Optical Control of Actin Network Assembly on the Supported Lipid Bilayer
支持脂质双层上肌动蛋白网络组装的光学调控
发布: 2026年04月20日第16卷第8期 DOI: 10.21769/BioProtoc.5656 浏览次数: 509
评审: Munenori IshibashiSupreet BhattacharyaAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Aug 2025
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Abstract
The spatiotemporal dynamics and density of actin networks are key determinants of actin cytoskeleton–mediated cellular functions. In vitro reconstitution systems have been widely used to study actin cytoskeletal dynamics; however, many existing approaches offer limited flexibility in controlling the geometry, thickness, and density of the assembled actin networks. Here, we present an in vitro optogenetic protocol that enables precise control of actin network assembly on supported lipid bilayers using an improved light-induced dimer (iLID)-SspB-based light-inducible dimerization system. In this system, His-mEGFP-iLID is anchored to a Ni-NTA-containing lipid bilayer, while SspB-mScarlet-I-VCA, a nucleation-promoting factor fused with SspB, together with other actin cytoskeletal proteins, is supplied in bulk solution. Upon blue light illumination, SspB-mScarlet-I-VCA is recruited to the membrane in a spatially and temporally defined manner, inducing localized actin polymerization. By tuning illumination patterns and duration, actin networks with defined density, thickness, and geometry can be generated, and polymerization can be rapidly halted by stopping illumination. This protocol provides a versatile platform for reconstructing actin networks with controlled spatial organization and density, enabling quantitative analysis of density-dependent interactions between actin networks and actin-binding proteins.
Key features
• Actin networks with varying densities and arbitrary shapes can be formed on the same supported lipid bilayer by controlling blue light illumination through the objective lens.
• Actin polymerization can be stopped simply by turning off blue light illumination, enabling the formation of actin networks with defined thicknesses.
• This protocol requires purified actin and actin-binding proteins.
Keywords: Optogenetics (光遗传学)
Actin cytoskeleton (肌动蛋白细胞骨架)
Lipid bilayer (脂质双层)
In vitro reconstitution (体外重构)
Light-induced dimerization (光诱导二聚化)
Graphical overview
Background
The dynamic network of the actin cytoskeleton underlies a wide range of cellular functions, including the lamellipodia, blebs, and contractile rings [1,2]. The formation of these specific structures relies on interactions between actin filaments and actin-binding proteins [3–6]. To understand the functions of actin-binding proteins, in vitro reconstitution systems using purified proteins—where actin is mixed with actin-binding proteins—have been widely employed. In particular, to mimic actin polymerization under physiologically relevant conditions, nucleation-promoting factors (NPFs) need to be spatially confined to two-dimensional substrates such as glass surfaces or lipid membranes [7,8]. Patterned UV illumination on polyethylene glycol (PEG)-coated glass has enabled the immobilization of NPFs on glass surfaces with arbitrary geometries [9–13]. Furthermore, techniques for forming spatially patterned lipid membranes have also been reported [14,15]. These techniques have enabled spatial patterning of NPFs under conditions that are closer to the in vivo environment by eliminating the height gap between PEG molecules and lipid membranes, reducing binding of NPFs with random orientation to the substrate, reducing energetic instability of lipid membranes at PEG–lipid boundaries, and minimizing heterogeneous lateral diffusion of lipid molecules [15]. However, it remains challenging to stop actin polymerization during observation, to design more complex three-dimensional structures, or to generate actin networks with different densities on the same lipid membrane. Thus, further flexibility in controlling actin polymerization is still required in in vitro reconstitution systems.
