Stefan Grimm

The permeability transition pore signals apoptosis by directing Bax translocation and multimerization

Mitochondria are key players of apoptosis and can irreversibly commit the cell to death by releasing cytochrome c (Cyt.c) to the cytosol, where caspases 9 and 3 subsequently get activated. Under conditions of oxidative stress, opening of the mitochondrial permeability transition pore (PTP) represents an early trigger and is crucial in causing Cyt.c release. To account for the latter, current models propose that PTP gating would result, as is the case in vitro, in the rupture of the outer mitochondrial membrane caused by mitochondrial matrix swelling. Using live cell imaging and recombinant fluorescent probes based on the green fluorescent protein (GFP) and its mutants, we report that directed repetitive gating of the PTP triggers a delayed Cyt.c efflux, which is not associated with mitochondrial swelling. Instead, subcellular imaging shows that PTP opening signals the redistribution of the cytosolic protein Bax to the mitochondria, where it secondarily forms clusters that appear to be a prerequisite for Cyt.c release. Fluorescence resonance energy transfer imaging further reveals that Bax clustering coincides with the formation of Bax multimers. We conclude that the PTP is not itself a component of the Cyt.c release machinery, but that it acts indirectly by signaling Bax translocation and multimerization.

Cyclophilin D, a Component of the Permeability Transition-Pore, Is an Apoptosis Repressor

The permeability transition (PT)-pore is an important proapoptotic protein complex in mitochondria. Although it is activated by many signals for apoptosis induction, the role of its various subunits in cell death induction has remained largely unknown. We found that of its components, only the voltage-dependent anion channel in the outer mitochondrial membrane and the adenine nucleotide translocator-1 (ANT-1), a PT-pore subunit of the inner membrane, are apoptosis inducers. We also report that ANT-1’s direct interactor, cyclophilin D, can specifically repress ANT-1-induced apoptosis. In addition, cotransfection experiments revealed that for a diverse range of apoptosis inducers, cyclophilin D shows the same repression profile as the compound bongkrekic acid, a specific inhibitor of the PT-pore. This activity seems to be independent of its chaperone activity, the only known function of cyclophilin D to date. Importantly, cyclophilin D is specifically up-regulated in human tumors of the breast, ovary, and uterus, suggesting that inhibition of the PT-pore via up-regulation of cyclophilin D plays a role in tumorigenesis.

The art and design of genetic screens: mammalian culture cells

Given the wealth of sequence information from the Human Genome Project, many open reading frames urgently require assignment of function. Whereas genetic model organisms, such as yeast, Drosophila and Caenorhabditis elegans, have successfully been used in genetic screens for a long time, mammalian culture cells have only recently emerged as a suitable screening system to elucidate gene function. Diverse cellular activities, such as apoptosis, senescence, tissue-specific differentiation and oncogenic transformation, can be studied in cell culture. There is a plethora of functional assays that can provide a link between genes and physiology. The number of genetic elements to be tested necessitates the use of miniaturization strategies or robotic instrumentation for effective screens that use mammalian cell lines.

Spike, a novel BH3-only protein, regulates apoptosis at the endoplasmic reticulum

We have isolated Spike, a novel and evolutionary conserved BH3‐only protein. BH3‐only proteins constitute a family of apoptosis inducers that mediate proapoptotic signals. In contrast to most proteins of this family, Spike was not found to be associated with mitochondria. Furthermore, unlike the known BH3‐only proteins, Spike could not interact with all tested Bcl‐2 family members, despite its BH3 domain being necessary for cell killing. Our findings indicate that Spike is localized to the endoplasmic reticulum. The endoplasmic reticulum is an organelle that has only recently been implicated in regulation of apoptosis. At this locale, Spike interacts with Bap31, an adaptor protein for pro‐caspase‐8 and Bcl‐XL. In doing so, Spike is able to inhibit the formation of a complex between Bap31 and the antiapoptotic Bcl‐XL protein. Furthermore, Spike transmits the signal of specific death receptors. Its down‐regulation in certain tumors suggests that Spike may also play a role in tumorigenesis. Our findings add new insight for how BH3‐only and antiapoptotic Bcl‐2 proteins regulate cell death.

The metastasis suppressor gene C33/CD82/KAI1 induces apoptosis through reactive oxygen intermediates

Here we describe the isolation of C33/CD82/KAI1 in a screen for apoptosis‐inducing genes. C33 is a gene that is downregulated in many metastatic tumor cells and the expression of which can attenuate the process of metastases formation in a variety of tumors. In accordance, we observed cell death induction by C33 in many different cell types. C33 seems to promote cell death by the generation of reactive oxygen intermediates (ROIs). These ROIs, however, are not derived from the mitochondrial respiratory chain as in most other scenarios leading to apoptosis. We observed that C33 renders cells sensitive to ROIs by causing the specific release of the intracellular antioxidant glutathione (GSH) from cells. Moreover, C33 activates the GTPase Cdc42, which mediates GSH release and apoptosis induction and allows to detect the formation of ROIs.

A high-throughput screen for single gene activities: isolation of apoptosis inducers.

We describe a novel genetic screen that is performed by transfecting every individual clone of an expression library into a separate population of cells in a high-throughput mode. The screen allows one to achieve a hitherto unattained sensitivity in expression cloning which was exploited in a first read-out to clone apoptosis-inducing genes. This led to the isolation of several genes whose proteins induce distinct phenotypes of apoptosis in 293T cells. One of the isolated genes is the tumor suppressor cytochrome b(L) (cybL), a component of the respiratory chain complex II, that diminishes the activity of this complex for apoptosis induction. This gene is more efficient and specific for causing cell death than a drug with the same activity. These results suggest further applications, both of the isolated genes and the screen.