Visualizing Yeast Organelles with Fluorescent Protein Markers

Riepilogo

Here we describe the use of a set of fluorescent protein-based organelle markers in live-cell imaging of the budding yeast, Saccharomyces cerevisiae.

Abstract

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have constructed fluorescent protein-based markers for major organelles and endomembrane structures, including the nucleus, endoplasmic reticulum (ER), Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, lipid droplets, and autophagosomes. The protocol presented here describes the procedures for using these markers in yeast, including DNA preparation for yeast transformation, selection and evaluation of transformants, fluorescent microscopic observation, and the expected outcomes. The text is geared toward researchers who are entering the field of yeast organelle study from other backgrounds. Essential steps are covered, as well as technical notes about microscope hardware considerations and several common pitfalls. It provides a starting point for people to observe yeast subcellular entities by live-cell fluorescent microscopy. These tools and methods can be used to identify protein subcellular localization and track organelles of interest in time-lapse imaging.

Introduzione

Subcellular compartmentalization into membrane-bound organelles is a common principle in the organization of eukaryotic cells. Each organelle fulfills specific functions. Like in many other aspects of eukaryotic biology, the budding yeast, Saccharomyces cerevisiae, has been a classic model system in elucidating the basic principles of organelle organization and dynamics. Examples include the seminal discoveries in the protein secretion pathway, the peroxisomal protein import pathway, and the autophagy pathway^1,2,3.

In typical nutrient-rich conditions, fast-growing yeast cells contain endoplasmic reticulum (ER), early Golgi, late Golgi/early endosomes, late endosomes, vacuoles, and mitochondria. Some peroxisomes, lipid droplets, and autophagosomes (even fewer than the first two, mainly of the Cvt vesicle type, which are present in nutrient-rich conditions⁴) are also present, but not as prominent as it would be under specific culturing conditions (lipid-rich media, starvation media, etc.). Compared to other common eukaryotic models, yeast cells are quite small; the diameter of a typical yeast cell is around 5 µm, compared to tens of micrometers for most animal and plant cells. As a result, in the same imaging field that normally contains a single adherent animal cell, one normally sees tens of yeast cells at various cell cycle stages. Besides the size difference, yeast organelle morphology also has some peculiar features. At the ultrastructural level, yeast ER is composed of sheets and tubules, like in other systems. Under fluorescent microscopy, yeast ER manifests as two rings with some interconnecting structures in between. The inner ring is the nuclear ER, which is continuous with the nuclear envelope, and the outer ring is the peripheral ER, which is a tubular network lying beneath the plasma membrane⁵. Similar to plant cells but different from animal cells, a hybrid organelle, the late Golgi/early endosome, sits at the intersection between the secretory pathway and endocytic pathway^6,7. Morphologically, yeast Golgi apparatuses are dispersed in the cytoplasm. Vacuoles are functionally analogous to lysosomes in animal cells. They often occupy large portions of the cytoplasm and undergo frequent fission and fusion. Besides the use of fluorescent colocalization markers, the vacuolar membrane can be distinguished from the nuclear ER by at least two criteria: The vacuolar membrane is generally more rounded than the nuclear ER, and the concaving appearance of the vacuole in DIC is also more pronounced than that of the nucleus.

Routinely, we use a set of fluorescent protein-based markers to visualize the aforementioned organelles in live yeast cells (Table 1). The fidelity and functionality of these organelle markers have been experimentally verified^7,8. These marker constructs are intended to introduce fluorescent protein chimera cassettes into the yeast genome. As outlined below, in preparation for yeast transformation, linear DNA fragments are generated either by enzymatic digestion or PCR amplification^7,8. The linear DNA fragments get integrated into the genome via homologous recombination. For plasmids described in this protocol, three types of design are employed. In the first type, covering the majority of the plasmids, it is often possible to obtain transformants carrying multiple copies of the construct. This is usually undesired because it introduces expressional and possibly functional variations across transformants. Single-copy transformants need to be identified through imaging as described in this protocol, by immunoblotting or by carefully designed PCR tests. In the second type, covering GFP-Sed5, GFP-Pep12, and GFP-Atg8, only single-copy integration is produced in haploid yeast cells. Both the first type and the second type keep the endogenous copy of the marker gene intact in the genome. A third type of plasmid design, covering Sec7-2GFP and Vph1-2GFP, is intended to introduce C-terminal knock-ins, leading to the chimeras being the sole copy of the corresponding marker gene.

Here we describe the procedure to utilize these organelle markers, provide exemplary microscopy images, and discuss precautions geared toward researchers new to yeast organelle imaging.

