Direct Visualization of Cellular Protein Complexes in situ by Fluorescence-Guided Cryo-FIB-SEM and Cryo-ET

Citation

Wang, Jue (Phyllis) (2025) Direct Visualization of Cellular Protein Complexes in situ by Fluorescence-Guided Cryo-FIB-SEM and Cryo-ET. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/rmdd-5t54. https://resolver.caltech.edu/CaltechTHESIS:02252025-210356380

Abstract

Cryogenic electron tomography (cryo-ET) is a technique that can reconstruct three-dimensional volumes of large protein complexes in situ at sub-nanometer resolution. In addition to imaging proteins extracted from cells, cryo-ET also allows direct visualization of macromolecular complexes in their native environment. To reveal molecular details buried deeply inside thick eukaryotic cells, cryogenic focused ion beam milling with scanning electron microscopy (cryo-FIB-SEM) has been established as the leading approach for preparing thin sections of cells suitable for cryo-ET. Recent advances in cryo-FIB-SEM systems integrate fluorescence microscopy (cryo-FM-FIB-SEM) to help direct the milling to specific labeled regions of interest. This method has had success localizing large organelles and protein aggregates. Unfortunately, it is difficult to localize small and rare targets along the optical axis of the cryo-FIB. This thesis work pioneered a customized integrated tri-coincident imaging system (ENZEL) that allows for simultaneous fluorescence imaging and cryo-FIB milling. This novel method allows precise targeting of small and rare structures with a high success rate compared to other systems. To demonstrate the imaging workflow, we applied this approach to visualize the microtubule organizing center (MTOC), a crucial organelle responsible for cell division and cellular transport in mammalian cells. It presents as a single fluorescent punctum expanding approximately 1 µm in diameter in live cells, making it a challenging target for cryo-FM-FIB-SEM. Our cryo-tomograms resolved the molecular architecture of the MTOC and revealed molecular details at the microtubule nucleation sites. We then used the ENZEL to explore more complicated biological systems. Here we chose the NLRP3 inflammasome, a master mediator of innate immunity colocalized with the MTOC. We captured the first in-situ image of the NLRP3 inflammasome and showed new mechanistic insights that this complex forms a condensate at the MTOC, halting cell division and inducing drastic organelle changes.

Thesis Files