Donna E. Goldhawk

Using the magnetosome to model effective gene‐based contrast for magnetic resonance imaging

Formation of iron biominerals is a naturally occurring phenomenon, particularly among magnetotactic bacteria which produce magnetite (Fe(3) O(4) ) in a subcellular compartment termed the magnetosome. Under the control of numerous genes, the magnetosome serves as a model upon which to (1) develop gene-based contrast in mammalian cells and (2) provide a mechanism for reporter gene expression in magnetic resonance imaging (MRI). There are two main components to the magnetosome: the biomineral and the lipid bilayer that surrounds it. Both are essential for magnetotaxis in a variety of magnetotactic bacteria, but nonessential for cell survival. Through comparative genome analysis, a subset of genes characteristic of the magnetotactic phenotype has been found both within and outside a magnetosome genomic island. The functions of magnetosome-associated proteins reflect the complex nature of this intracellular structure and include vesicle formation, cytoskeletal attachment, iron transport, and crystallization. Examination of magnetosome genes and structure indicates a protein-directed and stepwise assembly of the magnetosome compartment. Attachment of magnetosomes along a cytoskeletal filament aligns the magnetic particles such that the cell may be propelled along an external magnetic field. Interest in this form of magnetotaxis has prompted research in several areas of medicine, including magnetotactic bacterial targeting of tumors, MR-guided movement of magnetosome-bearing cells through vessels and molecular imaging of mammalian cells using MRI, and its hybrid modalities. The potential adaptation of magnetosome genes for noninvasive medical imaging provides new opportunities for development of reporter gene expression for MRI.

Biophysical features of MagA expression in mammalian cells: implications for MRI contrast

We compared overexpression of the magnetotactic bacterial gene MagA with the modified mammalian ferritin genes HF + LF, in which both heavy and light subunits lack iron response elements. Whereas both expression systems have been proposed for use in non-invasive, magnetic resonance (MR) reporter gene expression, limited information is available regarding their relative potential for providing gene-based contrast. Measurements of MR relaxation rates in these expression systems are important for optimizing cell detection and specificity, for developing quantification methods, and for refinement of gene-based iron contrast using magnetosome associated genes. We measured the total transverse relaxation rate (R2*), its irreversible and reversible components (R2 and R2′, respectively) and the longitudinal relaxation rate (R1) in MDA-MB-435 tumor cells. Clonal lines overexpressing MagA and HF + LF were cultured in the presence and absence of iron supplementation, and mounted in a spherical phantom for relaxation mapping at 3 Tesla. In addition to MR measures, cellular changes in iron and zinc were evaluated by inductively coupled plasma mass spectrometry, in ATP by luciferase bioluminescence and in transferrin receptor by Western blot. Only transverse relaxation rates were significantly higher in iron-supplemented, MagA- and HF + LF-expressing cells compared to non-supplemented cells and the parental control. R2* provided the greatest absolute difference and R2′ showed the greatest relative difference, consistent with the notion that R2′ may be a more specific indicator of iron-based contrast than R2, as observed in brain tissue. Iron supplementation of MagA- and HF + LF-expressing cells increased the iron/zinc ratio approximately 20-fold, while transferrin receptor expression decreased approximately 10-fold. Level of ATP was similar across all cell types and culture conditions. These results highlight the potential of magnetotactic bacterial gene expression for improving MR contrast.

Microscopic and biochemical analysis of the viability and permeability of guinea pig amnion and chorion laeve in vitro

Tissue viability and permeability of guinea pig amnion and chorion leave were analyzed microscopically and biochemically. The vital dyes T1111 and fluorescein diacetate were used to locate and determine the integrity of cell plasma membranes in early and late tissue in vitro using confocal laser scanning microscopy and scanning electron microscopy. Early amnion and chorion laeve were each found to contain a single epithelial cell layer, composed of membrane-intact cells. In contrast, plasma membrane lesions were present throughout the epithelium of late amnion. Late chorion laeve contained both regions of intact and damaged epithelial cells on its maternal side. There was also a layer of membrane-intact squamous cells on the fetal side of late chorion laeve. ATP measurements confirmed that early fetal membranes were viable after incubation in isotonic salt solutions at physiological pH. Late amnion was depleted of ATP stores while late chorion laeve retained its capacity for generating energy. These viability markers indicate that late guinea pig amnion is not a viable tissue in vitro, while late chorion laeve is a viable but probably degenerating tissue. Confocal X-Z scans were used to trace the movement of T1111 through the tissue as an indication of permeability to free solutes. Whereas dye will permeate across the main thickness of early amnion and chorion leave, it did not pass between cells, but was blocked, presumably by a line of tight junctions. Late amnion was characterized by the complete permeability to T1111. Late chorion leave contained regions where solute migration was blocked, but overall was a permeable tissue. These results provide an important context for the interpretation of molecular movement across fetal membranes.

Transfer of steroidal and nonsteroidal compounds across guinea pig fetal membranes.

