Going Deeper: Biomolecular Tools for Acoustic and Magnetic Imaging and Control of Cellular Function

Most cellular phenomena of interest to mammalian biology occur within the context of living tissues and organisms. However, today’s most advanced tools for observing and manipulating cellular function, based on fluorescent or light-controlled proteins, work best in cultured cells, transparent model species, or small, surgically accessed anatomical regions. Their reach into deep tissues and larger animals is limited by photon scattering. To overcome this limitation, we must design biochemical tools that interface with more penetrant forms of energy. For example, sound waves and magnetic fields easily permeate most biological tissues, allowing the formation of images and delivery of energy for actuation. These capabilities are widely used in clinical techniques such as diagnostic ultrasound, magnetic resonance imaging, focused ultrasound ablation, and magnetic particle hyperthermia. Each of these modalities offers spatial and temporal precision that could be used to study a multitude of cellular processes in vivo. However, connecting these techniques to cellular functions such as gene expression, proliferation, migration, and signaling requires the development of new biochemical tools that can interact with sound waves and magnetic fields as optogenetic tools interact with photons. Here, we discuss the exciting challenges this poses for biomolecular engineering and provide examples of recent advances pointing the way to greater depth in in vivo cell biology

Quantitative Analysis of Relaxation Rate Dependence on Interecho Time in MagA-expressing, Iron-labeled Cells

Reporter gene-based methods of labeling cells with iron is an emerging method of providing magnetic resonance imaging (MRI) contrast for long-term cell tracking and monitoring of cellular activities. This thesis investigates 9.4 T NMR properties of mammalian cells over-expressing a magnetotactic bacterial putative iron transport gene, MagA, and the associated untransfected parental cells. Cells were cultured in medium alone or supplemented with 250 μM ferric nitrate. Using the Carr-Purcell-Meiboom-Gill sequence, the relationship between R2 and interecho time was analyzed for each of the cell types using a model based on water diffusion in weak magnetic field inhomogeneities (Jensen and Chandra, 2000) as well as a fast-exchange model (Luz and Meiboom, 1963). Iron levels were assessed with inductively-coupled plasma mass spectrometry. As expected from previous work, the iron content in iron-supplemented, MagA-expressing cells was higher than the unsupplemented or parental cell lines. With regard to NMR, increases in R2 with increasing interecho time were typically greatest in the cells containing higher iron content. The dependence of R2 on interecho time in iron-supplemented, MagA-expressing cells was better represented by the Jensen-Chandra model compared to the Luz-Meiboom model, which is consistent with comparisons of these models in iron-containing tissues. On the other hand, the Luz-Meiboom model performed better than the Jensen-Chandra model for the remaining cell types. These findings provide insight into the high field relaxation mechanisms present in cells expressing a candidate MR reporter gene, which should be valuable for optimizing MRI contrast for long-term cell tracking and monitoring of cellular activities

Genetic Manipulation of Iron Biomineralization Enhances MR Relaxivity in a Ferritin-M6A Chimeric Complex

Ferritin has gained significant attention as a potential reporter gene for in vivo imaging by magnetic resonance imaging (MRI). However, due to the ferritin ferrihydrite core, the relaxivity and sensitivity for detection of native ferritin is relatively low. We report here on a novel chimeric magneto-ferritin reporter gene – ferritin-M6A – in which the magnetite binding peptide from the magnetotactic bacteria magnetosome-associated Mms6 protein was fused to the C-terminal of murine h-ferritin. Biophysical experiments showed that purified ferritin-M6A assembled into a stable protein cage with the M6A protruding into the cage core, enabling magnetite biomineralisation. Ferritin-M6A-expressing C6-glioma cells showed enhanced (per iron) r2 relaxivity. MRI in vivo studies of ferritin-M6A-expressing tumour xenografts showed enhanced R2 relaxation rate in the central hypoxic region of the tumours. Such enhanced relaxivity would increase the sensitivity of ferritin as a reporter gene for non-invasive in vivo MRI-monitoring of cell delivery and differentiation in cellular or gene-based therapies

