STORM Super-Resolution Visualization of Self-Assembled γPFD Chaperone Ultrastructures in Methanocaldococcus...

Area of Science:

• Microscopy
• Structural Biology
• Archaea Biology

Background:

• Gamma-prefoldin (γPFD) is a unique chaperone protein identified in the thermophilic archaeon Methanocaldococcus jannaschii.
• γPFD is known to self-assemble into filaments in vitro, previously visualized by electron microscopy techniques.
• Understanding the in vivo structure and localization of γPFD is crucial for elucidating its cellular functions.

Purpose of the Study:

• To resolve the ultrastructure of gamma-prefoldin (γPFD) both in vitro and in vivo using super-resolution microscopy.
• To determine the cellular distribution and organization of γPFD filaments within Methanocaldococcus jannaschii cells.
• To establish 3D-STORM as a viable method for studying nanoscale archaeal components.

Main Methods:

• Utilized three-dimensional stochastic optical reconstruction microscopy (3D-STORM) for high-resolution imaging.
• Employed CF647 NHS ester labeling for in vitro visualization of purified γPFD filaments and bundles.
• Implemented immunofluorescence labeling of a 3xFLAG-tagged γPFD strain for in vivo imaging within M. jannaschii cells.

Main Results:

• Achieved approximately 20 nm resolution, enabling precise localization of individual fluorescent molecules.
• Successfully visualized filamentous and bundled structures of purified γPFD in vitro.
• Demonstrated the widespread distribution of filamentous γPFD structures within M. jannaschii cells, co-localizing with DNA.

Conclusions:

• 3D-STORM microscopy provides unprecedented nanoscale resolution for visualizing γPFD ultrastructure in vitro and in vivo.
• γPFD forms extensive filamentous structures within M. jannaschii cells.
• This study opens new avenues for investigating nanoscale structures in archaea and other microorganisms using super-resolution imaging techniques.