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

Pushpanjali Soppina; Dipeshwari J. Shewale; Pradeep K. Naik; Virupakshi Soppina

doi:10.3791/63837

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Method Article

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

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DOI:

10.3791/63837

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July 27th, 2022

Pushpanjali Soppina¹^,² , Dipeshwari J. Shewale¹ , Pradeep K. Naik² , Virupakshi Soppina¹

¹Discipline of Biological Engineering, Indian Institute of Technology Gandhinagar, ²Department of Biotechnology and Bioinformatics, Sambalpur University

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....