CHMP5 was originally co-purified with the cytosolic NF-B:IB complex from rabbit lung tissue extracts by Dr. Sankar Ghosh when he purified the NF-B complex in Dr. David Baltimore's lab.

Despite enormous efforts, we could not find any direct involvement of CHMP5 in NF-B signal transduction. Later on, CHMP5 was found to be homologous to the yeast Vps60/Mos10 gene and members of the ESCRT-III complex. We therefore, changed our direction to analyze the role of CHMP5 in endocytic pathway from its role in NF-B signal transduction. To analyze the biological role of CHMP5 in endocytosis, we have deleted the CHMP5 gene in mice, with the resulting phenotype being early embryonic lethality, reflecting defective late endosome function and dysregulation of signal transduction.

These mutant phenotypes are different from those of NF-B component knock out mice (i.e., p65, IKKß, IKK/NEMO) that exhibit increased cell death of hepatocytes in the mutant embryos around E12 (embryonic day 12), implying genetically that CHMP5 is not involved in NF-B signal transduction. In the context of endocytosis, Chmp5-/- cells exhibit enlarged late endosomal compartments containing abundant internal vesicles expressing both late endosomal and lysosomal proteins. This is in contrast to Hrs-deficient animal cells, or ESCRT-III mutants in yeast, which are defective in multivesicular body (MVB) formation. The degradative capacity of Chmp5-/- cells was reduced, with undigested proteins accumulating in the enlarged MVBs that failed to fuse efficiently with lysosomes. Therefore CHMP5 regulates late endosome function downstream of MVB formation and the activity of ESCRT-III complex and CHMP5 deficiency leads to enhanced signal transduction by inhibiting the downregulation of internalized receptors.

We, recently, found that CHMP5 exists in a protein complex and is connected to other CHMP family of proteins through a coherent protein network. This prompted us to test whether the CHMP5 complex can interact with NF-B signaling components.

Indeed, one of the CHMP5 complex components interacts with IBß. Thus, we are back to investigate the role of the CHMP5 complex in NF-B signal transduction and we are on the process of generating Chmp5 conditional knock-out mice to circumvent embryonic lethality by CHMP5 deletion during embryonic development.

- Could you please tell us the main difficulties you had in the laboratory work and how you overcame them?

When I began this project, I coped with two main difficulties. First, CHMP5 is not involved in NF-B signal transduction, but in late endocytic trafficking, which is well known to be a topic of the core cell biology. Finally, Chmp5 mutant embryos die at early embryonic stage, which prevents us to generate and culture Chmp5-/- embryonic fibroblasts (MEFs).

With these difficulties, I first decided to analyze the role of CHMP5 in mouse embryonic development. In the condition of collaboration, I went to Dr. Brigid Hogan's lab (a pioneer of mouse embryonic development) at Vanderbilt University, Tennessee and stayed for 6 weeks to learn experimental skills and analyze defects of Chmp5 embryos. With a huge help with Dr. Bettina Wilm, a postdoctoral fellow, I found that Chmp5 mutant embryos exhibit a defect of ventral folding morphogenesis, which is similar to the mutant phenotype of embryos lacking Hrs, a mammalian endocytic protein (Komada and Soranio, Genes and Dev., 1999).

However, I could not further investigate the mutant phenotypes of Chmp5 embryos at Yale, because our lab was not capable of researching mouse embryonic development due to deficient experimental systems. Therefore, I have switched my focus on analyzing the role of CHMP5 in endocytosis.

Since I should learn most of the experimental skills for cell biology and the experimental system has not been well established to research cell biology in our lab, we collaborated with a few of cell biology experts, including Dr. Ira Mellman and Dr. Marc Pyaert, Yale and Dr. Tamotsu Yoshimori, Japan. After two year of struggling with experiments, we were able to finish our paper with the help of our collaborators in mouse embryonic development and cell biology and with incessant support from Sankar.

