Membrane-permeable cygnets: rapid cellular internalization of fluorescent cGMP-indicators

Area of Science:

• Cell biology
• Molecular biology
• Biochemistry

Background:

• Genetically encoded cyclic guanosine monophosphate (cGMP) indicators (cygnets) allow study of intracellular cGMP dynamics.
• Traditional methods for introducing molecular reporters are laborious and time-consuming, requiring cell culture.
• Primary mammalian cells are difficult to maintain in undifferentiated culture for genetic reporter expression.

Purpose of the Study:

• To develop a rapid, non-genetic method for introducing cGMP indicators into cells and intact tissues.
• To overcome limitations of existing genetic and laborious single-cell reporter introduction techniques.
• To enable faster and more efficient studies of intracellular cGMP signaling.

Main Methods:

• Expression and purification of five different 125 kDa membrane permeable peptide (MPP)-cygnets from insect SF9 cells.
• In vitro characterization of cGMP-dependent Förster Resonance Energy Transfer (FRET) in MPP-cygnet constructs.
• Application of the most effective MPP-cygnet (Ant7-Cygnet) in cultured smooth muscle cells and intact cerebral arteries.

Main Results:

• Three MPP-cygnet constructs exhibited high-level cGMP-dependent FRET in vitro.
• Ant7-Cygnet showed emission ratio changes comparable to the unmodified indicator.
• Ant7-Cygnet was rapidly (3 hours) and efficiently internalized in cultured smooth muscle cells and intact cerebral arteries.
• Internalized Ant7-Cygnet detected nitric oxide-mediated cGMP elevations in smooth muscle cells and CNP-induced cGMP increases in intact arteries.

Conclusions:

• Membrane permeable peptide-cygnets offer a novel, rapid, and efficient non-genetic approach for introducing cGMP indicators.
• This technique overcomes the limitations of genetic methods and laborious single-cell delivery.
• MPP-cygnets provide a powerful tool for studying intracellular cGMP dynamics in both cultured cells and intact tissues.