Sample NSF Fellowship Proposal Essays

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Nov 7, 2002 ... Sample NSF Fellowship Proposal Essays. In this document are essays from biology graduate students who received NSF fellowships.
Sample NSF Fellowship Proposal Essays In this document are essays from biology graduate students who received NSF fellowships. We thank the following students who agreed to share their essays. Indicated below are their names, entering class, advisor, and if they applied for their fellowship while an MIT graduate student. Lourdes Aleman (2001, Sharp)

Applied as a 2nd year MIT graduate student

Calvin Jan (2004, Bartel)

Applied as a 1st year MIT graduate student

Keren Hilgendorf (2007, Lees)

Applied as a 1st year MIT graduate student

Laralynne Przbyla (2006, Voldman) Jennifer Ricks (2008, Hemann) Jeremy Rock (2006, Amon) Jason Sheltzer (2008, Amon)

Applied as a 1st year MIT graduate student

Kevin Wang (2003, Baker)

Applied as a 1st year MIT graduate student

Matthew Wohlever (2008, Sauer/Baker) Joshua Wolf (2004, Fink)

Applied as a 1st year MIT graduate student

Lourdes M. Alemán

In choosing graduate school programs, I found the MIT Biology Department’s commitment to its students’ education and progress extremely appealing. I wanted my educational journey while at graduate school to be an active learning process, and MIT’s emphasis on teaching and training matched my expectations exceptionally well. Another aspect of MIT’s Biology graduate program that was very attractive to me was the collaborative nature of the graduate student community and the personal relationships that I saw between graduate students and faculty. MIT would not only be able to provide me with a broad range of research experiences and excellent facilities, but also with a research community that would support and guide my graduate school career. Coming to graduate school, I knew I wanted to study basic processes governing cell function and development in a genetically tractable organism. I also wanted to work under the guidance of an advisor who took a primary interest in my scientific development. In the Solomon lab at MIT, I have been able to find exactly that. Our laboratory is primarily interested in the regulation of microtubule assembly. We are using the budding yeast S. cerevisiae as an experimental system to study microtubules because of its accessibility to biochemistry as well as classical and molecular genetics. Research Proposal Microtubules play an essential role in many cellular functions including cell division, chromosome segregation, cell growth, and cell movement. Perturbations of microtubules can have deleterious consequences to the cell. Incorrect partitioning of DNA during mitosis or meiosis due to defects in microtubules, for example, causes aneuploidy, which can lead to abnormal cell growth and development. Proper microtubule morphogenesis is dependent on proper expression, folding, and stable association of the alpha and beta tubulin monomers. Regulation of microtubule assembly can occur at any step along this pathway. Maintaining comparable levels of α- and β-tubulin monomers, for example, is crucial for normal microtubule activity and cell survival. A large excess of α-tubulin has only modest consequences for S. cerevisiae; conversely excess levels of β-tubulin cannot only affect microtubule assembly, but also cause lethality (1). We are using β-tubulin associated lethality as a probe to identify genes involved in early steps of microtubule morphogenesis in S. cerevisiae. Our laboratory has identified a gene in S. cerevisiae, PLP1, whose absence rescues the lethality associated with βtubulin overexpression (2). PLP1 could be contributing to β-tubulin lethality by altering α- to β-tubulin ratios or by rendering the excess β-tubulin non-toxic. The first possibility was ruled out because deletion of plp1 does not cause a decrease in the ratio of β to α-tubulin since tubulin polypeptide levels are completely normal in plp1Δ cells. Instead PLP1 appears to contribute to the production of the toxic form of β-tubulin. In plp1 deleted cells, a substantial portion of tubulin is found in a large aggregate. Aggregated β-tubulin in plp1Δ cells cannot bind its partner α-tubulin, which suggests that the ability of plp1Δ to rescue cells with higher levels of β-tubulin than α-tubulin is likely due to a misfolding of β-tubulin. Genetic data from our laboratory also suggests that PLP1 acts at an early step in β-tubulin folding. Consistent with this idea is that a significant portion of Plp1 is found associated with polysomes (2). To further elucidate the role of PLP1 in the proper folding of β-tubulin I propose to: (1) identify interactors of Plp1 and (2) test whether PLP1 functions specifically in β-tubulin folding. Identifying Interactors of Plp1 The PLP1 gene codes for a 27 kDa protein. Although a substantial fraction of Plp1 associates with polysomes, more than 50% of Plp1 is found in a large complex that is several hundred thousand daltons in size (2). To gain further insight into Plp1’s role and regulation, I propose to identify proteins that interact with Plp1 and are components of this same complex. I will modify the PLP1 chromosomal allele to encode a tagged Plp1 protein using a tandem affinity purification (TAP) tag. The TAP method utilizes two affinity tags spaced by a cleavage site of tobacco etch virus (TEV) proteinase. Compared with more traditional tags used for immunoprecipitation, the TAP method gives better yields of affinity purified proteins, along with lower background of none-specifically associated proteins (3). I will determine whether this fusion protein is functional by testing whether the strain containing tagged Plp1 is sensitive to overexpression of β-tubulin under an inducible galactose promoter just like strains containing untagged wildtype Plp1 are. To identify Plp1-associated proteins, extracts of cells expressing TAP tagged Plp1 will be subject to precipitation by the TAP method. Although there are several versions of the TAP tags, the one I will use consists of two IgG binding domains, Staphylococcus aureus protein A (Prot A) and a calmodulin binding peptide (CBP), separated by a TEV protease cleavage. This tag has been previously used for the successful tagging and precipitation of small proteins (down to 9 kDa in size) and of proteins present at low concentrations (4). I will purify the candidate Plp1-associated proteins on a series of affinity columns based on established procedures (4). The identified proteins will be digested with a mixture of proteases and the resulting peptides will be fractionated in two dimensions on a strong cation exchange resin followed by a reverse-phase resin prior to sequence analysis by mass spectrometry (5). Mass spectrometry facilities are available at my academic institution. This novel method differs from more conventional techniques for determination of protein identity by mass spectrometry as it eliminates the need to identify proteins on SDS-polyacrymalide gels stained with silver, prior to sequence analysis. Consequently, this method is more sensitive for proteins found in substoichiometric amounts and for identifying proteins that stain poorly with silver or