Transfer of steroidal and nonsteroidal compounds across guinea pig amnion and chorion laeve was investigated as a function of stage of gestation, tissue orientation, steroid specificity, and molecular size. Each fetal membrane was examined at early and late stages of gestation, before and after pubic symphysis relaxation. Early amnion was impermeable to macromolecules and small charged molecules while [3H]estrone and [3H]pregnenolone were transferred, the latter depending on tissue orientation and involving conjugation at the basolateral interface. After symphysis dilation, amnion transferred all substrates tested with the exception of BSA; the molecular weight cutoff was approximately 5,000. Unlike amnion, early chorion transferred both free and conjugated steroids as well as inorganic sulfate. Transfer of estrone involved conjugation and depended on tissue orientation. Transfer of [3H]estrone-sulfate, [3H]estrone-glucuronide, and [3H]pregnenolone-sulfate was similar despite selective deconjugating activity toward estrone-sulfate. Near term, chorion was impermeable to inorganic sulfate and transfer of estrone-glucuronide depended on tissue orientation, involving deconjugation in the maternal to fetal direction. At no stage of gestation did chorion transfer macromolecules. These results suggest that the transfer of free and conjugated steroids across fetal membranes is differentially regulated by tissue, its stage of development, and direction of transfer.

The Interface Between Iron Metabolism and Gene-Based Iron Contrast for MRI

Using a gene-based approach to track cellular and molecular activity with magnetic resonance imaging (MRI) has many advantages. The strong correlation between transverse relaxation rates and total cellular iron content provides a basis for developing sensitive and quantitative detection of MRI reporter gene expression. In addition to biophysical concepts, general features of mammalian iron regulation add valuable context for interpreting molecular MRI predicated on gene-based iron labeling. With particular reference to the potential of magnetotactic bacterial gene expression as a magnetic resonance (MR) contrast agent for mammalian cell tracking, studies in different cell culture models highlight the influence of intrinsic iron regulation on the MRI signal. The interplay between dynamic regulation of mammalian iron metabolism and expression systems designed to sequester iron biominerals for MRI is presented from the perspective of their potential influence on MR image interpretation.

β-glucuronidase is not required for transfer of [3H]-estrone-[14C]glucuronide across guinea pig fetal membranes

To understand the means whereby a charged, estrogen conjugate may be transferred across guinea pig amnion and chorion, the permeability to [3H]estrone-[14C]glucuronide was examined at 45 days and near term. No evidence of deconjugation was obtained in either early or late amnion, despite significantly greater transfer near term. Early amnion was virtually impermeable, regardless of ATP depletion. In contrast, early chorion transferred estrone-glucuronide without any requirement for deconjugation or ATP. No effect of tissue orientation was observed in amnion; whereas, incubations from maternal to fetal side of late chorion exhibited beta-glucuronidase activity. Inhibition of the latter demonstrated that hydrolysis was concomitant with but not required for transport. [3H]Estrone produced by deconjugation was enzymatically reduced after pubic symphysis relaxation, although beta-glucuronidase activity began prior to this stage. Transport across late fetal membranes was not saturable and chorion incubations from maternal to fetal side demonstrated a lower transport capacity. In either tissue orientation, late chorion displayed a lower rate of transfer than amnion. These results indicate that fetal membranes possess distinct abilities for transferring intact estrone-glucuronide, depending on stage of development and tissue orientation. The passive nature of transport and its dependence on structural characteristics is consistent with possible regulation of tight junctions.

Transfer of [3H]estrone-[35S]sulfate across guinea pig fetal membranes

The possible role of fetal membrane deconjugating activity in the movement of a charged steroid conjugate between fetal and maternal compartments was investigated. The ability of amnion and chorion laeve to transfer [3H]estrone-[35S]sulfate was assessed in both orientations of guinea pig tissue at 45 days and near parturition. While early amnion was impermeable, late tissue transferred approximately 50% (w/w) of the substrate in a bidirectional process that was non-saturable and independent of either deconjugation or ATP. Transfer across early chorion was similar to late amnion. Saturation curves from each tissue were superimposable, as were those of the time course. Transfer across both early and late chorion proceeded in the absence of deconjugation, with no effect of tissue orientation or ATP depletion. However, late chorion exhibited a decrease in estrone-sulfate transfer, as verified by concentration dependency and time course analyses, though transport across the tissue remained non-saturable. The results in amnion were congruous with the presence and absence of tight junctions in the epithelium of early and late tissue, respectively. However, sulfoconjugate transfer across early chorion proceeded in the presence of a paracellular barrier, suggesting specialized regulation of the transport process which extended late into gestation.

Forming Magnetosome-Like Nanoparticles in Mammalian Cells for Molecular MRI

To identify molecular activities that define the early stages of disease progression, there is a critical need to noninvasively image these processes. For this, the magnetosome is an ideal structure by which cellular and molecular magnetic resonance imaging (MRI) may be refined. Within the design of this magnetotactic bacterial compartment lies the genetic machinery to fashion versatile and effective gene-based magnetic resonance (MR) contrast in mammalian systems. Review of the current understanding of magnetosome formation indicates that a subset of bacterial genes provides a rudimentary compartment within which iron biomineral synthesis may be tailored. Based on the success of MagA and Mms6 expression in a variety of mammalian cells and tissue, future applications using a combination of magnetosome genes are envisioned, to build magnetosome-like nanoparticles in mammalian cells that will provide distinct MR signatures for reporter gene expression. An estimate of the limits of sensitivity of MRI reporter gene expression based on a magnetosome-like nanoparticle is provided. Such a tool is expected to have broad application, including molecular imaging of iron metabolism and magnetic particle imaging.