Tuning the Contractility and Deformation Modes of Active Actin-Based Assemblies In Vitro: From Two-Dimensional Active Networks to Liquid Crystal Drops

Summary

Here, we present methods for preparing quasi-two-dimensional (2D) entangled, cross-linked, and liquid crystal actin assemblies from purified proteins.

Abstract

Actin cytoskeleton-based materials are widely investigated as model cellular materials to elucidate physical mechanisms of cell mechanics, such as shape regulation and force production, as well as intriguing soft polymeric materials. In this method, we detail creating actin-based assemblies in vitro using purified protein for fluorescence microscopy studies. We polymerize long actin filaments in a sample chamber and use a polymer depletant to crowd filaments into a two-dimensional (2D)-entangled network against a surface passivated with a surfactant layer. Adding skeletal muscle myosin II filaments in the presence of adenosine triphosphate (ATP) induces contraction of the actin network. By bundling actin filaments with a crosslinker, we tune the contractility of the assembly, transitioning from a material that buckles to a material that slides at the microscale. By reducing the length of the actin filaments through co-polymerizing actin in the presence of capping protein, we tune the material from being a 2D network to a liquid crystal. Cross-linking of dispersed short actin filaments results in three-dimensional (3D) liquid crystal droplet formation.

Introduction

Active biological materials underly mechanical processes in a variety of physiological processes, including intracellular transport, cell migration, cell shape regulation, and biological force generation^1,2,3. In vitro assemblies of cytoskeletal systems, constructed from purified and engineered protein components self-assembled in buffer, are an established tool for characterizing fundamental biophysical and biochemical processes^4,5,6,7. Th....

Protocol

NOTE: Proteins and labeling: For the purposes of this protocol, it is assumed that the researcher begins with stocks of purified proteins, which are fluorescently labeled when appropriate for fluorescence microscopy investigation. These proteins can be purchased or purified and fluorescently labeled in lab^{31,32,33,34,35,36,37}. In this protocol, stocks of proteins that have been frozen in liquid nitrogen and stored at -80 °C were used....

Results

A broad range of actin assemblies can be formed in model systems using purified proteins by following the general strategy described in these methods, where actin is polymerized into filaments in the presence of accessory proteins that modify the assembly architecture (Figure 1). When actin is polymerized into filaments without crosslinkers or capping protein, it forms entangled filament networks (Figure 1A). Adding crosslinker to the entangled networks results .......

Discussion

There are many considerations to producing reproducible actin assemblies. One of the most critical to producing reproducible, analyzable data is the coverglass surface of the sample chamber. The proteins in in vitro actin samples, particularly myosin, are extremely sticky and will adhere to untreated or poorly treated glass surfaces, rendering a sample that is unusable (Figure 5A). We have discussed a standard way of prepping the surface to yield reproducible samples through passiva.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.6 mL microcentrifuge tube	Fisher Scientific	05-408-123
008-Fluorosurfactant	RAN Biotechnologies	00115081361-6525	Alternate to premixed solution
008-FluoroSurfactant in HFE7500	RAN Biotechnologies	008-FluoroSurfactant-2wtH-50G	Premixed solution
2-mercaptoethanol (β-mercaptoethanol)	Sigma Aldrich	63689	CAS Number: 60-24-2 Stock solution: 25 mM in MilliQ water, stored at 4 °C
4-(2-Hydroxyethyl)piperazine-1-ethane-sulfonic acid (HEPES)	Sigma Aldrich	H3375	CAS Number: 7365-45-9
5 min Epoxy	Devcon	14250
Actin	Cytoskeleton, Inc.	AKL99-A	>99% pure rabbit skeletal muscle (Alternate to purifying)
Actin (rhodamine labeled)	Cytoskeleton, Inc.	AR05-A	Rabbit skeletal muscle (Alternate to purifying)
Adenosine 5′-triphosphate (ATP)	Sigma Aldrich	A6419	CAS Number: 34369-07-8 Stock solution: 25 mM in MilliQ water, store at -20 °C Keep frozen to avoid hydrolysis
Calcium Chloride (CaCl2)	Sigma Aldrich	C5670	CAS Number: 10043-52-4
Capping Protein (Mouse)			Purified from overexpression in E.coli with a HisTag Stock solution: 20 mM in capping protein buffer at -80 °C
Catalase from bovine liver	Sigma Aldrich	C9322	CAS Number: 9001-05-2 Stock solution: 85 ku/mL, store aliquoted with glucose oxidase at -20°C
Cover Glass	Fisherbrand	12544C	Borosilicate, #1.5, 24 mm x 40 mm Fisherbrand not Fisher Finest
D-(+)-Glucose	Sigma Aldrich	G8270	CAS Number: 50-99-7 Stock solution: 225 mg/mL in MilliQ water, store at -20 °C
Dithiothreitol (DTT)	Sigma Aldrich	3860-OP	CAS Number: 3483-12-3 Stock solution: 1 M in MilliQ water, store at -20 °C
Double Sided Tape	3M	3136
Ethyl alchohol 200 Proof	PHARMCO	111000200	CAS Number: 64-17-5 200 proof
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA)	Sigma Aldrich	E3889	CAS Number: 67-42-5
Ethylenediaminetetraacetic acid (EDTA)	Sigma Aldrich	E9884	CAS Number: 60-00-4
Glucose Oxidase	Sigma Aldrich	345386	CAS Number: 9001-37-0 Stock solution: 135 mg/mL in MilliQ water, store aliquoted with catalase at -20 °C
Glycerol	Sigma Aldrich	356352M	CAS Number: 56-81-5
Imidazole	Fisher Bioreagents	BP305-50	CAS Number: 288-32-4
Magnesium Chloride (MgCl2)	Fisher Bioreagents	BP214	CAS Number: 7786-30-3, 7791-18-6
Methylcellulose	Sigma Aldrich	M0512	CAS Number: 9004-67-5 15 centipoise
Microscope Slides Plain	Electron Microscopy Sciences/Gold Seal	63710-05	3"x1", 1 mm thick
MilliQ water	Millipore	CUFBI001	Ultrapure water
Novec-7500 Engineered Fluid	3M	7100134816	Alternate to premixed solution
Potassium Chloride (KCl)	Sigma Aldrich	P3911	CAS Number: 7447-40-7
Potassium Phosphate (KPO4)	Sigma Aldrich	P3786	CAS Number: 7758-11-4
Rabbit Skeletal Muscle Acetone Powder	Pel-Freez Biologicals	41995-1	Used when purifying actin (alternate to purchasing) Store around 30 mM in actin Buffer G at -80 °C
Sodium Azide	Sigma Aldrich	71289	CAS Number: 26626-22-8
Tetramethylrhodamine-C6-maleimide	AnaSpec	AS-81445	CAS Number: 174568-68-4 Used when purifying actin (alternate to purchasing) Store around 30 mM in actin Buffer G at -80 °C. Used when labeling actin (alternate to purchasing)
Tris Hydrochloric Acid (Tris HCl)	Sigma Aldrich	648317	CAS Number: 1185-53-1
Ultrasonic Bath	Emerson Branson	CPX2800H