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Lourdes M. Alemán

comigrate with subunits of the complex under investigation (5). To rigorously validate the inferred composition of the putative Plp1 complex, I will create TAP tagged versions of them as previously done with Plp1, and perform another round of purification and identification by mass spectrometry. In addition I will perform co-immunoprecipitation experiments with Plp1 and the identified proteins to further confirm that they physically interact. To study which of the proteins that I identify also associate with polysomes, cells will be fractionated using a sucrose gradient and the extent of co-fractionation with Rpl3, an integral ribosomal component, determined as previously done with Plp1 (2). One possible result is that all of the components of this putative complex associate with polysomes, which would indicate that the Plp1 complex as a whole interacts with polysomes. Another possible outcome of this experiment is that only a subset of the proteins that associate with Plp1 also interact with polysomes. This would suggest that Plp1 probably exists in multiple complexes, some associated with polysomes. A third possible result would be that none of the components associated with Plp1 also associate with the polysomes, in which case it would suggest that Plp1 has multiple associations corresponding to multiple functions. To probe the role of different components of the Plp1 complex in tubulin folding, strains containing deletions for each component will be made. First I will ask whether the genes that code for these components when deleted phenocopy the plp1 deletion. In other words, I will determine if deletion of these genes rescues cells that are either overexpressing β-tubulin under an inducible galactose promoter or are sensitive to modest excess of β-tubulin due to their genetic background. If none of the proteins forming this complex with Plp1 phenocopy the plp1 deletion, then it is possible that to see such effects, it might be necessary to delete more than one at a time, which would indicate that deletion of each gene by itself is not enough to rescue βtubulin associated lethality. If some or all of the components are involved in rescuing cells that are overexpressing β-tubulin, then this would indicate that Plp1 as well as the new components identified are also necessary for proper folding of β-tubulin in the cell. It is also important to note that some if not all of the components might be known genes, in which case information about their function will be already available. The proposed experiments outlined in this section form part of the major effort in the lab to understand early steps in microtubule morphogenesis. However, I formulated these experiments independently and they constitute my own project. Specificity of PLP1 in β-tubulin folding Plp1 function is relatively specific for β-tubulin since deletion of PLP1 has no apparent effects on α-tubulin folding (2). It is known that the GimC/prefoldin complex and the cytosolic chaperonin TriC are important for tubulin folding (2). Genetic studies in our lab determined that PLP1 acts at a step upstream of the GimC/prefoldin complex. Consistent with that ordering, Plp1 is associated with polysomes. Based on these findings, one hypothesis for Plp1 function is that it specifically facilitates the transfer of nascent β-tubulin polypeptides to the folding apparatus. To test this hypothesis, I will determine whether the ratio of Plp1 to a ribosomal component such as Rpl3, that is the relative amount of Plp1, increases when polysomes are further fractionated by immunoprecipitation with an anti-β-tubulin amino terminus antibody. As controls, I will use two other antibodies: one directed against the amino terminus of α-tubulin and one directed against the amino terminus of Cpy, a carboxy peptidase which we know is not affected by Plp1 levels. If Plp1 acts as a mediator between the transcription machinery and the folding apparatus specifically for β-tubulin relative to these other proteins, then its relative amount should be enriched in ribosomes that are associated with β-tubulin mRNA. If there is no enrichment of Plp1 in β-tubulin immunoprecipitated lysates vs. controls, then the experiment would suggest that Plp1 might act to affect βtubulin folding in a different manner or that Plp1 is a more general component of polysomes. The proposed experiments outlined in this section were formulated in collaboration with my advisor Frank Solomon and Soni Shimoda, a graduate student in our laboratory. Understanding what proteins interact with Plp1 and the specific function of PLP1 in proper folding of β-tubulin will lead us to a better understanding of how production of toxic β-tubulin is regulated in the cell and therefore how early steps in microtubule morphogenesis affect essential cell functions. 1. Weinstein, B., and Solomon, F. (1990). Phenotypic Consequences of Tubulin Overproduction in Saccharomyces cerevisiae: Differences Between Alpha-Tubulin and Beta-Tubulin. Mol. Cell. Biol. 10:5295-5304. 2. Shimoda, S. and Solomon, F. An Early Step in β-tubulin Folding Affects Tubulin Heterodimer Levels. (MS submitted, Genes and Development). 3. Shevchenko, A., Schaff, D., Roguev, A., Pim Pijnappel, W.W.M., Stewart, A. F., and Shevchenko, A. (2002). Deciphering Protein Complexes and Protein Interaction Networks by Tandem Affinity Purification and Mass Spectrometry. Mol. Cell Proteomics 1:214-212. 4. Puig, O., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm M., and Seraphin, B. (2001). The Tandem Affinity Purification (TAP) Method: A General Procedure of Protein Complex Purification. Methods 24:218-229. 5. Deshaies, R.J., Seol, J.H., McDonald, W.H., Cope G., Lyapina S., Shevchenko, A., Shevchenko, A., Verma, R., and Yates, J.R. (2002). Charting the Protein Complexome in Yeast by Mass Spectrometry. Mol. Cell Proteomics 1:3-10.

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Reference Code: G42456

NSF Graduate Research Fellowships

2003-05344

Nov 7 2002 4:24PM FASTLANE_SUBMITTED

APPLICATION FORM Deadline for filing: November 7, 2002 READ INSTRUCTIONS BEFORE COMPLETING THIS FORM. PLEASE TYPE.

Name: Aleman

Lourdes last

Maria first

middle

Jr,II,etc

15. Describe any personal, professional, or educational experiences or situations that have contributed to your desire to pursue advanced study in science, mathematics, or engineering. See attached response 16. Describe experiences integrating research and education, advancing diversity in science, enhancing scientific and technical understanding, and benefiting society. See attached response

17. Mark the one choice that most appropriately describes your current status: I have not yet completed a baccalaureate degree. I have completed a baccalaureate degree but since then have not completed and am not currently enrolled in any graduate level courses or courses for graduate credit. I am currently enrolled in the first semester/quarter of graduate school and have not yet completed any graduate level courses or courses for graduate credit since receipt of a baccalaureate degree. I have completed some graduate level courses or courses for graduate credit since receipt of a baccalaureate degree, but not more than one year full time or the equivalent. I have completed more than one year of full time graduate study or the equivalent since receipt of a baccalaureate degree but am still eligible for the program. (Respond to Question 18 below.) 18. Applicants who selected the last choice in Question 17 must answer Question 18. Others should not. Explain the circumstances that you believe demonstrate that you are in the early stages of your proposed graduate program. Be sure to read the passage on "Earned Graduate Study" in the Guidelines. No response provided

By submission of this application, I hereby certify that I am a United States citizen or national or a permanent resident alien legally admitted to the United States and that the information I have provided on the Fellowship application is correct to the best of my knowledge.

