Clayton J. Brown; Robert M. Hughes

doi:10.21769/BioProtoc.5841

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Peer-reviewed

Applications of OptoProfilin in Living Cells for the Imaging of Focal Adhesions and Stress-Associated Phenotypes

CB Clayton J. Brown

RH Robert M. Hughes email

发布: 2026年10月20日第16卷第20期 DOI: 10.21769/BioProtoc.5841 浏览次数: 25

评审: KM Rifat FaysalAnonymous reviewer(s)

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Cover of ChemBioChem, featuring study using the protocol.

May 2024

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Abstract

Cellular stress induces profound changes in cytoskeletal organization and biomolecular condensate formation. Traditional approaches for monitoring cellular stress often require multi-component biosensors, endpoint staining procedures, or indirect biochemical measurements. Here, we describe a protocol for the use of OptoProfilin, a genetically encoded single-component optogenetic biosensor derived from Profilin-1 fused to Cryptochrome 2 (Cry2) and mCherry. Following transient expression in mammalian cells, OptoProfilin exhibits light-dependent localization to focal adhesions under non-stressed conditions and transitions to punctate condensates under energetic, oxidative, osmotic, and senescence-associated stress conditions. The protocol includes transient transfection, induction of cellular stress, live-cell imaging, immunofluorescence validation, and quantitative image analysis. While this protocol describes imaging on a Leica widefield fluorescence microscope, it can readily be extended to other microscopy platforms. As a stand-alone biosensor that produces visually distinct responses in stressed versus non-stressed cells, OptoProfilin provides a convenient platform for investigating stress-associated cytoskeletal remodeling and biomolecular condensate formation.

Key features

• Single-component optogenetic biosensor for monitoring cellular stress.

• Distinguishes stressed and unstressed cells through distinct localization phenotypes.

• Compatible with live-cell imaging, fixed-cell analysis, and immunofluorescence.

• Applicable to energetic, oxidative, osmotic, and senescence-associated stress models.

Keywords: OptoProfilin

Biomolecular condensates

Graphical overview

OptoProfilin applications in cells. Figure created using BioRender.

Background

The actin cytoskeleton is highly responsive to environmental and physiological stress. Stress-induced remodeling of actin-associated proteins contributes to processes including biomolecular condensate formation, stress granule assembly, senescence, neurodegeneration, and disease progression. Profilin-1 is a multifunctional actin-binding protein that regulates actin polymerization and interacts with numerous cytoskeletal regulatory proteins, including vasodilator-stimulated phosphoprotein (VASP).

OptoProfilin was developed by fusing mouse Profilin-1 to the photolyase homology region of Arabidopsis thaliana Cryptochrome 2 and mCherry [1]. Upon blue-light activation, Cry2 undergoes oligomerization, promoting localization of OptoProfilin to VASP-containing structures; the interaction is reversible in the absence of blue light. Under non-stress conditions, OptoProfilin localizes primarily to focal adhesions. Following cellular stress, OptoProfilin transitions into punctate condensates that serve as a visual indicator of stress-associated cytoskeletal remodeling. OptoProfilin was first investigated using a Leica DMi8 live cell imaging system; thus, this system and the associated LasX software are featured prominently in this protocol. However, these methods could reasonably be extended to other microscopy platforms.

Compared to multi-component biosensors, OptoProfilin requires expression of only a single construct, simplifying experimental implementation and increasing compatibility with multiplexed imaging approaches. This protocol is best suited to energetic (ATP depletion), oxidative, osmotic, and senescence-associated stress paradigms; it is not appropriate for detecting heat stress, as OptoProfilin does not form clusters under heat shock conditions. VASP (vasodilator-stimulated phosphoprotein) is an actin-associated protein and a known component of focal adhesions. Mutations to the VASP-binding region of Profilin-1 within OptoProfilin eliminate both its focal adhesion and stress cluster phenotypes. In cell lines with higher VASP expression (e.g., N2a), the assay may lose its ability to discriminate stressed from unstressed cells. In cell lines with lower endogenous VASP expression (e.g., HEK293T, NIH 3T3), only the stress-induced clustering phenotype is informative, whereas the dual-phenotype readout (focal adhesion recruitment versus stress-induced clustering) is most robust in HeLa cells.

