Visualizing Yeast Organelles with Fluorescent Protein Markers

Summary

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.

Introduction

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.

Protocol

1. Yeast strain construction 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 or…

Representative Results

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…

Discussion

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…

Materials

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