Iron-Sequestering Nanocompartments as Multiplexed Electron Microscopy Gene Reporters

Abstract

Multi-colored gene reporters such as fluorescent proteins are indispensable for biomedical research, but equivalent tools for electron microscopy (EM), a gold standard for deciphering mechanistic details of cellular processes 1,2 and uncovering the network architecture of cell-circuits 3,4 , are still sparse and not easily multiplexable. Semi-genetic EM reporters are based on the precipitation of exogenous chemicals 5-9 which may limit spatial precision and tissue penetration and can affect ultrastructure due to fixation and permeabilization. The latter technical constraints also affect EM immunolabeling techniques 10-13 which may furthermore be complicated by limited epitope accessibility. The fully genetic iron storage protein ferritin generates contrast via its electron-dense iron core 14-16 , but its small size complicates differentiation of individual ferritin particles from cellular structures. To enable multiplexed gene reporter imaging via conventional transmission electron microscopy (TEM), we here introduce the encapsulin system of Quasibacillus thermotolerans (Qt) as a fully genetic iron-biomineralizing nanocompartment. We reveal by cryo-electron reconstructions that the Qt monomers (QtEnc) self-assemble to nanospheres with T=4 icosahedral symmetry and an~44 nm diameter harboring two putative pore regions at the fivefold and threefold axes. We furthermore show that the native cargo (QtIMEF) auto-targets to the inner surface of QtEnc and exhibits ferroxidase activity leading to efficient iron sequestration inside mammalian cells. We then demonstrate that QtEnc can be robustly differentiated from the non-intermixing encapsulin of Myxococcus xanthus 17 (Mx,~32 nm) via a deep-learning model, thus enabling automated multiplexed EM gene reporter imaging in mammalian cells. Encapsulins are a class of proteinaceous spherical nanocompartments naturally occurring in bacteria and archaea, so far described as icosahedral structures with either T=1 (60 subunits,~18 nm diameter) or T=3 (180 subunits,~30 nm) symmetry, which can encapsulate cargo proteins with a wide range of functions 18-21. It has also been shown that foreign cargos such as fluorescent proteins or

Encapsulins—Bacterial Protein Nanocompartments: Structure, Properties, and Application

“…Another promising approach is based on encapsulin-based reporters, which can be expressed in mammalian cells using bioengineering [ 79 , 80 ]. Thus, encapsulins allow iron sequestration via the formation of 10–30-nm particles, which could possibly enable the MRI imaging of the cells.…”

“…In one study [ 79 ], Quasibacillus thermotolerans (Qt) and Myxococcus xanthus (Mx) encapsulin expression was achieved by transient co-transfection in HEK293T cells. The cells were transfected with plasmid DNA encoding the encapsulin shell, its cargo protein (ferroxidase enzyme), and iron transporter, which is required for more efficient iron transport.…”

Introduction of Surface Loops as a Tool for Encapsulin Functionalization

“…Previous studies already showed how encapsulin can be employed for liver targeting, vaccine development, imaging or as nanoreactors. 6,20,[32][33][34] Therefore, it is crucial to deepen our knowledge about encapsulin modification and to optimize their production and usability. Hence, this study paves the way for the development of improved encapsulin engineering and production.…”

“…4 Previous studies on protein cages can be found for a variety of different applications such as targeted drug delivery, gene reporting and vaccines. [5][6][7] Protein cages are well-defined monodisperse, hollow structures and they are biocompatible thanks to the fact that they are protein based, with a spherical shape, in the range of 1-100nm size. [8][9][10] Among the variety of protein cages, our work focuses on encapsulin cages which were discovered in some bacteria and archaea 11 and may have viral origin shared with the capsid proteins of tailed bacteriophages.…”

The Emerging Trend of Bio-Engineering Approaches for Microbial Nanomaterial Synthesis and Its Applications

“…Upon expression in HEK293T cells, this encapsulin protein system self-assemble into nanospheres having icosahedral symmetry that further sequesters and encapsulates ferritin like proteins into it. The mammalian cells harboring this sequestered cargo protein enables its multiplexed gene reporter imaging using the conventional transmission electron microscopy (Sigmund et al, 2019). In magnetotactic bacteria, magA gene encodes for a protein involved in iron sequesteration and transport into the cells.…”