The focus of this proposal is to investigate biologically important mechanisms of membrane receptor mobility regulation and how they relate to T cell activation in the human immune system. Specifically, we will examine the mobility control mechanisms and molecular associations of the receptor-like tyrosine phosphatase CD45, a key regulator of T cell activation. CD45 is necessary for T cell stimulation by antigen, and this requirement involves the dynamic regulation of CD45 mobility. There is biochemical evidence that populations of CD45 reside in lipid microdomains and associate with the cytoskeleton, and it is proposed that these interactions regulate CD45 function. To better understand the dynamic regulation of CD45, we will characterize this receptor's lateral mobility in a T cell model using high-resolution single-molecule techniques. We will measure the effects on CD45 mobility of cell activation and of pharmacological inhibitors predicted to disrupt CD45 molecular interactions (e.g., cytochalasin, jasplakinolide, colchicine). We will also investigate the role of specific molecular partners (e.g., spectrin, ankyrin) in CD45 mobility regulation. Together, these studies will provide a detailed molecular picture of CD45 interactions and function and provide insight into general mechanisms of receptor mobility regulation. Relevance of research to public health: Among other functions, the human immune system protects our bodies from a variety of infections. In order to perform these functions effectively without causing damage to body tissues, the immune system must maintain a delicate balance in which immune effector cells (for example, T cells) are activated only when needed. The specific molecular mechanisms that allow the activation of T cells are not completely understood, but it is clear that a critical aspect of activation is the regulation of receptor mobility in the plane of the T cell membrane. The focus of this proposal is to investigate biologically important mechanisms of membrane receptor mobility regulation and how they relate to T cell activation in the human immune system. Improved understanding of this process is likely to lead to safer and more effective therapies for infectious diseases, autoimmune diseases, and cancer. Introduction to Revised application: This is a revised application which is now on its second submission. In response to the previous review, the applicant, Quentin Baca, has revised the research plan to address criticisms that it was overly ambitious and that the biological problem to be addressed was poorly specified. There were also concerns that recent publications by the mentor were not as the corresponding author and that the mentor did not adequately spell out a detailed training plan for the Applicant. Applicant's Preparation for Graduate Study: As noted in the previous review, Mr. Baca is a promising student. His undergraduate record at Stanford in the sciences was generally excellent and he received a BS in chemistry in 2004. Almost all the courses taken at Harvard in the MD/PhD program were Medical School courses that were only offered on a pass fail basis and these grades were all P's. In three of the graduate courses at Harvard Mr. Baca received A's. The MCAT scores were excellent. The applicant's undergraduate research experience was in the Zare lab studying attachment of biologically active enzymes to a solid support matrix. This work resulted in a publication in Anal. Chem. on which he was a middle author. Mr. Baca has received numerous awards for his undergraduate work. At Harvard Mr. Baca did 2 rotations first in Thomas Look's lab working on making antibodies to oncogenic transfection factors involved in Human leukemias and then in Dr. David Golan's lab studying transmembrane receptor mobility. This will be the subject of his PhD thesis work. The letters of recommendation are very strong and attest to the applicant's talents as a creative future physician-scientist. The Quality of the Training Program/Institution: The environment at the Harvard Medical School will provide the applicant with an outstanding environment for the combined MD-PhD degree. Dr. Golan's lab will provide excellent facilities for the proposed work involving demanding single particle tracking techniques required for his thesis project. Additional facilities include the Imaging Center at the Medical School. Numerous seminars and journal clubs and opportunities for the applicant to present his work are also available. To address a concern in the previous review, the revised application states more explicitly how the mentor through frequent meetings will be involved in Mr. Baca's training. The Proposed Research: In the previous review of this application there were concerns that some parts of the application were overly ambitious and that the scientific problem to be addressed was not clearly stated. In response the applicant has revised Aim 3 to narrow the number of CD45 interaction partners to a study of 1-3 cytoskeletal proteins. He has also more explicitly stated the significance of the biological problem to be studied as how cells control transmembrane receptor mobility. The applicant will use technologically sophisticated single particle tracking of individual receptors to examine how mobility of CD45, a receptor which is a key regulator of T cell activation, is differentially regulated in resting vs. activated T cells. Using as a model system, the Jurkat T cell line, the applicant has interesting data for aim 1 to show the feasibility of these approaches and for aim 2 preliminary evidence that CD45 lateral mobility in the membrane is regulated by association with the spectrin-ankyrin cytoskeleton. He will experimentally examine associations of the CD45 receptor with other cytoskeletal elements such as actin and microtubules to test their role in regulation of receptor mobility.
In aim 3 the applicant will use proteomic screening to identify several interaction proteins that associate differentially with CD45 in activated and resting T cells. This is a well laid out proposal to address an important question regarding molecular mechanisms by which CD45 modulates the immune response. Given the quality of the preliminary data and the expertise available in Dr. Golan's lab, the applicant is likely to produce an excellent thesis that will provide an outstanding training experience. The Thesis Mentor: In response to concerns about the mentor's recent publications as corresponding author, the revised application has updated Dr. Golan's CV. Dr. Golan has had a productive 20 year career on the Harvard faculty where he is a Professor of Biological Chemistry and Molecular Pharmacology. His publications in the area of protein and lipid mobility in cell membranes have appeared in high profile journals and he has received an NIH MERIT award which terminated in March 2007. It is not mentioned whether this grant will be renewed. Dr. Golan has trained 18 postdocs and 12 graduate students and 4 of the 5 students listed have tenure track faculty positions at research universities. The mentor has also been a Co-director of the Harvard MD-PhD program. Overall Recommendation: This is a well written application that addresses the concerns of the previous review. The goals of the work are clearly stated and the mentor provides a more detailed training program. The applicant is an excellent candidate for this award and has outstanding potential to combine basic science with clinical applications as an MD-PhD.
|Chiasson-MacKenzie, Christine; Morris, Zachary S; Baca, Quentin et al. (2015) NF2/Merlin mediates contact-dependent inhibition of EGFR mobility and internalization via cortical actomyosin. J Cell Biol 211:391-405|
|Alenghat, Francis J; Baca, Quentin J; Rubin, Nooreen T et al. (2012) Macrophages require Skap2 and Sirp? for integrin-stimulated cytoskeletal rearrangement. J Cell Sci 125:5535-45|
|Cairo, Christopher W; Das, Raibatak; Albohy, Amgad et al. (2010) Dynamic regulation of CD45 lateral mobility by the spectrin-ankyrin cytoskeleton of T cells. J Biol Chem 285:11392-401|