CHAPTER ONE
Actin Filament Dynamics Using Microfluidics Marie-France Carlier1, Guillaume Romet-Lemonne1, Antoine Jégou1 Laboratoire d’Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette, France 1 Corresponding authors: e-mail addresses: [email protected]; [email protected]; [email protected]
Contents 1. Introduction 2. Key Technical Aspects of the Microfluidic Method 2.1 Overview 2.2 Controlling the flow 2.3 Controlling surface properties 2.4 Calibrating the applied force 2.5 Caveats and control experiments 3. Assets for the Study of Actin Dynamics 3.1 Improving the observation of single filaments 3.2 New experimental capabilities 4. Perspectives 4.1 Expanding the setup 4.2 Beyond single-filament actin dynamics References
4 5 6 7 8 8 9 10 10 10 13 13 14 15
Abstract We describe how combining microfluidics with TIRF and epifluorescence microscopy can greatly facilitate the quantitative analysis of actin assembly dynamics and its regulation, as well as the exploration of issues that were often out of reach with standard single-filament microscopy, such as the kinetics of processes linked to actin selfassembly or the kinetics of interaction with regulators. We also show how the viscous drag force exerted by fluid flowing on the filaments can be calibrated in order to assess the mechanosensitivity of end-binding protein machineries such as formins or adhesion proteins. We also discuss how microfluidics, in conjunction with other techniques, could be used to address the mechanism of coordination between heterogeneous populations of filaments, or the behavior of individual filaments during regulated treadmilling. These techniques also can be applied to study the assembly and regulation of other cytoskeletal polymers such as microtubules, septins, intermediate filaments, as well as the transport of cargoes by molecular motors under a flow-produced load.
Methods in Enzymology, Volume 540 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-397924-7.00001-7
#
2014 Elsevier Inc. All rights reserved.
3
4
Marie-France Carlier et al.
1. INTRODUCTION Actin self-assembly into filaments has been extensively studied in vitro, following Fumio Oosawa’s pioneering works, using bulk solution kinetic and thermodynamic measurements. However, in cellular motile processes, actin filaments cannot be simply considered as free polymers in solution in dynamic equilibrium with monomers and soluble regulators. Instead, they are assembled into higher order, modular, structurally, and functionally distinct meshworks, by protein machineries that operate like “factories” (Small, Stradal, Vignal, & Rottner, 2002) in a cell signaling controlled fashion. The resulting motile processes that produce force and movement by actin assembly at the cell scale are understood through in vitro reconstitution studies, which bridge a wide gap from molecular to macroscopic scales (Loisel, Boujemaa, Pantaloni, & Carlier, 1999). However, the intermediate stages of understanding how force and movement are generated by single filaments assembled in a site-directed fashion, or how a collective behavior arises from specific interactions between a small number of polymers and soluble regulators, are not well understood. In particular, in live cells, filament nucleation and polarized growth are mediated by membrane-bound regulators of barbed end dynamics. A complete understanding of actin-based motile processes, therefore, requires the observation and quantitative analysis of actin assembly dynamics and its control by immobilized end-binding proteins at the scale of individual filaments. At a higher scale, thermodynamics systems consisting of a few filaments will be built as an intermediate step preceding the reconstitution of larger self-organized systems mimicking the in vivo context. Total Internal Reflection Fluorescence (TIRF) microscopy emerged as a significant advance for visualizing single molecules and single filaments (Funatsu, Harada, Tokunaga, Saito, & Yanagida, 1995; Vale et al., 1996). Visualizing single filaments using actin labeled with an extrinsic fluorophore provided a vivid illustration of the polymerization time course (Kuhn & Pollard, 2005), kinetics of filament barbed end capping (Kuhn & Pollard, 2007), and processive growth mediated by formins (Breitsprecher, Kiesewetter, Linkner, & Faix, 2009; Kovar & Pollard, 2004; Romero et al., 2004). In vivo, TIRF microscopy monitored actin filaments and microtubules localized at the basal plasma membrane of adherent cells (Manneville, 2006). TIRF and epifluorescence single-molecule microscopy also were used as a more accurate alternative to FRAP and photoactivatable
Actin Filament Dynamics Using Microfluidics
5
fluorophores to monitor actin filament dynamics in motile structures such as lamellipodia and focal adhesions (Adams et al., 2004; Watanabe, 2012). In vitro analysis of single-filament assembly by TIRF faces technical limitations, which can introduce artifacts and impose conditions that differ from the cellular context. To be constrained within the TIRF field (