NSF Form 289, September 2002 Form Approved OMB No. 3145-0023

Question 15 

Aleman,L 

2003-05344 

                            

Question 16 

Aleman,L 

2003-05344 

                                                        





Proposed Plan of Research 

Aleman,L 

2003-05344 



                      αβ αβ β   β βαβ β βαΔ β βΔαΔ βαβ β   β β            β           





Proposed Plan of Research 

Aleman,L 

2003-05344 



               β β  β β β      β βα    β  β α  β β ββ    β β     β        





Previous Research Experience 

Aleman,L 

2003-05344 

                                                                





Previous Research Experience 



Aleman,L 

2003-05344 

                                                      





Hilgendorf, Keren Nature of interaction of pRB, E2F1, and histone modifying enzymes in response to DNA damage Key Words: pRB, E2F1, DNA damage, pro-apoptotic During college, I became increasingly interested in molecular biology, particularly in the mechanism of interaction of proteins during various cell processes. While my primary undergraduate research was more genetic in nature, a summer undergraduate research experience allowed me to explore this area of molecular biology research. Various classes I took during college and during my current graduate education have further solidified this interest. While I won’t start lab rotations till the spring semester of my 1st year as part of the MIT biology graduate program, I am interested in following up on recent unpublished observations made by the Jacqueline Lees Lab regarding the positive role the retinoblastoma protein (pRB) plays in inducing apoptosis in response to DNA damage. Background: pRB belongs to the family of pocket proteins that are involved in cell cycle regulation.(1,2) It interacts with E2F1, a member of the E2F family of transcription factors that plays a key role in activation of genes required for cell cycle progression to S phase.(1,2) During G0/G1 pRB binds E2F1, inhibiting the function of E2F1 by both masking its transactivation domain and by recruiting HDACs (histone deacetylase) to the complex and silencing cell cycle progression genes.(1) Phosphorylation of pRB by cyclin/CDKs results in release of E2F1 and progression to S phase (Fig. 1a).(1) E2F1 also functions in inducing apoptosis in response to DNA damage. The Lees Lab has recently shown that pRB plays a positive role in this DNA damage response: DNA damage results in phosphorylation of pRB and, rather than resulting in release of E2F1, the phospho-pRB/E2F1 complex associates with promoters of known proapoptotic genes and recruits P/CAF (histone acetyltransferase), resulting in induction of apoptosis (unpublished data). The Lees Lab also showed that pRB is concurrently complexed with E2F1, binds HDAC and represses cell cycle progression genes (unpublished data) (Fig. 1b). This proposal focuses on understanding how pRB can mediate these two distinct roles in response to DNA damage. I hypothesize that the nature of the pRB/E2F1 complex differs from that of the phospho-pRB/E2F1 complex and this difference allows the former to recruit HDAC and associate with cell cycle gene promoters while the latter recruits P/CAF and associates with pro-apoptotic gene promoters. Prior observations (by the Lees Lab and others) suggest two mechanisms that could modulate the composition and activity of the pRB/E2F1 complex: (1) DNA damage induces post-transcriptional modifications in the E2F1 and/or pRB proteins; and (2) DNA damage alters the interaction between pRB and E2F1. My initial experiments will focus on testing these non-mutually exclusive hypotheses. Specific Aims: Aim 1: To establish the role of post-transcriptional modifications in the formation of the pro-apoptotic pRB/E2F1 complex. As described above, the Lees Lab has already shown that DNA damage allows phosphorylated pRB to participate in the pro-apoptotic pRB/E2F1 complex. However, the sites of phosphorylation have not been determined yet; thus, it is remains unclear whether the modified sites are the same sites phosphorylated by cyclin/CDKs during normal cell cycle progression or whether novel sites are involved. Furthermore, it is unknown if the phosphorylation of pRB is required for its ability to function in the pro-apoptotic pRB/E2F1 complex. I will address each of these questions: To determine the sites of phosphorylation, I will recover pRB complexes

Hilgendorf, Keren

from proliferating T98G cells that have been treated with or without doxorubicin, a DNA damaging agent. Specifically, I will recover three species: Total pRB – by immunoprecipitating (IPing) with antibodies against pRB; Repressive pRB – by IPing for HDAC, dissociating the complex and then re-IPing for pRB; Activatory pRB – by IPing for P/CAF, dissociating the complex and then re-IPing for pRB. I will then determine the sites of phosphorylation by mass spectrometry (MS). MIT has a proteomics facility and a number of professors (e.g. Forest White) with expertise in mapping phosphorylation sites by MS who can help with these experiments. To determine whether phosphorylation of pRB is required for its ability to function in the proapoptotic pRB/E2F1 complex, I will transfect a pRB mutant lacking the identified phosphorylation site(s) into a pRB-deficient cell line. I will then test the ability of mutant pRB to participate in the pro-apoptotic complex (by IP), its ability to activate target genes (by RTPCR), and its ability to induce apoptosis (by FACS analysis). A pRB mutant already exists in which all the known CDK phosphorylation sites have been mutated. If I identify novel sites by MS, I will generate additional pRB mutants. I also wish to examine the potential role of acetylation in the complex formation, since it has been reported that DNA damage results in acetylation of three lysine residues in E2F1, increasing its apoptotic activity.(3) Since the role of pRB has not been addressed, I will transfect E2F1 mutants in which these sites have been mutated (to either prevent or mimic acetylation) and test the ability of mutant E2F1 to participate in the pro-apoptotic pRB/E2F1 complex. Aim 2. To determine whether DNA damage alters the interaction between pRB and E2F1 to allow formation of the pro-apoptotic pRB/E2F1 complex. In addition to the C-terminal pocket domain with which pRB binds to E2Fs during cell cycle repression, pRB also has an E2F1-specific binding site.(4) Similarly, E2F1 can bind to pRB through two domains, one located in the transactivation domain that is shared with the other E2Fs and one that is unique to E2F1.(4) (Interestingly, the E2F1 residues that are acetylated in response to DNA, map within this second pRB binding domain.) These observations raise the possibility that pRB and E2F1 use these alternate interaction domains to form the transcriptional active, pro-apoptotic pRB/E2F1 complex. I will test this model by transfecting deletion mutants of pRB and E2F1 into pRB-deficient cells and testing their activity in a similar manner to the experiments described above. I will include HA- or Flag-tags in the E2F1 constructs to distinguish from endogenous E2F1. If my experiments confirm that the alternate interaction domains are used, I will determine the ability of this complex to bind P/CAF versus HDAC (by IP). Depending on the outcome of aim 1, I can also assess how phosphorylation and/or acetylation influence the affinity of the specific versus the classic pRB-E2F interaction domains. Conclusion: This study focuses on understanding the difference in the nature of the two pRB/E2F1 complexes formed in response to DNA damage. A logical follow-up is determining the mechanism of differential recruiting of the formed complexes to cell-cycle progression and pro-apoptotic gene promoters. Understanding the molecular basis of the pRB/E2F1 complexes is important, as the differential complexes can affect chemotherapeutic responses. Originality Statement: This research proposal reflects my own work. 1. J. M. Trimarchi, J. A. Lees, Nat Rev Mol Cell Biol 3, 11 (Jan, 2002). 2. J. H. Dannenberg, H. P. te Riele, Results Probl Cell Differ 42, 183 (2006). 3. M. A. Martinez-Balbas, U. M. Bauer, S. J. Nielsen, A. Brehm, T. Kouzarides, Embo J 19, 662 (Feb 15, 2000). 4. L. M. Julian, O. Palander, L. A. Seifried, J. E. Foster, F. A. Dick, Oncogene (Sep 24, 2007).

