Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

Summary

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

Abstract

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

Introduction

An immensely crowded cell environment poses many challenges in sorting destined proteins and molecules. This intense workload of organization and spatiotemporal distribution of molecules within the cytoplasm is facilitated by molecular motors and cytoskeletal tracks. Molecular motors are the enzymes that hydrolyze the energy currencies such as ATP and utilize that energy during motion and force generation¹. Based on the amino acid sequence similarity, kinesins are grouped into 14 families and despite this similarity, each motor contributes uniquely to the functioning of a cell. Kinesin-3 family motors constitute one of the largest, comprising f....

Protocol

1. Sf9 culture, transfection, and virus generation

NOTE: Maintain Sf9 cells in 30 mL of Sf-900/SFM medium in 100 mL sterile, disposable conical flask without any antibiotic/antimycotic at 28 °C. Keep the suspension culture in an orbital shaker at 90 rpm. Supply of CO₂ and humidity maintenance is not required. Cells are usually subcultured every fourth day by inoculating 0.5 x 10⁶ cells/mL to reach 2.0 x 10⁶ cells/mL density on the fourth day.......

Discussion

The Sf9-baculovirus expression system is one of the most versatile and successful methods for high-throughput protein production^19,36,37. The posttranslational modification ability of Sf9 cells is highly comparable to the mammalian system¹⁵. A considerable disadvantage of using this system is that it is slow and sensitive to contamination. One of the most critical steps is efficient infection and successf.......

Materials

Name	Company	Catalog Number	Comments
Sf9 culture and transfection materials
anti-FLAG M2 affinity	Biolegend	651502	For motility purification
Aprotinin	Sigma	A6279	For motility assay and purification
Cellfectin	Invitrogen	10362100	For Sf9 transfection
DTT	Sigma	D5545	For motility assay mixture
FLAG peptide	Sigma	F3290	For motility purification
Glycerol	Sigma	G5516	For motility purification
HEPES	Sigma	H3375	Preparing lysing Sf9 cells
IGEPAL CA 630	Sigma	I8896	Preparing lysing buffer for Sf9 cells
KCl	Sigma	P9541	For motility purification
Leupeptin	Sigma	L2884	For motility assay and purification
MgCl2	Sigma	M2670	For preparing lysis buffer
NaCl	Sigma	S7653	For preparing lysis buffer
PMSF	Sigma	P7626	For motility purification
Sf9 cells	Kind gift from Dr. Thomas Pucadyil (Indian Institute of Science Education and Research, Pune, India).		For baculovirs expression and purification
Sf9 culture bottles	Thermo Scientific	4115-0125	For suspension culture
Sf-900/SFM medium (1X)	Thermo Scientific	10902-096 -500ml	For culturing Sf9 cells
Sucrose	Sigma	S1888	Preparing lysing buffer for Sf9 cells
Unsupplemented Grace’s media	Thermo Scientific	11595030 -500ml	For Sf9 transfection
Mirotubule Polymerization and Single molecule assay materails
ATP	Sigma	A2647	For motility and gliding assay
BSA	Sigma	A2153	For blocking motility chamber
Catalase	Sigma	C9322	For motility and gliding assay
DMSO	Sigma	D5879	For dissolving Rhodamine
EGTA	Sigma	3777	For preparing buffers
Glucose	Sigma	G7021	For motility and gliding assay
Glucose oxidase	Sigma	G2133	For motility and gliding assay
GTP	Sigma	G8877	For microtubule polymerization
KOH	Sigma	P1767	Preparing PIPES buffer pH 6.9
PIPES	Sigma	P6757	For motility and gliding assay
Microtubule gliding assay materials
26G  needle	Dispovan		For shearing microtubules
Casein	Sigma	C3400	For microtubule glidning assay
GFP nanobodies	Gift from Dr. Sivaraj Sivaramakrishnan (University of Minnesota, USA)		For attaching motors to the coverslip
Rhodamine	Thermo Scientific	46406	For preparing labelling tubulin
Microscope and other instruments
0.5ml, 1.5 and 2-ml microcentrifuge tubes	Eppendorf		For Sf9 culture and purification
10ml  disposable sterile pipettes	Eppendorf		For Sf9 culture and purification
10ul, 200ul, 1ml micropipette tips	Eppendorf		For Sf9 culture and purification
15ml concal tubes	Eppendorf		For Sf9 culture and purification
35mm cell culture dish	Cole Palmer	15179-39	For Sf9 culture
Balance	Sartorious		0.01g-300g
Benchtop orbial shaking incubator	REMI		For Sf9 suspenculture at 28oC
Camera	EMCCD Andor iXon Ultra 897		For TIRF imaging and acquesition
Double sided tape	Scotch		For making motility chamber
Glass coverslip	Fisherfinest	12-548-5A	size; 22X30
Glass slide	Blue Star		For making motility chamber
Heating block	Neuation		Dissolving paraffin wax
Inverted microscope	Nikon Eclipse Ti- U		To check protein expression
Lasers	488nm (100mW)		For TIRF imaging
Liquid nitrogen			For sample freezing and storage
Microcapillary loading tip	Eppendorf	EP022491920	For shearing microtubules
Microscope	Nikon Eclipse Ti2-E with DIC set up		For TIRF imaging
Mini spin	Genetix, BiotechAsia Pvt.Ltd		For quick spin
Objective	100X TIRF objective with 1.49NA oil immersion		For TIRF imaging
Optima UltraCentrifuge XE	Beckman Coulter		For protein purification
Parafilm	Eppendorf
pH-meter	Corning	Coring 430	To adjust pH
Pipette-boy	VWR		For Sf9 culture and purification
Sorvall Legend Micro 21	Thermo Scientific		For protein purification
Sorvall ST8R centrifuge	Thermo Scientific		Protein purification
ThermoMixer	Eppendorf		For microtubule polymerization
Ultracentrifuge rotor	Beckman coulter		SW60Ti rotor
Ultracentrifuge tubes	Beckman		5 mL, Open-Top Thinwall Ultra-Clear Tube, 13 x 51mm
Vortex mixer	Neuation		Sample mixing
Wax	Sigma	V001228	To seal motility chamber