Benedikt Westphalen  

Home Institution
Universität Hamburg, Zentrum fuer molekulare Neurobiologie

Host Institution(s)
University of Pennsylvania, Philadelphia
Department of Physiology
Mentors: Axel Methner, M.D., J. Kevin Foskett, Ph.D.

E-Mail: benedikt.westphalen@zmnh.uni-hamburg.de
Research Topic
see Abstract
Personal Reactions to the U.S. Experience
I had been to the US and to Philadelphia before as a tourist, traveling the Northeastern States with my parents. But this prolonged stay offered so many possibilities to learn, to make new friends and to grow. Philadelphia is a great place to be, not as extreme as New York, easier to discover and therefore it is easier to feel at home. Living and working at and around the University of Pennsylvania (“Penn”) was tremendous and inspiring. I met so many interesting people from all over the world, learned more than I could ever tell and was able to establish valuable contacts for my future.
Greatest Difficulties Encountered
The most difficult part was to obtain the necessary forms to receive the visa. Everything had to happen quite quickly after I received my DS-2019, but the U.S. embassy was booked up for weeks. This resulted in a three-week lag before I was allowed to enter the US. Besides this, everything went pretty well, because “Penn” takes excellent care of exchange students and my mentor in the States was fabulous in organizing things for me. While I was working in the lab we had some serious problems with cells desperately needed for our experiments, because every single batch was already sick when we got them from the companies supplying them. This resulted in some two months of frustrating tissue culture until “our babies” got back on track.
Most humorous incident
There was good laughter basically every other day. One of my roomates, for example, used to clear his throat quite noisily while being in the bathroom.
When another roomate moved into the house and heard him doing his “daily routine,” she was absolutely sure that he was encountering serious medical problems. She ran down the stairs, asking if he needed immediate help, or if she should call 911. After that day he was unofficially named “Moby Dick,” for his famous “bathroom sounds”.
Helpful Hints for Future Students
  • Try to do all your paperwork as soon as possible and force your host institution to do so as well, because everything depends on your DS-2019.
  • Most universities have excellent programs for foreign students. Be sure to join as many as possible, since you will visit interesting places, while making new friends simultaneously.
  • Don't put too much pressure on yourself! Work hard but don't forget to live as well, since your time in the US is precious not just in terms of your research!

  table of contents

 

Abstract on Research Topic
Effect of Bax Inhibitor-1 on Cellular Calcium Homeostasis
Authors:
*Benedikt Westphalen, #Axel Methner, *J. Kevin FoskettInst

*University of Pennsylvania, School of Medicine, Department of Physiology, Philadelphia Pennsylvania, # Heinrich Heine Universität Düsseldorf, Department of Neurology, Germany

Aim of Study and Background:
Xu and Reed isolated Bax inhibitor-1 (BI-1) by screening clones in yeast for their ability to convey resistance against Bax-induced cell death. It was shown that BI-1 can protect mammalian cells from different apoptotic stimuli [1]. High conservation between species and its functional interchangeability revealed a role of BI-1 as an ancient suppressor of cell death [2, 3]. Recently it was shown that BI-1 regulates an apoptosis pathway linked to endoplasmic reticulum (ER) stress and that BI-1 alters ER Calcium (Ca2+) handling [4]. Our group was able to show that the antiapoptotic properties of BI-1 depend on its ability to decrease agonist induced Ca2+ release from the ER by lowering the Ca2+ content of the ER [5].
In order to gain more insight into the function of BI-1 in terms of cellular calcium handling we wanted to conduct experiments two clarify which of the following two main hypotheses holds true:
a) Bax Inhibitor is an ion channel itself [1, 6] or contributes to the formation of a new ion channel.
b) Bax Inhibitor functionally interacts with the inositol 1,4,5-trisphosphate receptors (IP3R) or sarcoplasmic/endoplasmic reticulum calcium ATPase (Serca) and alters their functions similar to Bcl-2[7-10].

Materials and Methods:
Co-immunopreciptiation of BI-1 and IP3R or Serca: Overexpression of BI-1 and either IP3R or Serca in Sf9 insect and mammalian cells followed by co-immunoprecipitation.
Overexpression of BI-1 in Sf9 insect cells and nuclear patch clamping: Sf9 cells were transfected with BI-1 or infected with a bacculovirus producing BI-1 and used in patch clamp experiments after nuclei isolation. In these experiments different pipette solutions (with or without IP3) were used to see possible effects on the IP3R itself or on the ER membrane.
Overexpression of BI-1 in DT40 cells: We transiently and stably overexpressed BI-1 EGFP in wild type DT40 and DT40 IP3R knock out cell lines. These cells were used for single cell calcium imaging to address the question if the function of BI-1 is dependent on the IP3R and to gain more insight into the effects of BI-1 on intracellular calcium handling. The same cells were used for nuclear patch clamping experiments as described above.

Results and Conclusion:
BI-1 did not form any new channel in the ER-membrane after transient transfection or viral infection in SF9 insect cells or stable overexpression in DT40 cells. Preliminary results indicate that transient overexpression and viral infection of cells with BI-1 increases the sensitivity of IP3R to (suboptimal doses) of IP3. Furthermore it was possible to coimmunoprecipitate the Type 1 IP3R using an antibody against a V5 tag fused to BI-1. These preliminary results indicate that BI-1 might influence the intracellular calcium homeostasis due to interaction with IP3R.

References:
1. Xu, Q. and J.C. Reed, Bax inhibitor-1, a mammalian apoptosis suppressor identified by functional screening in yeast. Mol Cell, 1998. 1(3): p. 337-46.
2. Chae, H.J., et al., Evolutionarily conserved cytoprotection provided by Bax Inhibitor-1 homologs from animals, plants, and yeast. Gene, 2003. 323: p. 101-13.
3. Huckelhoven, R., BAX Inhibitor-1, an ancient cell death suppressor in animals and plants with prokaryotic relatives. Apoptosis, 2004. 9(3): p. 299-307.
4. Chae, H.J., et al., BI-1 regulates an apoptosis pathway linked to endoplasmic reticulum stress. Mol Cell, 2004. 15(3): p. 355-66.
5. Westphalen, B.C., et al., BI-1 protects cells from oxygen glucose deprivation by reducing the calcium content of the endoplasmic reticulum. Cell Death Differ, 2005. 12(3): p. 304-6.
6. Bolduc, N. and L.F. Brisson, Antisense down regulation of NtBI-1 in tobacco BY-2 cells induces accelerated cell death upon carbon starvation. FEBS Lett, 2002. 532(1-2): p. 111-4.
7. Erin, N. and M.L. Billingsley, Domoic acid enhances Bcl-2-calcineurin-inositol-1,4,5-trisphosphate receptor interactions and delayed neuronal death in rat brain slices. Brain Res, 2004. 1014(1-2): p. 45-52.
8. Chen, R., et al., Bcl-2 functionally interacts with inositol 1,4,5-trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5-trisphosphate. J Cell Biol, 2004. 166(2): p. 193-203.
9. Dremina, E.S., et al., Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA). Biochem J, 2004. 383(Pt 2): p. 361-70.
10. White, C., et al., The endoplasmic reticulum gateway to apoptosis by Bcl-X(L) modulation of the InsP3R. Nat Cell Biol, 2005. 7(10): p. 1021-8.