The overall goal of this methodology is to identify the Kinesin-1 cargos using fluorescence microscopy. This method helps us answer key questions in the field of molecular motors, such as which cargos are being carried by kinesins and myosins. The main advantage of this technique is that the dominant negative KIF5B mutants can help us visually identify this cargo using fluorescent microscopy.
To begin, for live cell or indirect fluorescence imaging at 40X or below, feed 0.2 to 0.3 million HeLa cells in one milliliter of complete medium in each well of a six well plate. Grow the cells at 37 degrees Celsius with five percent carbon dioxide. For indirect immunofluorescent studies with magnifications above 40X, in a biosafety cabinet to avoid contamination, use absolute alcohol to wash cover glasses and stand them up in the wells of a six well plate to air dry.
When the cover slips have dried, lay them down in the wells and seed 0.2 to 0.3 million cells in one milliliter of complete DMEM in each well of the plate. Culture the cells overnight. The following day, inside a biosafety cabinet, Use 0.1 milliliters of transfection medium to dilute 0.6 micrograms of a tdTomato-tagged wild type or motorless KIF5B expression plasmid with or without a plasmid expressing a candidate cargo protein.
In another 1.6 milliliter microcentrifuge tube, add 1.8 microliters of the transfection reagent to 0.1 milliliters of transfection medium. Then incubate at room temperature for five minutes. Next, add 0.1 milliliters of the diluted transfection reagent to the diluted DNA samples.
Invert the tubes to mix the contents and spin down for five seconds. Then incubate the samples at room temperature for 45 minutes. During the incubation period for the formation of the transfection complex, use PBS to wash the cells three times.
Then add 0.8 mililiters of pre-warmed transfection medium to each well, and return the plates to the incubator. After the diluted transfection reagent has incubated for 45 minutes, add 0.2 mililiters of the solution dropwise in each well of the plate. Gently rock the six well plate for five seconds and return it to the 37 degree Celsius incubator for six to eight hours.
Then use complete DMEM to replace the medium. To carry out live cell imaging after an additional 16 hours of incubation, add the DNA intercalating stain Hoechst 33342 to the medium to a final concentration of 1 micromolar and incubate the cells at 37 degrees Celsius for 10 minutes. Then, using a 40X objective, examine the cells for the expression of fluorescent proteins.
To perform indirect immunofluorescence on cells that have been incubated with transfection reagent:After preparing paraformaldehyde according to the text protocol, use room temperature PBS to wash the cells by swirling the six well plate for five seconds. Add one milliliter of freshly prepared 4%paraformaldehyde and PBS per well and incubate at room temperature for 30 minutes. Then use PBS to wash the cells once by swirling the plate and discard the solution.
Next, add one mililiter of 0.1%Triton-X 100 in PBS to each well and incubate at room temperature for 30 minutes to permeabilize the cells. Then use PBS to wash the cells four times, discarding the solution after each wash. Add primary antibodies and incubate the cells at room temperature with rocking for four hours.
Following the incubation, use PBS to wash the cells four times by swirling the plate. Then add one milliliter of fluorescent dye conjugated secondary antibodies and incubate in the dark at room temperature with rocking for two hours. After washing the cells four times with PBS, add one milliliter of DAPI and incubate in the dark at room temperature for ten minutes.
If using cover glasses with the samples, apply 10 microliters of mounting solution at the center of the microscope slide and mount a cover glass over the mounting solution. Then incubate in the dark at room temperature overnight. The next day, use nail polish to seal the edges and incubate in the dark in a fume hood over night.
To carry out fluorescence microscopy, use a 40X objective with the filter sets for DAPI, CFP, FITC, and Cy3 where applicable. As demonstrated here, exogenously expressed wild-type KIFB appeared homogeneously in the cytoplasm, while c-MYC appeared mainly in the nucleus. The motorless KIF5B mutant, however, formed aggregates in the cytoplasm and this led to the aggregation of c-MYC.
In addition, the motorless KIF5B also induced aggregation of the endogenous c-MYC and the transcription factor p53, indicating that c-MYC and p53 are both the cargos of Kinesin-1 and KIF5B regulates the subcellular localization of both endogenous proteins. This figure illustrates that the movement of transcription factors by KIF5B appears to be specific because the expression of the motorless KIF5B mutant did not affect the subcellular localization of c-Fos or cause it to aggregate. These data reveal that the method demonstrated in this video allows for the high specificity identification of Kinesin-1 cargos.
While attempting this procedure, this is important to remember that the kinesins most suited for cells that has a lot of volume of cytoplasm so that aggregating in the cytoplasm can easily be observed. After this development, this technique paved the way for researchers in the field of molecular motors to construct similar dominant negative mutants of other kinesin and myosins in order to identify their particular cargos. After watching this video, you should have a good understanding of how to use the dominant negative mutant of a molecular motor to identify this cargo, using fluorescent microscopy.
Don't forget that working with pheno, ethidium bromide, and paraformaldehyde can be extremely hazardous and precautions should always be taken to avoid direct skin contact with them while performing this procedure. Thanks for watching. Good luck with your experiment.
