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
Nanomaterials are of enormous value for biomedical applications because of their customizable features. However, the material properties of nanomaterials can be altered substantially by interactions with tissue thus making it important to assess them in the specific biological context to understand and tailor their effects. Here, a genetically controlled system is optimized for cellular uptake of superparamagnetic ferritin and subsequent trafficking to lysosomes. High local concentrations of photoabsorbing magnetoferritin give robust contrast in optoacoustic imaging and allow for selective photoablation of cells overexpressing ferritin receptors. Genetically controlled uptake of the biomagnetic nanoparticles also strongly enhances third-harmonic generation due to the change of refractive index caused by the magnetiteprotein interface of ferritins entrapped in lysosomes. Selective uptake of magnetoferritin furthermore enables sensitive detection of receptor-expressing cells by magnetic resonance imaging, as well as efficient magnetic cell sorting and manipulation. Surprisingly, a substantial increase in the blocking temperature of lysosomally entrapped magnetoferritin is observed, which allows for specific ablation of genetically defined cell populations by local magnetic hyperthermia. The subcellular confinement of superparamagnetic ferritins thus enhances their physical properties to empower genetically controlled interrogation of cellular processes with deep tissue penetration.The interest in overexpressed ferritin has recently been revived in attempts to create biomagnetic actuators aimed at evoking controlled cellular responses to magnetic fields. In this regard, ferritin was used to manipulate cellular processes via magnetic hyperthermia or magnetomechanical transduction, [4] although the biophysical mechanisms underlying these effects are so far not understood well on a theoretical level. [5] Fully genetically encoded sensors and actuators, which do not necessitate supplementation of synthetic components, have become the frequently preferred solutions for, e.g., fluorescent Ca 2+ imaging and optogenetics. However, with regard to noninvasive techniques with deep tissue penetration such as optoacoustic (OA) imaging or MRI, synthetic nanostructures often still possess superior material properties as compared to genetically encodable biomaterials.Semigenetic approaches, which consist of a genetic component that interacts with an exogenous compound, can exploit the superior physical properties of synthetic nanostructures for remote cell actuation and deep tissue imaging and at the same time benefit from genetic targetability. [6] A recent study showcased the use of ferritin in a semigenetic approach to obtain cellular MR-contrast. [7] It was hypothesized that the enhanced contrast resulted from ferritin agglomeration inside lysosomes after cellular uptake through murine T cell immunoglobulin domain and mucin domain 2 (Tim-2) receptor. This contrastenhancing effect could be exploited even more effectively if the...
Loss mechanisms in fluid heating of cobalt ferrite (CFO) nanoparticles and CFO-Pd heterodimer colloidal suspensions are investigated as a function of particle size, fluid concentration and magnetic field amplitude. The...
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] .
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