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....

Protocol

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 n....

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

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