Mar 12, 2024
Live cell Imaging of yeast cells expressing human PINK1-GFP and the TOM complex subunits
- Kenneth Ehses1,2,
- Olawale Raimi3,4,
- Ruben Fernandez-Busnadiego1,2,4
- 1Institute of Neuropathology, University Medical Center Göttingen, 37099 Göttingen, Germany;
- 2Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), University of Göttingen, Germany;
- 3MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK;
- 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
Protocol Citation: Kenneth Ehses, Olawale Raimi, Ruben Fernandez-Busnadiego 2024. Live cell Imaging of yeast cells expressing human PINK1-GFP and the TOM complex subunits. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp3p71vzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 20, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 95585
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Abstract
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].
Attachments
1022-2635.pdf
351KB
Materials
Medium
Standard yeast medium you use for liquid culture- In this specific case:
- Yeast Synthetic Drop-out Medium Supplements without tryptophanMerck MilliporeSigma (Sigma-Aldrich)Catalog #Y1876 Yeast Nitrogen Base without Amino Acids Wickerham formula, classification of yeasts based on amino aBecton Dickinson (BD)Catalog #291940
- D(+)-Raffinose pentahydrateCarl RothCatalog #5241.3
Consumables and reagents
- Concanavalin ACarl RothCatalog #7246.1
- MitoSpy™ Red CMXRosBioLegendCatalog #424801
- Cover glasses, thickness no. 1, 20 mm x 20 mm, square shape, pure whiteBRANDCatalog #470050
- Glass slides (Epredia, #AD00008432E01MN250)
- PBS pH 7.4Thermo Fisher ScientificCatalog #10010023
Instruments
- Leica DMi8 with HCPL APO 63x/1.40 lens
- Incubator (Thermo Fisher Scientific, #10519912)
- Tabletop centrifuge
Software
- Leica Application Suite X, ImageJ
Yeast Cell culture
Yeast Cell culture
Grow yeast cells Overnight in liquid medium.
Note
In our case, we picked a colony on a respective plate and inoculated 3 mL of liquid medium and let them incubate at 30 °C and 180 rpm Overnight .
On the next day, check OD600 and dilute to OD600=0.1.
Note
Here, we again aim for a total volume of 3 mL and continue the incubation.
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 03:00:00 .
3h
For the staining of mitochondria, add 500 nanomolar (nM) of the Mito-Tracker into the medium and let it incubate for 00:45:00 up to the completion of the incubation period.
45m
Concanavalin A coating
Concanavalin A coating
Prepare a stock solution of 1 mg/ml Concanavalin A using sterile water.
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 00:30:00 at Room temperature .
30m
Remove excessive Concanavalin A solution and wash 3x with sterile water for about 00:05:00 .
Note
Note that you may want to collect and reuse the excessive Concanavalin A for future use. Storage is at -20 °C .
5m
Leave the coverslips under the air at Room temperature until fully dry.
Mounting of the cells for imaging
Mounting of the cells for imaging
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.
Note
In our case, we took 2 mL of the liquid culture and washed the pellet with 1 mL of 1x PBS.
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.
Note
Using the 2 mL of liquid culture, we typically resuspend the pellet in 60 µL prior to mounting.
Imaging procedure
Imaging procedure
Place the sample in the microscope and navigate to a group of yeast cells for setting the correct imaging parameters.
Note
For navigation and identification, we recommend using differential interference contrast (DIC), as it has given us better contrast than simply brightfield.
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.
Note
Ideally, the signal should be completely reflected in your histogram without being too close on either the lower or upper end to avoid under- and overexposure respectively. Try to keep the laser intensity as low as possible and rather increase the exposure time, if possible.
Images were acquired in Z-stacks to enable later deconvolution and/or maximum intensity projection (MIP).
Note
We normally use a Z-range of 10 µm and a step-size of 0.5 µm for yeast, resulting in a stack of 20 frames per image.
Basic image processing
Basic image processing
For denoising the image stacks, we used the 'Small Volume Computational Clearing' in the Leica Application Suite X (LAS X) software.
Note
Here, we applied the adaptive strategy with a set reflective index of 1.33.
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.
Note
The various steps are color-coded according to the respective topic and labelled with respect to the relevant chapter number in the protocol:
Figure 1: Schematic representation of the entire workflow
Note
This image shows a cropped and processed live-cell image of Saccharomyces cerevisiae. It shows colocalization of PINK1-GFP (green) with the MitoTracker (red) in the merged composite-image:
Figure 2: Exemplary image following the protocol
Protocol references
[1] Morais, V. A. et al. Parkinson’s disease mutations in PINK1 result in decreased Complex I activity and deficient synaptic function. EMBO Mol. Med. 1, 99–111 (2009).
[2] Lazarou, M., Jin, S. M., Kane, L. A. & Youle, R. J. Role of PINK1 Binding to the TOM Complex and Alternate Intracellular Membranes in Recruitment and Activation of the E3 Ligase Parkin. Dev. Cell 22, 320–333 (2012).
[3] Sekine, S. & Youle, R. J. PINK1 import regulation; a fine system to convey mitochondrial stress to the cytosol. BMC Biol. 16, 1–12 (2018).
[4] Florian Wilfling, Fabian Fiedler, Anna Bieber, Cristina Capitanio 2022. Yeast cells live fluorescence imaging. protocols.io
https://dx.doi.org/10.17504/protocols.io.n92ldzqjnv5b/v1