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. 2019 Oct 9;19(10):6955-6963.
doi: 10.1021/acs.nanolett.9b02266. Epub 2019 Sep 25.

Engineering a Genetically Encoded Magnetic Protein Crystal

Thomas L Li  1   2 , Zegao Wang  3 , He You  4 , Qunxiang Ong  1 , Vamsi J Varanasi  1 , Mingdong Dong  3 , Bai Lu  4 , Sergiu P Paşca  2 , Bianxiao Cui  1
Affiliations

Engineering a Genetically Encoded Magnetic Protein Crystal

Thomas L Li et al. Nano Lett. .

Abstract

Magnetogenetics is a new field that leverages genetically encoded proteins and protein assemblies that are sensitive to magnetic fields to study and manipulate cell behavior. Theoretical studies show that many proposed magnetogenetic proteins do not contain enough iron to generate substantial magnetic forces. Here, we have engineered a genetically encoded ferritin-containing protein crystal that grows inside mammalian cells. Each of these crystals contains more than 10 million ferritin subunits and is capable of mineralizing substantial amounts of iron. When isolated from cells and loaded with iron in vitro, these crystals generate magnetic forces that are 9 orders of magnitude larger than the forces from the single ferritin cages used in previous studies. These protein crystals are attracted to an applied magnetic field and move toward magnets even when internalized into cells. While additional studies are needed to realize the full potential of magnetogenetics, these results demonstrate the feasibility of engineering protein assemblies for magnetic sensing.

Keywords: Magnetogenetics; ferritin; magnetic; protein crystal.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
(a) Schematic of the three plasmids used in ftn-PAK4: control inkabox-PAK4cat, GFP-tagged plasmid, and ferritin-linked plasmid, alongside a schematic of how the ferritin subunits might fit inside of the crystals’ hollow channel. (b) Phase-contrast and GFP images of ftn-PAK4 and inka-PAK4 growing in HEK293T cells over 72 h. White arrows highlight individual crystals. (c) Quantification of the length of ftn-PAK4 and inka-PAK4 crystals over 72 h, with n > 150 crystals and p < 0.0001 for all conditions. (d) Quantification of the proportion of GFP-positive cells that grew ftn-PAK4 or inka-PAK4 crystals over time. Data beyond 24 h for inka-PAK4 crystals was omitted, as it became impossible to assign crystals to individual cells. Error bars are the standard deviation of 3 fields, containing between 150 and 700 cells each. (e) Confocal images of cells cotransfected with the membrane marker CAAX-mCherry and either inka-PAK4 or ftn-PAK4, imaged 72 h after transfection. The left inset images are averaged XZY cross-sections along the length of the crystal, for inka-PAK4, and along the portion of the crystal protruding beyond the cell body, for ftn-PAK4. The right inset images are slices taken from the same Z-stack as the ftn-PAK4 image, zoomed in on the boxed region to demonstrate membrane wrapping around the tapered tip.
Figure 2.
Figure 2.
(a) Representative images of ftn-PAK4 and inka-PAK4 crystals after being isolated from cells. (b) Prussian blue staining of isolated ftn-PAK4 and inka-PAK4 crystals after exposure to iron. (c) STEM imaging of isolated ftn-PAK4 and inka-PAK4 crystals after iron exposure. The inset shows zoomed-in images. (d) EDX spectra of ftn-PAK4 and inka-PAK4 crystals after iron exposure.
Figure 3.
Figure 3.
(a) Schematic and picture of the chamber built to image magnetic pulling of crystals. (b) Time-lapse imaging of ftn-PAK4 and inka-PAK4 crystals in the magnetic imaging chamber, alongside bright-field images to indicate the position of the magnet. (c) Time-lapse of magnetic pulling of ftn-PAK4 and inka-PAK4 crystals acquired using confocal imaging. Each image is an XZ cross-section of a portion of the imaging area, with the magnet at the top of the image. (d) Plot of the frame-by-frame changes in z positions of representative ftn-PAK4 and inka-PAK4 crystals over time. (e) Plot of average z-velocity during movement, as measured by confocal imaging. n = 104 for iron-loaded ftn-PAK4, n = 73 for non-iron-loaded ftn-PAK4, n = 65 for iron-exposed inka-PAK4, and n = 69 for non-iron-exposed inka-PAK4.
Figure 4.
Figure 4.
(a) Photograph of the setup used for magnetic pulling of crystals on cells. (b) Cropped time-lapse of magnetic pulling. HEK 293T cells were labeled with Cellmask orange and incubated with a crystal suspension before trypsinization. White arrows indicate ftn-PAK4 crystals that have moved during the time-lapse. (c) Prussian blue stain of HEK 293T cells with ftn-PAK4 crystals exposed to different iron-loading conditions.
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