However, we had our paper rejected in Nature and Nature Cell biology, and it took one and half years to get our paper accepted after three rounds of revision in Journal of Cell Biology.

2. Please introduce your laboratory, university or organization to bio researchers in Korea.

Yale University is located in New Haven, Connecticut (east coast area) and is close to New York City and Boston (It takes 2-3 hours by car).

My PI, Dr. Sankar Ghosh is a pioneer scientist who originally purified the cytosolic NF-B:IB complex (NF-B and IBß) in Dr. David Baltimore's lab.

Research in our laboratory is focused on understanding how engagement of receptors of both the innate and adaptive immune system lead to the activation of appropriate cellular responses through the inducible transcription factor, NF-B. NF-B plays a critical role in regulating the expression of a large number of genes involved in immune, inflammatory and apoptotic processes. NF-B can be activated by different stimuli such as microbial products, proinflammatory cytokines, T and B cell mitogens and physical and chemical stresses. NF-B in turn regulates the inducible expression of many cytokines, chemokines, adhesion molecules, acute phase proteins and anti-microbial peptides. Therefore NF-B plays a central, evolutionarily conserved role in coordinating immune and inflammatory responses.

In unstimulated cells, NF-B is retained in the cytoplasm through its interaction with the inhibitory IB proteins. Stimulation of cells with different inducers leads to the phosphorylation and subsequent degradation of the IB proteins. Upon degradation of IB, the free NF-B enters the nucleus, however translocation of NF-B to the nucleus is, in itself, not sufficient to drive transcription of target genes. Instead, specific phosphorylation of one of the NF-B subunits, p65/RelA, is required for both efficient DNA-binding and transcriptional activity of the nuclear NF-B.

We wish to understand the mechanisms that operate in the signal transduction pathways that lead to NF-B activation, as well as the regulatory mechanisms that control the activity of NF-B in the nucleus. Specific projects that are underway at present are listed below.

1. Understanding the mechanism by which signals from Toll/IL-1 receptors, TNF receptor and the T-cell receptor lead to NF-B activation.
2. Characterizing the mechanism by which the transcriptional activity of nuclear NF-B is regulated.
3. Exploring the dysregulation of NF-B activity in diseases such as arthritis and cancer.
4. Understanding the biology of novel Toll-like receptors in response to infection.

(http://info.med.yale.edu/immuno/fac_ghosh.html)

3. Please tell us your experiences and your thoughts related to research activities abroad.

Most of Korean researchers in Yale, including postdoctoral fellows and graduate students are doing well with their research and study, and they are also productive in publication. However, most of time, we feel isolated from other people and frustrated with your research by lacking communication and expression problem in English, especially when we really need some help from lab members and other department members. Although you are very busy with working and studying in the lab, if you take some time to prepare for English, I am sure that you will enjoy researching and living in U.S.

4. Could you provide some advice for scientists who have plans to study in your research field?

From this paper, I have learned a valuable lesson. When you choose a project in the new lab, you would better pick up the project that is related to your PI's expertise and you can easily work on in the lab.

5. Your Plans or wishes

I would like to pursue my research in the combined field of cell biology and immunology for my post-doctoral fellowship, and then apply cell biology techniques to the NF-B signal transduction in immune systems.

6. Do you have anything else that you'd like to tell Korean scientist?

In my opinion, science is not easy to work on, because we should sacrifice too many things in our life and we are stuck in the laboratory day and night to prove our hypothesis. In addition, we are always frustrated with frequent failures and routine repetition of our experiments. However, I believe that these are worth to invent our time, if we are the person who finds happiness from searching for the truth in science at all costs and we are the person who can forget everything around us while we are absorbed in making hypothesis, performing experiments, and proving our hypothesis.

I hope that you have an opportunity to study abroad as a postdoctoral follow or as a graduate student, because it is very helpful to make my mind and eyes wide on science and world. Last of all, I would like to thank my wife for her continuous support, endurance and understanding of the frustrations and excitements intrinsic to the process of scientific discovery.