High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Summary

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Abstract

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Introduction

Microtubules are cytoskeletal polymers made of αβ-tubulin protein subunits and are used in a wide variety of cellular contexts, including intracellular transport, cell division, morphogenesis, and motility. To build microtubule networks for these diverse roles, cells must carefully regulate where and when microtubules form. This regulation is accomplished by controlling the reactions that either assemble αβ-tubulin subunits into microtubule polymers or disassemble microtubules into free subunits; this is known as microtubule dynamics.

A major goal of the microtubule field is to elucidate the molecular mechanisms that reg....

Protocol

1. Preparing -LEU Dropout Plates

1. Add 800 mL of sterile, deionized water (DiH₂O) to a 2 L flask. Add 26.71 g of dropout base (DOB) powder, 0.69 g of CSM-Leu, and 20 g of agar. Mix with a magnetic stir bar. Bring to 1 L with DiH₂O.
2. Autoclave at 121 °C and 19 psi for 30 min.
3. Remove the flask from the autoclave and allow it to cool to ~65 °C with gentle stirring.
4. Pour 30 mL/plate into 100 mm diameter plates. Let these sit at room temperature for 36-48 h before storing at 4 °C.

2. Integrating GFP-Tub1 for the Constitutive Expression of GFP-labeled Tubulin

Results

Measuring microtubule dynamics in living yeast cells provides a compelling tool to assess how mutations in genes encoding microtubule regulators or tubulin subunits impact polymerization and depolymerization rates, as well as the frequency of transition between these states. Figure 1 displays a time series of astral microtubule dynamics in a wild-type cell and a mutant cell with a mutation in β-tubulin (tub2-430Δ). Microtubules are labeled with GFP-tagged α-tubul.......

Discussion

The budding yeast model offers major advantages for gathering high-resolution measurements of microtubule dynamics in an in vivo setting, including the ability to image single microtubules over time and the ability to manipulate tubulins and microtubule regulators using the tools of yeast genetics.

The Concanavalin A-coated chambers provide a number of advantages over previously described apparatuses, including molten agar pads. Slides with chambers can be pre-made and stored long ter.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DOB (dropout bases)	Sunrise science	1650
CSM-Leu	Sunrise science	1005
Agar	Ameresco	N833
100 mm polystyrene plates	Fisher Scientific	FB0875713
ssDNA (Samon Sperm)  in sterile DiH2O	Sigma-Aldrich	D7656	resuspend at 10 mg/mL in DiH2O. Store aliquots at -20 ºC
Synthetic Complete Media	Sunrise science	1459-100
Concanavalin A	Sigma-Aldrich	L7647	resuspend at 2 mg/mL in DiH2O. Store aliquots at -20 ºC
Microscope slides	Fisher Scientific	12-544-1
Microscope Coverslips	Fisher Scientific	12-541-B
Parafilm	Fisher Scientific	13-374-12	paraffin film
VALAP (Equal parts of Vaseline, lanolin and paraffin)			melt at 75 ºC before use
forceps	Fisher Scientific	16-100-106
Poyethylene glycol (PEG) 3350	Sigma-Aldrich	202444
Name	Company	Catalog Number	Comments
Microscope
Ti E inverted Perfect Focus microscope	Nikon Instruments	MEA53100
1.45 NA 100X CFI Plan Apo objective	Nikon Instruments	MRD01905
Piezo electric stage	Physik Instrumente	P-736
Spinning disk scanner	Yokogawa	CSU10
Laser combiner module	Agilent Technologies	MCL400B
EMCCD camera	Andor Technology	iXon Ultra 897
Name	Company	Catalog Number	Comments
Software
NIS Elements software	Nikon Instruments	MQS31100
Microsoft Excel software	Microsoft
MATLAB software	MathWorks, Inc
ImageJ64	NIH		Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2016.
Bio-Formats Importer plug-in	Open Microscopy Environment
Name	Company	Catalog Number	Comments
Plasmids
pUC19-LEU2::GFP-TUB1		pSK1050	reference: Song, S. and Lee, K. S. A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis. J Cell Biol. 152 (3), 451-469 (2001)