Tuesday, December 12, 2017

The Protein Gemologists That Shape Nature's Living Gemstones

The intricate splendor of the Thalassiosira pseudonana frustule
viewed under a scanning electron microscope. (Adapted from here.)

By Ishmael Al-Ghalith    

            Alive, inert. The variety of both microbial and mineral species stands testament to the resplendence of our earthly abode. Despite the nigh overwhelming diversity in these two seemingly disparate categories of chemical systems, they are not strictly delimited to two discrete, static categories independent of one another.  From the azure watery abyss of the Atlantic to the tranquil shores of Lake Superior lurks a peculiar organism that straddles the intersection of microbe and mineral. This organism is responsible for the production of a magnificent crystalline shell, the intricacy of which eclipses the most elaborate of the Sassanian glass sculptures. The organism responsible for this wondrous display is known to microbiologists as Thalassiosira pseudonana. This fascinating microorganism is a member of a group of tiny, single-celled organisms called diatoms (1). The members of this large grouping of microscopic organisms are able to manufacture cell walls called “frustules” out of the mineral silica, the structure of which is determined at the genetic level of each member (1). Together, these organisms form a plethora of dazzling contours and shapes almost as impressive as the crystal structures formed by silicate minerals in the bowels of the Earth. Within their crystalline sarcophagi, the diatoms are afforded refuge from small predators that graze on plankton. Central to the production of this remarkable shell is the process known as biomineralization (2). This process is carried out by multiple kinds of proteins made by T. pseudonana. How could these proteins possibly be responsible for producing such an elaborate and durable mineral structure?
            Well, that's where the true cunning of mother nature yet again lends its hand. To find out, we ask prominent phycologist Nicole Poulsen and her team. According to Poulsen, “Biominerlaization is pretty much the process of converting inorganic, chemical materials such as silicic acid into mineral structures that serve a specific function.” This process is much more common then one might assume – in fact it is occurring in your body as you read this post. It is through this process that our teeth and bones are formed. If this process is so common, why should T. pseudonana garner such special attention? What sets the production of the T. pseudonana frustule apart from other biominerals is the scale and intricacy of its design. However, in order to fathom the construction of such a fantastic display of nature's craftsmanship, one must first acquaint themselves with the process behind which their smallest subcomponents are produced. The mineral silica is obtained in the form of a mild acid called silicic acid. This acid is hoarded by the T. pseudonana cell, which pumps it in from its watery environs via special silicic acid transport proteins (2). Once gathered in the cell, this acid is essentially gobbled up by membranous compartments called silica deposition vacuoles (SDVs) that are formed with the aid of a mysterious organic protein matrix thought to underlie the frustule. These vacuoles form tiny acidic compartment that serve as nano-factories where the sillica is patterned into a plethora of different shapes, the likes of which vary depending on the species of diatom (2). Special silica-forming proteins are suspected by Poulsen to shape the silica crystals and set them into proteins. It is with this very process that the boundary between biology and mineralogy is breeched, for the frustule structure is composed of a hybrid material of the two categories referred to quite appropriately as a “silicate biomineral.”
            However, the contribution of the proteins involved in this magnificent architectural feat remain as enigmatic as the those performed by the laborers charged with the construction of Khufu's pyramid. Despite this dearth of information, what little that is known is enough to provide at least a rough sketch of the process. Multiple protein groups are involved in silica biomineralization. Of these proteins, the silaffin proteins are currently the best understood (2), which makes them an attractive candidate for experimental manipulation. In order to shed light on some of the mechanisms involved in the process of frustule formation, Poulsen and her crew decided to have the silaffin proteins themselves literally light the way. This step involved the gene for a protein called GFP (short for “green fluorescent protein”) that is able to impart a vivid green fluorescence upon examination under a fluorescence microscope. By inserting the GFP gene next to the silaffin gene in the T. pseudonana DNA, she and her team were able to make the cells cobble together a hybrid GFP-silaffin protein. In a sense, the silaffin protein was given a shiny green nose to find where it spent most of its time in the T. pseudonana cell. What they found was that the silaffin proteins were themselves embedded in the biomineral frustule. However, the silaffins would need some sort of appendage that would allow them access to the frustule after they are made by the cell.
            To find out, Poulsen and her crew scrambled specific amino acid sequences that form part of the silaffin protein and tested them for their ability to reach and embed themselves in the biosilica frustule. The ones that weren't able to embed offered tantalizing clues into which specific sequence was needed because this sequence would have been crucial to the proper formation of the appendage. It turns out that a cluster of 5 lysine amino acids form a sort of positively charged key that enable the silaffins to embed themselves in the negatively charged silica of the frustule. Poulsen calls this special key a “pentalysine cluster”. This pentalysine key allowed the silaffins access to the silica of the frustule, which in turn afforded them the ability to incorporate into a vast network of proteins that together comprise the organic matrix. This structure forms a meshwork underneath the silica that is thought to function as an assembly nexus for the individual silica biomolecules by lining the interior of the SDV factories. The positive charges of the pentalysine key allow it to act as a sort of magnet to attract and gather the silica from the negatively charged silicic acid molecules (2).
