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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Abstract

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.

In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

Introduction

Cryo-electron tomography (cryoET) is a powerful imaging technique that allows three-dimensional (3D) visualization of cells and tissues and provides insights into the organization of native organelles and cellular structures at molecular resolution in a close-to physiological state1. However, the inherently low contrast of unstained frozen-hydrated specimen, combined with their radiation sensitivity, makes it difficult to locate areas of interest inside a cell and subsequently performing the tilt series successfully without damaging the target area. In order to overcome these problems, a correlative approach that combines light and electron microscopy is ne....

Protocol

1. HeLa Cell Culture on Carbon-coated, Gold EM Finder Grids

  1. Glow discharge the carbon-side of a 200-mesh R2/2 Quantifoil gold EM finder grid under 25 mA for 25 sec.
  2. Coat the EM grid with fibronectin, by floating it, carbon-side down, onto a 40 μl droplet of 50 μg/ml fibronectin solution, then disinfect it under UV light for 2 hr in a tissue culture hood.
  3. Plate HeLa cells onto the grid at a density of 2 x 104 cells/ml (total 2 ml culture) in a glass-bottom culture dish and .......

Representative Results

To characterize the dynamic behavior of the virus particles, HeLa cells infected with HIV-1 were imaged by high-speed confocal live-cell microscopy and the particle movements were analyzed by automated 3D particle tracking (Figure 1). To avoid the time lapse of several minutes that can occur between collection of the last confocal live-cell image and plunge-freezing (which may be long enough to lose correlated HIV-1 particles), a cryo-fluorescence light microscopy stage (Figure 3) was de.......

Discussion

We have presented a straightforward set of protocols to provide an advanced correlative approach to analyze dynamic cellular events using time-lapse confocal, live-cell fluorescence imaging followed by cryoET. Our methodological development to correlate 3D live-cell imaging with high-resolution cryoET is critical to investigate many challenging biological problems, such as visualizing rare, dynamic (not static, as previously reported), and diffraction limited viral particles and their interactions with host cells. The sp.......

Disclosures

The authors declare no competing financial interests.

Acknowledgements

The authors would like to thank Travis Wheeler and the machine shop at the Department of Cell Biology and Physiology, University of Pittsburgh for construction of the cryo-fluorescence sample stage, Changlu Tao and Cheng Xu at the University of Science and Technology of China for technical assistance, and Dr. Teresa Brosenitsch for critical reading of the manuscript. This work was supported by the National Institutes of Health (GM082251 & GM085043).

....

Materials

NameCompanyCatalog NumberComments
Reagents
DMEM 4.5 g/L Glucose w/ L-GlutamineAtlanta Biologicals, Inc. Lawrenceville, GA12-604F 
FibronectinSigma, St. Louis, MOF1141-1MGHeLa cell culture on EM grids
Cell trackerInvitrogen Corporation, Carlsbad, CAC34552Red CMTPX
Protein A gold conjugatesTed Pella, Inc., Redding, CA15822-115 nm diameter
0.2 μm fluorescent microspheresInvitrogen Corporation, Carlsbad, CAF8811Yellow-green fluorescent
Heat-inactivated fetal calf bovine serumInvitrogen Corporation, Carlsbad, CA10082-139 
Penicillin-StreptomycinInvitrogen Corporation, Carlsbad, CA15140-122 
Glass-bottom culture dishMatTek CorporationP35G-1.5-14-C 
Gold quantifoil finder EM gridsQuantifoil Micro Tools, Jena, GermanyR2/2 Au NH2 200 mesh 
PBSInvitrogen Corporation, Carlsbad, CA70011-044 
Equipment
Glow-discharge device 100XEMS, Hatfield, PA  
Tecnai Polara G2 electron microscope with a Field Emission GunFEI, Hillsboro, OR 300 keV
Vitrobot Mark IIIFEI, Hillsboro, OR  
Olympus IX 71 microscopeOlympus America Inc., Center Valley, PA LUCPlanFLN 40X/0.6 NA (2.7-4 mm working distance) objective lens
Nikon TiE microscopeNikon Instruments, Melville, NY using a 60X/1.35 NA oil immersion objective lens
Sweptfield confocal microscopePrairie Technologies, Middleton, WI  
Tokai Hit live cell chamberTokyo, Japan  
Cryo-fluorescence sample stageHome-made Homebuilt by machine shop, see reference 15 for the design.

References

  1. Leis, A., Rockel, B., Andrees, L., Baumeister, W. Visualizing cells at the nanoscale. Trends in Biochemical Sciences. 34, 60-70 (2009).
  2. Maurer, U. E., Sodeik, B., Grunewald, K. Native 3D intermediates of membrane fusion in herpes simplex virus....

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Correlative MicroscopyCryo electron TomographyLive cell ImagingHIV 1Virus host Interactions3D Structural AnalysisFluorescence MicroscopyCryo fluorescence Microscopy

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