wash one well of cells. And collect in a 1.5-milliliter tube.
Centrifuge the cells at 350 g for 5 minutes. Then discard the supernatant. Dislodge the pellet by adding 100 microliters of fixing solution.
Mix well and incubate at room temperature for 15 minutes. Next, use 1 milliliter of 1%BSA in PBS to wash the cells three times. Then use 0.5 milliliters of 70%ethanol to fix the cells at 4 degrees Celsius overnight.
When performing EdU labeling with cells transfected with GFP and RFP tagged proteins, it is critical to quench the fluorescent signals from GFP and RFP. To achieve this, we add an extra step of overnight cell fixing using 70%ethanol. The following day, after washing and permeabilizing the cells according to the text protocol, add 0.5 milliliters of reaction cocktail into each tube and mix well.
Following the incubation in the dark and another wash, use 50 micrograms per milliliter of propidium iodide or PI in 1%BSA PBS to stain the DNA. Analyze the cells by flow cytometry to follow the EdU-positive population. Present a scattered dot plot of EdU-labeled cells stained for DNA content and EdU.
Use the APC channel for Alexa 647 EdU utilizing a 670 30-bandpass filter with all light present, less than 685 nanometers hitting that filter and the phycoerythrin channel for PI with a 581 15-bandpass filter in front of it, with all light present less than 600 nanometers hitting that filter. With a standard gating strategy for acquistion, plot FSC area by SSC area for morphology, followed by PI by Alexa 647 EdU for cell staining. Record data for all tubes one by one, acquiring 10, 000 events per sample.
In this figure, mitochondrial matrix protein Hsp60 and intermembrane space protein Timm13 were used as submitochondrial localization markers. Similar with Hsp60, but unlike Timm13, cyclinB1 and Cdk1 were protected from trypsin digestion, indicating that they localize to the mitochondrial matrix. As shown here, by Western blotting of the isolated mitochondrial fractions, using the MTS and GFP-tagged cyclinB1 and Cdk-1 constructs, overexpression of cyclinB1 and/or Cdk-1 was achieved in the mitochondria.
Using an EdU pulse chase assay, it was demonstrated that labeled S phase cells progressed through G2M phase and appeared in G1 phase as fast as 4 hours in cells expressing wild-type mitochondrial cyclinB1 Cdk-1. As compared to 6 hours, in cells transfected with a vector control or mutant cyclinB1 Cdk-1, indicating that enhancement of mitochondrial cyclinB1 Cdk-1 accelerates cell cycle progression. Following this procedure, other methods like mitochondrial ATP generation, oxygen consumption, membrane potential, and reactive oxygen species can be measured in order to answer additional questions like how mitochondrial localization of cell cycle kinase Cdk-1 alters mitochondrial respiration and energy output.
After watching this video, you should have a good understanding of how to study the mitochondrial localization and the mitochondria-specific functions of a nuclear kinase.
