Nicholas Bishop and Dr. Marc D Hansen, Physiology and Developmental Biology
Purpose and Protocol
My research aims to elucidate the protein interactions at cell-cell contacts (adherens junctions or AJ’s) by studying the fluorescence recovery of three known AJ proteins (zyxin, VASP, and actin) after photobleaching. At AJ’s, zyxin interacts with VASP (Vasioukhin et al., 2000) which in turn encourages the polymerization of actin (Vasioukhin et al., 2000). Mutation of certain regions of zyxin may reveal their role in zyxin-VASP binding and regulation. The effects of altering VASP activity should be evident in actin turnover rate. In Fluorescence Recovery After Photobleaching (FRAP), regions of cell-cell adhesions with fluorescently labeled zyxin, VASP, or actin are bleached with a high intensity laser. The time it takes for the regions of interest to reach half of their maximum fluorescence after bleaching (T1/2) provides clues as to how these proteins interact at the junction. Understanding normal AJ protein interactions may lead to models for how cell adhesion malfunctions in cancer metastasis.
Progress, Preliminary Results, and Publication
Before use in a FRAP experiment, a cell must first receive a piece of DNA that encodes for a fluorescent version of zyxin, VASP, or actin. This process is called transfection. In order for the transfection to function efficiently, the conditions must be optimized for each cell line and DNA plasmid through trial and error. After the process of optimization, the transfections resulted in up to 40% of cells expressing fluorescently tagged proteins. To show the interaction of two proteins a cell may be transfected with both proteins displaying different fluorescent tags. After trying to produce cell lines that permanently expressed the two different fluorescently tagged proteins, my mentor suggested that transiently transfected cells would suffice for the purposes of my experiments.
I learned to conduct FRAP experiments using a confocal microscope and imaging software that records the fluorescence measured in each bleached region. I performed numerous experiments and generated a large quantity of data. Dr. Woodbury (another professor in my department) helped me work out the mathematics needed to understand the data. Now, I am able to generate recovery curves of the expected shape and calculate realistic recovery half-times.
My results reveal information about the ability of zyxin to regulate actin dynamics via VASP. The 4A region of zyxin recruits VASP to the junction, and the LIM domain of zyxin inhibits actin polymerization by VASP (Hansen and Beckerle, 2006). Zyxin 4A ΔLIM fails to associate with VASP resulting in less actin polymerization as evidenced by the significantly longer half time to fluorescence recovery. One would expect actin recovery in the ΔLIM cells to be faster, yet it varies little from actin turnover in cells transfected with normal zyxin. This suggests either that the LIM domains do not bind VASP as predicted, or that this association lasts only briefly. Over-expression of zyxin in the double transfectants causes turnover differences between singly and doubly transfected cells.
These results will be included in posters from the Hansen lab at the conference of the American Society for Cell Biology in December of this year. Due to exam conflicts, I will be unable to attend the conference, but my work will be presented by other students from the Hansen Lab. My data will also appear in a manuscript to be submitted for publication in the journal Nature Cell Biology.
Problems & Plans
While the data seem to confirm the role of zyxin in regulating actin polymerization at AJ’s, further investigation is required. In many cases, the differences shown fail to demonstrate statistical significance. The technical challenges of consistent image capture make reproducible experiments difficult. Also, improper data analysis may contribute to the error observed. In some cases, small regions of cytoplasm were included in the analysis of the data. More careful isolation of the cell junction will eliminate the experimental variability and confirm the significance of what these data now only tenuously suggest. I plan to reanalyze much of the data already collected with greater specificity in an effort to decrease error in the data.
Poor bleaching of the junction could also explain the variability of the data. Ideally, all fluorescence disappears from the region of interest in a FRAP experiment. I never succeeded in bleaching the cell-cell junction completely. The computer must save the images of the bleach sequence before it begins the recovery sequence. During this interval of time, fluorescent proteins recover and could perhaps mask the efficiency of our photobleaching. I am currently corresponding with a live-cell imaging expert from the microscope manufacturer to troubleshoot this technical problem. If I were able to achieve total bleaching in each experiment, the data could be compared with more certainty. I will continue to work with the microscope technician to solve this issue.
My experience in the Hansen lab has proved richly rewarding. I am currently registered for mentored research credit and plan to continue in the lab until I graduate in April, 2009. I will submit a final report on this work before the deadline on December 31, 2007.