Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Summary

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Abstract

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

Introduction

Dynein and kinesin are cytoskeletal motor proteins responsible for myriad functions in eukaryotic cells¹. By converting the chemical energy of ATP hydrolysis into productive work, these motors translocate on microtubules to haul and distribute various intracellular cargos. They also coordinate in the massive intracellular rearrangements associated with mitosis, where they exhibit orchestrated forces that contribute to the positioning and separation of chromosomes. Structural, biochemical, and biophysical assays, including single molecule observations, have revealed the mechanisms of these motors at the individual level (well-reviewed in previou....

Protocol

1. Growth, expression and harvesting of motor proteins controlled by a galactose induced promoter

1. Using a yeast-peptone-dextrose (YPD) culture plate and a sterile inoculating stick, streak desired frozen yeast strain and incubate for 3-4 days at 30 °C.
2. Day 1 of culture growth: In the afternoon, add 10 mL of YP culture media with 2% dextrose to a 1" diameter glass culture tube and inoculate it with a single colony from the plate. Grow in a rapidly rotating roller drum at 30 °C overnight.......

Discussion

The molecular construction techniques of DNA origami provide a unique way to construct motor ensembles with defined architectures, motor numbers, and types, enabling studies of how emergent behavior arises from specific motor configurations³¹. As structural and cellular studies continue to elucidate examples of cytoskeletal motors working in teams, techniques for isolating and investigating the biophysical and biochemical mechanisms of motors in ensembles are growing in utility. For example, cryo-.......

Materials

Name	Company	Catalog Number	Comments
2 mL Round Bottom Tube	USA Scientific	1620-2700
Biotin labeled tubulin protein: porcine brain, >99% pure	Cytoskeleton.com	T333P-A
Biotin-BSA	Sigma	A8549-10MG
Bottle Assembly, Polycarbonate, 250 mL, 62 x 120 mm	Beckman Coulter	356013
Bottle, with Cap Assembly, Polycarbonate, 10.4 mL, 16 x 76 mm	Beckman Coulter	355603
Centrifugal Filter Unit	Millipore Sigma	UFC30VV00
IgG Sepharose 6 Fast Flow, 10 mL	GE Healthcare	17096901
Micro Bio-Spin Chromatography Columns, empty	Bio-Rad	7326204EDU
P8064 Scaffold	Tilibit	2 mL at 400nM
Poly-Prep Chromatography Columns	Bio-Rad	731-1550
ProTev Protease	Promega	V6101
Scotch Double Sided Tape with Dispenser	amazon.com	N/A
Sephacryl S-500 HR	GE Healthcare	17061310
Streptavidin	Thermo Fisher	434302
SYBR Safe DNA stain	Invitrogen
Tubulin protein (>99% pure): porcine brain	Cytoskeleton.com	T240-B
Tubulin, HiLyte 647	Cytoskeleton.com	TL670M-A
Ultra-Clear Centrifuge Tubes	Beckman Coulter	344090