4D Microscopy of Yeast

Summary

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

Abstract

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

Introduction

Compartments of the endomembrane system are in constant flux, and their full characterization requires live cell imaging. Described here is a protocol that employs 4D (time-lapse 3D) confocal microscopy to visualize fluorescently labeled compartments in budding yeasts. The method was developed to track the dynamics of secretory compartments in Pichia pastoris and Saccharomyces cerevisiae^1,2,3. This protocol focuses on S. cerevisiae, which has a nonstacked Golgi in which the individual cisternae are optically resolvable⁴. The ....

Protocol

1. Preparation

1. Make nonfluorescent minimal NSD medium². The absence of riboflavin is expected to reduce background intracellular green fluorescence and associated phototoxicity.
2. Grow the yeast strain overnight at ~23 °C to logarithmic phase in 5 mL NSD in a 50 mL baffled flask with good aeration. About 3-4 h prior to analysis, dilute the yeast culture in fresh NSD so that the final OD₆₀₀ will be 0.5-0.8 at the time of imaging.
3. Prepare a 2 mg/mL solution of concanavalin A (ConA). If desired, freeze aliquots of this solution in liquid nitrogen and store at -80 °C.
4. Spin the ConA solution for 5....

Results

The example given here documents and quantifies the maturation of two yeast Golgi cisternae as visualized by dual-color 4D confocal microscopy³. A yeast cell contains on the order of 10-15 Golgi cisternae, each of which matures over a time course of approximately 2-4 min. Maturation can be visualized by tagging the early Golgi marker Vrg4 with GFP and by tagging the late Golgi marker Sec7 with a red fluorescent protein such as mCherry or mScarlet. An individual cis.......

Discussion

4D confocal imaging of yeast organelles requires careful tuning of multiple parameters. The major concern is photobleaching and phototoxicity. A typical 4D movie involves collecting thousands of optical sections, so the laser illumination must be kept as low as possible. Tandem fluorescent protein tags can be used to boost the signal without increasing expression of the tagged protein^16,17. Maximizing the scan speed helps to limit photodamage, and also allows Z-s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35 mm glass bottom dishes No. 1.5	MatTek	P35G-0.170-14-C	Imaging dishes
Concanavalin A powder	Sigma-Aldrich	C2010
Trolox	Vector Laboratories	CB-1000
Leica SP8 confocal microscope	Leica Microsystems
Leica Application Suite X	Leica Microsystems		Microscope software
Huygens Essential software, version 17.04	Scientific Volume Imaging		Deconvolution software
ImageJ	NIH		Image processing and analysis software