Jan, Calvin Introduction: Small double stranded RNAs have recently surfaced as key negative regulators of gene expression. They are roughly classified as either small interfering RNAs (siRNAs) or microRNAs (miRNAs) based on their origin. siRNAs are derived from perfectly complementary dsRNAs or extended stem loops, and are processed to form a heterogeneous population of small RNAs. siRNAs are best known for their role in guiding cleavage of perfectly complementary target 1,2 mRNAs in a process known as RNA interference (RNAi) . In contrast, miRNAs are derived from stem loop structures that define a specific small RNA product. miRNAs are better known for their function in development, where they regulate gene expression by either directing cleavage of perfectly complementary target mRNAs, or repressing 3 translation of partially complementary target mRNA . To better understand small RNA effector complexes, several groups have used antibody affinity chromatography to purify microRNA containing ribonucleoprotein complexes (miRNP) and siRNA programmed RNA 4,5 Induced Silencing Complexes (RISC) . Interestingly, analysis of these complexes shows that microRNAs lacking endogenous cleavage targets can perform RNAi on perfectly complementary heterologous/synthetic targets. Purified miRNPs also share a large number of proteins with purified RISC, notably the argonaute proteins. These data taken together imply that there may be one common effector complex, which by convention is referred to as RISC. How RISC is programmed to perform RNAi and/or translation repression is not yet understood. One model is that different RISCs contain different effector proteins that determine the mode of action of that complex. A second model is that all RISCs are of equal functional potential, and that commitment to cleavage or repression is decided upon substrate binding, particularly base pairing and degree of complementation of the target mRNA with the resident small RNA. Looking for differences in heterogeneous RISC populations could potentially test these two models. In mammals, there are four members of the argonaute family2. These argonautes are integral RISC components and presumably associate one per RISC. Microarray analysis of miRNAs associated in complex with the various argonautes shows the 6 miRNAs are unbiased in which argonaute they complex with . However, recent experiments have demonstrated that ago2 provides RISC’s endonucleolytic activity; moreover, it appears that ago2 is the only argonaute capable of directing 7,8 cleavage in mammals . These data favor the first model, where RISC components ultimately decide the functional capacity of a given complex. What then, are the functions of the other argonautes? Many studies characterizing RISC assembly and activity were done in drosophila melanogatster, and while much of the basic biochemistry of RISC is conserved between flies and mammals, there are emerging differences in many aspects of the RNAi pathway. For example, drosophila ago2 is essential for siRNA directed RNAi but is entirely 9 dispensable for cleavage of miRNA targets . In mammals there is only one dicer homolog that processes miRNAs and siRNAs, whereas drosophila has two paralogs that vary subtly in their specificity. While studies in drosophila and plants have provided a wealth of basic RNAi knowledge, it appears that detailed mechanisms may vary between systems. These studies will focus on characterization of mammalian RISC using mammalian cell culture and cell free systems. Specific Aims: 1. To separate cleavage-competent RISC from non-cleavage RISC on the basis of target RNA interactions, and characterize these RISCs biochemically. 2. To evaluate the interactions between RISC, target mRNAs, and known mRNA processing bodies in vivo. An in vitro system for analyzing RISC:mRNA complexes To understand differences between RNAi RISC and translation repression RISC, it is necessary to capture complexes committed to either cleavage or repression. Established purification techniques using antibodies against members of RISC may not be sufficient to make this distinction. A more functionally unbiased approach is to analyze all complexes performing either RNAi or translation repression in vitro. A purification scheme will be used that takes advantage of base pair interactions between RISC and target mRNAs. Briefly, “bait” RNA containing a perfectly or partially complementary miRNA recognition site will be tagged with an MS2 hairpin sequence. This structure is bound by the MS2 phage coat protein, which when fused with maltose binding protein can be affinity purified on an amylose 10 column . Intact complexes can then be eluted in maltose. By this method, RISC programmed with endogenous miRNAs can be purified from cell extracts on the basis of target recognition. Control columns will use nonspecific bait sequences. When using a perfectly complementary column, I will select against RNAi RISC by allowing cleavage to occur on the column, releasing RNAi RISC into solution. This selection can be controlled since RNAi is ATP dependent at multiple steps4. It is unclear if RISC binding to target RNA has an ATP dependent helicase step. This can be tested using both perfect and partially complementary columns depleted for, or with excess ATP. If binding is not ATP dependent, then ATP can be used with a perfectly complementary column to create cleavage permissive conditions. Binding to the RNA column under cleavage conditions does not prove that these complexes perform translation repression. To prove this, eluted complexes must repress translation of a target reporter mRNA in vitro. The reporter RNA can be assayed for cleavage by electrophoresis

