STORM Super-Resolution Visualization of Self-Assembled γPFD Chaperone Ultrastructures in <i>Methanocaldococcus jannaschii</i>.

Abstract

Gamma-prefoldin (γPFD), a unique chaperone found in the extremely thermophilic methanogen <i>Methanocaldococcus jannaschii</i>, self-assembles into filaments <i>in vitro</i>, which so far have been observed using transmission electron microscopy and cryo-electron microscopy. Utilizing three-dimensional stochastic optical reconstruction microscopy (3D-STORM), here we achieve ∼20 nm resolution by precisely locating individual fluorescent molecules, hence resolving γPFD ultrastructure both <i>in vitro</i> and <i>in vivo</i>. Through CF647 NHS ester labeling, we first demonstrate the accurate visualization of filaments and bundles with purified γPFD. Next, by implementing immunofluorescence labeling after creating a 3xFLAG-tagged γPFD strain, we successfully visualize γPFD in <i>M</i>. <i>jannaschii</i> cells. Through 3D-STORM and two-color STORM imaging with DNA, we show the widespread distribution of filamentous γPFD structures within the cell. These findings provide valuable insights into the structure and localization of γPFD, opening up possibilities for studying intriguing nanoscale components not only in archaea but also in other microorganisms.