Daniel Lew - US grants

Cancer cells must acquire multiple mutations to evade the many restraints that prevent uncontrolled proliferation. Normal cells are vigilant in maintaining genome integrity (i.e. preventing mutations), but one hallmark of cancer cells is a defect in such self- surveillance and hence genome instability. Checkpoint controls that govern entry into mitosis are essential for maintaining genome integrity during cell proliferation. The Wee1-family kinases that inhibit Cdc2 are among the targets for these checkpoints both in mammalian cells and in yeast, but the basis for their regulation remains unclear. In budding yeast, the Wee1-relative Swe1p is employed by the morphogenesis checkpoint, responding to perturbations of cell shape and the actin cytoskeleton. We have identified a novel pathway controlling Swe1p degradation, whose initiation depends on a family of cytoskeletal proteins called septins. Septin organization appears to respond to cell shape, and can regulate a checkpoint kinase called Hsl1p. One aim of this proposal is to understand how septins are organized and how that organization responds to cell geometry to act as a shape sensor. In addition to cell shape, the checkpoint responds to several physiological stresses, and a second aim of the proposal is to understand how these stresses regulate swe1p degradation and hence cell cycle progression. Although a morphogenesis checkpoint has not yet been identified in cells other than yeast, appropriate responses of mammalian cells to geometrical and mechanical features of their microenvironment are thought to involve sensing of similar types of cytoskeletal information. The metastatic potential of cancer cells increases upon derangement of these pathways, and the proposed studies will therefore provide new insight into cancer progression, and may yield potential targets for therapeutic intervention.

A novel idea is presented which combines the information from the yeast genome with available technologies to generate a new research tool called genomeCPA. The basic idea behind genomeCPA is to produce a set of 60 96-well plates in which each well contains a single distinct immobilized protein of known identity from the yeast genome. The set of 60 plates would then constitute a spatially ordered complete protein array of all the proteins in a yeast cell. Any desired biochemical activity assay could then be performed on the entire array, identifying in one step all of the proteins in the cell possessing the desired activity. The specific aim of this application is to prove the feasibility of the genomeCPA concept. If it is feasible, genomeCPA has the potential to be an extremely powerful new tool for identifying proteins with particular biochemical properties. In the long term, its development should speed the pace of biochemical research and open new horizons of technical feasibility for larger scale projects.

Hydroxyapatite cement (HAC) has thus far proven to be an effective material to serve as a bone substitute in non-load bearing applications associated with congenital malformations, oncology surgery and traumatic injury. In this technology transfer project, we propose to expand the use of HAC by continuing to explore its use in bone defects in the oral cavity. Preliminary information indicates that HAC is effective in supporting bone repair in surgically created mandibular bone defect sites, as long as the material remains stabilized in the defect site. In this project we have partnered with Howmedica-Leibinger to expand on our earlier findings to study the effectiveness of HAC in supporting bone implants adjacent to dental implants. Additionally it is proposed to study straightforward methodology to prevent washout of the cement and thereby maintain its stability in clinical situations. We plant to expand on our current animal model to conduct these studies. These studies will further develop the myriad of clinical applications for which HAC may be useful. It is anticipated that pending the results of this work and other ensuing projects from the Center, clinical trials may be developed through the Clinical Resources and Outreach Center.

DESCRIPTION (provided by applicant): Cdc42 plays a key role in the polarization of cells towards a variety of signals (e.g., T cell polarization towards antigen-presenting cells, fibroblas polarization towards wound sites, or yeast bud formation). Human CDC42 can functionally substitute for its yeast counterpart, suggesting that key functions of Cdc42 have been highly conserved, and the ability to apply genetic, biochemical, and cell biological approaches makes yeast a very powerful system for delineating the mechanism of Cdc42 action in cell polarization. The goal of the proposed research is to understand how Cdc42 polarization is regulated, and how the process is restricted so that cells only form one polarization front. Cancer cells display alterations of cell shape, cell-cell adhesion, and cell motility (all actin-dependent processes regulated by Cdc42), which are likely to be important for numerous aspects of malignant transformation. Deregulation of Cdc42 in mammalian cells promotes anchorage-independent growth, and is necessary for the morphological changes (as well as anchorage independence) that occur in Ras-transformed cells. Thus, Cdc42 deregulation affects the proliferation as well as the metastatic potential of cancer cells. Understanding the normal regulation and function of Cdc42 is an important first step towards addressing how their misregulation might promote cancer.

