Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

Summary

We present protocols for simple actin filament microfluidic assays, in combination with fluorescence microscopy, that allow one to accurately monitor individual actin filaments in real-time while sequentially exposing them to different protein solutions.

Abstract

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor individual reactions live in well-controlled conditions. To do so, live single-filament experiments have emerged over the past 20 years, mostly using total internal reflection fluorescence (TIRF) microscopy, and have provided a trove of key results. In 2011, in order to further expand the possibilities of these experiments and to avoid recurring problematic artifacts, we introduced simple microfluidics in these assays. This study details our basic protocol, where individual actin filaments are anchored by one end to the passivated coverslip surface, align with the flow, and can be successively exposed to different protein solutions. We also present the protocols for specific applications and explain how controlled mechanical forces can be applied, thanks to the viscous drag of the flowing solution. We highlight the technical caveats of these experiments and briefly present possible developments based on this technique. These protocols and explanations, along with today's availability of easy-to-use microfluidics equipment, should allow non-specialists to implement this assay in their labs.

Introduction

The assembly and disassembly of actin filaments and actin filament networks are controlled by several biochemical reactions and depend on the mechanical context. In order to gain insight into these complex mechanisms, it is invaluable to be able to observe individual reactions on individual filaments (in sufficiently large numbers). Over the past decades, the observation of dynamic actin filaments in real-time, mostly using total internal reflection fluorescence (TIRF) microscopy, has emerged as a key technique and has provided an impressive list of results that could not have been obtained with bulk solution biochemical assays¹.

Protocol

1. Microfluidic chamber preparation

1. Select a SU-8 master mold with several chamber patterns. Typical chambers are cross-shaped with three inlets and one outlet, 20 µm high and 800 µm wide (Figure 1). Such master molds can be purchased from external companies or made in academic laboratories (e.g., Gicquel, Y. et al.⁵).
2. Place tape around the edge of the mold.
   1. Put ~50 cm long, 19 mm wide, standard transparent office tape (see Table of Materials) on a bench, sticky side up. Place the mold vertically at one end and along the midline of the tape.
   ....

Results

For all the experiments described above, fluorescently labeled actin filaments should be clearly visible, with good contrast, indicative of low background fluorescence from the surface (Figure 4, see Supplementary File 1 for troubleshooting of common issues). Actin filaments should also not stick to the surface: when the dominant flow rate is low, the actin filaments' lateral fluctuations should be perceptible when observing them live and allow one to clearly determine that .......

Discussion

Compared to standard single-filament methods where actin filaments are anchored to the surface by multiple points along their length or maintained close to it by a crowding agent such as methylcellulose, microfluidics offers a number of advantages. As interactions with the surface are minimal, the artificial pauses these interactions can induce during both elongation and depolymerization are avoided. The filaments are aligned by the flow, parallel to each other, easing their monitoring and the measurement of their length.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
β-Casein	Merck	C6905	Used at 8 mg/mL
Biopsy punch (with plunger)	Ted Pella	15115-2	ID 0.75 mm, OD 1.07 mm
Biotin-BSA	Merck	A8549	Used at 1 mg/mL
BSA	Merck	A8022	Used at 50 mg/mL
Coverslip Mini-Rack Teflon holder	Invitrogen	C14784	for 8 coverslips
Coverslips 22x40mm Thickness #1.5	Menzel Gläser	631-1370
DABCO	Merck	D27802	component in f-buffer
DTT	Euromedex	EU0006-D	component in f-buffer
Ester NHS Alexa Fluor 488	Invitrogen	A20000	Fluorophore for actin labeling on Lys328.
EZ-Link Sulfo-NHS-Biotin	Thermo Scientific	21338	To biotinylate actin on Lys328
Hellmanex III	Hellma	9-307-011-4-507	Glass cleaning detergent
ImageJ	NIH	N/A	open source software
Laboport	KNF	811kn.18	vacuum pump (ultimate vacuum: 240 mbar)
Magic invisible tape	Scotch	7100024666	standard transparent office tape
Micrewtube	Simport	T341-6T	2 mL microfluidic reservoir tubes
Microfluidic device Part 1: Flow Unit S	Fluigent	FLU-S-D-PCKB	Flowmeter
Microfluidic device Part 2: Fluiwell-4C-2 mL	Fluigent	14002001PCK	Reservoir holder
Microfluidic device Part 3: MFCS-EZ	Fluigent	EZ-11000001 EZ-00345001	Pressure controller
Model 42 - UVO-Cleaner	Jelight Inc.	42-220	Ultraviolet cleaner
N6-(6-Aminohexyl)-ATP-ATTO-488	Jena Bioscience	NU-805-488	ATP-ATTO used to label actin
neutravidin	Thermo Scientific	31000
PLL-PEG	SuSoS	PLL(20)-g[3.5]- PEG(2)	Use at 1 mg/mL in PBS.
Polydimethylsiloxane (PDMS) Sylgard 184 Silicon Elastomer	Dow Corning	1673921	Contains PDMS base and curing agent
Polyetheretherketone (PEEK) tubing	Merck	Z226661	“Blue” : I.D. = 0.25 mm
Safety blow gun	Coilhose Pneumatics	700-S	filtered air
Silicon tubing	VWR	228-0701P	connect PEEK to coupler
Stainless steel catheter coupler	Prime Bioscience	SC22/15	Inserted into PDMS inlets and outlet to connect to PEEK tubing
Thermoplastic film	Sigma Aldrich	PM996	Standard "parafilm"
Ultrapure ethanol	VWR	64-17-5
Ultrasonic cleaning bath	VWR	USC200TH	To accomodate 1 L beakers
Vacuum dessicator	SP Bel-Art	F42022-0000	to degas the PDMS or solutions