Protocollo

1. Yeast strain construction

1. Obtain marker plasmids and a suitable yeast strain.
   NOTE: The plasmids are available from Addgene. This protocol utilizes TN124 (MATa ura3 trp1 pho8Δ60 pho13Δ::LEU2), BY4741(MATa leu2Δ0 ura3Δ his3Δ1 met15Δ0), and DJ03 (BY4741 trp1Δ::MET15) as examples. One important consideration for strain choice, other than the nature of the scientific question, is the compatibility of selection markers. The organelle marker plasmids described here use URA3 and TRP1 as the selection markers. Therefore, the genotype of the recipient strain needs to be ura3 and trp1. Otherwise, one needs to modify the strain or the plasmids.
2. Prepare yeast media according to the following recipes.
   NOTE: SMD (synthetic minimal dextrose; 2% glucose, 0.67% yeast nitrogen base (YNB) without amino acids, 30 mg/L adenine, 30 mg/L lysine, 30 mg/L methionine, 20 mg/L histidine, 20 mg/L uracil, 50 mg/L tryptophan, 50 mg/L leucine). SMD+CA (SMD with the addition of 0.5% casamino acids). YPD (Yeast extract peptone dextrose; 1% yeast extract, 2% peptone, 2% glucose). SD-N (synthetic dextrose without nitrogen; 0.17% YNB without amino acids and ammonium sulfate, 2% glucose). Please refer to the Discussion section for considerations on the choice of the medium.
3. Prior to the transformation step, generate linearized DNA fragments by enzymatic digestion or PCR amplification of an organelle marker plasmid.
   NOTE: Table 1 lists the restriction enzyme sites and PCR primers to generate linear fragments from the organelle marker plasmids.
4. Culture yeast cells in liquid YPD medium and transform yeast with linear DNA fragment.
   NOTE: Yeast transformation can be performed using the conventional LiAc-based method⁹ or other methods of choice. It is advisable to include appropriate controls for transformation, in particular, a negative control with no plasmid or plasmid-derived DNA.
5. Incubate on an appropriate selection plate (e.g., for URA3 selection, use SMD-Ura medium) at 30 °C for 2-3 days.
   NOTE: It takes about 2 days for single colonies to show up.
6. To verify fluorescent chimera expression and integration copy number, pick eight colonies from the selection plate, re-streak on fresh selection plates, and incubate at 30 °C.

2. Fluorescence microscopy: general procedures and single time-point imaging

1. Culture yeast cells overnight in SMD liquid medium at 30 °C with shaking.
2. The next morning, measure the optical density of yeast culture at 600 nm (hereafter referred to as OD₆₀₀) in a spectrophotometer or plate reader.
   NOTE: With some practice on inoculating yeast, one can usually ensure that OD₆₀₀ of all samples is lower than 2 at this stage. If it ends up higher, it is advisable to dilute more in the next step and allow more time for yeast cells to recover.
3. Dilute yeast culture to approximately 0.2 OD₆₀₀ using a fresh medium.
4. Continue culturing till OD₆₀₀ reaches approximately 0.8-1.0.
   NOTE: The doubling time of yeast is about 1.5-2 h.
5. Put a cover glass on top of tissue paper on a flat surface, spread 5 µL of 1 mg/mL concanavalin A on the top side of the cover glass, and wait for 5 min (Figure 1A)¹⁰.
   NOTE: This preparation step can be done ahead of time.
6. Transfer 100 µL of yeast culture to the top side of the cover glass, and wait for 5 min.
7. Combine cover glass with a supporting glass slide, with the yeast cells sandwiched in-between; press with appropriate force to secure the attachment.
   NOTE: It takes some practice to find the right pressure, so that yeast cells form an immobile single layer but are not crushed (Figure 1B). The liquid medium will be pushed out and absorbed by the underlying tissue paper during this step.
8. Mount the sample slide on the microscope stage. Locate and focus on a patch of yeast cells using differential interference contrast (DIC) or phase contrast (PC) illumination.
   NOTE: Do not use fluorescence to locate cells; otherwise, the signal may get bleached prior to data collection.
9. Manually configure three sets of parameters to collect image z-stacks: z-sectioning, imaging channels, and exposure parameters.
   NOTE: See Supplementary Figure 1 for GUI examples of parameter settings in one commercial software. The exact look differs across software platforms, but the options are more or less the same.
   1. Z-sectioning: Collect slices at 0.5 µm stepping for 15 slices (covering 7 µm depth, generally sufficient for normal haploid yeast cells).
   2. Imaging channels: Select DIC or PC for cell contours and appropriate fluorescence channels as needed.
   3. Excitation light intensity and exposure time: Set the excitation light intensity and exposure time as appropriate for the sample.
      NOTE: As a starting point, use 100% for excitation light intensity and 100 ms for exposure time; decrease light intensity if photobleaching becomes obvious with the progression of z-stack collection or if the signal exceeds camera recording capacity (i.e., signal saturation); increase the light intensity if the signal-to-noise ratio is low.
10. Write down the imaging settings and use the same settings for all samples to be compared.
    NOTE: Do not use the "auto" setting for data collection and image visualization. It is important to record the settings in a lab note so that the exact same parameters can be re-applied in future experimental repeats. Some microscope controlling software applications have the ability to save and re-apply settings; even so, it is advisable to have the settings recorded independently because software profiles may get deleted or modified unintentionally by co-workers.
11. Save the data in 16-bit multichannel stack format.
    NOTE: Do not save as 8-bit RGB pictures (or 24 bit, if counting all three colors). See the image visualization section (Section 4) for limitations of the 8-bit format.
12. Move to a completely different area for the next image stack collection.
    ​NOTE: Adjacent areas may have experienced photobleaching and phototoxicity. As a result, the image stack collected from these areas may be misleading.

