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St. Jude Team Uncovers Key Event in Apoptosis Using Real-Time, Single-Cell Confocal Imaging

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A group led by scientists from St. Jude’s Children’s Research Hospital has published research showing how they used real-time, single-cell microscopy and image-analysis software to observe how specific mitochondrial proteins are released into the cytosol during apoptosis.
 
The research provides for the first time evidence that the permeabilization of the outer mitochondrial membrane is a relatively rapid process that allows the simultaneous release of a variety of proteins involved in apoptosis, and underscores the importance of targeting this event in drug development.
 
The research is also an example of how sophisticated image-analysis software continues to fill an important niche in basic image-based cell biology research despite the recent focus on its utility in high-content automated microscopy.
 
Douglas Green, immunology chair at Memphis, Tenn.-based St. Jude, was lead author on the study, which appears in the Aug. 1 issue of the Proceedings of the National Academy of Sciences. Scientists from the La Jolla Institute for Allergy and Immunology in California also contributed to the work.
 
Mitochondrial outer membrane permeabilization, otherwise known as MOMP, had already been established as a critical event during apoptosis. What’s more, scientists have known that the event is controlled by pro- and anti-apoptotic Bcl-2 –family proteins, and that the end result is a release of several mitochondrial intermembrane space proteins (MISPs) into the cytosol, which orchestrate apoptosis.
 
But most scientific studies have suggested that the proteins are released at different times, suggesting that membrane permeabilization is protein-specific – that is, different pores are formed in the mitochondria for different proteins.
 
Green and colleagues found evidence pointing to the contrary – that MOMP is a one-time event resulting in the release of all MISPs – and believe that the evidence is especially compelling because of their use of a confocal, real-time, single-cell microscopy technique that allowed them to directly observe the temporal relationships between the release of individual proteins and other parameters of cell death.
 
As described in the PNAS paper, the researchers fused several MISPs – including Smac, adenylate kinase 2, Omi, and AIF – to small tags containing a tetracysteine motif that is specifically recognized by either cell-permeable fluorescein- or resorufin-based fluorescent dyes. They expressed these proteins in cells that also expressed cytochrome c – another MISP – along with a green fluorescent protein tag.
 
They induced apoptosis in the cells using staurosporine, and then took images of the cells every two minutes using a homemade confocal system that featured a Nikon microscope body, Bio-Rad confocal head, and Molecular Devices MetaMorph image-analysis software.
 
They witnessed a near-simultaneous release of cytochrome c, Smac, Omi, and adneylate cyclase from the mitochondria, and found that only the AIF protein released on a much longer time scale and more incompletely, though its release did commence with the other MISPs.
 
“A number of papers have suggested that differential release of proteins not only occurs, but is important for different types of death,” Green wrote to CBA News in an e-mail. “Our work does not support that conclusion.
 
“Our work provides evidence that the formation of the MOMP pore is a key event in apoptosis, and provides further data on the nature of that pore,” Green added. “Drug discovery focusing on enhancing or inhibiting such pore formation – mediated by Bcl-2 family proteins – is an important goal.”
 
The researchers hypothesize that the AIF protein release, which took place over the course of a few hours as opposed to a 3- to 10-minute window for the other MISPs, is likely not directly involved with the induction of apoptosis, but may play a part in the process through a secondary event.
 
“There is a large body of work on AIF, but nearly all of it is controversial,” Green wrote in his e-mail. “The relationships between its release and that of cytochrome c [had] not been established until now. Clearly, AIF release involves additional mechanisms to those of cytochrome c, although at this point we simply do not know if this release is important. Some would say it is very much so, but I’d have to say that we are keeping an open mind.”
 
Regarding his group’s imaging technique, Green said that it used the MetaMorph 4.0 image-analysis program because it was “convenient for our analysis, although it is somewhat cumbersome.” Basically, the group manually drew regions around individual cells using the program, which then computed the standard deviation of the intensity of pixels and integrated brightness.
 
Green said that the group did explore some modicum of automation, but that in the end, manual delineation of cells was the most efficient method for its particular needs.
 
Their desire for more sophisticated image-analysis software underscores the important market that still exists for this type of product in basic cell biology labs. In fact, MetaMorph was originally designed at Universal Imaging with these types of applications in mind, and only over the past few years has Molecular Devices made the program the cornerstone of its high-content analysis platforms. However, the company has said that it still sells a significant number of MetaMorph packages to cell biologists conducting basic imaging research.
 
The MetaMorph 4.0 package is several generations old. The newest Molecular Devices MetaMorph package is 7.0. Green said that his group is currently exploring other image-analysis packages.
 
The researchers will now turn their attention to further elucidating the MOMP event because of its seemingly critical role in apoptosis.
 

“We are very actively exploring the events that accompany MOMP … at the molecular, biochemical, structural, and cellular levels, using a variety of techniques,” Green wrote in his e-mail. “The few minutes during which MOMP occurs are, we believe, the critical moments in the life and death of the cell, and we wish to understand this event in detail, including upstream and downstream processes.”

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