Actin Filament Dynamics Using Microfluidics Marie-France Carlier1, Guillaume Romet-Lemonne1, Antoine Jégou1 Laboratoire d’Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette, France 1 Corresponding authors: e-mail addresses: [email protected]; [email protected]; [email protected]
Contents 1. Introduction 2. Key Technical Aspects of the Microfluidic Method 2.1 Overview 2.2 Controlling the flow 2.3 Controlling surface properties 2.4 Calibrating the applied force 2.5 Caveats and control experiments 3. Assets for the Study of Actin Dynamics 3.1 Improving the observation of single filaments 3.2 New experimental capabilities 4. Perspectives 4.1 Expanding the setup 4.2 Beyond single-filament actin dynamics References
4 5 6 7 8 8 9 10 10 10 13 13 14 15
Abstract We describe how combining microfluidics with TIRF and epifluorescence microscopy can greatly facilitate the quantitative analysis of actin assembly dynamics and its regulation, as well as the exploration of issues that were often out of reach with standard single-filament microscopy, such as the kinetics of processes linked to actin selfassembly or the kinetics of interaction with regulators. We also show how the viscous drag force exerted by fluid flowing on the filaments can be calibrated in order to assess the mechanosensitivity of end-binding protein machineries such as formins or adhesion proteins. We also discuss how microfluidics, in conjunction with other techniques, could be used to address the mechanism of coordination between heterogeneous populations of filaments, or the behavior of individual filaments during regulated treadmilling. These techniques also can be applied to study the assembly and regulation of other cytoskeletal polymers such as microtubules, septins, intermediate filaments, as well as the transport of cargoes by molecular motors under a flow-produced load.
Methods in Enzymology, Volume 540 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-397924-7.00001-7
#
2014 Elsevier Inc. All rights reserved.
3
4
Marie-France Carlier et al.
1. INTRODUCTION Actin self-assembly into filaments has been extensively studied in vitro, following Fumio Oosawa’s pioneering works, using bulk solution kinetic and thermodynamic measurements. However, in cellular motile processes, actin filaments cannot be simply considered as free polymers in solution in dynamic equilibrium with monomers and soluble regulators. Instead, they are assembled into higher order, modular, structurally, and functionally distinct meshworks, by protein machineries that operate like “factories” (Small, Stradal, Vignal, & Rottner, 2002) in a cell signaling controlled fashion. The resulting motile processes that produce force and movement by actin assembly at the cell scale are understood through in vitro reconstitution studies, which bridge a wide gap from molecular to macroscopic scales (Loisel, Boujemaa, Pantaloni, & Carlier, 1999). However, the intermediate stages of understanding how force and movement are generated by single filaments assembled in a site-directed fashion, or how a collective behavior arises from specific interactions between a small number of polymers and soluble regulators, are not well understood. In particular, in live cells, filament nucleation and polarized growth are mediated by membrane-bound regulators of barbed end dynamics. A complete understanding of actin-based motile processes, therefore, requires the observation and quantitative analysis of actin assembly dynamics and its control by immobilized end-binding proteins at the scale of individual filaments. At a higher scale, thermodynamics systems consisting of a few filaments will be built as an intermediate step preceding the reconstitution of larger self-organized systems mimicking the in vivo context. Total Internal Reflection Fluorescence (TIRF) microscopy emerged as a significant advance for visualizing single molecules and single filaments (Funatsu, Harada, Tokunaga, Saito, & Yanagida, 1995; Vale et al., 1996). Visualizing single filaments using actin labeled with an extrinsic fluorophore provided a vivid illustration of the polymerization time course (Kuhn & Pollard, 2005), kinetics of filament barbed end capping (Kuhn & Pollard, 2007), and processive growth mediated by formins (Breitsprecher, Kiesewetter, Linkner, & Faix, 2009; Kovar & Pollard, 2004; Romero et al., 2004). In vivo, TIRF microscopy monitored actin filaments and microtubules localized at the basal plasma membrane of adherent cells (Manneville, 2006). TIRF and epifluorescence single-molecule microscopy also were used as a more accurate alternative to FRAP and photoactivatable
Actin Filament Dynamics Using Microfluidics
5
fluorophores to monitor actin filament dynamics in motile structures such as lamellipodia and focal adhesions (Adams et al., 2004; Watanabe, 2012). In vitro analysis of single-filament assembly by TIRF faces technical limitations, which can introduce artifacts and impose conditions that differ from the cellular context. To be constrained within the TIRF field (