In recent years, optogenetic approaches have been increasingly applied in in vivo experimental systems using living cells and tissues to manipulate actin cytoskeleton dynamics with high spatial and temporal resolution. Among these approaches, a light-induced dimerization system, iLID-SspB, has been widely used due to its rapid association upon blue light illumination and reversible dissociation in dark conditions [16–21]. Taking this advantage, many iLID-SspB-based systems are designed so that iLID is localized to the plasma membrane or organelle membranes, and blue light illumination activates iLID to recruit SspB-fused actin cytoskeletal regulators from the cytoplasm onto the lipid membrane. The increased density of these regulatory factors on the membrane subsequently alters downstream actin cytoskeletal dynamics. Another advantage of the iLID-SspB system is that the proteins are relatively easy to purify compared to other optogenetic proteins, making them well-suited for incorporation into in vitro reconstitution assays [22,23].
Here, we present a protocol that combines actin cytoskeleton regulatory proteins with the iLID-SspB system to induce light-dependent assembly of actin networks on a lipid bilayer that mimics the plasma membrane. To generate Arp2/3 complex-mediated branched actin networks, we utilize the VCA domain located at the C-terminus of one of the major NPF, WAVE [24–26]. The V and CA domains are known to interact with G-actin and the Arp2/3 complex, respectively, inducing actin branching [24]. In this protocol, His-tagged iLID is pre-anchored to lipid membranes containing Ni-NTA, and the SspB-fused VCA domain is recruited from bulk solution onto the membrane in a light-dependent manner, thereby increasing VCA density on the membrane. Because the accumulated VCA domains are positioned in close proximity to each other, the stochastic binding of two VCA domains with Arp2/3 complexes and fragments of actin filament in bulk solution is facilitated, triggering actin polymerization [27,28]. According to previous studies on actin network reconstitution, profilin and capping protein are mixed together with the Arp2/3 complex and SspB-fused VCA [9,11,29]. Profilin and capping protein suppress actin nucleation in bulk solution and prevent excessive filament elongation, respectively. Collectively, under these conditions, light illumination triggers the assembly of actin networks in a light-dependent manner [30]. Using this system, both the shape and density of actin networks can be precisely controlled by modulating the pattern and intensity of light. Furthermore, actin polymerization can be immediately stopped by turning off the illumination. Thus, this protocol provides enhanced flexibility for actin network reconstitution and can be applied to studies of actin-binding protein function in a density-dependent manner [30], as well as to the design of three-dimensional structures of biomolecules.
Materials and reagents
Purified proteins
1. His-mEGFP-iLID (see [30] for the purification method)
2. SspB-mScarlet-I-VCA (see [30] for the purification method)
3. Actin (10% labeled with Alexa Fluor 647) (see [30,31] for the purification method)
4. Arp2/3 complex (Cytoskeleton, catalog number: RP01P)
5. Profilin (see [30,32] for the purification method)
6. Capping protein (CP) (see [30] for the purification method)
Note: Plasmids required for protein expression are available from the authors upon request. Plasmids used for the expression of His-mEGFP-iLID and SspB-mScarlet-I-VCA will be deposited at Addgene.
Reagents
1. 18:1 DGS-NTA(Ni) [DGS-NTA(Ni)] (Avanti, catalog number: 790404); product format: 10 mg/mL in chloroform
2. 16:0–18:1 PC (POPC) (Avanti, catalog number: 850457); product format: powder
3. Chloroform (Wako, catalog number: 038-02606)
4. KCl (Wako, catalog number: 163-03545)
5. NaCl (Wako, catalog number: 191-01665)
6. Na2HPO4 (Wako, catalog number: 194-02875)
7. KH2PO4 (Wako, catalog number: 166-04255)
8. 99.5% ethanol (Wako, catalog number: 057-00451)
9. NaOH (Wako, catalog number: 194-18865)
10. HEPES (Sigma, catalog number: H3375)
11. EGTA (Wako, catalog number: 342-01314)
12. MgCl2 (Wako, catalog number: 136-03995)
13. Riboflavin 5′-monophosphate sodium salt hydrate (FMN) (Sigma, catalog number: F8399)
14. Creatine phosphokinase from rabbit muscle (CPK) (Sigma, catalog number: C3755)
15. Phosphocreatine disodium salt hydrate (Pcr) (Sigma, catalog number: P7936)
16. Adenosine 5’-triphosphate disodium salt n-hydrate (ATP) (Wako, catalog number: 019-09672)
17. (+/-)-Dithiothreitol (DTT) (Wako, catalog number: 042-29222)
18. (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) (Sigma, catalog number: 238813)
19. KOH (Wako, catalog number:168-21815)
Solutions
1. 1× PBS, pH 6.5 (see Recipes)
2. Glass washing solution (see Recipes)
3. 10× A50 buffer (see Recipes)
4. SspB mixture (see Recipes)
5. 20× energy mix (see Recipes)
6. Polymerization mixture (see Recipes)
Recipes
1. 1× PBS, pH 6.5
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| NaCl | 137 mM | 8 g |
| KCl | 2.7 mM | 0.2 g |
| Na2HPO4 | 8.1 mM | 1.15 g |
| KH2PO4 | 1.5 mM | 0.2 g |
| Total | 1 L |
Mix all reagents and adjust the pH to 6.5 using HCl. Adjust the final volume to 1 L with Milli-Q water.
