photoactivated-localization-microscopy-with-bimolecular-fluorescence

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

Oregon Health and Science University

Wall shear stresses (WSS) exerted by blood flow on cardiac tissues modulate growth and development of the heart. To study the role of hemodynamic conditions on cardiac morphogenesis, here, we present a methodology that combines imaging and finite element modeling to quantify the in vivo blood flow dynamics and WSS in the cardiac outflow tract (OFT) of early chicken embryos (day 3 out of 21-day incubation period). We found a distinct blood flow field and heterogeneous distribution of WSS in the chicken embryonic heart OFT, which have physiological implications for OFT morphogenesis.

Quantifying blood flow and wall shear stresses in the outflow tract of chick embryonic hearts.

Bimolecular fluorescence complementation (BiFC) has been widely used to visualize protein-protein interactions (PPIs) in cells. Until now, however, the resolution of BiFC has been limited by the diffraction of light to ∼250 nm, much larger than the nanometer scale at which PPIs occur or are regulated. Cellular imaging at the nanometer scale has recently been realized with single molecule superresolution imaging techniques such as photoactivated localization microscopy (PALM). Here we have combined BiFC with PALM to visualize PPIs inside cells with nanometer spatial resolution and single molecule sensitivity. We demonstrated that PAmCherry1, a photoactivatable fluorescent protein commonly used for PALM, can be used as a BiFC probe when split between residues 159 and 160 into two fragments. PAmCherry1 BiFC exhibits high specificity and high efficiency even at 37°C in detecting PPIs with virtually no background from spontaneous reconstitution. Moreover, the reconstituted protein maintains the fast photoconversion, high contrast ratio, and single molecule brightness of the parent PAmCherry1, which enables selective PALM localization of PPIs with ∼18 nm spatial precision. With BiFC-PALM, we studied the interactions between the small GTPase Ras and its downstream effector Raf, and clearly observed nanoscale clustering and diffusion of individual KRas G12D/CRaf RBD (Ras-binding domain) complexes on the cell membrane. These observations provided novel insights into the regulation of Ras/Raf interaction at the molecular scale, which would be difficult with other techniques such as conventional BiFC, fluorescence co-localization or FRET.

Photoactivated localization microscopy with bimolecular fluorescence complementation (BiFC-PALM) for nanoscale imaging of protein-protein interactions in cells.

Article	Year
Quantifying blood flow and wall shear stresses in the outflow tract of chick embryonic hearts. Computers & structures\| PubMed ID: 21572557	2011
Photoactivated localization microscopy with bimolecular fluorescence complementation (BiFC-PALM) for nanoscale imaging of protein-protein interactions in cells. PloS one\| PubMed ID: 24963703	2014

Andrew Nickerson

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