My research focuses on the structured function relationship of mammalian zinc transporters with special emphasis on the regulation mechanism. Due to the importance of zinc ion to cellular function, it is challenging to obtain accurate measurement of cellular zinc dynamics. Our protocol addresses the low signal to noise ratio and low temporal resolution of zinc dynamics.
This method allows testing of zinc influx across the cell membrane with high temporal resolution and considerably less background noise and cross interactions by other divalent cation. We found that the unstructured extension of the mammalian transporter ZnT-1, C terminal domain is not involved in the zinc transport function. This allows researchers to focus on other domains of the ZnT-1 protein for zinc transport regulation.
Begin By analyzing transfected HEK293T cells using an inverted fluorescence microscope. Once the cells are focused, using brightfield light switch to fluorescent mCherry excitation wavelength of 587 nanometers and emission wavelengths of 610 nanometers. After turning off the brightfield light, check for the fluorescence of the cells to confirm the expression of ZnT-1 mCherry.
To prepare dye loading solution, take four microliters of zinc specific fluorescent dye solution. Then add four microliters of 10%Pluronic acid. Mix the solution by pipetting up and down, and transfer it to a 1.5 milliliter tube.
Add 750 microliters of washing solution into the prepared solution and vortex the tube vigorously. Again, add 750 microliters of wash solution. Once vortexed, add 750 microliters of this solution into two wells of a new six-well culture plate and cover it with aluminum foil.
Using tweezers transfer a 13 millimeter cover slip from the previously cultured HEK293T cell plate into a six well plate. After covering the plate with foil, gently shake it for 15 to 20 minutes and replace the dye loading solution with the prepared washing solution. Once the plate is covered, shake it again for 20 minutes.
To begin, turn on the microscope, light source, camera, and suction system. For washing the perfusion system use Ringer's solution in the first chamber and Ringer's zinc solution supplemented with pyrithione in the second chamber. After closing the tap, fill the chambers with the same solutions.
To begin sample preparation, place the perfusion chamber with the narrow side of the groove facing up and apply a ceiling silicone around the groove. Then using a fine tweezer transfer a 13 millimeter cover slip from the washing solution on top of the groove with cells facing down. Place a 22 millimeter cover slip on top of the 13 millimeter cover slip and tighten it using the tweezer, taking care not to crack the cover slips.
Flip the chamber and press it to release the liquids. Then fill the groove with 100 microliters of Ringer's washing solution. And press again, ensuring no leakage.
Mount and secure the perfusion chamber onto the platform. Then attach the perfusion and suction tubes for perfusion over the cells in the groove. Change the perfusion rate to approximately two milliliters per minute and turn on Ringer's solution perfusion.
For measurements, open the imaging software. Set the microscope magnification to 10X using the left side button of the microscope. After choosing live view, use the joystick to move the platform and focus on the cells in the groove.
Select the appropriate wavelength for mCherry. Once the cells are focused, move the platform for the selection of appropriate cell patches. Select the Draw ROIs dropdown menu.
Choose Draw circular ROIs and draw ROIs on cell clusters. with the mCherry expressing cells. Create new ROIs beyond the first by holding down the shift button and then by pressing and dragging the existing ROIs.
Select Draw Square Background ROI from the dropdown menu and adjust the location and size of the background ROI where cells are not present. After ensuring the absence of cells in the background ROI go to the ND Acquisition tab. Select the Wavelength sub tab and mark its checkbox.
Ensure that the checkbox for GFP wavelength is also marked. Click on the Time sub tab and define the measurement interval as every five seconds and duration as 30 minutes. On the microscope panel, press the On to enable the perfect focus system or PFS.
Under ND Acquisition ensure PFS On is ticked. Adjust the focus and click Run now. Start baseline period measurement for 90 seconds using Ringer's solution perfusion.
After 90 seconds switch from the Ringer's solution perfusion to the Ringer's zinc solution perfusion. And click the red flag on the bottom right of the main interface panel. Once the fluorescence rise starts to saturate, change back to perfusion with Ringer's solution until the experiment time expires.
To export the data with baseline subtraction, press the Background correction button and view the changes in the ND Acquisition window. Click the Export button to save the data in the desired location. An increase in fluorescence resulted from increased intracellular zinc concentration, while a decrease indicated the activity of ZnT-1 transporting zinc across the cell membrane.
The wild type ZnT-1 had a smoother fluorescence curve than the mutant relating to the variability in the cellular responses to zinc load. A box plot comparing the activities of wild type and Delta-USCTD showed similar average transport rates indicating a lack of difference between the wild type and the mutant. However, the difference in the spread may indicate a functional difference.