Jan, Calvin to determine if the effect on reporter gene expression was due to translation repression or RNAi. Notably, attempts to 3,11 recapitulate translation repression in vitro have been unsuccessful . If an in vitro translation repression assay becomes feasible, then this purification scheme can be used to correlate protein constituents identified by mass spec to functional complexes. Currently however, these techniques can be used to look for differences between cleavage RISC and noncleavage RISC by performing mass spectrometry on cleavage-released complexes and retained complex eluted in maltose. Any factors found specifically in retained complexes may be candidate proteins involved in mediating translation repression. Primary cells from ago2 knockout mice will be used to test if translation repression RISC still functions in vivo when RNAi RISC is impaired. First, miRNA expression patterns in these cells will be identified by small RNA cloning and sequencing, or by microarray. These cells will then be transduced with lentivirus carrying a GFP reporter containing appropriate miRNA binding sites. If translation repression still occurs, it can be inferred that amongst the purified nonRNAi RISCs, there are at least some translation repression RISCs. If ago2 is required for translation repression, then I will proceed to purify ago2 complexes retained on a partially complementary column. These complexes can then be eluted, and ago2 complexes specifically purified by antibody chromatography. These complexes can be compared by mass spec to ago2 complexes that cleaved from the perfectly complementary column. An in vivo system for monitoring localization of miRNP:mRNA complexes 3,11 Thus far attempts to recapitulate translation repression in vitro have been unsuccessful . This may be due to technical challenges, or fundamental problems with the assay. While RNAi has worked in cell free systems, the fact that translation repression failed implies that there may be cellular requirements for repression not present in extracts. In S. cerevisiae there is evidence that mRNAs can enter a translationally dormant state when colocalized to 12 discrete cytoplasmic foci called processing bodies (p-bodies) . In some mammalian cells there is evidence for RNA granule structures resembling p-bodies. An intriguing possibility is that RISC shuttles target mRNAs to p-bodies or other similar structures, where they become translationally silent. P-bodies are rich in exonucleases, which would also facilitate rapid degradation of RISC-cleaved target mRNAs. However, they are also known to contain dormant mRNA that are neither translated nor degraded. These quiescent mRNAs are in a state similar to that expected of miRNArepressed mRNAs. This hypothesis does not necessarily conflict with the existing model that translation repression occurs at a step after translation initiation, since mRNAs may be shuttled to p-bodies after having initiated 12,13 . translation To test if p-bodies play a role in miRNA translation repression, colocalization studies can be done with p-body markers, RISC markers, fluorescent siRNAs, and tagged reporter mRNAs. This experiment would require 4 flours: CFP-dcp2 (p-body marker), YFP argonaute (RISC marker), texas red labeled synthetic let-7 siRNAs, and GFP-MS2bp, which will bind to MS2-tagged reporter mRNA. The experiment will be repeated for each argonaute, as well as a general member of RISC. The reporter will be luciferase to monitor translation repression, and carry MS2 sites and a let-7 miRNA-binding site. The experiments will be done in cells lacking let-7 miRNA expression. All fluorescent markers will be tested individually to ensure no adverse effect on function. All markers will be built stepwise into a stable cell line, and fluorescent let-7 introduced by transfection. These interactions can be monitored in real time by confocal microscopy. Though interaction between RISC, miRNA and target mRNA is expected, colocalization to discrete loci would imply a functional body. Colocalization of these molecules with p-bodies would imply p-bodies have a function in translation repression. Treatments known to dissociate p-bodies can test the importance of this structure for repression by monitoring luciferase expression under dissociating conditions. 1

P. D. Zamore, T. Tuschl, P. A. Sharp, D. P. Bartel, Cell 101, 25 (Apr 31, 2000). G. Meister, T. Tuschl, Nature 431, 343 (Oct 16, 2004). 3 D. P. Bartel, Cell 116, 281 (Feb 23, 2004). 4 G. Hutvagner, P. D. Zamore, Science 297, 2056 (Oct 20, 2002). 5 Z. Mourelatos et al., Genes Dev 16, 720 (Apr 15, 2002). 6 M. A. Carmell, Z. Xuan, M. Q. Zhang, G. J. Hannon, Genes Dev 16, 2733 (Dec 1, 2002). 7 J. Liu et al., Science 305, 1437 (Oct 3, 2004). 8 G. Meister et al., Mol Cell 15, 185 (Aug 23, 2004). 9 K. Okamura, A. Ishizuka, H. Siomi, M. C. Siomi, Genes Dev 18, 1655 (Aug 15, 2004). 10 Zhou Z, Licklider LJ, Gygi SP, Reed R, Nature 419, 182 (Sep 12, 2002). 11 Hannon G, personal communication (2004). 12 Sheth U, Parker R, Science 300, 805 (Jun 2, 2003). 13 P. H. Olsen, V. Ambros, Dev Biol 216, 671 (Dec 15, 1999). 2

Przbyla, Laralynne Studying interactions of Sindbis virus capsid protein using atomic force microscopy Key Words: Sindbis virus, atomic force microscopy, virus capsid, alphavirus, DNA oligomer Introduction Sindbis virus consists of an envelope and a nucleocapsid containing a single RNA strand. Sindbis infects mainly mammals and birds, and is not highly dangerous to humans but is a member of the alphavirus family, which contains many dangerous viruses including several encephalitis viruses (1). The Sindbis nucleocapsid consists of a spherical 240-mer of a capsid protein bound around the RNA. The formation of the virus capsid is highly dependent on the presence and size of the nucleic acid oligomer, based on studies using analytical ultracentrifugation (AUC) and surface plasmon resonance (SPR). Although the wild type virus encloses RNA, the capsid will still assemble with single stranded DNA, which is what will be used in experimental procedures due to its stability and ease of manipulation. The method of initiation and the strength of the reactions between the nucleic acid and the virus capsid proteins will be studied further using atomic force microscopy (AFM), a nanoscale imaging method consisting of a very small silicon tip at the end of a cantilever. A surface map of the interactions can be creating by measuring the magnitude of the deflection of a laser beam shining down onto the cantilever (2). Hypothesis I expect that a single molecule of Sindbis virus capsid protein (SVCP) will bind to single stranded DNA with different interaction energies that are dependent on the DNA sequence, number of bases, and the environment of the oligomer. Also, the binding energies of different engineered mutants of SVCP to a DNA molecule will be dependent on the sequence and size of SVCP. Research plan SVCP will be purified using an expression system and a GST tag. The DNA oligomers will be purchased, made single stranded by boiling and cooling quickly, and biotinylated. DNA oligomers will be attached to an AFM tip that is covered in biotin labeled beads (purchased from Novascon) by incubating the AFM tips with avidin, then taking advantage of the multiple binding sites of avidin by incubating the biotin-avidin tips with biotinylated ssDNA (3). A selection of DNA oligomers based on AUC experiments will be investigated. These include 6-mer, 12-mer, 36-mer, and 48-mer, to see what effect the length of the DNA strand will have on formation of a complete virus capsid. SVCP will be immobilized on a glass slide, and the interaction of these molecules with the DNA oligomers attached to AFM probes will be analyzed. Liquid cell AFM will be used, so the effects of DNA exposure to different chemical environments can be studied. The controls will include using a complimentary DNA strand on the glass slide as a positive control, as it will be expected to interact with the DNA oligomer on the AFM tip. A glass slide with only buffer will be used as a negative control, to set a baseline for nonspecific interactions. Another negative control will allow the capsid protein to interact with ssDNA before placing it on the glass slide, so that the DNA on the AFM tip will not interact further. Another set of experiments will involve attaching different forms of SVCP to an AFM probe using the method of Avci et.al. (4), using an ethanolamine linker and a flexible tether. DNA oligomers will be immobilized on a glass slide, and binding energies measured. Different forms of SVCP will include truncations and mutations, to determine the important residues in nucleic acid binding and capsid formation. Similar controls to the above set of experiments will be used.

