Altered cellular membrane integrity is one of the earliest signs of apoptosis.1,2 One key change during this event is the movement of phosphatidylserine (PS) from the inner leaflet of the cell membrane towards the cell surface. This process, due to the inactivation of flippase- and activation of scramblase-enzymes, is inducible, reversible and dependent upon calcium release from the endoplasmic reticulum.1 Exposure of PS acts as an "eat me" signal, prompting phagocytosis of apoptotic cells.3 In the absence of PS exposure during apoptosis, dying cells would escape the immune system ensuing an inflammatory response.2 Thus, PS externalization has emerged as a useful event and target for monitoring the progression of apoptosis in real-time.
The discovery of a natural ligand, Annexin A5, which binds reversibly, selectively and with high-affinity to exposed PS prompted its development as a biological tool for the assessment of apoptosis. In the initial assay from the early 1990s, Annexin A5 was conjugated with fluorescein (FITC) and used to detect apoptotic cells by flow cytometry.4 Overall, Annexin A5 was validated as an effective tool for identifying cells at an early stage in the apoptotic process, coincidental with DNA fragmentation. Nevertheless, the high background fluorescence associated with the use of FITC represented a major caveat preventing the use of this tool for monitoring apoptosis in real-time.
In 2010 a new Annexin based tool was developed, further facilitating live-cell imaging of apoptosis.5 Rather than Annexin B5, the new tool used Annexin B12 conjugated to polarity-sensitive fluorophores. pSIVA (polarity-sensitive indicator of viability and apoptosis), conserves the reversible calcium dependent binding to PS, but has the added benefit of the fluorophore tag, IANBD*. pSIVA lacks fluorescence in solution or polar environments, but is "turned on" upon interaction with PS in the membrane, thus serving as a biological switch to monitor apoptosis in real-time and at a single cell level.5,6 Because PS externalization is reversible, pSIVA can be used to study the factors and conditions that contribute to cell death recovery.
Figure 6. Real-time live cell imaging of apoptosis: COS-7 cells were induced to undergo apoptosis with etoposide (100 mM) or left untreated [DMSO (-) control]. pSIVA-IANBD + PI was added directly to the culture media and cells were imaged (37oC, 5% CO2) by time lapse microscopy of the same fields over time. Green fluorescence indicates pSIVA-IANBD binding to PS exposed on the outer leaflet of the plasma membrane, and orange-yellow fluorescence indicates PI staining of nuclei. *Time point when PI staining was first seen in the cell; PI staining indicates loss of membrane integrity, pSIVA-IANBD stained cells prior to PI staining.
Recently, Röttgermann et al. demonstrated the power of pSIVA as a biological sensor to evaluate nanoparticle-induced apoptosis.6 Applications of nanoparticles in medicine are ample including delivery of drugs to treat cancer and the delivery of enzymes to treat metabolic diseases. However, the mechanism by which nanoparticles interact with cells and associated toxicities remain poorly understood. Therefore, Röttgermann et al. devised "micro-patterned single-cell arrays" to monitor the progress of apoptosis using pSIVA and propidium iodide (PI) as early and late markers of apoptosis, respectively. Polystyrene nanobeads functionalized with amino groups (PS-NH2) were added to cells under physiological conditions and cells were monitored using time-lapse microscopy every 10 minutes for 48 hours. Using this approach, investigators established a dose response curve, demonstrating a delay in the onset of pSIVA fluorescence with decreased nanoparticle dose.
pSIVA is the next step in the annexin saga. As an advanced biological sensor, pSIVA monitors the progression of apoptosis in real-time and furthers our understanding of individual cell responses, which often get lost in the noise of the crowd.
*IANBD (N,N′-di-methyl-N(iodoacetyl)-N′-(7nitrobenz-2-oxa-1,3-diazol-4-yl)ethyleneamine)
