Imaging of proteolytic activity using a conditional cell surface receptor.

Abstract

Programmed cell death (apoptosis) is a ubiquitous means utilized by multicellular organisms for elimination of unwanted cells during development and homeostasis. Dysregulated apoptosis is implicated in an array of clinical disorders including cancer, autoimmune diseases, neurodegenerative disorders, and ischemia. During programmed cell death, a series of proteases, known as caspases, with different specificities play crucial roles in the apoptotic process. Caspase-3, a group II cysteine aspartate protease, recognizes and cleaves substrates harboring the amino acid sequence aspartic acid-glutamic acid-valine-aspartic acid (DEVD), and it plays an important role in the terminal phase of apoptosis. Here we report the development of a novel imaging platform for sensing the activation of cellular proteases. A recombinant chimeric protein was constructed, composed of a cell-surface-targeted single-chain antibody (sFv) fused to a Golgi retention signal. The DEVD tetrapeptide sequence was included between the single-chain antibody and the Golgi retention signal as a caspase-3 protease cleavage site. When expressed in cultured cells this fusion protein was localized to Golgi bodies and was not detected on the cell surface. Induction of apoptosis resulted in cleavage of the fusion protein releasing the single-chain antibody from the Golgi retention signal in a caspase-dependent manner. As a result, in cells undergoing apoptosis the single-chain antibody was visualized at the cell surface by immunofluorescence microscopy. The expression of sFv on the surface of cells in a protease-dependent manner provides a unique opportunity for real-time imaging through the use of targeted nanoparticles. This methodology may provide for a multimodal noninvasive real-time imaging of apoptosis and a new opportunity for high-throughput screening of cell-death-modulating therapeutic agents.