Iron-sequestering nanocompartments as multiplexed Electron Microscopy gene reporters

Abstract

Multi-coloredgene 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][15][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 enzymes can be genetically targeted into the encapsulin lumen in bacterial and mammalian hosts [22][23][24][25][26] .

Advances in encapsulin nanocompartment biology and engineering

“…(c) Schematic representation of the heterologous expression and self‐assembly of encapsulins in mammalian cells, with performance of automated TEM image classification by decision tree and matrices for MxEnc + MxBCD (Mx), QtEnc + QtIMEF (Qt), and H‐chain ferritin (F) via a deep learning approach. Reprinted with permission from (Sigmund et al, 2019). Copyright 2019 American Chemical Society.…”

“…Another recent study involved the use of multiple iron‐sequestering encapsulins as multiplexed electron microscopy gene reporters, or “EMcapsulins” (Sigmund et al, 2019). Here, two different iron‐sequestering encapsulin systems were employed due to their ability to generate high contrast in TEM experiments: the T = 4 encapsulin from Q. thermotolerans (QtEnc) with IMEF cargo (QtIMEF), and the T = 3 encapsulin from M. xanthus (MxEnc) with ferritin‐like cargo proteins (MxBCD).…”

Bioengineering a light-responsive encapsulin nanoreactor: a potential tool for photodynamic therapy

“…Fe 2+/3+ ) into and out of its internal cavity. 3,13 As depicted in Figure 2a, we hypothesized that molecular oxygen (O2) substrate can diffuse through the open surface pores of Enc-mSOG, enabling its interaction with the mSOG cargo within. Enc-mSOG can then be activated "on demand" with blue light to photoconvert O2 into 1 O2.…”

“…organelles) into designer 'nanoreactors' that can augment/enhance existing biological reactions, or enable the creation of entirely new synthetic reactions. [1][2][3] This includes protein compartments that encase enzymes within a selectively permeable protein shell that self-assembles from multiple protein subunits. 4,5 This design isolates and promotes specialized reactions by placing catalytic proteins, their substrates and cofactors within close proximity of each other;…”

“…6 Encapsulins are a newly established class of prokaryotic protein nanocompartments, which self-assemble from identical protein subunits into semi-permeable icosahedral protein shells with T = 1 (60 subunits, 20-24 nm), T = 3 (180 subunits, 30-32 nm) or T = 4 (240 subunits, 43 nm) symmetries. 3,[7][8][9] In nature, they encase functional cargo proteins that help their microbial hosts maintain iron homeostasis, cope with oxidative and nitrosative stress, and/or safely derive energy from ammonium. [10][11][12] Moreover, encapsulins recognise and selectively encapsulate native cargo proteins that display a short encapsulation signal peptide (ESig), a distinct mechanism that can be adapted to instead package foreign cargo, reprogramming the nanocompartments' functionality.…”