During clathrin mediated endocytosis, a polyhedral lattice of clathrin is linked to membrane proteins or lipids via clathrin binding adapta proteins such as SL one, SL one binds clathrin through its variant clathrin box or VCB. In this video, three methods are used for analysis of clathrin SLA one interactions. First yeast cells expressing GFP tagged native and VCB mutant mutants SLA one are imaged using spinning disc confocal microscopy.
Time-lapse movies are then analyzed to determine the effect of mutation on the kinetics of endocytosis. Second yeast cytosolic extracts are prepared from wild type cells as a source of clathrin and G-S-T-V-C-B fusion protein pull down assays are performed to examine clathrin VCB interaction in vitro. Third total extracts are prepared from yeast cells expressing native and VCB mutants.
SLA one and coun precipitations are performed to examine the effective mutation on the affinity of SLA one clathrin binding in vivo. Ultimately, the data obtained show that mutation of the VCB delays endocytosis and that binding of SLA one to clathrin is lost. In these mutants, Though this method can provide insight into clattering adaptor dynamics in endocytosis.
It can also be applied to other protein protein interactions within vesicles in the secretory pathway. Generally, individuals knew this method will struggle because photobleaching can di the fluorescent signal from the lower expression of tagged proteins from an endogenous locus. Because this protocol combines image analysis with biochemical studies of physical interactions, it's a very powerful approach to study the mechanisms that control endocytosis To study clathrin adaptor interactions during endocytosis yeast strains are generated that express GFP tag native and mutant versions of the clathrin adapto protein SL one first A GFP tag is added to the SL one C terminus by homologous recombination.
Next, an intermediate yeast strain is generated by disrupting the SLA one gene in a selective manner using a fragment containing the RA three gene with specific SL one flanking sequences to regenerate a functional gene for the expression of mutant SL one from the chromosomal locus. URA three is replaced by a SL one fragment containing the clathrin binding motif or VCB in which point mutations have been incorporated into VCB. The resulting yeast strains are then used for microscopic analysis of SLA one dynamics.
At endocytic sites, a similar approach is utilized for introducing VCB mutation in untagged SLA one strains such as TVY 6 1 14, which are suitable for biochemical experiments begin by growing the yeast strain SLA one GFP and the SLA one mutant strain SLA one A-A-A-G-F-P in four milliliters of supplemented SD media at 30 degrees Celsius in a rotator in the dark. Once the yeast reach an OD 600 of 0.1 to 0.4, transfer one milliliter of each culture to a micro centrifuge tube and spin at 4, 000 G for one minute at room temperature following the spin, discard about 950 microliters of SUP natant resuspend cells in the remaining liquid, which should be about 50 microliters. Deposit three microliters of the cell suspension onto a microscopy slide and cover it with a glass cover slip.
Protect the sample from light on a spinning disc on focal microscope. Mount the slide on a 100 x oil immersion objective. Locate the correct focal plane using bright field illumination or a 488 nanometer laser with appropriate filters to excite and detect GFP capture time-lapse images of SLA one GFP and SLA one A-A-A-G-F-P in cells with an exposure time of 500 milliseconds at an interval of one image per second for 150 seconds.
GFP labeled Punte will appear on the inner surface of the plasma membrane. Persist for several seconds and move toward the center of the cell as the signal quickly disappears indicating coat disassembly. Using the software, select a portion of a time-lapse image of a wild type SLA one GFP in which spots are seen to appear.
Remain for several seconds and disappear. Crop this section of the image for each frame of the time-lapse images. Next, use the software to generate a chime graph representing this section in each frame of the 152nd time lapse image by aligning the frames along an axis corresponding to the time elapsed here, this is demonstrated using Adobe Photoshop.
Calculate the dwell time of each spot by manually counting the number of time lapse frames in which it is present. In the chime graph, record the findings in a spreadsheet. Note that each spot should curve toward the interior of the cell as it is disappearing.
Reflecting endocytosis and disassembly of the coat. Analyze five spots per cell for approximately 40 cells. Then repeat this process with SLA one A-A-A-G-F-P to obtain yeast cytosolic extracts for use in GST protein affinity assays.
Begin by preparing three liters of the appropriate yeast strain according to the instructions in the accompanying text. Resuspend the pellet in three to five milliliters of sterile water and pet up and down until the pellet is completely resuspended. Next, pour about 150 milliliters of liquid nitrogen in a 250 milliliter plastic beaker.Flash.
Freeze the yeast by adding it dropwise into the liquid nitrogen in a circular pattern. To avoid clumping, do not let the pellets thaw add more liquid nitrogen If necessary, pour liquid nitrogen into a stainless steel blender container to chill it. Allow the liquid nitrogen to evaporate almost completely.
Then add the frozen yeast pellets. Close the blender with a cold rubber stopper and grind the yeast pellets for 10 seconds. Invert three to four times to mix contents and repeat the grinding step twice the ground.
Frozen yeast will have a powder like appearance chiller funnel with a 50 milliliter conical tube with liquid nitrogen. Using the cold funnel, transfer the ground yeast to the tube. The frozen ground yeast can be stored at minus 80 degrees Celsius or used immediately.
To obtain a cytosolic extract, weigh three grams of frozen ground TV Y 6 1 4 yeast in a 15 milliliter conical tube and resus suspend in three milliliters of room temperature. Buffer a containing protease inhibitor cocktail cap and invert the tube several times until the contents are completely thawed. Place it on ice.