Non-invasive imaging using reporter genes altering cellular water permeability

Non-invasive imaging of gene expression in live, optically opaque animals is important for multiple applications, including monitoring of genetic circuits and tracking of cell-based therapeutics. Magnetic resonance imaging (MRI) could enable such monitoring with high spatiotemporal resolution. However, existing MRI reporter genes based on metalloproteins or chemical exchange probes are limited by their reliance on metals or relatively low sensitivity. Here we introduce a new class of MRI reporters based on the human water channel aquaporin 1. We show that aquaporin overexpression produces contrast in diffusion-weighted MRI by increasing tissue water diffusivity without affecting viability. Low aquaporin levels or mixed populations comprising as few as 10% aquaporin-expressing cells are sufficient to produce MRI contrast. We characterize this new contrast mechanism through experiments and simulations, and demonstrate its utility in vivo by imaging gene expression in tumours. Our results establish an alternative class of sensitive, metal-free reporter genes for non-invasive imaging

Non-invasive imaging using reporter genes altering cellular water permeability

Non-invasive imaging of gene expression in live, optically opaque animals is important for multiple applications, including monitoring of genetic circuits and tracking of cell-based therapeutics. Magnetic resonance imaging (MRI) could enable such monitoring with high spatiotemporal resolution. However, existing MRI reporter genes based on metalloproteins or chemical exchange probes are limited by their reliance on metals or relatively low sensitivity. Here we introduce a new class of MRI reporters based on the human water channel aquaporin 1. We show that aquaporin overexpression produces contrast in diffusion-weighted MRI by increasing tissue water diffusivity without affecting viability. Low aquaporin levels or mixed populations comprising as few as 10% aquaporin-expressing cells are sufficient to produce MRI contrast. We characterize this new contrast mechanism through experiments and simulations, and demonstrate its utility in vivo by imaging gene expression in tumours. Our results establish an alternative class of sensitive, metal-free reporter genes for non-invasive imaging

Biomolecular MRI reporters: Evolution of new mechanisms

Magnetic resonance imaging (MRI) is a powerful technique for observing the function of specific cells and molecules inside living organisms. However, compared to optical microscopy, in which fluorescent protein reporters are available to visualize hundreds of cellular functions ranging from gene expression and chemical signaling to biomechanics, to date relatively few such reporters are available for MRI. Efforts to develop MRI-detectable biomolecules have mainly focused on proteins transporting paramagnetic metals for T_1 and T_2 relaxation enhancement or containing large numbers of exchangeable protons for chemical exchange saturation transfer. While these pioneering developments established several key uses of biomolecular MRI, such as imaging of gene expression and functional biosensing, they also revealed that low molecular sensitivity poses a major challenge for broader adoption in biology and medicine. Recently, new classes of biomolecular reporters have been developed based on alternative contrast mechanisms, including enhancement of spin diffusivity, interactions with hyperpolarized nuclei, and modulation of blood flow. These novel reporters promise to improve sensitivity and enable new forms of multiplexed and functional imaging

Biomolecular Tools for Noninvasive Imaging and Manipulation of Engineered Cells

Today’s most advanced tools for imaging and controlling cellular function are based on fluorescent or light-controlled proteins, which have limited utility in large organisms or engineered living materials due to the scattering of photons. Deeply penetrant forms of energy such as magnetic fields and sound waves, while routinely used to monitor and treat diseases on the tissue and organism level, do not process the equivalent set of biomolecular tools for interfacing with biology on the molecular and cellular level. Emerging technologies discussed in this thesis aim to bridge this gap by harnessing biomolecules that have the appropriate physical properties to interact with sound waves or magnetic fields in such a way that enables the visualization and control of specific cells (Chapter 1). We describe two additions to the expanding toolkit for noninvasive imaging and control. In the first case, we show that gas vesicles, a class of hollow protein nanostructures naturally found in aquatic single-cell organisms, can be used as acoustic actuators to enable the control of cellular forces, movement, and patterning using ultrasound (Chapter 2). In the second case, we show that aquaporins, a class of membrane water channels, can be used to alter cellular permeability and serve as genetic reporters for magnetic resonance imaging (Chapter 3). These tools provide critical capabilities for interfacing with cellular function noninvasively and could open the door to applications in various research, biomedical, and industrial settings