Quantitative Super-Resolution Imaging Reveals Protein Stoichiometry and Nanoscale Morphology of Assembling HIV-Gag Virions

Abstract

The HIV structural protein Gag assembles to form spherical particles of radius ~70 nm. During the assembly process, the number of Gag proteins increases over several orders of magnitude, from a few at nucleation to thousands at completion. The challenge in studying protein assembly lies in the fact that current methods such as standard fluorescence or electron microscopy techniques cannot access all stages of the assembly process in a cellular context. Here, we demonstrate an approach using super-resolution fluorescence imaging that permits quantitative morphological and molecular counting analysis over a wide range of protein cluster sizes. We applied this technique to the analysis of hundreds of HIV-Gag clusters at the cellular plasma membrane, thus elucidating how different fluorescent labels can change the assembly of virions. Keywords: Super-resolution imaging; protein assembly; protein counting; HIV-GagPage 2 of 12 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 3 Viruses represent a major class of pathogens, whose assembly in the cellular context contains important information about the complex processes governing viral infection. Viruses are nanoscale objects that assemble from small nucleation complexes to ensembles containing thousands of molecules. In the case of human immunodeficiency virus (HIV), the viral components are targeted to the plasma membrane of infected cells where they assemble and eventually form spheres ~70 nm in radius. Viral assembly is widely studied using HIV-Gag, the main structural protein of HIV, which is sufficient to drive the assembly of virus-like particles (VLPs) in the absence of other viral components 1,2 . Fluorescence imaging has been used to follow the time-dependent increase in the intensity of Gag clusters in living cells 3,4 , revealing the time scale of virion formation. Electron microscopy (EM) has elucidated the spatial arrangement of Gag in fully formed virions [5][6][7][8] . However, studying the complete assembly process requires nanoscale resolution over a large dynamic range, since the size of a cluster ranges from a few molecules to several thousand. This cannot be achieved with standard fluorescence imaging methods since they lack both the necessary resolution to determine cluster morphology, and the sensitivity to detect smaller clusters. EM-based methods in turn lack information on protein identity; thus, complexes composed of small numbers of Gag proteins are difficult to identify, precluding the first step toward quantitative analysis. As an alternative approach, super-resolution fluorescence imaging based on single molecule localization (SR) 9-11 offers nanoscale resolution of structures formed by specific proteins. Here, we use an SR-based approach to quantitatively image hundreds of forming HIV-Gag virions in different stages of cluster formation. With this inf...

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

“…Single molecule counting-PALM is used to determine protein stoichiometry when single step photobleaching is not feasible (such as high density of proteins) [11][12][13][14] . Fluorescent proteins are photoactivated one at a time, detected and subsequently photobleached (Fig.…”

“…Imaging schemes have been proposed for controlled photoactivation of fluorescent proteins 11,13 . Furthermore, the cumulative number of fluorescent proteins detected in each frame has been used to determine whether the fluorescent proteins have been exhaustively imaged 11 .…”

“…Furthermore, the cumulative number of fluorescent proteins detected in each frame has been used to determine whether the fluorescent proteins have been exhaustively imaged 11 . The cumulative plot has an initial rise that reaches a plateau once the majority of fluorescent proteins have been detected.…”

“…These probes can be activated in sparse numbers over time such that their images are optically resolvable and can be precisely localized 4 . These techniques have been exploited to image sub-cellular structures [5][6][7] , visualize protein (co)-organization [8][9][10] and quantify protein stoichiometry [11][12][13][14][15] .…”

Fluorescence nanoscopy. Methods and applications

“…This protein has received much attention since the early proof-of-concept reports of superresolution, studying the assembly and dynamics of tdEosGag [34,41]. Gag clusters have been imaged at subdiffraction resolution to quantitatively characterise their size and morphological characteristics in different stages of their formation [209]. Importantly, the authors also found that fusion proteins Gag-mEos and Gag-tdEos organised differently, confirming previous evidence that fluorescent proteins (or at least large ones as tdEos) interfere with the assembly [210].…”