In the Lew lab, we study the mechanisms underlying cell polarity establishment and maintenance. We use the budding yeast, Saccharomyces cerevisiae, as the model organism because 1) it is genetically tractable, and 2) the genes and processes we study are highly conserved among various organisms. Our approaches include molecular genetics, light microscopy, and mathematical modeling. We have been able to ask fundamental questions in cell biology, such as how yeast makes sure to make one and only one bud at each cell cycle.
My current project focuses on how polarization processes are temporally regulated. Just like humans, yeast has a “time schedule” for activities. My research has revealed that in yeast, polarity establishment, targeted exocytosis, septin assembly, and bud formation occur in a consistent order with predictable intervals. In everyday language, the questions I ask the budding yeast sound like this: “Why did you have lunch at 12pm today? Was it because you noticed that it was time for lunch ? Or, was it because it had been a few hours since breakfast and you were hungry?” In other words, I aim to understand if the timing of later events (eating lunch, or budding) depends on that of earlier events (hours after breakfast, or minutes after polarization). Alternatively, the timing of later events could be triggered by an independent clock (12pm, or the “lunch time” equivalent for a cellular timer). The results of my research will shed light on how cell polarity and morphogenesis are regulated in a timely manner.
We work with genetically engineered mice to recapitulate a human neurovascular disease called cerebral cavernous malformations. By developing these mouse models we hope to gain a fundamental understanding of how this disease develops and test the efficacy of proposed therapeutics.
Research in the Sullivan lab focuses on the genetics and epigenetics of the centromere, a chromosomal region vital for maintaining genomic stability. Human centromeres are assembled on repetitive alpha satellite DNA arrays that span multiple megabases and are organized into CEN chromatin, a distinct type of chromatin containing marks of active transcription. My research explores the non-coding RNAs produced from the repetitive alpha satellite DNA underlying centromeres, with a particular focus on defining the differences and similarities between alpha satellite transcripts produced from different chromosomes, as well as their overall role in centromere function. Although alpha satellite arrays on all chromosomes produce non-coding RNAs, we have discovered that each array produces distinct transcripts that are localized to the centromere in cis and stably associated with the chromosome throughout the cell cycle. An improved understanding of this unique class of non-coding RNA will shed light on how centromeres are formed, maintained, and, in some cases, inactivated to ensure chromosome stability.
Monica Alvarez is looking at the functional intersect of human genetic variation and susceptibility to Typhoid fever. Specifically, she studies how human genetic variation that decreases expression of VAC14, a host phosphoinositide scaffolding protein, modulates membrane lipids and subsequently increases Salmonella Typhi invasion. This association is not only seen in tissue culture but was also confirmed in a Typhoid Susceptibility study in Vietnam. Additionally, Monica has created a Zebrafish model to study early Salmonella infection. This model has shown that pretreating the Zebrafish with lipid-modifying drugs has a protective effect against Salmonella Typhi infection. This project has added to our understanding of basic host-pathogen interactions and may also have implications for prophylactic therapeutics against Typhoid Fever.
As a graduate student in MGM, I have been working in David Tobin’s laboratory on discovering and modeling bacterial factors in Mycobacterium tuberculosis (Mtb) that promote dissemination and skeletal infection. Using comparative genomics and a zebrafish-Mycobacterium marinum infection model, we have uncovered a genetic feature in ancient strains of Mtb that promotes disseminated disease. We believe that this may reveal a key step in the evolution of Mtb into a specialized pulmonary pathogen.