            The biosilica chemical building blocks in turn compose the basic structural units of the frustule (3). Termed girdle bands and valves, these structural units are involved in the construction of the unique patterns in the frustule called nanopaterns and micropatterns. By investigating the composition of the underlying organic matrix, renowned phycologist Alexander Kotzsch and his team hope to piece together the mechanisms by which these elaborate patterns are made and how they contribute to the nigh unearthly strength and durability of the frustule. In order to understand the role of this unusual organic matrix in the construction of the frustule, Kotzsch and his team first decided to take a more destructive approach. This approach involved dousing the entirety of the organic matrix in a mildly acidic ammonium fluoride solution. In so doing, he was able to separate the two major groups that compose the matrix: the portion that dissolved in the acid consisted of proteins such as the silaffins studied by Poulsen and her team, whereas an additional portion consisted of proteins that did not dissolve in the acid. This ill-understood undissolved portion turned out to be far more complex in both its composition and in its pattern. It was within this portion of the matrix that another key silica forming protein was observed.
An image of the T. pseudonana organic
matrix captured using a scanningelectron
microscope. The white arrows point
to the micro-plate structures.
            Originally discovered in a study of the silaffin genes during a previous bout of experimentation (3), cingulins proved to be a valuable target for further research by Kotzsch and his team. Like Poulsen before him, Kotzch and his team decided to have the cingulins enlighten them as to where they spend their time in the cell. This involved genetically engineering the T. pseudonana cells to produce GFP-cingulin hybrid molecules. When the T. pseudonana cells were observed under the fluorescence microscope, the GFP-cingulin hybrids were found to congregate within a most unusual structure. Along with other proteins of the organic matrix, the cingulins appeared as if to form a tiny ring structure that Kotzsch called a “micro-ring.” These rings were in turn attached to the girdle band structures of the frustule. If he could understand the role of these ring structures in the process of biomineralization, Kotzsch believes that phycologists can revolutionize their understanding of silica biomineralization in T.   pseudonana frustule formation. According to him, “These rings are like the loophole in our current understanding of how the process of biomineralization can lead to the stunning intricacy and superb physical resilience of the  Thalassiosira pseudonana frustule”. In addition to these bizarre micro-rings, the cingulins were also found to be incorporated within unique plate-like protein structures found underneath of the valve structures of the frustule.
            It is within both of these structures that the cingulins contribute their fair share to the silica arrangements that ultimately compose the frustule (3). Kotzsch and his team speculate that cingulin proteins could somehow clump together within these structures in order to gather silica into high density clusters to form the basic pattern of the frustule. The tightly knit silica nanopatterns and micropatterns produced by these cingulin clusters would in turn provide the frustule its superb durability (3). However, the inability to dissolve the cingulin proteins out of the micro-rings and micro-plates makes it difficult to get a closer look at the cingulin components alone. In order to surmount this challenge, Kotzsch and his team devised a way to genetically engineer E. coli cells, which don't make the other protein components of the microrings and plates, to make two important types of cingulin proteins in place of the T. pseudonana cells: a  Y-shaped version and a W shaped version (3). The E. coli cells can be thought of as a sort of surrogate of the two major flavors of cingulin protein. However, it was up to Kotzsch and his team to shed light on how these proteins clump together once they were made.
            Using a technique called dynamic light scattering, which involved shining a laser at a droplet of salt water containing either the Y or W shaped cingulins, Kotzsch discovered that both varieties clump together on their own when the solution they are in is slightly acidic and the salt concentration is neither too high nor too low. Under these favorable conditions, the cingulins were not only observed to clump together with cingulins of the same shape – they were even able to huddle together with cingulins of the other shape! Furthermore, subtle differences in the acidity and salt concentration, in line with those found near different parts of the organic matrix in the T. pseudonana cells, allowed the cingulins to clump together in different ways. This dynamic camaraderie amongst the cingulins in acidic environments is reminiscent of the organization noted by Poulsen to be found within the SDVs. Since these compartments form as extensions of the organic matrix underlying the girdle bands and valves of the frustule (2), the shape of the cingulin clumps is likely to vary depending on whether they are part of the micro-ring or micro-plate structures, respectively. The newfound insight into the composition of the previously mystifying organic matrix enabled Kotzsch and his team to entertain the notion of specialized location- specific silica assembly plants. 
            Depending on where they are formed, the little SDV silica factories weave together different silica patterns. The unique ways in which the cingulins clump together in the micro-ring and micro-plate portions of the organic matrix allow the silica molecules to be shaped into their characteristic patterns before they are donated to the frustule. The integration of these different silica patterns afford the magnificently molded T. pseudonana frustules their mechanical fortitude (3). In this sense, the cingulins are like specialized gemologists that work together in different ways depending on which factory they are employed in. With their strong, positively charged  “pentalysine keys”, the silaffins studied by Poulsen and her team function as the mineral miners by gathering silica from the environment, whereas the more weakly positively charged cingulins function collectively as certified gemologists to mold the magnificent silica patterns that are incorporated into the greater frustule. Thus with the aid of the silafin and singulin silica forming proteins, T. pseudonana is able to encapsulate itself in a most wondrous display of Nature's cunning and thereby bejewel our oceans and not the bellies of predatory grazers. 


1. Round, F. E., Crawford, R. M., & Mann, D. G. (1990). Diatoms: biology and morphology of the genera. Cambridge University Press.

2. Poulsen, N., Scheffel, A., Sheppard, V. C., Chesley, P. M., & Kröger, N. (2013). Pentalysine clusters mediate silica targeting of silaffins in Thalassiosira pseudonana. Journal of Biological Chemistry, 288(28), 20100-20109.

3. Kotzsch, A., Pawolski, D., Milentyev, A., Shevchenko, A., Scheffel, A., Poulsen, N., ... & Kröger, N. (2016). Biochemical composition and assembly of biosilica-associated insoluble organic matrices from the diatom Thalassiosira pseudonana. Journal of Biological Chemistry, 291(10), 4982-4997.

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