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Research

High resolution imaging of virus infection and antiviral signaling in single cells

Unlike the adaptive immune system, the cell’s innate immune response is effective in most mammalian cell types and mounted within several hours of infection. During viral infection, several pathogen recognition receptors, including RIG I Like Receptors (RLR) and Toll-Like Receptors (TLR), are responsible for viral recognition and activation of several hundred antiviral genes involved in innate immunity.  Retinoic acid Inducible Gene I (RIG I), is the major cytosolic receptor which detects human pathogens including Influenza, Sendai and Rabies Virus. RIG I distinguishes viral RNA from cellular RNA by the unique viral features including 5’ triphosphate and double stranded RNA. Upon activation, RIG I initiates the antiviral signaling cascade through IRF3 dimerization (Interferon regulatory factor 3) that induces the expression of several hundred antiviral genes including over expression of RIG I itself.  Using Sendai virus as a model pathogen, our goal is to characterize the interaction of RIG I with viral RNA in mammalian cells. Employing the recently developed method single molecule Fluorescent In Situ Hybridization (FISH), we can localize genomic viral RNA in infected cells. Here, multiple fluorescently labeled DNA probes are annealed to complementary regions of target viral RNA allowing detection and quantification of viruses in single cells.  Combined with conventional Immunofluorescence, the location of RIG I and other host proteins can be detected simultaneously. Some important questions we are addressing include localization patterns of viral RNA, proteins and RIG I, and rate of viral replication as a function of time, and receptor concentration.

 

 

 

 

 

 

 

 

 

 

 

Sendai Virus RNA FISH
HeLa cells

 

New single molecule fluorescence assay development and calibration: Protein Induced Fluorescence Enhancement

Single molecule FRET has been widely used for monitoring protein-nucleic acids interactions.  Direct visualization of the interactions, however, often requires a site specific labeling of the protein, which can be circuitous and inefficient.  In addition, FRET is insensitive to distance changes in the 0-3 nm range.  Recently, we developed a single molecule fluorescence assay termed Protein Induced Fluorescence Enhancement (PIFE) and calibrated it on several protein systems.  This method circumvents protein labeling and displays a marked distance dependence below 4 nanometer distance range.  The enhancement of fluorescence is based on the photophysical phenomenon whereby the intensity of a fluorophore increases upon proximal binding of a protein.  Our data reveals that the method can resolve as small as a single base pair distance at the extreme vicinity of the fluorophore, where the enhancement is maximized. In addition, we have demonstrated the general applicability and distance sensitivity of PIFE on multiple protein-nucleic acid test systems including but not limited to BamHI restriction enzyme binding to double stranded DNA,  DExH enzyme RIG‐I  translocation on RNA, and filament dynamics of RecA on single stranded DNA.  The high spatio-temporal resolution data and sensitivity to short distances combined with the ability to bypass protein labeling makes this assay an effective alternative or a complement to FRET.

 

DNA damage repair and genome integrity by recombinase/anti-recombinase balance

DNA is constantly exposed to genotoxic elements such as UV light and environmental toxins. Therefore, DNA repair system remains active at all times, responding to the frequent needs.  Aberrantly repaired DNA, however induces chromosomal rearrangement and mutations which lead to cancer.  Currently we do not have a thorough understanding of the molecular mechanism that governs the DNA repair process. What are the key steps involved in detecting and responding to DNA damage?  To best address this question, we will elucidate the initial steps of homologous recombination, which is one of the major DNA repair pathways.  Recombinase (Rad51) forms a filament on damaged/resected single stranded DNA to perform homologous recombination.  This activity is counterbalanced by the anti-recombinase which uses its ATP-dependent translocation to dismantle the recombinase filament.  The balance between the two opposing activities is crucial for maintenance of the genome integrity.  In collaboration with Tom Ellenberger laboratory (Washington University in St. Louis) and Patrick Sung laboratory (Yale University), we seek to use single-molecule fluorescence assays to monitor real time dynamics of individual Rad51 nucleoprotein filament and its interaction with Srs2 (as well as its human homologs).  We aim to uncover the mode of Rad51 formation and removal by the anti-recombinases which will reveal how a balance between recombinase and anti-recombinase is established in yeast and human systems.  In addition, we plan to perform fluorescence cell imaging to quantitatively analyze the DNA damage and repair process in vivo.

 

Gene silencing: microRNA processing by Dicer-TRBP

microRNA is a genome-encoded small RNA which plays a major role in gene silencing mediated by RNA interference pathway.  Once transcribed in the nucleus and exported to cytosol as a primary microRNA (pri-miRNA), it undergoes three major steps which lead to the disruption of mRNA.  First, an RNAseIII enzyme, Dicer cleaves the long stem-looped pri-miRNA into a short double stranded RNA.  Second, the double stranded RNA is unwound into two single stranded RNA termed passenger and guide strand and the guide strand is preferentially loaded to the RNA induced silencing complex (RISC).  Third, the endonuclease, Argonaut in RISC searches, finds and cleaves the mRNA target and thereby down regulating the protein expression.  Although much details have been revealed about the first and the third steps, the second step remains unclear.  In collaboration with Jennifer Doudna laboratory (University of California, Berkeley, HHMI), we seek to determine the mechanistic details involved in selection of the guide strand and concomitant or subsequent unwinding of siRNA mediated by Dicer-TRBP and RISC.

 

Double-strand RNA Binding Protein Profiling

The dsRNA binding protein (DRBP) family comprises a growing number of eukaryotic, prokaryotic, and viral-encoded products that share an evolutionarily conserved dsRNA binding domain (DRBD)and they have been identified to function in a diverse range of critically important roles in the cell, including RNA editing, cleavage and silencing as in RNA interference and anti-viral immunity pathway. My project is to study proteins with one or multiple DRBDs at the single molecule level, aiming to characterize them in terms of substrate specificity, binding affinity, motion upon binding dsRNA substrates and multiple DRBD collaboration/redundancy. I clone DRBP genes from Human Open Reading Frame Library, overexpress them in human A549 cells, immobilize the proteins on quartz slide surface by Single Molecule Pull Down and investigate their dynamic property upon biding dsRNA substrates using Protein Induced Fluorescence Enhancement (PIFE). For instance, PIFE data below is showing the intensity fluctuation of Cy3, which we used to label dsRNA substrates, indicating that this DRBP, TRBP, is sliding back and forth along dsRNA strand (Fig.c and d).