José Vargas-Muñiz is a 5th year Ph.D. candidate in the Steinbach lab. The Steinbach lab specialize in the etiological agent of invasive aspergillosis, the filamentous fungus Aspergillus fumigatus. Invasive aspergillosis is a leading cause of death in immunocompromised patients and carries a mortality rate of 40-60%. For clinical disease and invasive growth into host tissue, A. fumigatus requires hyphal growth and these hyphae are compartmentalized by the septum. Although the full function of the septum is unknown, it has been suggested that it is involved in increasing hyphal rigidity, limiting mechanical damage, and facilitating differentiation. Despite the probable role of septation in hyphal growth, the regulation of septum formation in Aspergillus fumigatus is unknown. Understanding septum formation would allow a better comprehension of fundamental disease pathogenesis and facilitate development of new, targeted fungal-specific therapeutics. In order to provide a critical insight into septum formation we decided to study a conserved family of GTP-binding proteins called septins. Septins are involved in a myriad of cellular processes, including septation, cell wall organization, and cytokinesis. A. fumigatus contains five septin genes: aspA, aspB, aspC, aspD and aspE. The first four, known as core septins, have orthologs in the model yeast Saccharomyces cerevisiae, but aspE is only found in filamentous ascomycetes. Septins organize in higher-order structures, yet it remains unclear how these complexes are regulated. Previous work in S. cerevisiae has shown that mutation of phosphorylation sites affects septin ring organization. However, the role of septin post-translational modification in the Aspergillus proteins has not been studied before my dissertation. Our overall goal is to delineate the role of septins and their post-translational modification in A. fumigatus growth and pathogenesis.
We have deleted all five septins genes in A. fumigatus, and found that AspA, AspB, AspC and AspE are required for regular septation. Only the deletion of core septin genes significantly reduced conidiation. the ΔaspB strain was also sensitive to anti-cell wall agents. Moreover, using a strain expressing AspB-GFP we were able to observe alteration of AspB localization patterns as well as a 7-fold increase in the number of structures found in the apical compartment after 2 hours of caspofungin, a b-glucan synthase inhibitor, treatment. While infection with the ΔaspB strain in a Galleria mellonella model of invasive aspergillosis showed hypervirulence, no virulence difference was noted when compared to the wild-type strain in a murine model of invasive aspergillosis. Due to the pleotropic role of AspB in A. fumigatus growth, we decided to study how AspB is regulated. We have deleted two non-essential kinases (Gin4 and Cla4) and one protein phosphatase 2A subunit (ParA). This approach revealed that deletion of these genes in a strains expressing AspB-GFP fusion protein resulted in altered AspB. LC-MS/MS phosphoproteomics show that AspB is phosphorylated in vivo at 7 residues. Additionally, deletion of the parA gene resulted in AspB being phosphorylated at two additional sites: T68 and S447. Phosphomimetics and non-phosphorylatable alleles of AspB were created. Mutation of T68 or S447 altered the localization of AspB but only the mutation of T68 resulted in an increased apical compartment. Taken together, these results point out the importance of AspB phosphorylation/dephosphorylation in the regulation of septation.
Throughout my time as a graduate student in Dr. Cullen’s lab my research has focused on two primary areas. First, I am investigating the use of the DNA editing CRISPR-Cas9 system as a means to target HIV-1. We have shown that by targeting highly conserved regions of HIV-1 (namely the TAR/Tat signaling system) we can effectively inhibit HIV-1 infection by expressing Cas9 and its cognate sgRNA in cells normally permissive for HIV-1 infection. Similarly, we are able to inactivate an integrated HIV-1 provirus in a latently infected cell line by transducing cells with a lentiviral vector expressing Cas9 and well as the cognate sgRNA. Currently, my research on this project focuses on investigating where in the cell Cas9 is cleaving the proviral DNA. Is cleavage occurring before integration or after integration has occurred? We have evidence that in the majority of cases cleavage may be occurring before integration of the provirus takes place. Finally, we are in the process of developing lentiviral vectors to express the CRISPR-Cas9 components in a humanized mouse model of HIV-1.
The second project I have spent much of my time on has been the development of novel technology to facilitate CRISPR-Cas9 vector design. There has been significant innovation in the development of smaller Cas9 proteins that are more amenable to vector packaging limits. However, little progress has been made to design a more compact system for sgRNA expression. Previously, our lab has shown that human tRNAs can be used to express micro RNAs. I have adapted this technology to show that a variety of tRNAs can be used to express sgRNAs at levels comparable to the traditional U6 promoter based expression. The use of tRNAs is advantageous because of their small size (~70 bp) relative to the U6 promoter (~250 bp). This allows us, for example, to include two sgRNA expression cassettes as well as a Cas9 expression cassette within a single AAV vector. Currently, we are adapting this novel technology into both our lentiviral and AAV vectors.