Ultracentrifuge the extract at four degrees Celsius for 20 minutes at 300, 000 G.Following the spin, use perpet to carefully transfer the supinate or cytosolic extract to a conical tube on ice. Cut the end of a 200 microliter tip, then use it to add 100 microliters of a 50%glutathione sphero slurry in buffer A to the cytosolic extract. Place the tube on a rotator at four degrees celsius after the sample has rotated for 15 minutes.
Spin down the beads by centrifugation at four degrees Celsius for two minutes At 1000 G.Transfer the snat, which is the cytosolic extract to a fresh tube reserve 50 microliters for the input control sample. Freezing is not recommended at this point. Keep the extract on ice at all times and continue with the experiment.
To perform GST fusion protein Affinity assays, add one milliliter of PBS 30 microliters of a 50%glutathione Sphero slurry in buffer A and 50 micrograms of dialyzed G-S-T-G-S-T-V-C-B or mutant G-S-T-V-C-B to labeled fuge tubes. Rotated room temperature for 30 minutes to allow for binding. Wash the beads two times with PBS and one time with buffer a centrifuge.
10 seconds at 5, 000 G after each wash, then add one milliliter of yeast cytosolic extract. Place the tube on a rotator at four degrees celsius after the sample has rotated for one hour. Spin down the beads by centrifugation at four degrees Celsius for 10 seconds At 5, 000 G.Wash the beads three times with buffer A containing 0.1%tritton X 101 time with buffer A after each wash centrifuge at four degrees Celsius for 10 seconds.
At 5, 000 G after the last spin. Use the gel loading tip to remove as much liquid as possible. At this point, the samples can be stored at minus 20 degrees Celsius.
To continue at a later time, add 15 milliliters of two times lamb lee sample buffer to each tube. Place the samples on a heat block and incubate at 96 degrees Celsius for five minutes. Then centrifuge the samples for 10 seconds at 5, 000 G to bring the sample to the bottom of the tube.
Analyze the samples by immuno blotting using an antibody against the clathrin heavy chain to obtain total cell extracts for co precipitation experiments. Prepare frozen ground yeast samples as before this time using strain TVY six 14 expressing wild type SL one, the SL one A A a strain carrying an L-L-D-L-Q to A-A-A-L-Q mutation in TY six 14. Background and a SL one delta strain carrying a deletion of the SL one gene such as GPY 3 1 3 0 way.
Two grams of the frozen ground yeast in conical tubes and reus. Suspend them in two milliliters of room temperature buffer a containing protease inhibitor cocktail and 2%triton X 100 cap and invert the tube several times until completely thawed and then incubate on ice for 10 minutes. Periodically mixing by inversion.
Transfer the material to micro centrifuge tubes and spin at four degrees Celsius for 15 minutes at 16, 000. G carefully without disturbing the pellet. Transfer the sup, natant or total cell extract to a conical tube on ice.
Using a pipette add 100 microliters of a 50%protein, a sphero slurry in buffer, a rotate at four degrees Celsius for 15 minutes. Then spin down the beads by centrifugation at four degrees Celsius for two minutes at 1000 G and transfer the S supernatant to a fresh tube. Reserve 50 microliters of extract for input control To perform co immunoprecipitation assays.
Add one milliliter of PBS 30 microliters of a 50%protein. A sphero slurry in buffer A and two micrograms of a rabbit anti sla. One antibody to labeled micro centrifuge tubes.
Rotate at room temperature for 30 minutes. Wash the beads twice with PBS and one time with buffer. A containing 1%tritton X 100.
Add one milliliter of the total yeast extracts. SLA one, SLA one A A A and SLA one delta. Rotate for one hour at four degrees Celsius.
Spin for 10 seconds at 5, 000 G to pellet the beads following the spin. Discard the SUP natant and quickly wash the beads three times with buffer A containing 0.1%Tritan X 101 time with buffer. A spin down the beads one more time and using a gel loading tip, remove as much liquid as possible.
At this point, the samples can be stored at minus 20 degrees Celsius. To continue at a later time, add 15 microliters of two times laly sample buffer to each tube. Incubate at 96 degrees Celsius for five minutes and spin for 10 seconds at 5, 000 G.Analyze the samples by immuno blotting using an antibody to the clathrin heavy chain.Here.
Live cell imaging results from wild type and mutant SL one are compared on the left. A single yeast cell is shown with SLA one positive endocytic regions at the cell periphery shown in green. The area between white lines corresponds to the region from which krafts shown on the bright were generated.
Note that endocytic sites in the mutant persist significantly longer, about 80 seconds than wild, type about 30 seconds indicating a defect in coat formation. The physical interaction between clathrin and the sla one variant clathrin box was assessed by GST Fusion Protein. Affinity assays, GST fused to SLA one fragment AA 7 98 to 8 1 3 containing the sequence L-L-D-L-Q.
The corresponding A-A-A-L-Q mutant or GST alone were bound to glutathione, sero beads, and incubated with a cytosolic extract from wild type yeast cells. The associated proteins were alluded and analyzed by immuno blotting for clathrin heavy chain as seen here. Clathrin binds the G-S-T-V-C-B for not to GST or G-S-T-V-C-B mutant, suggesting that an intact variant clathrin box is required for SLA one binding to clathrin.
While attempting this procedure, it's important to work always close to the flame to avoid contamination of your GS cultures Following this procedure. Tagging of other fluorescent proteins such as RFP can be performed to examine colocalization of different proteins. After watching this video, you should have a good understanding of how to study protein protein interactions using biochemical and fluorescent cell imaging techniques.
