**3.2.2 A new view of zinc from XFM**

One of the first findings regarding zinc (utilizing sub-micron X-ray fluorescence imaging) was that it may be involved in cell differentiation, particularly looking at HL-60 cells (Glesne, Vogt et al. 2006). In examining the growth of human embryonic stem cells, taking a systems biology approach to examining entire colonies of cells and all the first row transition metals, we also found that the amount of zinc present in cells directly correlated with their differentiation (Wolford, Chishti et al. 2010). The images in Figure 1, of stem cells differentiated with retinoic acid, are particularly illustrative. Loss of Oct4 (pink) is associated with higher zinc (red in 'Zn' panel). This was found to be true regardless of the method of differentiation, or whether the cells at the outer edge or at the center of the colony

Inorganic Signatures of Physiology: The X-Ray Fluorescence Microscopy Revolution 83

maturation. While the reason for this is still unclear, the team has recently reported that experiments utilizing extra-cellular optically-fluorescent zinc indicators have shown that some of this zinc is exported upon fertilization (Kim, Bernhardt et al. 2011). This may be an

Copper is widely used in biology for enzymatic chemistry. Its ability to cycle between (I) and (II) oxidation states makes it particularly useful for reduction and oxidation chemistry. It is used to activate oxygen, detoxify radicals, and in mitochondrial function. Yet, for as much as is known about copper, direct X-ray fluorescence imaging is revealing new roles, and changes in distributions that may have the potential to serve as biomarkers of the

It was reported in 2006 by Gitlin et al. that copper in hippocampal neurons appeared to be exported following NMDA-receptor stimulation (Schlief, Craig et al. 2005). The use of Cu-64 to try to measure this export made it somewhat difficult to determine exactly how much of the cellular copper was exported, or image exactly where the copper was. But, clearly, new

Shortly after this, Finney et al. reported that a dramatic efflux of copper occurs during the angiogenic process of tubulogenesis, or the process by which new capillaries are formed. As mentioned earlier, copper had long been known to be important to angiogenesis, and thus also to the growth of cancerous tumors that rely upon a growing blood supply. By directly imaging the tubulogenesis process, at fixed points, using X-ray fluorescence microscopy scientists found that between 80-90% of the cell's copper was exported at early points, and then taken back up later in the growth of capillary-like structures (Finney, Mandava et al. 2007). Exactly why this happens remains a mystery, and has sparked new efforts in the development of tools for metalloproteomics (Finney, Chishti et al. 2010). From this, one might speculate that a role for copper in intercellular signaling, of

Taking this technique, and applying it back to the same sort of systems which Gitlin et al. had examined, leads to another remarkable finding. As shown in Figure 2, a typical SH-sy5y cell, the majority of cellular copper is typically localized in the perinuclear area in neuronal cells. Upon stimulation, the copper may be seen to relocalize such that a significant increase in the fraction of cellular copper that is along the dendrites of the cell is seen. Not only can fluxes of copper be seen in hippocampal neurons, but they are dependent on calcium, and induced by extracellular stimulation (Dodani, Domaille et al. 2011). Clearly, undiscovered roles for copper as an important part of cell signaling exist. And X-ray fluorescence imaging is enabling our further understanding of them. As roles for metals such as this are further defined, they hold the potential to reveal patterns and signatures that may become the

example of the cell using metal bioavailability to regulate protein function.

**3.3 Case 3: The potential of copper as a dynamic, signaling molecule** 

**3.3.2 Viewing copper differently – Dramatic fluxes of copper** 

**3.3.1 Established, enzymatic roles for copper** 

roles for copper were emerging.

some sort, may exist.

biomarkers of the future.

future.

were the ones to differentiate first. This exciting finding brings us to a new understanding of how little we know about the majority of nuclear zinc, as well as the roles of metals during differentiation.

Fig. 1. Correlating XRF and immuno-fluorescence images of human embryonic stem cells.

At the same time, zinc plays many roles in the cell, as well as roles even outside of the cell. An elegant example of work utilizing X-ray fluorescence to better understand zinc physiology is reported by the Kelleher group in their studies of lactation (McCormick, Velasquez et al. 2010). In contrast to the coordination of zinc throughout much of the cell, zinc in milk is significantly associated with lower molecular weight molecules – at relatively high concentration relative to other essential metals. And it is not known how the mammary gland regulates the transfer of zinc into milk. By directly imaging the zinc in mammary tissue of both lactating and non-lactating mice, the researchers were able to demonstrate that zinc associates with a distinct peri-nuclear pool. Further, through experiments utilizing the chemical indictor Fluo-zin-3, which is a zinc indicator, together with dyes for the endoplasmic reticulum, mitochondrion, and Golgi, optical fluorescence microscopy indicated that this zinc was 'labile' on account with its ability to bind the zinc dye, and at least partially associated with the Golgi complex. This work represents some of the first Xray fluorescence imaging of mammary tissue at the sub-micron scale, and suggests that the pathways for zinc export during lactation likely are similar to those utilized in the prostate.

Another particularly surprising finding from direct X-ray fluorescence imaging of metals in cells relates to fertilization. Work by a team of scientists from Northwestern University has recently shown that the accumulation of zinc is essential for fertilization (Kim, Vogt et al. 2010). In this work, single-cell elemental analysis of mouse oocytes by X-ray fluorescence microscopy revealed a 50% increase in total zinc content within the 12-14-h period of meiotic maturation. While the reason for this is still unclear, the team has recently reported that experiments utilizing extra-cellular optically-fluorescent zinc indicators have shown that some of this zinc is exported upon fertilization (Kim, Bernhardt et al. 2011). This may be an example of the cell using metal bioavailability to regulate protein function.