Category: Antiviral Innate Immunity
My thesis research focuses on understanding how RNA viruses, in particular hepatitis C virus (HCV), avoids detection by the antiviral innate immune system, a host defense program. Human cells normally activate this antiviral defense during RNA virus infection to limit viral replication. However, many viruses have evolved ways to circumvent detection by this innate immune program, allowing them to establish infection. One of the major antiviral sensor proteins of RNA virus infection is RIG-I. When RIG-I senses virus infection, it activates a signaling pathway through the adaptor protein MAVS to induce the production of type I interferon. Interestingly, HCV can avoid this sensing pathway through the actions of its NS3-NS4A protein. My research focuses on how the NS3-NS4A protein blocks antiviral innate immune signaling of the RIG-I pathway. I’m studying (1) the molecular features that regulate the localization of NS3-NS4A and (2) how the factors that are regulated by NS3-NS4A are important for antiviral innate immune signaling.
Mission Statement: The MGM Outreach Group consists of graduate students passionate about increasing public awareness and appreciation for science. We are committed to improving public knowledge of scientific concepts through hands on workshops because we believe that the best way to learn and understand science, is to do it! Our mission is to include the greater triangle area in the exciting field of microbial science and to connect with anyone interested in learning more about STEM fields.
Outreach members: Alfred Harding, Allison Roder, Caitlin Esoda, Calla Telzrow, Caitlyn Mitchell, Christine Vazquez, Firas Midani, Hannah McMillan, Hilary Renshaw, Jared Brewer, Jeff Bryant, Kaila Pianalto, Kayla Sylvester, Kelly Hughes, Kyle Gibbs, Matt Sacco, Michael McFadden, Nicole Stantial, Rebekah Dumm, Zach Holmes, Heather Froggatt and Shannon McNulty
1. October 19th – Science Under the Stars from 6-8pm
A group of us from MGM outreach played a role in the North Carolina Collaborative (NCC) Summer Research Experience Program. This summer program provides a high-quality research experience for undergraduate students, high school students, and high school teachers during the summer academic break. The program capitalizes on world-renowned training programs in place at Duke University under the leadership Dr. Danny Benjamin, professor of pediatrics at Duke and faculty associate director of the Duke Clinical Research Institute.
Our part was to introduce the participants to the basic sciences and explain to them how the research we do on a daily basis plays a role in clinical medicine and relates to translational studies. We conducted 4 different lab tours showing them 4 different pathogens (Cryptococcus, Tuberculosis, Influenza, and Malaria). Various MGM outreach participants volunteered their time to give presentations of their research on a broad level and to show demonstrations of various experiments (microscopy, egg influenza injections, and live imaging). The students toured the lab facilities and were able to interact with graduate students in small groups.
The event was a success and a week later when we went to talk with the students more about life as a graduate student, a lot of them expressed that touring the labs was their favorite part of the 8 week program!
Previous Outreach Leaders: Hannah Brown, Dora Posfai, Josh Messinger, Shannon Esher, Joe Saelens, Eric Walton, Katherine Rempe, Casey Perley, Kristen Smith
Student Run 2017
11 Graduate Students from MGM and one graduate student from Cell Biology teamed up in September to run a 200 mile relay race from Cumberland, Maryland to downtown Washington DC. The idea started with Shannon Esher and Allison Roder who heard about the race from a local running store. They recruited all of their friends and signed up for the race. The team, named ‘The Nerd Herd’ started a Monday evening running group and met for group runs in the months leading up to the race in order to get ready. The race, part of a series called Ragnar Relays, requires 12 runners to eat, sleep and live in 2 vans while teammates take turns running 3-12 miles at a time, working their way across the 200 mile stretch. Each runner runs three legs of the race, totaling anywhere from 14 to 28 miles over the entirety of the course. The team finished in about 36 hours and is hoping to complete another relay in Niagara Falls next year!
Students: Allison Roder, Shannon Esher, Hannah Brown, Jeff Bryant, Amy Hafez, Al Harding, Zack Holmes, Kaila Pianalto, Joe Saelens, Christine Vazquez, Eric Walton, and Kwabena Badu-Nkansah