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Super-resolution imaging of nuclear import of adeno-associated virus in live cells.

ABSTRACT: Adeno-associated virus (AAV) has been developed as a promising human gene therapy vector. Particularly, recombinant AAV vector (rAAV) achieves its transduction of host cells by crossing at least three physiological barriers including plasma membrane, endosomal membrane, and nuclear envelope (NE). So far, the AAV transduction mechanism has not been explored thoroughly at the single viral particle level. In this study, we employed high-speed super-resolution single-point edge-excitation sub-diffraction (SPEED) microscopy to map the events of single rAAV2 particles infecting live human cells with an unprecedented spatiotemporal resolution of 9-12?nm and 2-20?ms. Data reveal that rAAV2 particles are imported through nuclear pore complexes (NPCs) rather than nuclear membrane budding into the nucleus. Moreover, approximately 17% of the rAAV2 molecules starting from the cytoplasm successfully transverse the NPCs to reach the nucleoplasm, revealing that the NPCs act as a strict selective step for AAV delivery. This study lastly suggests a new pathway to improve AAV vectors for human gene therapy.

SUBMITTER: Kelich JM

PROVIDER: S-EPMC4667716 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Super-resolution imaging of nuclear import of adeno-associated virus in live cells.

Kelich Joseph M JM Ma Jiong J Dong Biao B Wang Qizhao Q Chin Mario M Magura Connor M CM Xiao Weidong W Yang Weidong W

Molecular therapy. Methods & clinical development 20151202

Adeno-associated virus (AAV) has been developed as a promising human gene therapy vector. Particularly, recombinant AAV vector (rAAV) achieves its transduction of host cells by crossing at least three physiological barriers including plasma membrane, endosomal membrane, and nuclear envelope (NE). So far, the AAV transduction mechanism has not been explored thoroughly at the single viral particle level. In this study, we employed high-speed super-resolution single-point edge-excitation sub-diffra ...[more]

PMID: 26665132

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Project description:The rapid development in fluorescence microscopy and imaging techniques has greatly benefited our understanding of the mechanisms governing cellular processes at the molecular level. In particular, super-resolution microscopy methods overcome the diffraction limit to observe nanoscale cellular structures with unprecedented detail, and single-molecule tracking provides precise dynamic information about the motions of labeled proteins and oligonucleotides. Enhanced photostability of fluorescent labels (i.e., maximum emitted photons before photobleaching) is a critical requirement for achieving the ultimate spatio-temporal resolution with either method. While super-resolution imaging has greatly benefited from highly photostable fluorophores, a shortage of photostable fluorescent labels for bacteria has limited its use in these small but relevant organisms. In this study, we report the use of a highly photostable fluoromodule, dL5, to genetically label proteins in the Gram-negative bacterium Caulobacter crescentus, enabling long-time-scale protein tracking and super-resolution microscopy. dL5 imaging relies on the activation of the fluorogen Malachite Green (MG) and can be used to label proteins sparsely, enabling single-protein detection in live bacteria without initial bleaching steps. dL5-MG complexes emit 2-fold more photons before photobleaching compared to organic dyes such as Cy5 and Alexa 647 in vitro, and 5-fold more photons compared to eYFP in vivo. We imaged fusions of dL5 to three different proteins in live Caulobacter cells using stimulated emission depletion microscopy, yielding a 4-fold resolution enhancement compared to diffraction-limited imaging. Importantly, dL5 fusions to an intermediate filament protein CreS are significantly less perturbative compared to traditional fluorescent protein fusions. To the best of our knowledge, this is the first demonstration of the use of fluorogen activating proteins for super-resolution imaging in live bacterial cells.

Dataset Information

Super-resolution imaging of nuclear import of adeno-associated virus in live cells.

Publications

Super-resolution imaging of nuclear import of adeno-associated virus in live cells.

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