RESUMO
Cell membrane stiffness is critical for cellular function, with cholesterol and sphingomyelin as pivot contributors. Current methods for measuring membrane stiffness are often invasive, ex situ, and slow in process, prompting the need for innovative techniques. Here, we present a fluorescence resonance energy transfer (FRET)-based protein sensor designed to address these challenges. The sensor consists of two fluorescent units targeting sphingomyelin and cholesterol, connected by a linker that responds to the proximity of these lipids. In rigid membranes, cholesterol and sphingomyelin are in close proximity, leading to an increased FRET signal. We utilized this sensor in combination with confocal microscopy to explore changes in plasma membrane stiffness under various conditions, including differences in osmotic pressure, the presence of reactive oxygen species (ROS) and variations in substrate stiffness. Furthermore, we explored the impact of SARS-CoV-2 on membrane stiffness and the distribution of ACE2 after attachment to the cell membrane. This tool offers substantial potential for future investigations in the field of mechanobiology.
Assuntos
Membrana Celular , Colesterol , Transferência Ressonante de Energia de Fluorescência , SARS-CoV-2 , Esfingomielinas , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Membrana Celular/metabolismo , Membrana Celular/química , Esfingomielinas/análise , Esfingomielinas/metabolismo , Colesterol/análise , Colesterol/metabolismo , Microscopia Confocal/métodos , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/análise , COVID-19/virologia , Enzima de Conversão de Angiotensina 2/metabolismo , Técnicas Biossensoriais/métodosRESUMO
We proposed a method to regulate nucleic acid polymerization by proximity and designed an ultrasensitive biosensor based on proximity-induced exponential amplification reaction for proximity assay of proteins (streptavidin) and small molecules (adenosine triphosphate), which allows us to detect a variety of interesting targets by simply changing the binding sites of DNA.