DESCRIPTION (provided by applicant): The CMB Training Program has been an important component of graduate education at Duke University, having trained a total of 432 students since its inception in 1975. The program is highly interdisciplinary, with 124 faculty members from all of the Basic Biomedical Sciences departments working in diverse areas of cell and molecular biology. CMB Program students benefit greatly from the outstanding research environment at Duke University. Students rotate through three or more labs during the first year, and are provided with extensive advising, orientation and mentoring experiences. Course work is mostly completed within the first two years, although the students continue to participate in the CMB Student Seminar Series throughout their graduate career. All CMB students affiliate with one or another PhD-granting unit that is smaller than CMB, providing students with both the broad interdisciplinary program experience and a more close-knit group of colleagues within one of the sub-disciplines. CMB Program students take great pride in their program, participating extensively in recruiting new students, orienting first-year students, planning the annual mini-symposium and picnic, and meeting with guest seminar speakers. The success of the program is evident from our track record. Considering 242 trainees who participated in the program within the last 10 years, 90% have completed or are on track to complete the PhD. The retention rate appears to be improving, since only one trainee has left the program from the last four entering classes (total of 70 trainees). The subgroup of 123 trainees that have completed their PhD within the last 10 years published 483 papers from their thesis training, for an average of 3.9 publications per trainee. This alumni subgroup currently includes 31 academic faculty (17 tenure-track), 21 research scientists in industry or institutes, 62 postdoctoral fellows or research associates, and 5 involved in science-related activities such as scientific writing or administration. These data strongly indicate that our training program is successfully preparing graduates for the rigors and demands of productive careers in modern biological sciences.

DESCRIPTION (provided by applicant): Cells are extraordinarily adept at detecting and tracking shallow gradients of chemicals of interest. Micro-organisms track gradients to find food or mates, and similar processes underlie axon guidance in the nervous system, homing of immune cells towards invaders, crawling of repair cells towards wound sites, and guidance of sperm towards the egg during conception. Gradient tracking also contributes to metastasis in cancer, so understanding how cells track shallow chemical gradients is of medical relevance as well as fundamental interest. Upon detecting chemicals through cell-surface receptors, cells either move or grow towards the source of the signal. In many cases, the gradients of diffusible substances are shallow, resulting in minuscule concentration differences across the diameter of small cells. Gradient detection is made even more difficult by the randomness of individual receptor-ligand interactions, which leads to molecular noise that can mask the tiny spatial gradient signal. The mechanisms that allow cells to efficiently track even very shallow gradients despite noise are poorly understood. In this proposal, we use the uniquely tractable yeast model system to investigate these mechanisms. During mating, yeast cells polarize and grow up a gradient of pheromone to find and fuse with opposite-sex partners. We propose to use a combination of cutting-edge microscopy, genetics, and computational modeling to understand how it is that yeast cells track pheromone gradients.

DESCRIPTION (provided by applicant): We propose a broad IMSD Program for Duke University to promote student development and diversity at both the undergraduate and graduate levels. At the undergraduate level, we will provide both academic support and cultural engagement components, including research experiences. Undergraduate IMSD Scholars will experience independent mentored research for at least four semesters and one 10-week summer program, and present their research at the national ABCRMS or SACNAS conference. They will also benefit from additional mentoring, a unique first-year seminar course, and group learning during gateway courses. Graduate IMSD Scholars will participate in Early Start, beginning graduate school a month ahead of time with career development activities, community engagement, and an early start to research rotations. Graduate IMSD Scholars will also benefit from unusually early travel to a scientific conference or workshop/minicourse (e.g. Woods Hole Course) and from additional mentoring from an IMSD-connected faculty member and continued career and community engagement activities (beyond the Early Start). Several program activities are also designed to integrate the Undergraduate and Graduate IMSD Scholars and Duke faculty members into a more cohesive community and to facilitate connections between faculty/students at Duke and those at North Carolina institutions, particularly feeder institutions with a significant underrepresented (UR) undergraduate population. The goals of the Duke IMSD Program are to increase retention of undergraduate UR students in the biomedical/behavior fields, to eliminate achievement gaps between UR and non-UR undergraduates in gateway courses, to double the percentage of UR students in our biomedical/behavioral PhD programs, to create a more robust sense of community for UR students in these programs, and to improve communication with neighboring institutions of higher education.

ABSTRACT Our research is focused on fundamental questions related to cell polarity. Cell polarity describes the ability of cells to spatially organize their internal constituents along a specific axis. It is critical for cell migration (where cells need to generate a front and a back), and also for developing specialized cell shapes that are needed for many cells to function. In addition, derangements of the polarity machinery can contribute to several diseases, for example by enabling cancer metastases. Thus, an understanding of the mechanisms, regulation, and consequences of cell polarity is of both fundamental and medical interest. Studies on cell polarity have identified an evolutionarily ancient and conserved core machinery centered on a ?master regulator? of polarity called Cdc42. However, many of the most interesting questions remain unsolved. How is it that most cells only make a single ?front? enriched in Cdc42, but some cells with more complex shapes can specify several sites to act as fronts? How do cells read their environment to determine the direction in which they should orient the polarity axis? Once polarity is established, how is the precise downstream set of events orchestrated to give each cell type the right shape? And then, how do cells know what shape they are? We use the uniquely tractable yeast model system to investigate these questions, and apply a combination of cutting-edge microscopy, genetics, and computational modeling. Our previous work identified a positive feedback mechanism that explains how Cdc42 becomes concentrated at polarity sites to establish a polarity axis. Our recent work on polarization during yeast mating, when yeast cells orient in response to spatial gradients of pheromones, suggests a new paradigm for tracking chemical gradients. And our work on a yeast cell-cycle checkpoint responsive to cell shape now suggests a model for how cells know what shape they are. Based on these findings, we are poised to make significant advances on the questions posed above, and to exploit the answers to those questions to provide insights that extend well beyond the yeast system.