2. Glass washing solution
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 100% EtOH | 80% | 800 mL |
| NaOH | 50 g/L | 50 g |
| Total | 1 L |
Mix the reagents and adjust the final volume to 1 L with Milli-Q water.
3. 10× A50 buffer
| Reagent | Final concentration | Volume (for 1 L) |
|---|---|---|
| 1 M HEPES-KOH, pH 7.6 | 0.5 M | 500 mL |
| 2 M KCl | 0.5 M | 250 mL |
| 1 M MgCl2 | 50 mM | 50 mL |
| 0.2 M EGTA | 10 mM | 50 mL |
| Total | 1 L |
Mix the reagents and adjust the final volume to 1 L with Milli-Q water.
4. SspB mixture
| Reagent | Final concentration | Volume (for 20 μL) |
|---|---|---|
| 20 mM Trolox in A50 buffer | 2 mM | 2 μL |
| 200 μM FMN in A50 buffer | 10 μM | 1 μL |
| 4.3 μM SspB-mScarlet-I-VCA in A50 buffer | 150 nM | 0.7 μL |
| 1× A50 buffer | 16.3 μL | |
| Total | 20 μL |
Before mixing all components required for actin polymerization, we recommend first confirming that SspB-mScarlet-I-VCA is recruited to the lipid membrane upon light illumination in a simple composition without actin cytoskeletal proteins.
5. 20× energy mix
| Reagent | Final concentration | Volume (for 10 μL) |
|---|---|---|
| 0.2 M ATP in A50 buffer | 20 mM ATP | 1 μL |
| 0.4 M Pcr in A50 buffer | 0.2 M Pcr | 5 μL |
| 10 mg/mL CPK in A50 buffer | 2 mg/mL CPK | 2 μL |
| 1 M DTT in A50 buffer | 0.2 M DTT | 2 μL |
| Total | 10 μL |
The 20× energy mix is not stable for long-term storage and should therefore be freshly prepared for each experiment.
6. Polymerization mixture
| Reagent | Final concentration | Volume (for 20 μL) |
|---|---|---|
| 2× A50 buffer | 2 μL | |
| 20 mM Trolox in A50 buffer | 2 mM | 2 μL |
| 20× energy mix | 1× | 1 μL |
| 200 μM FMN in A50 buffer | 10 μM | 1 μL |
| 200 μM profilin in A50 buffer | 15 μM | 1.5 μL |
| 250 nM CP in A50 buffer | 25 nM | 2 μL |
| 2.8 μM Arp2/3 complex in A50 buffer | 100 nM | 0.7 μL |
| 4.3 μM SspB-mScarlet-I-VCA in A50 buffer | 150 nM | 0.7 μL |
| 50 μM actin in G-buffer | 5 μM | 2 μL |
| 1× A50 buffer | 7.1 μL | |
| Total | 20 μL |
To minimize actin polymerization in bulk solution, keep all solutions on ice and add actin last. Mix all reagents within 5 min and immediately load into a flow cell. Dilute capping protein (CP) immediately before use from a purified stock solution (30–50 μM) to a concentration of 250 nM in A50. Add the other proteins directly from their stock solutions, with mixing volumes adjusted according to stock concentrations. Adjust the total reaction volume to 20 μL with A50 buffer. Add 2× A50 buffer in a volume equal to that of actin.
