Iron-Sequestering Nanocompartments as Multiplexed Electron Microscopy Gene Reporters
journal contributionposted on 07.06.2019, 00:00 by Felix Sigmund, Susanne Pettinger, Massimo Kube, Fabian Schneider, Martina Schifferer, Steffen Schneider, Maria V. Efremova, Jesús Pujol-Martí, Michaela Aichler, Axel Walch, Thomas Misgeld, Hendrik Dietz, Gil G. Westmeyer
Multicolored gene reporters for light microscopy are indispensable for biomedical research, but equivalent genetic tools for electron microscopy (EM) are still rare despite the increasing importance of nanometer resolution for reverse engineering of molecular machinery and reliable mapping of cellular circuits. We here introduce the fully genetic encapsulin/cargo system of Quasibacillus thermotolerans (Qt), which in combination with the recently characterized encapsulin system from Myxococcus xanthus (Mx) enables multiplexed gene reporter imaging via conventional transmission electron microscopy (TEM) in mammalian cells. Cryo-electron reconstructions revealed that the Qt encapsulin shell self-assembles to nanospheres with T = 4 icosahedral symmetry and a diameter of ∼43 nm harboring two putative pore regions at the 5-fold and 3-fold axes. We also found that upon heterologous expression in mammalian cells, the native cargo is autotargeted to the inner surface of the shell and exhibits ferroxidase activity leading to efficient intraluminal iron biomineralization, which enhances cellular TEM contrast. We furthermore demonstrate that the two differently sized encapsulins of Qt and Mx do not intermix and can be robustly differentiated by conventional TEM via a deep learning classifier to enable automated multiplexed EM gene reporter imaging.
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pore regionselectron microscopyMultiplexed Electron Microscopy Gene Reporters Multicolored gene reportersCryo-electron reconstructionsheterologous expressionlight microscopy4 icosahedral symmetryMxmultiplexed EM gene reporter imagingexhibits ferroxidase activitymultiplexed gene reporter imagingMyxococcus xanthusTEM contrastQt encapsulin shell self-assemblestransmission electron microscopyIron-Sequestering NanocompartmentsQuasibacillus thermotoleransnanometer resolutionencapsulin systemintraluminal iron biomineralization