Rapid Optogenetic Clustering in the Cytoplasm with BcLOVclust.
J Mol Biol
; 436(3): 168452, 2024 02 01.
Article
en En
| MEDLINE
| ID: mdl-38246410
ABSTRACT
Protein clustering is a powerful form of optogenetic control, yet remarkably few proteins are known to oligomerize with light. Recently, the photoreceptor BcLOV4 was found to form protein clusters in mammalian cells in response to blue light, although clustering coincided with its translocation to the plasma membrane, potentially constraining its application as an optogenetic clustering module. Herein we identify key amino acids that couple BcLOV4 clustering to membrane binding, allowing us to engineer a variant that clusters in the cytoplasm and does not associate with the membrane in response to blue light. This variant-called BcLOVclust-clustered over many cycles with substantially faster clustering and de-clustering kinetics compared to the widely used optogenetic clustering protein Cry2. The magnitude of clustering could be strengthened by appending an intrinsically disordered region from the fused in sarcoma (FUS) protein, or by selecting the appropriate fluorescent protein to which it was fused. Like wt BcLOV4, BcLOVclust activity was sensitive to temperature light-induced clusters spontaneously dissolved at a rate that increased with temperature despite constant illumination. At low temperatures, BcLOVclust and Cry2 could be multiplexed in the same cells, allowing light control of independent protein condensates. BcLOVclust could also be applied to control signaling proteins and stress granules in mammalian cells. While its usage is currently best suited in cells and organisms that can be cultured below â¼30 °C, a deeper understanding of BcLOVclust thermal response will further enable its use at physiological mammalian temperatures.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Proteínas Adaptadoras Transductoras de Señales
/
Criptocromos
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Optogenética
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Proteínas de la Matriz de Golgi
Límite:
Animals
Idioma:
En
Revista:
J Mol Biol
Año:
2024
Tipo del documento:
Article
País de afiliación:
Estados Unidos