Przbyla, Laralynne Analysis of the AFM data will consist of comparing force-frequency relationships over the duration of the AFM scan, which can be analyzed to give a measurement of the adhesion force between the molecule on the tip and the surface. Anticipated results and impact By determining the strength of interaction of SVCP with nucleic acid oligomers using atomic force microscopy, more about the formation of the viral nucleocapsid can be discovered. This information will be combined with ongoing studies using analytical ultracentrifugation and surface plasmon resonance. Results from this study will show what length of nucleic acid oligomer will be most successful at causing SVCP to bind, and which will show the lowest binding energy. In addition, the SVCP forms with the most favorable and least favorable binding to nucleic acid will be found. A mutation or truncation that increases or decreases nucleic acid binding and therefore affects core formation will help determine the mechanism of similar protein-protein and protein-nucleic acid interactions. In addition to helping elucidate the mechanics of the interaction between Sindbis capsid and the internal nucleic acid, this study can be applied towards helping to define a model system for studying other viral capsid-nucleic acid interactions. Such studies can be related to other alphaviruses that are more important to human disease, or to different viruses of which little is known about capsid formation, such as yellow fever virus and Dengue virus. References 1. Büchen-Osmond, C. (Ed), (2003). 00.073.0.01.001. Sindbis virus. In: ICTVdB – The Universal Virus Database, version 3. 2. Magonov, S.N., Reneker, D.H. (1997) Characterization of Polymer Surfaces with Atomic Force Microscopy. Annu. Rev. Mater. Sci. 27: 175-222 3. Sun, Z., Martinez-Lemus, L.A., Trache, A., Trzeciakowski, J.P., Davis, G.E., Pohl, U., Meininger, G.A. (2005). Mechanical Properties of the Interaction between Fibronectin and α5β1 Integrin on Vascular Smooth Muscle Cells Studied using Atomic Force Microscopy. Am J Physiol Heart Circ Physiol. 12 Aug. (not yet published) 4. Avci, R., Schweitzer, M., Boyd, R.D., Wittmeyer, J., Steele, A., Toporski, J., Beech, I., Arce, F.T., Spangler, B., Cole, K.M., McKay, D.S. (2003). Comparison of AntibodyAntigen Interactions on Collagen Measured by Conventional Immunological Techniques and Atomic Force Microscopy. Langmuir 20: 11053-11063. This research proposal is original, and was conceived, written, and submitted by -------------.

Ricks, Jennifer DEATH MECHANISMS IN ATR-DEFICIENT LYMPHOMA CELLS KEYWORDS: ATR, mitotic catastrophe, G2/M arrest, DNA damage RNA-interference (RNAi) technology is a powerful tool currently used to explore cell signaling pathways including the cellular response to DNA damage. One of the genes under investigation by this method is ATR (ATM and Rad3-related). In in vitro studies, lymphoma cells that have undergone RNAi-mediated suppression of ATR are moderately resistant to the frontline chemotherapy drug doxorubicin (DOX), which causes double-stranded DNA breaks (1). However, when those same cells are injected into a mouse model, the resulting in vivo tumors are sensitive to DOX treatment—the complete opposite of the in vitro result. After a series of preliminary experiments exploring this difference, I concluded that currently unidentified microenvironmental factor(s) present in vivo (but absent in the cell culture) were affecting the tumor’s response to DOX. Through a flow cytometry DNA content analysis of ATR-deficient cells treated with DOX both in vitro and in vivo, I found that the in vitro cells arrested at the G2/M checkpoint while the in vivo cells lacked this arrest response. Furthermore, in vitro cells deficient in ATM (a protein similar in function to ATR) displayed a stronger G2/M arrest and a higher level of resistance to DOX treatment. HYPOTHESIS: The presence or absence of a G2/M arrest determines the overall response of ATR-deficient lymphoma cells to DOX-induced DNA damage. The in vitro cells undergo arrest at the G2/M checkpoint and repair the damaged DNA before proceeding through mitosis. The in vivo cells are somehow unable to arrest at this point and attempt to proceed through mitosis with damaged DNA. As a result, these cells die through a process called mitotic catastrophe that leads to apoptotic cell death. RESEARCH PLAN: The hypothesis will be tested by 1) determining if the tumor cells treated in vivo show upregulation of proteins that drive cells through the G2/M checkpoint, 2) determining if these tumors undergo mitotic catastrophe leading to apoptosis, and 3) determining if the cells treated in vitro repair their damaged DNA, undergo mitosis, and reenter the G1 phase. I. When phosphorylated at certain amino acids, Cdc25B and C signal the cell to proceed with mitosis (2). If the treated ATR-deficient cells are being driven through the G2/M checkpoint in vivo, then they should display higher levels of activation of these proteins than treated in vitro cells that arrest at G2/M, or untreated cells. Immunoblots will be performed to determine the levels of phosphorylated Cdc25B/C in ATR-deficient in vivo lymphoma cells, ATR-deficient in vitro cells, and ATM-deficient in vitro cells. All three groups will be treated with DOX and cells will be collected 12 hours later, with untreated cells from all three populations used as controls. Cell lysates from each group will be run on two SDSpolyacrylamide gels, one gel per protein tested, along with an appropriate loading control (βactin). The gels will be run through the standard electrophoresis and membrane transfer protocols, probed with antibodies specific for the phosphorylated proteins, and detected via chemiluminescence. II. Mitotic catastrophe is a cell death mechanism that is characterized by the formation of micronuclei that arise from multipolar spindles and incorrect chromosome segregation during anaphase (3). Mitotic catastrophe eventually leads to apoptotic cell death through a p53independent pathway (4). In order to show that the ATR-deficient tumors undergo mitotic catastrophe and not canonical (p53-dependent) apoptosis, DOX-treated ATR-deficient in vitro and in vivo cells will be stained with antibodies specific for α- or γ-tubulin, followed by fluorescently-labeled secondary antibodies. α-tubulin staining will identify multipolar spindles,

