Probing Myosin Ensemble Mechanics in Actin Filament Bundles Using Optical Tweezers

Summary

Formation of actomyosin bundles in vitro and measuring myosin ensemble force generation using optical tweezers is presented and discussed.

Abstract

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle contraction. To understand their force-generating mechanisms, myosin II has been investigated both at the single-molecule (SM) level and as teams of motors in vitro using biophysical methods such as optical trapping.

These studies showed that myosin force-generating behavior can differ greatly when moving from the single-molecule level in a three-bead arrangement to groups of motors working together on a rigid bead or coverslip surface in a gliding arrangement. However, these assay constructions do not permit evaluating the group dynamics of myosin within viscoelastic structural hierarchy as they would within a cell. We have developed a method using optical tweezers to investigate the mechanics of force generation by myosin ensembles interacting with multiple actin filaments.

These actomyosin bundles facilitate investigation in a hierarchical and compliant environment that captures motor communication and ensemble force output. The customizable nature of the assay allows for altering experimental conditions to understand how modifications to the myosin ensemble, actin filament bundle, or the surrounding environment result in differing force outputs.

Introduction

Motor proteins are essential to life, converting chemical energy into mechanical work^1,2,3. Myosin motors interact with actin filaments by taking steps along the filaments similar to a track, and the dynamics of actin-myosin networks carry out muscle contraction, cell motility, the contractile ring during cytokinesis, and movement of cargo inside the cell, among other essential tasks^3,4,5,6,7,

Protocol

1. Etching coverslips

1. Dissolve 100 g of KOH in 300 mL of 100% ethanol in a 1,000 mL beaker. Stir with a stir bar until the majority of the KOH has dissolved.
   CAUTION: Concentrated KOH solution can cause burns and damage to clothing. Wear gloves, eye protection, and a lab coat.
2. Place coverslips individually in coverslip cleaning racks.
   NOTE: Racks are designed with slits that hold single coverslips spaced apart to allow for etching and rinsing on each face of the coverslip, drain holes in the bottom, and made of material that can withstand the harsh etching conditions. They can be custom-made or purchased commerciall....

Results

Flow cells containing the actomyosin bundle systems are of a standard design, consisting of a microscope slide and an etched coverslip separated by a channel made from double-sided sticky tape (Figure 1). The assay is then built from the coverslip up using staged introductions as described in the protocol. The final assay consists of template rhodamine-labeled actin filaments; the desired myosin concentration (1 μM was used for the representative results in Figure 2.......

Discussion

An in vitro study using optical tweezers combined with fluorescence imaging was performed to investigate the dynamics of myosin ensembles interacting with actin filaments. Actin-myosin-actin bundles were assembled using muscle myosin II, rhodamine actin at the bottom of the bundle and on the coverslip surface, and 488-labeled, biotinylated actin filaments on the top of the bundle. Actin protein from rabbit muscle was polymerized and stabilized using general actin buffers (GAB) and actin polymerizing buffers (APB.......

Materials

Name	Company	Catalog Number	Comments
Actin protein (biotin): skeletal muscle	Cytoskeleton	AB07-A	Biotinylated actin protein
Actin protein, rabbit skeletal muscle	Cytoskeleton	AKL99-A	Actin protein
Alexa Fluor 488 Phalloidin	Invitrogen	A12379	Actin stabilizer and Alexa Fluor 488 stain
ATP	Fisher scientific	BP413-25	Required for actin assembly and myosin motility
Beta-D-glucose	Fisher scientific	MP218069110	Part of oxygen scavenging system used to reduce photobleaching during fluorescence imaging
Blotting Grade Blocker (casein)	Biorad	1706404	Used to block surface from non-specific binding
CaCl2	Fisher scientific	C79500	Calcium chloride, provides the necessary control over the dynamics of actin myosin network
Catalase	Fisher scientific	ICN10040280	Part of oxygen scavenging system used to reduce photobleaching during fluorescence imaging
Coverslips	Fisher scientific	12544C	Used to make flow cells
DTT	Fisher scientific	AC327190010	Used for buffer preparation
Ethanol	Fisher scientific	A4094	Regent used for cleaning coverslips
Glucose oxidase	Fisher scientific	34-538-610KU	Part of oxygen scavenging system used to reduce photobleaching during fluorescence imaging
KCl	Fisher scientific	P217-500	Used for buffer preparation
KOH	Fisher scientific	P250-1	Used to etch coverslips and adjust buffer pH
MgCl2	Fisher scientific	M33-500	Used for buffer preparation
Microscope slides	Fisher scientific	12-544-2	Used to make flow cells
Myosin II protein: rabbit skeletal muscle	Cytoskeleton	MY02	Full length myosin motor protein isolated from rabbit skeletal muscle
Nanotracker2	Bruker/JPK	NT2	Optical trapping instrument
Poly-l-lysine	Sigma-Aldrich	P8920	Facilities adhesion of actin filaments onto glass surface of the coverslip
Rhodamine Phalloidin	Cytoskeleton	PHDR1	Actin stabilizer and rhodamine fluorescent stain
Streptavidin beads, 1 μm	Spherotech	SVP-10-5	Optical trapping handle
Tris-HCl	Fisher scientific	PR H5121	Used for buffer preparation