Each year the Astbury Centre holds a research retreat to promote interdisciplinary activity, to discuss and present latest findings and to promote and encourage interdisciplinary research at the highest level. I communicated my results from a Chikungunya virus project in a 15-min presentation. Slides from this presentation are shown here.
Shining a light on virus infection with high-resolution microscopy
1. SHINING A LIGHT ON VIRUS INFECTION WITH
HIGH-RESOLUTION MICROSCOPY
Roland Remenyi, Hong Zhou#, Ren Sun#, Mark Harris
#: UC Los Angeles
School of Molecular and Cellular Biology
FACULTY OF BIOLOGICAL SCIENCES
Mark Harris Laboratory
2. 1. Bioimaging of Chikungunya virus (CHIKV) replication
A. Live-cell fluorescence imaging
B. Confocal Microscopy with Airyscan
Molecular Interactions in Cells -
Preview of today’s talk
3. 2.5µm 250nm 50nm
Cellular level Subcellular level
Supramolecular &
Protein level
HCV-infected
hepatocyte
Mito-
chondria ER
Golgi
3-D or 4-D
Light/Laser
Microscopy 2-D or 3-D Electron Microscopy
Super-resolution Light
Microscopy
Viral Particles
Investigating virus infection with
bioimaging at different resolutions
6. CHIKV genome organization and
fluorescent reporter design
Untranslate
d region nsP1 nsP2 nsP3 nsP4
Noncoding
region
Subgenomic
promoter
(-) strand RNA
Synthesis, RNA
capping
helicase
proteinase
RNA-dependent
RNA
polymeraseRNA synthesis
Engineered reporter constructs: Subgenomic
promoter
Role of nsP3 protein?
7. Fluorescent reporter protein reveals
dynamics of viral replication in living cells
Imaged with Incucyte Zoom, Stonehouse Lab
8. ZsGreen
mCherry Overlay
Live cell transfected
with CHIKV replicon
Imaged with Confocal
Microscope in Cat3
Facility,
Dr. Jamel Mankouri
nsP3 forms both punctae and tubules
9. Airyscan unprocessed Airyscan processed FBS Bioimaging Facility,
Prof. Michelle Peckham
Dr. Brian Jackson
Dr. Sally Boxall
Imaging replicating cells with LSM880 with
Airyscan technology
11. Future plans: Investigating the role of nsP3
in the CHIKV life cycle
Examine changes in nsP3 localization in the presence of
mutations and small-molecule inhibitors
Track formation of nsP3-positive clusters over time
Determine nsP3 localization by Stochastic Optical
Reconstruction Microscopy (STORM)
12. Charles Rice, Rockefeller
Steven Foung, Stanford
Confocal microscope in Cat3 Facility, Dr. Jamel Mankouri
FBS bioimaging facility, LSM880 Airyscan, Prof. Michelle Peckham, Dr. Brian Jackson, Dr. Sally
Boxall
Incucyte Zoom System, Prof. Nicola Stonehouse
Electron Imaging Center for Nanomachines, Dr. Ivo Atanasov, Dr. Xing Zhang
Advanced Light Microscopy and Spectroscopy Facility, Dr. Laurent Bentolila, Dr. Matt Schibler
Electron Microscopy Laboratory, Dr Sergey Ryazantsev
Reagents
Facilities/Microscopes
Funding
NIH (National Institutes of Health) Virology/Gene therapy training grant
Seed Grant for AIDS Related Malignancy Research
Wellcome Trust Investigator and Multi-user equipment Award
Dr. Hangfei Qi, Dr. Vaithi Arumugaswami (former member)
Ren Sun lab HCV team
Predoctoral Researchers
Tamar Stokelman, Jerry Lo
Francis Chisari, Scripps
California Nanosystems
Institute
University of Leeds
SMCB Virology Chikungunya Team
Dr. Mark Harris, Dr. Andrew Tuplin
Grace Roberts, Lauren Branfield, Raymond Li
Andres Merits, University of Tartu
Dr. Niluka Goonawardane, Dr. Hazel Stewart, Carsten Zothner, Christopher Bartlett, Lorna Kelly, Joseph Lattimer, Tracy
Yin
Harris Lab
Acknowledgements
Contact: r.g.remenyi@leeds.ac.uk
Twitter: rol_rem
Editor's Notes
Task: Viruses are intracellular parasites whose pathogenicity depends on the interaction with infected cells. We use a bioimaging approach to investigate infection at different resolutions.
Goal: to establish a research portfolio to investigate Chikungunya virus, a re-emerging member of the alphavirus family. It’s transmitted through mosquitoes. Disease occurs in Africa, Asia and the Indian subcontinent, but currently there’s a serious outbreak in Latin America and the Caribbean.
This improvement in resolution is achieved by the use of a multichannel area detector with 32 elements –each detector element functions as a single pinhole and allows more light to be collected. This differs from a classical confocal microscope, which illuminates one spot on the sample and employs a single pinhole to reject out of focus light.
The size of the pinhole determines how much light reaches the detector, so whilst a smaller pinhole increases resolution, it also means less light gets through and the signal-to-noise ratio (SNR) decreases significantly. By using a series of pinholes, more light is collected in total, maintaining a good SNR, whilst the resolution can be improved by using 32 smaller pinholes in the detector.