Ricks, Jennifer

while γ-tubulin staining will visualize multiple centrosomes, both hallmarks of mitotic catastrophe (3). The absence or presence of the canonical apoptotic pathway will also be determined by performing an immunoblot with in vivo and in vitro ATR-deficient cell lysates. The primary antibodies used will be specific for phosphorylated proteins involved in this pathway (e.g. p53, Puma, Noxa). Mitotic catastrophe does not involve the activation of these proteins, so the presence of phosphorylated (activated) versions of these proteins will indicate cell death via the canonical apoptotic pathway. In both of these experiments, untreated ATRdeficient in vivo and in vitro cells will be used as controls. III. Finally, I plan to show that the treated ATR- and ATM-deficient cultured cells successfully repair damaged DNA during G2/M arrest, proceed through mitosis, and reenter G1. DNA content will be examined by flow cytometry for these treated cell populations prior to treatment and 12, 24, 48, and 72 hours after DOX treatment (3). If the G2/M arrest is transient, then DNA content analysis should demonstrate a reversion to the normal cell cycle with the majority of cells in G1 within this 72 hour window. Treated in vivo ATR-deficient cells as well as untreated populations of these three cell types will be used as controls. EXPECTED RESULTS: I expect to find upregulation of G2/M arrest inhibitors (e.g. Cdc25B/C), hallmarks of mitotic catastrophe, and the absence of indicators of p53-induced apoptosis (e.g. p53, Puma, Noxa) in the treated ATR-deficient tumors in vivo. I also expect to see a transient G2/M arrest and an eventual return to a normal cell cycle in the ATR-deficient cells treated in culture. Together, these results will point to a drug response mechanism in which the lymphoma cells are able to sense the DOX-induced DNA damage and arrest at the G2/M checkpoint in vitro. While undergoing arrest, the damage is repaired and the cells proceed through mitosis successfully. Conversely, the cells treated in vivo either do not recognize the DNA damage or cannot arrest at G2/M in order to repair it. These cells attempt to proceed through mitosis with heavily damaged DNA, which results in mitotic catastrophe and eventually apoptosis. The goal of future investigation should be to identify the extracellular factor(s) that prevent the in vivo population from either recognizing DOX-induced DNA damage or arresting at the G2/M checkpoint. BROADER IMPACTS: At the conclusion of this project, I will have gained a better understanding of the mechanisms behind the response of ATR-deficient cells to DOX-induced DNA damage. Performing research on a basic, central part of cell biology like cell death will solidify my background in this field and strengthen my ability as an instructor. Despite the development of new drugs and treatments for cancer, chemotherapy-resistant tumors remain a major obstacle to successfully treating this disease. Understanding how specific genetic mutations lead to drug resistance or sensitivity will help scientists design treatments that are more effective. Physicians and cell biologists will also be better able to predict if existing drugs will be successful against a tumor containing certain genetic mutations. I plan to not only build up this understanding, but also disseminate this information to physicians. I hereby attest that this proposal is my own work, with reviews from faculty advisors. CITATIONS: 1. Hemann, et al. (unpublished results) 2. Hermeking H. 2003. The 14-3-3 cancer connection. Nature Reviews: Cancer 3:931-43. 3. Eriksson D, et al. 2007. Cell cycle disturbances and mitotic catastrophes in HeLa Hep2 cells following 2.5 to 10 Gy of ionizing radiation. Clinical Cancer Research 13(18):5501s-8s. 4. Castedo M, et al. 2004. Cell death by mitotic catastrophe: A molecular definition. Oncogene 23:2825-37.

Rock, Jeremy Subcellular Localization Dependent Regulation of TAB1 Mediated Autophosphorylation of p38α KEYWORDS: p38, transforming growth factor-β-activated protein kinase 1 (TAK1)- binding protein 1 (TAB1), Toll-like receptor 9 (TLR9), CpG DNA The content of this research proposal is, except where specifically cited, the work of this author alone. No relationship exists between this work and the author’s previous research, with the technical exception that the author proposes to utilize his knowledge of zinc finger nucleases (ZFNs) (see Previous Research Experience statement; 1) as a tool for somatic cell genetics. Cells recognize and react to changes in their environment by initiating signal transduction pathways, some of the most important of which involve the activation of mitogen activated protein kinases (MAPKs). MAPK signal cascades are composed of three sequentially acting protein kinases. In the central dogma of MAPK activation, an upstream signaling event activates a MAP kinase kinase kinase (MAPKKK), which in turn phosphorylates and activates a MAP kinase kinase (MAPKK), which then phosphorylates and activates the MAPK. Five distinct families of MAPK pathways are currently recognized in mammals, one of the most extensively studied of which is p38. p38 plays an essential role in a diverse repertoire of cellular functions ranging from apoptosis to inflammation to cellular differentiation. Fundamental biological consequences aside, p38 is also intriguing in that, in addition to being activated by the canonical method of MAPK activation, there is evidence for a non-canonical, non-phosphorelay dependent method of p38 activation (2). Moreover, this alternative activation pathway seems to be important in a number of biological processes. Ge et al. (2002) discovered that p38α (there are four known splice isoforms: α, β, γ, and δ) was capable of binding to a scaffolding protein TAB1, and that this association resulted in the autophosphorylation and autoactivation of p38α. Surprisingly, short of the findings that this pathway may be broadly biologically significant mentioned above, little has been discovered about this alternative method of p38α activation. The goal of this research project is to determine how the direct activation of p38α by TAB1 is regulated. An experimentally tractable system in which to study this interaction is Toll-like receptor 9 (TLR9) signaling in human B lymphocytes. The TLR9 pathway is involved in the detection of CpG DNA, the binding

Rock, Jeremy of which can induce B cells to proliferate, class switch and differentiate (3). In an abbreviated form, the conventional wisdom for TLR9 signaling in B cells proceeds as follows (3): extracellular CpG DNA is internalized after binding to anti-DNA B cell receptors. TLR9 encounters this DNA in the lysosomal/late endosomal compartments, where it then transduces the signal by way of a physical interaction with a protein complex containing, among other components, TAB1, the MAPKKK TAK1, and TRAF6. This interaction results in the phosphorylation and activation of TAK1, which in turn phosphorylates and activates a number of substrates, including the upstream p38α MAPKKs MKK3 and MKK6, the activation of which leads to the transphosphorylation and activation of p38α. I would alternatively propose, first, that p38α activation in TLR9 signaling in B cells proceeds by a direct interaction between TAB1 and p38α and is independent of MKK3/6 activation. Moreover, I would hypothesize that the TAB1-p38α activation pathway is regulated by their distinct subcellular localizations in the absence of a signaling event. The model proposed above is supported by two additional pieces of evidence: first, Ge et al. (2002) found that SB203580, a pharmacological agent that specifically inhibits autophosphorylation of p38α but does not prevent transphosphorylation, prevented the phosphorylation of p38α in response to CpG oligonucleotides but did not prevent its phosphorylation in response to lipopolysaccharides in a B cell line; and second, there is some evidence that TAB1 may be membrane associated in a basal state (4). I propose to address this model experimentally in either the B cell line RPMI 8226 or the Burkitt lymphoma cell line Namalwa (5) for reasons of ease of use and ability to use ZFN driven genetics (1). If TAB1 mediated autophosphorylation of p38α is indeed the sole mechanism by which p38α is activated in TLR9 signaling in B cells, loss of function of the p38α MAPKKs (MKK3/6, potentially MKK4) should not affect p38α activation under these conditions. One can use RNAi to knockdown the expression of these MAPKKs and assay for levels of phosphorylated p38α (Western blot with phospho-p38α specific antibody) in the presence and absence of TLR9 activation. Activation of the IFN-I signaling pathway, in which MKK3/6 are essential for p38α activation, could be used as a control for RNAi efficacy. If RNAi of the

