The Tsien lab creates versatile tools that enable visual examination of cell signaling and disease processes. Most of their recent work concerns activatable cell-penetrating peptides (ACPPs), which allow detection of enzyme activities or other reactive species in living systems. These peptides are not only useful in basic research, but also promise translation to the clinic to guide tumor resection. In addition, Tsien’s team is engineering variations on the fluorescent protein concept to allow imaging at greater depths in living tissues and label-free electron microscopic detection of specific proteins.
Whereas previous molecular fluorescent imaging agents were either too large to effectively penetrate tumors (e.g. antibodies) or had limited specificity (small molecules that bind to specific proteins), ACPPs are relatively small and accumulate in tissues only upon cleavage. This specific accumulation is possible because of a clever design: a fluorescently tagged cell-penetrating (polycationic) peptide is masked by a polyanionic peptide attached through a cleavable linker. The mechanism of activation is adaptable to numerous targets; the Tsien group has designed ACPPs that are cleaved by matrix metalloproteinases (MMPs) and elastase for tumor imaging, thrombin for detection of atherosclerotic plaques and ischemic damage in stroke, and by hydrogen peroxide for imaging of oxidative stress. Later generations of ACPPs incorporate an additional dye tag on the masking peptide that accepts fluorescence energy from the other dye; the ratio of the two signals eliminates the effect of imaging agent concentration and detection sensitivity.
After observing that fluorescent MMP-cleavable ACPPs allowed more thorough tumor removal and thus improved survival in mice, the group was inspired to tackle a related challenge in tumor surgery: avoiding nerves. Their development of a nerve-specific imaging agent (not an ACPP, but a dye-tagged peptide that binds a protein expressed only on nerve surfaces) suggests that surgery could be color-coded in the future.
Given the enormous utility of fluorescent proteins (for which Tsien won the Nobel prize in 2008), the lab is also developing new versions. Most recently, these include proteins that emit at far-red (named Neptune) and infrared wavelengths (IFPs, based on a protein from an extremophilic bacterium), to which biological tissue is relatively transparent, allowing whole-body imaging in live animals.
The Tsien group have also extended the concept of tagging proteins for visualization to electron microscopy (EM), allowing localization at the highest possible resolution. EM traditionally employs gold- or eosin-labeled antibodies, but the fixation necessary to preserve cellular structures impedes their diffusion into the tissue section. As eosin allows detection by generating singlet oxygen upon irradiation, which catalyzes polymerization of diaminobenzidine into a product detectable upon EM staining, Tsien’s team engineered a plant flavoprotein to generate maximal amounts of singlet oxygen. The resulting protein, miniSOG, has since proven useful not only for EM, but also for light-triggered inactivation of specific proteins.
M Whitney, EN Savariar, B Friedman, RA Levin, JL Crisp, HL Glasgow, R Lefkowitz, SR Adams, PA Steinbach, N Nashi, QT Nguyen, RY Tsien. Ratiometric activatable cell-penetrating peptides provide rapid in vivo readout of thrombin activation. Angew Chem Int 2013; 52 325–330.
X Shu, V Lev-Ram, TJ Deerinck, Y Qi, EB Ramko, MW Davidson, Y Jin, MH Ellisman, RY Tsien. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol 2012; 9:4.
QT Nguyen, ES Olson, TA Aguilera, T Jiang, M Scadeng, LG Ellies, RY Tsien. Surgery with molecular fluorescence imaging using activatable cell-penetrating peptides decreases residual cancer and improves survival. Proc Natl Acad Sci 2010; 107: 4317–4322.
X Shu, A Royant, MZ Lin, TA Aguilera, V Lev-Ram, PA Steinbach, RY Tsien. Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome. Science 2009; 324: 804-807.