Materials and reagents

Biological materials

1. OptoProfilin plasmids (Addgene plasmids, catalog numbers: 208286, 208287, 208288)

2. HeLa cells (ATCC, catalog number: CCL-2)

3. HEK293T cells (ATCC, catalog number: CRL-3216)

4. NIH 3T3 cells (ATCC, catalog number: CRL-1658)

5. Neuro-2a (N2a) cells (ATCC, catalog number: CCL-131)

6. Anti-VASP primary antibody (Cell Signaling, catalog number: 3132)

7. Anti-Paxillin primary antibody (Cell Signaling, catalog number: 12065)

8. Alexa Fluor 488-conjugated secondary antibody (Invitrogen, catalog number: A-11008)

Reagents

1. Dulbecco’s minimum essential medium (DMEM) with D-glucose and glutamine (Gibco, catalog number: 11965-092)

2. Fetalgro EX bovine growth serum (rmbio FGX-BBT) or fetal bovine serum (FBS) (Gibco, catalog number: A52568010)

3. Penicillin-streptomycin (Gibco, catalog number: 15140-122)

4. Trypsin-EDTA (0.25%), phenol red (Gibco, catalog number: 25200-056)

5. Calfectin transfection reagent (SignaGen, catalog number: SL100478)

6. Dulbecco’s phosphate-buffered saline (DPBS) containing Ca²⁺ and Mg²⁺ (Gibco, catalog number: 14040-133)

7. Sodium azide (NaN₃) (Fisher Scientific, catalog number: BP9221)

8. 2-Deoxy-D-glucose (2-DG) (Sigma, catalog number: D8375)

9. Sodium (meta)arsenite (NaAsO₂) (Sigma, catalog number: S7400)

10. Sorbitol (Fisher Scientific, catalog number: BP439)

11. Hydrogen peroxide (H₂O₂) (Sigma, catalog number: 216763)

12. Paraformaldehyde 16% (Fisher Scientific, Electron Microscopy Sciences, catalog number: 15710)

13. Triton X-100 (Acros, catalog number: 327372500)

14. Bovine serum albumin (BSA) (Fisher Scientific, catalog number: BP6711)

Solutions

1. ATP depletion medium (see Recipes)

2. Oxidative stress medium (see Recipes)

3. Osmotic stress medium (see Recipes)

4. Senescence induction medium (see Recipes)

5. Antibody dilution buffer (see Recipes)

6. 4% paraformaldehyde fixation solution (see Recipes)

Recipes

1. ATP depletion medium

Reagent	Final concentration	Quantity or volume
DPBS (with Ca²⁺/Mg²⁺)	1×	9.84 mL
NaN₃, 1 M stock	10 mM	100 μL
2-DG, 1 M stock	6 mM	60 μL
Total	n/a	10 mL

2. Oxidative stress medium

Reagent	Final concentration	Quantity or volume
NaAsO₂	0.5 mM	3.25 mg
DPBS	1×	to 50 mL
Total	n/a	50 mL

3. Osmotic stress medium

Reagent	Final concentration	Quantity or volume
D-sorbitol	200 mM	1.82 g
DPBS	1×	to 50 mL
Total	n/a	50 mL

4. Senescence induction medium

Reagent	Final concentration	Quantity or volume
H₂O₂, 9.79 M stock	200 μM	1.02 μL
DPBS	1×	49.999 mL
Total	n/a	50 mL

5. Antibody dilution buffer

Reagent	Final concentration	Quantity or volume
1× PBS	1×	9.97 mL
BSA	1% (w/v)	0.1 g
Triton X-100	0.3% (v/v)	30 μL
Total	n/a	10 mL

6. 4% paraformaldehyde fixation solution

Reagent	Final concentration	Quantity or volume
16% paraformaldehyde	4% (w/v)	10 mL
DPBS	0.75×	30 mL
Total	n/a	40 mL

Laboratory supplies

1. 35 mm glass-bottom dishes (MatTek, catalog number: P35G-1.5-14-C)

2. Sterile microcentrifuge tubes (1.5 mL)

3. Sterile serological pipettes

Equipment

1. Tissue culture incubator (37 °C, 5% CO₂)

2. Tissue culture microscope

3. Hemacytometer (Bright-Line^TM Hemacytometer; Cambridge Instruments, Inc.)

4. Widefield fluorescence microscope: Leica DMi8 Live Cell Imaging System, equipped with an OKOLab stage-top live cell incubation system, Infinity TIRF module, Leica HCX PL APO 63×/1.40–0.60 n.a. oil objective, Leica HC PL APO 100×/1.47 oil TIRF objective, Lumencor LED light engine, CTRadvanced+ power supply, and a Leica DFC900 GT camera

5. Confocal microscope (Zeiss LSM 700 laser scanning microscope using ZEN Black 2012 software)

6. Blue LED illumination source (Sunlite LED Par30 Reflector, Item #80021, 4 W, 120 V)

Software and datasets

1. FIJI/ImageJ (ImageJ 1.54p 17 February 2025)

2. GraphPad Prism (10.6.1 (892) 31 October 2026)

3. ZEN Black 2012 or updated equivalent

4. LAS X software (3.7.6.25997)

Procedure

文章信息

稿件历史记录

提交日期: Jul 6, 2026

接收日期: Sep 8, 2026

在线发布日期: Sep 20, 2026

出版日期: Oct 20, 2026

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如何引用

Brown, C. J. and Hughes, R. M. (2026). Applications of OptoProfilin in Living Cells for the Imaging of Focal Adhesions and Stress-Associated Phenotypes. Bio-protocol 16(20): e5841. DOI: 10.21769/BioProtoc.5841.