Rock, Jeremy MAPKKs is not technically feasible, one could use ZFNs to disrupt the coding sequence of the MAPKKs and thus completely ablate their functional expression. To demonstrate that p38α activation is truly autophosphorylation and not activation by some unknown MAPKK, one could construct a kinase dead allele of p38α using ZFNs and an engineered donor molecule to introduce a D-to-N mutation in the active site D in the catalytic loop. This mutation should abolish any possibility of autophosphorylation while allowing transphosphorylation. The phosphorylation state of the kinase dead allele could then be tested under TLR9 and IFN-I activation conditions discussed above. This approach would also eliminate any concerns of bystander effects of SB203580 in previous research. To further show that p38α is associated with TAB1 upon activation of TLR9, one could coimmunoprecipitate p38α and assay for the presence of TAB1 (by Western blotting). TAB1 should be pulled down upon activation TLR9, but it should not be pulled down in the absence of activation. Given that two isoforms of TAB1 exist (α and β) (6), both of which are capable of binding to and activating p38α but only one of which (α) is capable of binding to and activating TAK1 (a MKK3/6 MAPKKK), it would also be interesting at this point to investigate which TAB1 isoform p38α was interacting with (by way of isoform specific antibodies). Furthermore, to show that this p38α-TAB1 interaction is essential for p38α autophosphorylation, one could modify the endogenous TAB1 gene to disrupt key residues or simply delete the region involved in p38α binding. The TAB1 p38α docking site has been determined in vitro (2). One could then demonstrate that this TAB1 construct is no longer able to result in p38α activation in vivo, but importantly retains the ability to associate with other proteins in the signaling complex, e.g. TRAF6 (monitored by co-IP of TAB1 and Western blotting for TRAF6). Having demonstrated that TLR9 signaling in B cells is independent of MKK3/6 activation and is mediated by a direct interaction between TAB1 and p38α and, it would then be interesting to determine how this interaction is regulated. One method to analyze the subcellular distribution of proteins in live cells would be to tag p38α with GFP and the appropriate splice isoform of TAB1 with RFP. Their real-time localizations

Rock, Jeremy can then be observed in the presence and absence of TLR9 signaling. The prediction is that, in an unstimulated state, p38α is likely to stain diffusely in the cytoplasm and the nucleus, whereas TAB1 is predicted to be found bound to lysosomal/endosomal membranes and would thus produce a punctate staining pattern. Upon stimulation, TAB1 is then predicted to be released in the cytoplasm, likely in the form of a TAB1 containing protein complex, where it will interact with p38α. Finally, to show that this sequestered subcellular localization is essential for the regulation of TAB1 mediated p38α autophosphorylation, one could target p38α to the membrane with the introduction of a CAAX box or a lipid-binding MinD MTS sequence (7). Alternatively, it may be possible to disrupt TAB1 membrane association. Such modifications are predicted to show constitutive activation of p38α. p38 plays a fundamental role in a wide range of biological processes. Correct regulation of p38 activity, for example, allows for accurate neural progenitor development (8), whereas incorrect regulation seems likely to play a role in neurodegenerative diseases such as Alzheimer’s (9). The autoactivation of p38α mediated by interaction with the scaffolding protein TAB1 may be an important alternative activation pathway in these processes, and thus it is crucial to understand how this non-canonical signaling pathway is regulated. Final note: The use of SB203580 allowed Ge et al. (2002) to effectively distinguish p38α trans vs. autophosphorylation, and thus screen endogenously for pathways that may utilize this non-canonical method of p38α activation. Such agents are not available for most MAPKs. The use of ZFNs to engineer kinase dead alleles of all other human MAPKs would make it a simple task to identify how general this method of MAPK activation may be. 1. F.D. Urnov et al., Nature 435, 7042 (2005)

6. B. Ge et al., J. Biol. Chem. 278, 4 (2002)

2. B. Ge et al., Science 295, 5558 (2002)

7. T.H. Szeto, J. Biol. Chem. 278, 41 (2003)

3. S.L. Peng, Curr. Opin. Immunol. 17, 3 (2005)

8. G. Takaesu et al., Mol. Cell. Biol. 21, 7 (2001)

4. Z. Jiang, Mol. Cell. Biol. 22, 20 (2002)

9. A. Sun et al., Exp. Neurol. 183, 2 (2003)

5. M. Henault et al. J. Immunol. Methods 300, 1-2 (2005).

Sheltzer, Jason The Activity-Dependent Inactivation of a Yeast Permease Keywords: Yeast permease, Major Facilitator Superfamily, Gap1p, amino acid transport I. Background: The yeast amino acid permease Gap1p serves as a model system for studying the function and regulation of members of the major facilitator superfamily (MFS) of molecular transporters. In S. cerevisiae, Gap1p transports all naturally-occurring amino acids into the cell for use in nitrogen metabolism.1 Gap1p function is tightly regulated in response to the available nitrogen source, and high intracellular concentrations of amino acids cause Gap1p to be ubiquitinated and recycled to the vacuole.2 The stringent control of Gap1p activity is necessary for cell viability, as an intracellular overabundance of amino acids is cytotoxic.3 Risinger et al. (2006) have recently discovered a novel mechanism for permease regulation. When Gap1p is constitutively localized to the plasma membrane by point mutation of its ubiquitinated residues, Gap1p is inactivated by the uptake of amino acids. The inactivation of Gap1p specifically depends on substrate flux through Gap1p, and inactivation can be reversed by incubations in amino acid-free media. Gap1p inactivation decreases its activity to