Doctor of Philosophy
Goldhawk, Donna E.
Prato, Frank S.
With its superb spatial and temporal resolution, magnetic resonance imaging (MRI) has great potential to track cellular activities that define early stages of disease. To improve molecular imaging techniques, we are developing MRI reporter gene expression based on the magnetosome. In magnetotactic bacteria (MTB), magnetosome formation compartmentalizes iron biominerals in membrane-enclosed vesicles. We hypothesize that essential magnetosome proteins interact in any cell type to form rudimentary magnetosome-like nanoparticles, providing a genetically-controlled contrast agent for molecular MRI.
MTB genes mamE, mamB, mamI, and mamL were cloned from M. magneticum sp. AMB-1 genomic DNA by PCR and inserted into fluorescent vectors to create Mam fusion proteins then stably expressed in human MDA-MB-435 melanoma cells. Cines of fluorescent elements detected in intact cells were captured with confocal microscopy (Nikon A1R) and analyzed using both ImageJ and Mathematica for Brownian motion and velocity. To obtain longitudinal and transverse relaxation rates, cells stably expressing magnetosome proteins were supplemented with 250 µM ferric nitrate, harvested, mounted in a gelatin phantom, and scanned at 3 Tesla (Biograph mMR).
Tomato-MamL, Tomato-MamL/GFP-MamI, and Tomato-MamB all express punctate, mobile fluorescence, while GFP-MamE expresses punctate but stationary fluorescence. Analysis of motility revealed that magnetosome proteins have variable diffusion coefficients due to their variable sizes, but all magnetosome proteins travel at a velocity of around 0.2 µm/s. Relaxation rates of iron-supplemented cells expressing Tomato-MamB, GFP-MamI, or Tomato-MamL have significantly higher R2 and R2* than non-supplemented cells. Interestingly, iron-supplemented cells expressing GFP-MamE or co-expressing FLAG-MamL/GFP-MamI had relaxation rates comparable to unsupplemented cell types.
This is the first report characterizing essential magnetosome proteins MamE, MamB, MamI, and MamL in mammalian cells. Analysis of motion shows that magnetosome proteins travel at velocities comparable to the mammalian motor protein myosin. Expression of either MamB, MamI or MamL increases transverse relaxation rates; however, co-expression of MamI and MamL reduces them again, suggesting a regulatory effect of magnetosome gene combinations. Biosynthesis of magnetosome-like nanoparticles in mammalian cells would provide an endogenous magnetic resonance (MR) contrast agent under genetic control. This patented technology would provide long-term molecular imaging for tracking cellular and molecular activities throughout the cell's life cycle.
Summary for Lay Audience
Magnetic Resonance Imaging (MRI) is a medical imaging technology that is used worldwide to diagnose disease. It is very useful as it can show subtle differences in the soft tissues of the body. However, in its present form it is limited to detection of about one million cells. Special contrast agents are being developed with the goal of reducing the minimally detected cells to about one thousand. We have been developing such a contrast agent and our theoretical calculations suggest that if we are successful MRI will be able to detect as few as 3,000 cells.
We have been molecular engineering such a contrast agent that when introduced into a human cell will become MRI visible when the cell performs a specific function. This required us to find genes as raw material for this process. We isolated four genes from a special kind of bacteria that produces a form of iron that is the greatest MRI contrast agent known.
We have successfully shown that each of these four genes can be introduced into human cells. But for this to work, all four protein products from these genes must find each other and co-operate in making a special iron particle within the cell. Currently, we have introduced two genes in the cell and found that these proteins can find each other. What remains now is to first introduce three genes and evaluate their interactions, then finally introduce all four genes to reach our ultimate goal of MRI detection of about 1,000 cells.
Sun, Qin, "Building a Magnetosome-like Nanoparticle for Magnetic Resonance Imaging" (2023). Electronic Thesis and Dissertation Repository. 9281.
Timelapse of MamI (left) and MamL (right) individual expression in the cell.
mamI-mamL-coexp-sped-up-gif.gif (988 kB)
Timelapse of MamI and MamL co-expression in the cell.
mamltrunc-sped-up-gif.gif (695 kB)
Timelapse of MamL-trunc individual expression in the cell.
maml-trunc-red-spedup-punctate.gif (211 kB)
Timelapse of MamL-trunc and MamI co-expression in the cell (diffuse MamI).
mamltruncplusi-spedup-gif.gif (1237 kB)
Timelapse of MamL-trunc and MamI co-expression in the cell (punctate MamI)
psf-LplusI-spedup-gif.gif (660 kB)
Timelapse of FLAG-MamL and MamI co-expression in the cell.
mamB-gif.gif (192 kB)
Timelapse of MamB individual expression in the cell.