As the summer winds down, I have been reflecting on the project Dr. Steiner and I began working on in June. Dr. Steiner and I have been utilizing multi-transgenic zebrafish to deeply explore the regeneratory process of zebrafish sensory hair cells. The current body of zebrafish research suggests supporting mantle cells divide to produce hair cells during regeneration of neuromast sensory organs along the lateral line, although evidence of such is lacking. Together, we created a plan to manipulate three transgenic lines to image the regeneration of these neuromast cells, with the end-goal of better understanding this regeneratory process and the factors that control it.
With these research goals in mind, I’ve been learning about the complexities of conducting biological research with living specimens. Many times during the semester, my colleagues and I have sacrificed time to come in on weekends to feed the fish; there have been several weeks the fish didn’t cooperate with breeding, and we had to quickly work on a new plan for the week. Truthfully, though, working with live zebrafish brings an element of physicality and life to my research. It’s a pleasure starting my day seeing our entire population of zebrafish greet me with frantic swimming.
Also challenging, but extremely rewarding, is developing the Zebrabow process I’ve been using to visualize cells of zebrafish. At its best, this method makes the cells of zebrafish fluoresce a beautiful mosaic of colors, allowing a researcher to better understand the cell divisions that potentially lead to new hair cells. No single procedure will work for every lab; Dr. Steiner has greatly helped me in creating a Zebrabow process that works for our fish in particular. At the start of the summer, we had very little mosaic fluorescence amongst the cells. This provided me several opportunities to review the process we had used, and modify it for better performance in the future. Such modifications make this research project feel like it is truly our own; over the next few semesters, I hope to refine this procedure to obtain consistent and repeatable results.
This summer project has not only taught me about valuable lab techniques, such as confocal & fluorescent microscopy and taking care of living specimens; working with Dr. Steiner this summer made me increasingly comfortable with the research process and environment, which I hope to be a part of for years to come. Being able to pave my own way through a new set of procedures is fantastic experience for my future endeavors. Most of all, I’m excited to continue my research with Dr. Steiner over the upcoming year, building upon everything I’ve learned over the past months.
Included in this blog post is an image of one of the Zebrabow treatments I’ve done, in order for readers to better understand the process. This image was taken as numerous ‘slices’ of images from the confocal microscope, then compressed into a single image. On the bottom right is the neuromast, the organ containing hair cells that Dr. Steiner & I study. One can see numerous flourescent cells, as well as some macrophages (depicted by amorphous green-flourescent projections).
I am working on a project titled, “The Effects of the Vasodilator-stimulated Phosphoprotein on Mycobacterium bovis-BCG and the Macrophage Actin Filament Network.” It is a continuation of research that I started in September of 2011. The primary aim of the project is to determine if the Vasodilator-stimulated phosphoprotein (VASP) is involved in mediating cytoskeletal rearrangement in cells infected with Mycobacterium bovis-BCG, a model organism for the study of Mycobacterium tuberculosis. M. tuberculosis is the causative agent of tuberculosis. Tuberculosis is responsible for nearly 2 million deaths worldwide every year. There are about 10 million new cases of tuberculosis each year and about one-third of the world’s population is infected with the bacterium.
VASP is a scaffold protein involved in regulation of the host cell cytoskeleton (de Chastellier et al, 2000). VASP-dependent actin polymerization regulates membrane architecture, cell motility, and pathophysiologic processes such as metastatic invasion. Studies on VASP have shown that it is localized to the site of pathogen attachment on the host cell for enteropathogenic Escherichia coli and Cryptosporidium parvum and led to the intriguing possibility that VASP functionally contributes to the attachment and invasion of these pathogens. In addition, Listeria monocytogenes, Shigella flexneri, and Rickettsia rickettsia have all been show to exploit actin polymerization in the host cytoskeleton to aid in cell-to-cell spread of the organisms (Ball et al 2000). It has also been found that actin regulation in host macrophages is disrupted due to infection by some pathogenic species of mycobacteria, such as M. avium and M. marinum (de Chastellier et al, 2000). However, the role of VASP in the disruption of actin filaments has not been described for M. tuberculosis.
In order to understand the potential role that VASP and actin polymerization might have in M. tuberculosis infection, it is important to understand how the infection initiates. The airborne transmission of M. tuberculosis bacilli from an infected individual upon inhalation places the bacteria in the inner lining of the lungs. There, the bacilli are engulfed by alveolar macrophages through phagocytosis. M. tuberculosis is able to survive the innate antimicrobial defenses of the alveolar macrophages. Mycobacteria are distinct in that they are able to live inside host macrophages, rather than as free-living organisms in the host body. It is well established that rearrangement of the actin cytoskeleton is important to the early steps of phagocytosis by host macrophages (de Chastellier et al, 2000). Entry of a particle into a cell by the process of phagocytosis entails the reorganization of the actin cytoskeleton underlying the area of the plasma membrane of the host cell that is in contact with the particle. When a macrophage ingests a pathogenic mycobacterium, it becomes trapped in a phagosome, or a membrane-bound vesicle. This phagosome does not become fused with a lysosome, which occurs in cases with nonpathogenic bacteria, and thus the phagosome does not mature. The inhibition of association with a lysosome helps the pathogenic mycobacterium evade degradation by the potent, cytolytic environment of the phagolysosome. This occurs due to pathogenic disruption of the host macrophage’s actin cytoskeletal network in the phagosome (de Chastellier et al, 2000). This disruption suggests that VASP is being exploited by the pathogenic mycobacteria to evade normal, immune response by the host macrophages.
Based on the mentioned previous studies, and due to the fact that several pathogens have evolved to utilize the actin cytoskeleton of their host, we propose to determine whether M. tuberculosis is able to utilize VASP to alter the actin filament network during macrophage phagocytosis. In this project we will use an INF-gamma stimulated macrophage derived cell line infected with M. bovis-BCG to test for VASP-mediated cytoskeletal rearrangement. Uninfected cells will be used as a control. Immunofluorescent and time lapse confocal microscopy will be performed at selected time points after infection to determine if and how VASP plays a role in the infection process. Changes in macrophage morphology and VASP distribution will be examined and compared to the uninfected cells. Western blots will also be performed to test for concentration levels of VASP in the macrophages.
Successful completion of these experiments will lead to an understanding of the role of VASP in M. tuberculosis infection. It will lead to an increase in knowledge about how pathogenic bacteria infect host tissues as well as increasing knowledge about the regulation of actin filaments and VASP function. The overall goal from this work and the results that will be produced will provide more information to aid in the understanding of the M. tuberculosis infection process and aid in the development of vaccines or treatments to prevent the disease. I hope that my project will provide you with a fun and engaging opportunity to learn about this infectious disease!