Stem cells and apoptosis

Mitochondrial proteins involved in apoptosis

    - Functional characterization of PSAP/Mtch1 (Presenilin 1-associated protein/mitochondrial carrier homolog 1)

    - Novel interactions in Bcl-2 family members mediated by transmembrane domains

 

PSAP/Mtch1

We are attempting to characterize a mitochondrial protein with a so far elusive function. Presenilin 1-associated protein (PSAP) was first identified by the group of Xu and coworkers (University of Tennessee) in a two-hybrid screening designed to identify presenilin 1 interacting proteins. Presenilin is part of the gamma secretase complex, involved in Alzheimer´s disease through processing of the amyloid beta precursor protein. PSAP is also known as mitochondrial carrier homolog 1 (mtch1), since it contains a domain conserved in carriers of the inner mitochondrial membrane, although it is located in the outer membrane. The only activity assigned to mtch1 is its capacity to induce apoptotic cell death when expressed in cultured cells. We have reported that mtch1 has two proapoptotic isoforms generated by alternative splicing, which are targeted to the mitochondrial outer membrane by means of several internal targeting signals. Both isoforms contain two proapoptotic domains. Each of these domains is capable of inducing apoptotic cell death when targeted to the outer mitochondrial membrane by fusion to the carboxyl terminal transmembrane domain of Bcl-XL (which is involved both in outer membrane targeting and insertion). PSAP can induce apoptotic cell death in the absence of Bax and Bak, proapoptotic members of the Bcl-2 family of proteins.

We are characterizing self-interactions in PSAP by a combination of cross-linking and blue-native electrophoresis assays. We are also dissecting the function of different PSAP regions. Furthermore, we are analyzing the effect of knocking out PSAP in Drosophila, in the whole organism as well as in cultured fly cells.

 

Bcl-2 proteins

While using the Bcl-XL carboxyl terminal domain to target mtch1 fragments to mitochondria and analyzing protein interactions we discovered that this domain mediates self-interactions in Bcl-XL. Several experiments suggested that the domain GXXXXG, within the transmembrane helix, is mediating this interaction. We are studying the role of this domain in self-interactions as well as in interaction with other proteins involved in apoptosis or other mitochondrial activities.

 

Scheme of ongoing apoptosis-related research at the lab

 

Identification of bioactive molecules that induce specific stem cell apoptosis or directed differentiation by functional screening of chemical libraries

We are using chemical libraries and functional screenings to identify molecules that induce specific death in stem cells versus differentiated cells. We are also interested in compounds that induce cardiomyogenesis or neurogenesis. We use genetically modified mouse stem cells, expressing EGFP under the control of tissue-specific promoters. We also use human embryonic and induced pluripotent stem cells to confirm the results obtained with mouse cells. Compounds that induce specific stem cell death could be used to eliminate undifferentiated cells to prevent teratoma formation during stem cell therapy. Compounds that specifically kill differentiated cells could be used to isolate stem cells from tissues.

 

Approach for the identification of compounds with specific cell death activity on stem cells vs differentiated cells

 

 

Automated multiparametric analyses of multidimensional microscopy data

We are trying to develop software for supercomputing multiparametric analyses of multidimensional microscopy data. We use a fully automated Leica DMI6000B microscope with a cell culture chamber, structured illumination for semiconfocality and software Metamorph. We plan to use this approach to study different modes of cell death and also differentiation of stem cells.

 

The following video shows HeLa cells stained with Hoechst 33342 (blue nuclei) and Mitotracker Red  CMX ROS (red mitochondria) overlapped with bright field (Normarsky). Cells are dying by necrosis. Release of cytosolic content is evident as growing bubbles around the cells, whereas the nuclei keep their shapes. 400X.

 

The following video shows HeLa cells stained with Hoechst 33342 (blue nuclei), MitoTracker green (green mitochondria), and Cell Mask (red membranes). 200X.

 

 

 

 

 

The following video  shows HeLa cells dying by apoptosis. They are stained with Hoechst 33342 and MitoTracker green, and the color images ovarlapped with bright field images. 200X.

 

 

 

 

 

 

 

 

 

The following videos show movement of cytosolic component in HeLa cells. Movement of filopodia around the cells can also be observed. 630X.

 

 

 

 

 

The following video shows... well... just watch and listen.