Caenorhabditis elegans might not be an animal you’re particularly familiar with, but this humble little worm is one of the most important tools we have for studying metals in biological systems. These 1 mm long roundworms are self-fertilising, meaning you can raise literally millions of genetically identical offspring, and they were the first multi-cellular organism to have their entire genome mapped.
In a paper we’ve just published (coming from blog contributor Dr Gawain McColl) in Metallomics (grab it for free here), we partnered with researchers at the Australian Synchrotron’s X-ray Fluorescence Microscopy beamline and our new collaborator Dr Verena Wimmer, who runs the great Florey Advanced Microscopy Facility to look at how we can use a range of different techniques to examine metals in this worm at sub-micrometre levels.
The XFM beamline at the Australian Synchrotron is pretty unique, thanks to the contributions of some pretty talented physicists who designed a detector that allows use to zap these humble worms with light as bright as the sun and pick up the unique emission that is characteristic of each element in the periodic table at a speed that isn’t possible at most other facilities. This allowed us to focus incredibly intense X-rays into a beam nearly twenty-times narrower than the width of a human hair, and measure the metals and other bio-elements present within that area of the worm. Fly-scanning, where we zip the worm through the beam as quickly as possible allows us to build incredibly high resolution images; in the Supplementary Information (an example is here) these maps are more than 100 cm wide, meaning we can look at elemental distribution well below the dimensions of single cells. Matching the elemental distribution to known anatomical features of the worm helps us understand why metals and elements are located in certain cell types, giving us clues as to what role they play in development, life and death.
Here, you can see how anatomical features of the worm (Panel A, courtesy of WormAtlas, which is a great place to learn more about C. elegans) can be linked to not only the X-ray generated structure of the worm (called Compton here, after the type of scattering we measured that’s representative of the whole organism), but also the elemental content. Elements like sulfur (B) have shown us detail in the head we’ve previously never been able to see, whilst other metals like calcium and manganese once again highlight that it’s the intestine where most of the (metal) action is happening in C. elegans.
As you have probably gathered by the title of this blog, and our numerous other posts on the subject, iron is something we care a lot about here. XFM only gives us part of the story when it comes to metals like iron, as the maps we produce are simply a readout of the total metal levels. Iron might be just one metal, but it binds to hundreds of proteins that use it for all sorts of different roles. This is particularly important when looking at iron in disease (our favourite subject), as it’s typically not iron alone that’s misbehaving, but also the proteins responsible for keeping it in check.
So, we used a couple of different microscopy techniques to see if we could get more clues about what type of iron is the predominant form in the worm. The XFM image (A) shows that most of the detectable iron in the worm is, like calcium and manganese, concentrated along the gut. Thanks to the histology skills of Dr Ian Birchall at the Florey Institute, he managed to use Perls staining, a technique that’s been around for over 100 years, to stain for iron that’s not bound to heme (the most common iron-containing functional group in proteins like hemoglobin or several important enzymes). When comparing these images side-by-side (or top-to bottom in this case, Panel B), we were able to spot that the little ‘dots’ of iron in the XFM map matched up nicely with iron found in the nucleus of gut cells. Without the improved resolution of the XFM beamline we used in this study, this wouldn’t have been possible before.
To take it one step further, we approached Verena to use some of her particularly fancy microscopes to image a genetic strain of worm that makes ferritin, the protein responsible for storing iron when it’s not in use, with has an additional tag on it that emits a strong green fluorescence when excited by a laser. Once again, little defined deposits of the protein matched up nicely with our XFM and Perls images. This confirmed what we’ve suspected for a while, that most of the iron in C. elegans is bound to this protein, which we’ve previously talked about in a paper in Chemical Science having major implications for iron regulations going wrong in ageing. Verena also made this very cool movie showing the ferritin distribution in worms in three dimensions:
C. elegans are fast becoming a staple for us here at Ferrumblogger. Watch this space for some more news coming about other exciting experiments we’re doing with them.