Abstract
Caspases work as a double-edged sword in maintaining cell homeostasis. Highly regulated caspase activities are essential during animal development, but dysregulation might lead to different diseases, e.g. extreme caspase activation is known to promote neurodegeneration. At present, visualization of caspase activation has mostly remained at the cellular level, in part due to a lack of cell-permeable imaging probes capable of direct, real-time investigations of endogenous caspase activities in deep tissues. Herein, we report a suite of two-photon, small molecule/peptide probes which enable sensitive and dynamic imaging of individual caspase activities in neurodegenerative models under physiological conditions. With no apparent toxicity and the ability of imaging endogenous caspases both in different subcellular organelles of mammalian cells and in brain tissues, these probes serve as complementary tools to conventional histological analysis. They should facilitate future explorations of caspases at molecular, cellular and organism levels and inspire development of novel two-photon probes against other enzymes.
Highlights
Caspases are aspartate-specific cysteine proteases that have attracted significant attention because of their vital roles in apoptosis and inflammation[1,2]
Mindful of the current limit that many imaging studies of endogenous caspase activities rely heavily on organelle-specific fluorescent trackers to indirectly track the targeted caspase and its subcellular distribution[16], we further addressed such issues with the successful development of organelle-directed probes by using caspase-3/-7 as representative examples (Fig. 1b); with C3RM and C3RE, these two probes were directly delivered to their respective subcellular organelles of interest, where their two-photon fluorescence would be “Turned-On” only upon the presence of endogenous active caspase-3/-7
By linking a caspase WH to AAN via an amide linkage, the electron-withdrawing amide bond would effectively quench the fluorescence of AAN via internal charge transfer (ICT; Supplementary Fig. 1a)[29], rendering the probes non-fluorescent
Summary
Caspases are aspartate-specific cysteine proteases that have attracted significant attention because of their vital roles in apoptosis and inflammation[1,2]. Genetic manipulation with FPs and the two-step activation required to form fluorescent aggregates in the latter make these approaches ineffective for rapid imaging of endogenous caspase activation By virtue of their chemical tractability (e.g., different molecular structures/designs can be installed) and cell permeability, small molecule-based probes capable of being rapidly “Turned-On” in a single step in caspase activity-dependent manner, are highly desirable, especially if they can be readily converted into organelle-specific probes for subcellular investigations of caspase activation[15]. To show our newly developed two-photon caspase-detecting probes could provide complementary tools to commercially available coumarin-based peptide probes (which are commonly used in one-photon imaging experiments, but have very poor two-photon properties), representative probes were tested in live mammalian cells and fresh mouse brains of neurodegenerative models (Fig. 1c) Our results indicate these caspase-specifc probes could potientially be utilized to directly reveal the pathogenesis of diseased tissues by using two-photon fluorescence microscopy
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