Superresolution imaging of HIV in infected cells with FlAsH-PALM
Open Access
- 14 May 2012
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 109 (22), 8564-8569
- https://doi.org/10.1073/pnas.1013267109
Abstract
Imaging protein assemblies at molecular resolution without affecting biological function is a long-standing goal. The diffraction-limited resolution of conventional light microscopy (∼200–300 nm) has been overcome by recent superresolution (SR) methods including techniques based on accurate localization of molecules exhibiting stochastic fluorescence; however, SR methods still suffer important restrictions inherent to the protein labeling strategies. Antibody labels are encumbered by variable specificity, limited commercial availability and affinity, and are mostly restricted to fixed cells. Fluorescent protein fusions, though compatible with live cell imaging, substantially increase protein size and can interfere with their biological activity. We demonstrate SR imaging of proteins tagged with small tetracysteine motifs and the fluorescein arsenical helix binder (FlAsH-PALM). We applied FlAsH-PALM to image the integrase enzyme (IN) of HIV in fixed and living cells under experimental conditions that fully preserved HIV infectivity. The obtained resolution (∼30 nm) allowed us to characterize the distribution of IN within virions and intracellular complexes and to distinguish different HIV structural populations based on their morphology. We could thus discriminate ∼100 nm long mature conical cores from immature Gag shells and observe that in infected cells cytoplasmic (but not nuclear) IN complexes display a morphology similar to the conical capsid. Together with the presence of capsid proteins, our data suggest that cytoplasmic IN is largely present in intact capsids and that these can be found deep within the cytoplasm. FlAsH-PALM opens the door to in vivo SR studies of microbial complexes within host cells and may help achieve truly molecular resolution.This publication has 51 references indexed in Scilit:
- Fast, three-dimensional super-resolution imaging of live cellsNature Methods, 2011
- Flexible Use of Nuclear Import Pathways by HIV-1Cell Host & Microbe, 2010
- Light-Induced Dark States of Organic Fluochromes Enable 30 nm Resolution Imaging in Standard MediaBiophysical Journal, 2009
- Microscopy and its focal switchNature Methods, 2008
- Fluorescence nanoscopy by ground-state depletion and single-molecule returnNature Methods, 2008
- Live-cell photoactivated localization microscopy of nanoscale adhesion dynamicsNature Methods, 2008
- High-density mapping of single-molecule trajectories with photoactivated localization microscopyNature Methods, 2008
- HIV-1 DNA Flap formation promotes uncoating of the pre-integration complex at the nuclear poreThe EMBO Journal, 2007
- Electron cryotomography of immature HIV-1 virions reveals the structure of the CA and SP1 Gag shellsThe EMBO Journal, 2007
- Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)Nature Methods, 2006