Laboratory supplies
1. 1.5 mL glass tube (Maruemu, catalog number: 0407-03)
2. 24 × 60 mm coverslip (Matsunami, catalog number: C024601)
3. 18 × 18 mm coverslip (Matsunami, catalog number: C018181)
4. Double-sided tape; thickness: 0.3 mm (Scotch, catalog number: PBW-20)
5. VaLaP (Wako, catalog numbers: 224-00165, 128-00115, and 167-13335)
6. Membrane filter (Cytiva, catalog number: 10419504)
7. Filter support (Cytiva, catalog number: 230300)
8. Pipette tips (10 μL) (Eppendorf, catalog number: 0030000811)
9. Pipette tips (200 μL) (Eppendorf, catalog number: 0030000870)
10. Pipette tips (1,000 μL) (Eppendorf, catalog number: 0030000935)
11. 1.5 mL tube (Eppendorf, catalog number: 022363247)
12. Parafilm
Equipment
1. Vacuum desiccator (AS ONE, catalog number: 1-5801-23-20)
2. Glove bag (AS ONE, catalog number: 3-118-11)
3. Nitrogen gas line
4. Heat sealer (Fuji Impulse, model: P-300)
5. Vacuum sealing machine and associated bags and rolls (MagicVac, catalog number: V952S)
6. Vortex
7. Liquid nitrogen tank (Jecc Torisha, model: Cebell5)
8. Water bath (TAITEC, model: EX-B)
9. Microscope (Nikon, model: ECLIPSE Ti2)
10. Digital micromirror device (DMD) module (Nikon, model: TI-LA-DMD)
11. Objective lens (Nikon, model: CFI Plan Lamda D 60×, N.A. 1.42)
12. EMCCD camera (Andor, model: iXon Life 888)
13. Light source for DMD (Lumencor, model: SOLA)
14. Spinning disk confocal unit (Yokogawa Electric Corporation, model: CSU-W1)
15. ND filter (Thorlabs, catalog number: NE220B)
16. Dark microscopy room with red room light
17. Power sensor (Advantest, model: TQ8210)
18. Glass slide staining dish with stainless storage rack
19. Sonicator (AS ONE, catalog number: AS12GTU)
20. Plasma cleaner (Harrick Plasma, catalog number: PDC-32G)
21. Hot plate (Thermo Fisher Scientific, catalog number: HP2305BQ)
22. Small brush
23. Mini-extruder (Avanti, catalog number: 610000)
24. Humidity chamber (a plastic Petri dish filled with wet filter paper)
25. Deep freezer (-80 °C)
26. Freezer (-20 to 30 °C)
27. Fridge (4 °C)
28. Eppendorf pipette kit (Eppendorf, catalog number: 2231300002)
29. Weighing scale
Software and datasets
1. NIS-elements (Nikon, Version 5.42.03)
Procedure
文章信息
稿件历史记录
提交日期: Dec 28, 2025
接收日期: Mar 9, 2026
在线发布日期: Mar 18, 2026
出版日期: Apr 20, 2026
版权信息
© 2026 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
如何引用
Yamamoto, K. and Miyazaki, M. (2026). Optical Control of Actin Network Assembly on the Supported Lipid Bilayer. Bio-protocol 16(8): e5656. DOI: 10.21769/BioProtoc.5656.
Download Citation in RIS Format
分类
生物物理学 > 生物工程 > 光遗传学
生物化学 > 蛋白质 > 相互作用 > 蛋白质-脂质相互作用
生物化学 > 蛋白质 > 自组装
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