TY  - JOUR
TI  - Applications of OptoProfilin in Living Cells for the Imaging of Focal Adhesions and Stress-Associated Phenotypes
AU  - Brown, Clayton J
AU  - Hughes, Robert Murray
VL  - 16
IS  - 20
PY  - 2026
DA  - 2026/10/20
SP  - e5841
C1  - Bio-protocol 2026;16:e5841
DO  - 10.21769/BioProtoc.5841
UR  - https://doi.org/10.21769/BioProtoc.5841
AB  - 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.
KW  - OptoProfilin
KW  - Profilin-1
KW  - Cry2
KW  - Cellular stress
KW  - Optogenetics
KW  - VASP
KW  - Focal adhesions
KW  - Biomolecular condensates
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False