3. Time-lapse imaging

NOTE: The procedure of time-lapse imaging differs from the one for single time-point imaging in two areas: sample preparation and imaging parameters.

1. Sample preparation
   1. Coat a 35 mm glass-bottom dish with 1 mg/mL concanavalin A and wait 5 min or more.
   2. Add 1.5 mL of yeast liquid culture to the dish, and wait for 5 min to allow the yeast cells to settle to the glass surface.
   3. Using a pipet, aspirate the liquid medium from the edge of the dish, then gently rinse the patch of yeast sediment with about 1 mL of fresh medium to remove insecurely attached cells.
   4. Repeat the rinsing 2-3 times. Aspirate with a pipet, then gently add 2 mL of fresh culture medium.
2. Imaging parameters
   1. Z-sectioning: Collect slices at 0.5 µm stepping for 15 slices.
   2. Imaging channels: Select as needed.
   3. Excitation light intensity and exposure time: Use the minimal necessary to discern the subcellular structure under investigation.
      NOTE: For time-lapse imaging, photobleaching and phototoxicity from repeated illumination limit the total number of time points that can be collected. Therefore, excitation intensity and exposure time are generally set to low values so that more time points can be imaged.
   4. Imaging interval: Set the timing intervals appropriate for the biological process being investigated.
      NOTE: For example, the formation of an autophagosome under starvation conditions takes about 5-10 min; an interval of 1 min or less is preferred to track its dynamics. Under nutrient-rich conditions, the yeast cell cycle occurs on the hour scale; a longer interval, such as 10-20 min, can be utilized.
3. If proper hardware is available, maintain the dish temperature at 30 °C.
   ​NOTE: Most biological processes in yeast cells can also be imaged at room temperature (RT), except at a slower pace in general.

