Multimodal In Vivo Imaging and Blood Monitoring of Intrinsic and Extrinsic Apoptosis

Abstract

Noninvasive detection and in vivo imaging of apoptosis plays a critical role in the development of therapeutics in many different fields including cancer. We have developed an apoptosis biosensor by fusing green fluorescent protein (GFP) to the N-terminus of the naturally secreted Gaussia luciferase separated by a caspase-3 cleavage peptide consisting of aspartic acid (D), glutamic acid (E), valine (V), and aspartic acid (D) or DEVD. We showed that this fusion is retained in the cytoplasm of cells in an inactive form. Upon apoptosis, the DEVD peptide is cleaved in response to caspase-3 activation, freeing ssGluc, which can now enter the secretory pathway where it is folded properly and is released from the cells and can be detected in the conditioned medium in culture or in blood of live animals ex vivo over time. Because Gluc is secreted from cells via conventional pathway through the endoplasmic reticulum (ER), Golgi and vesicles, we showed that the presence of Gluc in these compartments in response to apoptosis can be visualized in vivo using bioluminescence imaging. This reporter provides a valuable tool for imaging and real-time monitoring of apoptosis and is compatible with high-throughput functional screening application in cultured cells and animal models.

zebraflash transgenic lines for in vivo bioluminescence imaging of stem cells and regeneration in adult zebrafish

“…Recently, technologies have been developed to localize the three-dimensional distribution of the underlying bioluminescent source and generate cross-sectional imaging with enhanced resolution (Chaudhari et al, 2005;Cronin et al, 2012;Collins et al, 2013). The reagents we describe should be amenable to these evolving bioluminescence tools and should also be useful for other applications to visualize and dissect in vivo protein-protein interactions (Paulmurugan et al, 2002), apoptosis (Niers et al, 2011;Scabini et al, 2011), inflammation (Van de Bittner et al, 2010), hypoxia responses (Lehmann et al, 2009) and bacterial and viral infections (Phelan et al, 2005;Rawls et al, 2006).…”

In vivo bioluminescence for tracking cell fate and function

“…Another advantage of Renilla and Gaussia luciferases over Photinus pyralis is the fact that they do not require ATP as a cofactor during bioluminescent reaction and thus can be used for imaging cells independent of their metabolic state (5,66). One particular characteristic of Gaussia luciferase is that it is naturally secreted (96) and therefore can be used as a reporter for quantitative assessment of cells in vivo by measuring its concentration in blood (61). However, Renilla and Gaussia luciferases produce shorter wavelengths of light (peaking at 480 nm), which are not transmitted through tissues as effectively as Photinus pyralis (100).…”

Hepatitis C Virus Is Released via a Noncanonical Secretory Route

“…8A). To directly compare the effects of the inhibitor on a cargo that is released via secretion through the Golgi apparatus, we used Huh7.5 cells, which were electroporated with a plasmid encoding Gaussia luciferase (35,52). This approach directly allows assessment of the efficiency of Gaussia secretion by measurement of luciferase activity in the supernatant (Fig.…”

“…5A). To exclude the possibility that the electroporation procedure might interfere with Golgi function or that there is an as-yet-unknown Golgi defect in the Huh7.5 cells used, we coelectroporated vectors expressing hepatitis B virus (HBV)-S-mCherry or Gaussia luciferase-YFP, each of which is known to be secreted through the Golgi apparatus (52,53), together with the GalT-CFP fusion. Both proteins showed strong colocalization with GalT-CFP as evident from the line profiles (Fig.…”