TITLE: Live cell Imaging of yeast cells expressing human PINK1-GFP and the TOM complex subunits
AUTHORS: Kenneth Ehses, Olawale Raimi, Ruben Fernandez-Busnadiego

[DESCRIPTION]
The PINK1-Parkin axis plays a major role in mitochondrial quality control and mutations have been closely associated with familial cases of Parkinson's disease [1]. To assess the correct functioning of mitochondria, PINK1 acts as a sensor by monitoring their import capabilities [2]. While proper import leads to degradation of PINK1, failed import causes PINK1 to form a complex with the translocon of the outer mitochondrial membrane, allowing it to be stabilized and activated by cross- phosphorylation [3]. Here we describe a protocol for live-cell imaging of Saccharomyces cerevisiae cells expressing human PINK1 with GFP tag at the C-terminus as well as all the human TOM complex subunits (TOMs 5, 6, 7, 20, 22, 40 and 70) immobilized using Concanavalin A coating. The aim of this experiment is to investigate if human PINK1 expressed in yeast cells is localized to the mitochondria. In this case, galactose was used to induce expression of the fluorescence-tagged protein-of interest and colocalization was tested using a respective mitochondrial tracker. The general workflow of this protocol is based on the one of [4].

[STEPS]
SECTION: Yeast Cell culture
1. Grow yeast cells   in liquid medium. 

 

SECTION: Yeast Cell culture
2. On the next day, check OD600 and dilute to OD600=0.1. 

 

SECTION: Yeast Cell culture
3. Allow the yeast cells to regrow into early exponential phase until they reach approx. OD600=0.3-0.6. At this state, we induce protein expression by addition of 2 % galactose into the medium. In our case, we were aiming for another incubation time of 180 min.

SECTION: Yeast Cell culture
4. For the staining of mitochondria, add 500 nanomolar (nM) of the Mito-Tracker into the medium and let it incubate for 45 min up to the completion of the incubation period. 

SECTION: Concanavalin A coating
5. Prepare a stock solution of 1 mg/ml Concanavalin A using sterile water.

SECTION: Concanavalin A coating
6. Place the coverslips into a suitable container of choice (e.g., 6-well, petri-dish), add 500 µL of the stock-solution on each coverslip and let them incubate for 30 min at Room temperature.

SECTION: Concanavalin A coating
7. Remove excessive Concanavalin A solution and wash 3x with sterile water for about 5 min.

 

SECTION: Concanavalin A coating
8. Leave the coverslips under the air at Room temperature until fully dry.

SECTION: Mounting of the cells for imaging
9. To remove excessive Mito-Tracker from the solution, spin down the yeast cells at 4000 x g, remove the supernatant and wash the pellet with 1x PBS. Repeat this step 2 more times.

 


SECTION: Mounting of the cells for imaging
10. Finally, add 8 µL of the resuspension to the Concanavalin A-coated coverslip and place it onto the glass slide. This volume should be sufficient for an air-tight closure.

 

SECTION: Imaging procedure
11. Place the sample in the microscope and navigate to a group of yeast cells for setting the correct imaging parameters.

 

SECTION: Imaging procedure
12. Dial in the appropriate laser and filter set for the fluorophore of your target-of-interest and start adjusting the exposure parameters according to the resulting brightness of your image.

 

SECTION: Imaging procedure
13. Images were acquired in Z-stacks to enable later deconvolution and/or maximum intensity projection (MIP).

 

SECTION: Basic image processing
14. For denoising the image stacks, we used the 'Small Volume Computational Clearing' in the Leica Application Suite X (LAS X) software.

 

SECTION: Basic image processing
15. When adjusting the histogram, make sure not to over- or under-expose the signal in any region. Apply adjustments to each channel individually and combine them afterwards for a composite image.