Department of Biology

Indiana University

Jordan Hall 142, 1001 E 3rd St.

Bloomington IN 47405-7005

Dear Dr. Brun and members of the faculty search committee --

I am pleased to write in support of DR. KE HU, a former graduate student in my group whom I understand has applied for a faculty position in your department. The basic point of this letter can be stated simply: Ke is an extremely talented cell biologistŠ an outstanding microscopist, and probably the most motivated and productive student that I have ever had. Although her application is perhaps slightly premature (she is now a 2nd year post-doc with Clare Waterman-Storer), I think that Ke would be an excellent fit with your department, and have therefore encouraged her to apply.

I have taken the liberty of providing you with a rather lengthy description of Ke's graduate work, for several reasons. First, because she left Penn relatively recently, and John Murray and I may therefore be in the best position to describe her abilities. Second, because Ke is proposing to continue working on apicomplexan parasites (focusing on Plasmodium, rather than the Toxoplasma parasites that she studied as a graduate student). And third, because it is important that you understand Ke as a person. I am confident that she will be a fantastic addition to any department that values imaginative science.

My laboratory studies intracellular protozoan parasites in the phylum apicomplexa, including Plasmodium (the causative agent of malaria) and Toxoplasma (notorious as a congenital pathogen, an opportunistic infection associated with AIDS, and more recently as a class B biodefense agent). We work on these organisms both because of their medical importance, and for the window they provide into eukaryotic organelles, processes, and evolution. Ke joined my group in 1999, as she was interested in using molecular techniques to explore basic cell biological problems.

As a rotation student, Ke set out to develop RNAi-based inhibition strategies for Toxoplasma. Although now a hot topic, RNAi was new at the time, and offered us great hope for regulating gene expression. Unfortunately, this project was completely unsuccessful, but it taught me several things about Ke. First, it demonstrated her enthusiasm and fearlessness in taking on new techniques. Second, it showed her technical competence and thorough approach to scientific problems: Ke selected three genes expected to yield varying phenotypes (HXGPRT as a native gene whose expression levels could be carefully titrated, tubulin as this had been shown to yield a dramatic phenotype in trypanosomes, and a GFP transgene because large numbers of mutants could easily be screened individually). She targeted all three using three different strategies (synthetic RNA, plasmids using T7 polymerase to drive expression of a hairpin structure, and plasmids containing two T7 promoters flanking the target sequence). These studies were completed rapidly, and were so thorough that Ke convinced everyone in the lab that Toxoplasma is not amenable to RNAi. Several other labs have since spent a great deal of time on the project, without success. Finally, this project clearly demonstrated Ke's insight: she instinctively recognizes not only what experiments to do next, but also when to drop a project -- a rare and valuable skill.

For her thesis research, Ke chose to study the dynamics of cytoskeletal assembly in Toxoplasma, a project that is interesting for several reasons. These parasites contain a remarkable diversity of morphologically, pharmacologically, and functionally distinct cytoskeletal elements -- including at least five distinct tubulin-based structures -- but these are constructed from a limited number of cytoskeletal proteins, which we can identify from the genome sequences now available. Parasite replication proceeds by assembling daughters on a cytoskeletal scaffolding, yet we knew little of the mechanisms involved. Motility is critical for host cell invasion, but remains poorly understood (the parasites possess no cilia, no flagella, no conventional myosins, and contain little F-actin). We had previously made extensive use of fluorescent protein reporters to study protein trafficking and organellar replication in living cells, but Ke was the first in my lab to recognize their potential for *quantitative* analysis of the cytoskeleton.

I will not dwell on Ke's results at any great length, as her publication record speaks for itself. A brief synopsis of Ke's accomplishments as a graduate student includes:

- Fluorescent labeling of the microtubule, microfilament, and actin-based cytoskeleton in transgenic T. gondii parasites, and visualization by time-lapse microcinematography.

- Development and implementation of quantitative methods for objective evaluation of wide-field vs. confocal imaging techniques.

- Demonstration that subpellicular microtubules grow from the apical to basal end of the parasite during daughter cell assembly (through fluorescence photobleaching studies).

- Quantitative analysis of tubulin content associated with various parasite cytoskeletal components.

- Showed that replication by endodyogeny in Toxoplasma and schizogony in Plasmodium are fundamentally related processes.

- Developed a fluorescently-tagged histone 2b marker, for quantitative studies of nuclear DNA content through the cell cycle.

- Single cell cloning of viable intracellular parasites by laser-capture microdissection, demonstrating that control of replicative strategies (endodyogeny/endopolyogeny/schzogony) is epigenetic.

- Identification of the conoid as comprised of a novel arrangement of tubulin protofilaments (thus resolving a long-standing controversy).

- Identification and partial characterization of previously unknown tubulin genes in T. gondii.

- Partial purification of the conoid, for proteomic analysis.

In addition to the publications that have already appeared, two manuscripts are now under review. In the first, Ke utilized a YFP fusion with histone 2b as a quantitative marker of DNA content to follow the unusual mitotic replicative mechanism of apicomplexan parasites. In the second (on which Ke is second author), we use a variety of markers to follow the complete process of organellar replication during endodyogeny. Two further manuscripts are in preparation, describing the identification and characterization of three alpha and three beta tubulins in T. gondii, and the proteomic characterization of the conoid.

In selecting a postdoctoral position, Ke wanted to work in a more 'mainstream' cell biology lab, and was particularly keen to expand her technical expertise. Given her impressive publication record and strong communication skills, she had her choice of post-doctoral opportunities, Ke

chose to work on focal adhesion plaques in the Waterman-Storer lab at Scripps, where she has settled in with characteristic rapidity, as I trust you will hear from Clare.

No letter of reference for Ke would be complete without recognizing her commitment to education. While at PENN, Ke was the teaching assistant for several undergraduate introductory and advanced courses including Biology 122 (S '00). Biology 122 is an introductory course for students (mostly pre-medical) who have advanced credit in biology. This course studies whole organism biology from an experimental point of view thus, required more thinking from the students. As the instructor for Biology 122, I can attest that Ke was an excellent TA and discussion facilitator. I would expect Ke to bring the same level of enthusiasm and excitement to all courses she teaches.

In closing, I hope it is clear that I think very highly of Dr. Hu. She is fully conversant with molecular genetic and microscopic techniques, a terrific experimentalist, and a stimulating colleague. In re-reading the above, however, I am afraid that this letter may not fairly capture the essence of Ke. The key thing is that she really thinks ... and thinks differently from others. If you are looking for a technically competent drone -- a cog in the machine -- then she is not for you. But if independent thinking is a characteristic that you value in your department, you could not do better than to take Ke on!

Please feel free to contact me directly, if there is any further information that I can provide.

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David S. Roos, Ph.D.

Merriam Professor of Biology

Director, Genomics Institute

Ellison Fdn. Sr. Scholar in Global Infectious Diseases

305 Goddard Laboratories, University of Pennsylvania

Philadelphia, Pennsylvania 19104-6018 USA