4. Visualization of image stacks and assessment of integration copy number

1. Install Fiji or ImageJ software for image visualization and analysis^11,12.
   NOTE: Fiji is a tool collection based on ImageJ, suitable for general purpose biological image data visualization and processing. For image processing listed in this protocol, ImageJ without additional plugins is sufficient.
2. Pick appropriate parameters for image display and comparison.
   1. In Fiji, open a couple of z-stack images.
   2. Drag the z-position in each stack to the mid-section (or other positions if the structure under investigation is better visualized there).
   3. Go to Image > Adjust > Brightness/Contrast and click on Reset.
   4. In the Brightness/Contrast (B&C) window, use the scroll bars to change two parameters, Minimum and Maximum values, to discern the structure under investigation clearly.
      NOTE: Generally, the Minimum value is set to be close to the average value in empty areas, and the Maximum is set to be close to the maximum in the image. Those values can be inferred from the displayed histogram (Figure 2A). If the imaging workstation is color-calibrated, the minimum value can be set slightly higher to hide more background signals.
   5. In the Brightness/Contrast (B&C) window, click on Set and select Propagate to all other Open Images checkbox in the popup window Set Display Range.
      NOTE: This operation applies the same brightness/contrast settings (including the minimum & maximum values) to all opened images so that the images can be cross-compared.
   6. Look across different images and scroll through the z-stack in each. If the images look good with a proper dark background and little overblown saturations, write down the Minimum and Maximum settings.
3. For multichannel images, pick image display settings for each channel:
   1. Go to Image > Color > Channels Tool, select the Greyscale mode in the popup window Channels.
   2. Go through each channel, pick the proper Brightness/Contrast for that channel, and write down the settings.
   3. With the Brightness/Contrast adjusted, go back to the Channels window and change to Composite mode for simultaneous visualization of multiple channels.
      NOTE: The pseudo color for each channel can be manually changed in Channels to suit personal preference. The checkboxes are for showing/hiding particular channels.
4. If needed, use the checkboxes in the Channels window to show or hide particular channels.
5. If desired, generate stack projections by going to Image > Stacks > Z Project.
   NOTE: Several projection modes are available. Max intensity is a good choice for quick assessment. The nature of the research should be considered to determine if a particular projection mode or individual slices should be used for quantification. Projection can also be limited to select z-slices to reduce the interference of out-of-focus light.
6. Screen for single-copy-integration transformants.
   1. Once the same Brightness/Contrast settings are applied across all opened images, compare the image intensities across samples to infer plasmid integration number.
      NOTE: Images should be acquired by following the general procedure for single time point imaging, beginning with overnight culturing in a liquid medium. Multi-copy integrations (See colony 2 & 3 in Figure 2B for examples) look substantially brighter than single-copy ones (colony 1 in Figure 2B). In contrast, single-copy ones look similar to each other (Figure 2B).
   2. Examine images from eight transformants for each strain, ensuring to use the same Brightness/Contrast settings.
   3. Re-streak and save three or more single-copy transformants for subsequent study.
7. Export images for presentation.
   1. Go to Image > Duplicate to make a copy of the image of interest, and give it a name of choice.
   2. Go to Image > Type > 8-bit to convert the duplicate copy from 16-bit to 8-bit and save it as needed.
      NOTE: Be aware that once this is applied, if the Brightness/Contrast settings are not written down previously, there is no easy way of retrieving that information, and thus no easy way of re-applying the same setting in subsequent image visualizations. This is also the reason why the previous duplication operation is recommended.
   3. To split a z stack into a collection of individual images, go to Image > Stacks > Stack to Images and save as needed.
      NOTE: Images can also be cropped to a smaller area in Fiji.

Risultati

Organelle morphology and dynamics are subject to change as yeast cells respond to external and internal signals. Here, we provide representative images of yeast organelles in the mid-log phase (Figure 3A,B). As mentioned previously, several organelles have their distinct morphological features, thus are easy to recognize without extensive comparison with other organelle markers. These include ER, mitochondria, and vacuoles. Note that in some laboratory strains, including the...

Discussione

The protocol described here provides a simple start for people entering from other research fields to explore imaging yeast organelles. Before moving on to specific topics, we would like to emphasize one more time that one needs to refrain from excessive use of automatic features in imaging software. Microscopy images are not just pretty pictures, they are scientific data, and therefore their acquisition and interpretation should be treated accordingly. It is especially important that image collection parameters be selec...

Materiali

Name	Company	Catalog Number	Comments
Adenine	Sangon Biotech	A600013
Casaminoacid	Sangon Biotech	A603060
Concanavalin A from canavalia ensiformis (Jack bean)	Sigma Aldrich	L7647
D-Glucose	Sangon Biotech	A501991
Fiji			https://fiji.sc/
Glass-bottom petri dish	NEST	706001	Φ35 mm
ImajeJ			https://imagej.net/
Inverted florescence microscope	Olympus		IX83 equipped with UPLXAPO 100X oil immersion objective, Lumencor Spectra X light source, and Hamamatsu Orca Flash4.0 LT camera.
L-Histidine	Sangon Biotech	A604351
L-Leucine	Sangon Biotech	A100811
L-Lysine	Sangon Biotech	A602759
L-Methionine	Sangon Biotech	A100801
L-Tryptophan	Sangon Biotech	A601911
Microscope cover glass	CITOTEST	10222222C	22 mm x 22 mm, 0.16–0.19 mm
Microscope slides	CITOTEST	1A5101	25 mm x 75 mm, 1–1.2 mm
Peptone	Sangon Biotech	A505247
Uracil	Sangon Biotech	A610564
Visiview	Visitron System GmbH		https://www.visitron.de/products/visiviewr-software.html
Yeast extract	Sangon Biotech	A100850
Yeast nitrogen base without amino acids	Sangon Biotech	A610507
YNB without amino acids and ammonium sulfate	Sangon Biotech	A600505