Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

Summary

The bi-directional mitotic kinesin-5 Cin8 accumulates in clusters that split and merge during their motility. Accumulation in clusters also changes the velocity and directionality of Cin8. Here, a protocol for motility assays with purified Cin8-GFP and analysis of motile properties of single molecules and clusters of Cin8 is described.

Abstract

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Introduction

A large number of motility events within eukaryotic cells are mediated by the function of molecular motor proteins. These motors move along the cytoskeletal filaments, actin filaments, and microtubules (MTs), and convert the chemical energy of ATP hydrolysis into kinetic and mechanical forces required to drive biological motility within cells. The MT-based S. cerevisiae Cin8 is a bipolar, homotetrameric kinesin-5 motor protein that crosslinks and slides spindle MTs apart¹. Cin8 performs essential functions during mitosis, in spindle assembly^2,3,4

Protocol

1. Preparation of buffers and reagents

1. Buffers
   1. -Leu aa dropout mix: Mix 2 g each of Adenine, Uracil, Tryptophan, Histidine, Lysine, and Methionine and store at room temperature.
   2. Yeast selective medium with raffinose (1 L): Mix 6.7 g of yeast nitrogen base (with ammonium sulfate), 2 g of -Leu aa dropout mix, and 20 g of raffinose in double-distilled water by stirring (without heating) until fully dissolved. Using a 0.22 µm filter, filter the solution into a sterile bottle.
   3. Lysis buffer: Prepare 25 mL of solution in triple distilled water (TDW) consisting of 50 mM Tris, 30 mM Pipes, 500 mM KCl, 10% glyc....

Results

The experiment aims to investigate the motility characteristics of bi-directional motor protein Cin8 of different cluster sizes on single MTs. Representative motility of Cin8-GFP is also evident from the kymographs in Figure 5A, where the spatial position of the motor over time is shown.

For the analysis of the motile properties of Cin8-GFP, first, the cluster size is assigned (step 4.3) to each MT-attached motile Cin8-GFP particle, and then the position of the ex.......

Discussion

In this work, a protocol for single-molecule motility assay with the bi-directional kinesin-5 Cin8 and the motility analysis are presented. The full-length Cin8¹⁸ including the native nuclear localization signal (NLS) at the C-terminal has been purified from the native host S. cerevisiae. As the Cin8 is a nuclear motor protein, grinding the S. cerevisiae cells under liquid nitrogen is found to be the most efficient method for cell lysis. After lysis, by combining metal affinity a.......

Materials

Name	Company	Catalog Number	Comments
Adenine	FORMEDIUM	DOC0230
ATP	Sigma	A7699
Biotinylated-BSA	Sigma	A8549
Casein	Sigma	C7078
Catalase (C40)	Sigma	C40
Creatine-Kinase	Sigma	C3755
Dithiothreitol (DTT)	Sigma	D0632
EDTA	Sigma	E5134
EGTA	Sigma	E4378
Fluorescence filter set for GFP	Chroma	49002: ET-EGFP (FITC/Cy2)
Fluorescence filter set for Rhodamine	Chroma	49004: ET-CY3/TRITC
Fluorescence inverted microscope	Zeiss	Axiovert 200M
Galactose	Tivan Biotech	GAL02
Glucose	Sigma	G8270
Glucose Oxidase	Sigma	G7141
Glycerol	Sigma	G5516
GlycylGlycine	Merck	G0674
GMPCPP	Jana Bioscience	Nu-405L
GTB	Cytoskeleton	BST01-010
GTP	Sigma	G8877
Histidine	Duchefa Biochemie	H0710.0100
ImageJ-FIJI software	https://imagej.net/plugins/trackmate/	version 2.1.0/1.53c; Java 1.8.0_172 [64-bit] for Windows 10
Imidazole	Sigma	I0125
InstantBlue Coomassie Protein Stain	Abcam	ab119211
Lens	Zeiss	100x/1.4 oil DIC objective
Lysine	FORMEDIUM	DOC0161
Magnesium Chloride	Sigma	M8266
Methionine	Duchefa Biochemie	M0715.0100
Neo	Andor Technologies	sCMOS camera
NeutraAvidin	Life	A2666
Ni-NTA Agarose	Invitrogen	R901-15
Phospho-Creatine	Sigma	P1937
Pipes	Sigma	P1851
Pluronic acid F-127 (poloxamer)	Sigma	P2443
Potassium Chloride	Sigma	P9541
Raffinose	Tivan Biotech	RAF01
Size Exclusion chromatography instument	GE Healthcare	AKTA Pure
Spectrophotometer	ThermoFisher Scientific	NanoDrop
Superose-6 10/300 GL	GE Healthcare	17-5172-01
Tris	Roshe	10708976001
Triton X-100	Sigma	T8787
Tryptophan	Duchefa Biochemie	T0720.0100
Tubulin protein	Cytoskeleton	T240
Tubulin, biotinylated	Cytoskeleton	T333P
Tubulin, TRITC Rhodamine	Cytoskeleton	TL530M
Uracil	Sigma	U0750-100G
Yeast nitrogen base	FORMEDIUM	CYN0401S
α-GFP antibody	Santa Cruz Biotechnology	SC8036
β-mercaptoethanol	Sigma	M3148