DOI
https://doi.org/10.25772/FMW0-BF45
Defense Date
2014
Document Type
Thesis
Degree Name
Master of Science
Department
Microbiology & Immunology
First Advisor
Dr. Jessica Bell
Abstract
During viral infections, Toll-like receptor-3 (TLR3) stimulation initiates signaling to activate transcription of pro-inflammatory cytokines and type-1 interferons. Suppressor of IKK-ε (SIKE) interacts with two kinases in the signaling pathway, IKK-ε and TANK-binding kinase 1 (TBK1), inhibiting the transcription of type I interferons. Recently, this laboratory discovered that SIKE blocks TBK1-mediated activation of type I interferons by acting as a high affinity, alternative substrate of TBK1. Co-immunoprecipitation (co-IP) assays suggested that the SIKE interaction network impinged upon the cytoskeleton and RNA transport. To characterize SIKE’s function within the antiviral response, SIKE’s role in RNA transport and cytoskeletal rearrangements was targeted for further study through immunofluorescence assays (IFAs), using traditional confocal microscopy. SIKE was found to colocalize with cytoskeleton components (β-actin and α-tubulin), endosomal and plasma membrane markers (Rab11a, LAMP-1, and LC3), and ribosomes (S6). Additionally, IFA labeling for actin cytoskeleton-associated proteins revealed that SIKE colocalized with α-actinin, β-catenin, ezrin, and Focal Adhesion Kinase (FAK) in both myeloid and epithelial cells. These results were consistent with the hypothesis that SIKE functions in trafficking related to the anti-viral innate immune response.
To further delineate the colocalization of α-actinin, α-tubulin, actin, and S6 colocalization with SIKE, super-resolution microscopy, Structured Illumination Microscopy (SIM), was used. In both cell lines, we found that SIKE colocalized with α-actinin, but did not consistently colocalize with the other markers. Therefore, SIKE localizes with actin, tubulin, and S6 at distances greater than the 85nm resolution achieved using SIM techniques.
To address SIKE function following pathogen challenge, SIKE interactions were examined following two distinct stimuli. Polyinosinic acid: polycytidylic acid (poly(I:C)) was used as a mimic for viral dsRNA challenge. Following poly(I:C) stimulation, SIKE localization from 15 minutes to 24 hours showed little to no change. In addition to mediating a response to viral challenge, TBK1, the kinase that phosphorylates SIKE, maintains Salmonella-containing vacuoles (SCVs) following S. enterica serovar typhimurium infection. Therefore, changes in SIKE colocalization during Salmonella enterica serovar typhimurium infection were examined. Because TBK1 is integral to maintenance of the Salmonella containing vacuole, the hypothesis was that SIKE may also contribute to the host cell response to this infection. SIKE colocalization with various markers for Salmonella-containing vacuoles changes during the time course of Salmonella infection (15 minutes to 18 hours) in a manner that suggested SIKE may play a role in signaling and trafficking related to the host cell’s response to this infection. These studies support a SIKE and α-actinin interaction that is consistent with SIKE functioning in the cytoskeleton. Furthermore, SIKE’s altered colocalization following Salmonella challenge suggest this protein may contribute to cytoskeletal structures that maintain these pathogen-containing vacuoles.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
12-18-2014