To Play or not to Play: A Story of the Arcade in Archaeal Cytoskeletons

Original Article: Ettema, Thijs J. G., Ann-Christin Lindås, and Rolf Bernander. 2011."An Actin-based Cytoskeleton in Archaea." Molecular Microbiology 80: 1052-061

Post By: Julie Castro

"Serious play is not an oxymoron; it is the essence of innovation." - Michael Schrage

 

 http://www.fastcompany.com/1716831/pac-man-reboot-bio-arcade-microorganisms-change-gameplay

 Many can agree that when entering an arcade one of the most difficult decisions is choosing what game to play first. However, regardless of the different colors and bright lights, many of the games are quite similar to each other in their general functioning and genres. We can share this feeling with a very special set of archaea, the Crenarchaeota and Thaumarchaeota, which also contain an “Arcade”, or the actin-related cytoskeleton in Archaea involved in shape determination.

Figure 1. Structural representation of an actin filament consisting of two protofilaments http://ghr.nlm.nih.gov/handbook/illustrations/actin

 

Although slightly different from the arcades that we attend, this “Arcade” also contains some choices to “play with”, and their decision to use this “Arcade” may be leading to some very innovative ideas in the area of eukaryogenesis. The main organism studied, Pyrobaculum calidifontis, belongs to the archaeal domain of life and contains a cytoskeleton regulated by these Arcade genes. The structure of the cytoskeleton in this organism is of upmost importance to researchers, as this may provide support for a possible archaeal origin of the cytoskeleton. The possible similarities between the inner components of archaeal and eukaryotic organisms may help point scientists in the right direction when trying to research common ancestors, as well as the evolutionary history of eukaryotic cells.

            Actin is an essential microfilament present in the cytoskeleton of eukaryotic cells. It forms a right-handed double helix, and is made up of two intertwining F-actin protofilaments (figure 1). Actin, along with the rest of the cytoskeleton, is essential for various cell functions such as cell growth, division, and formation of cell protrusions (figure 2).

Figure 2.  Depiction of some of actin’s essential roles in the cell including the formation of microvilli, contractile rings during cell division, appendages, and finally, protein bundles for cell contraction.

http://www.nature.com/scitable/topicpage/microtubules-and-filaments-14052932

The activity of Actin in the cell is of such importance that its structure is highly conserved across multicellular organisms, including the rabbit and yeast, which contain Actin filaments that share 88% homology.

            The concept of a bacterial cytoskeleton is a far more novel concept than that of Eukaryotes, however, many do contain a cytoskeleton that may perform similar functions to that of the eukaryotic cytoskeleton. Although bacteria do not use Actin as a structural filament, they are now known to use MreB, a protein that appears to contain a great deal of structural similarity to actin. Archaea, the last domain of life is also made up of organisms that contain distinct shapes, such as rod-like or filamentous. These organisms must maintain their shape, yet they do not contain Actin or MreB. Although there are some methanogenic rod-shaped archaea that contain MreB homologs, their presence in other members of the archaeal family have not been found. Interestingly, there is a set of cytoskeletal proteins that encompass two different orders within the archaeal domain, and contain a very closely resembling actin homolog (figure 3). The main component of the cluster Crenactin (actin homolog) is thought to be further assisted by other proteins in the gene cluster, known as Arcadins. Crenactin, along with the Arcadins provide for a spiral like cytoskeleton within a specific species, Pyrobaculum calidifontis. 

Figure 3. Stereo view of P. calidifontis Crenactin and eukaryotic yeast actin
http://www.sciencedirect.com/science/article/pii/S0014579314000581

The ability for archaea to use this cluster of genes or Arcade, and the large similarity of Crenactin to eukaryotic Actin may help shed insight on the possible evolution of the eukaryotic cytoskeleton.

Figure 4. Preservation of the Arcade gene cluster in different species of Archaea. Blue is the Crenactin gene.

            

After extensive phylogenetic studies involving P. calidifontis, and the archaeal homolog Crenactin, it was actually discovered that this protein’s closest relative is actually the commonly known eukaryotic actin (figure 3). These results suggest that there may be a common origin between these two proteins, and it is further supported by the fact that Crenactin actually shares some identical areas of its polypeptide with actin that no other ATPase has in common with actin.

One difference found in Crenactin however is its ability to respond to different types of triphosphates, while Actin only functions properly in the presence of ATP. In this aspect Crenactin does behave similarly to MreB, which also responds to a greater range of high-energy molecules. Further proof that Crenactin and the Arcade genes are involved in the cell shape determination of Crenactin containing archaea is present in their genome. There are no cell shape determining genes that code for MreB or a possible homolog in these organisms, however they are still able to maintain a rod shape. Further phylogenetic analysis (investigations relating to ancestral relationships between organisms) supported the theory that archaea of rod and filamentous like shapes in the Thermoproteales and Korarchaea orders use Crenactin to maintain their cytoskeleton.

Figure 5: Immunofluorescence microscopy of Crenactin in P. calidifontis cells. A. The helical shape present in the cells overall. B. A small group of cells which appeared to show Crenactin rings in the center of the cell body. C. Images of cells exposed to different cytoskeletal inhibitors, A22, Cytochalasin B and D for 4 hours. Note the helical shape is still highly prevalent.

     Immunohistochemical staining was performed on P. calidifontis cells using antibodies that were designed to tag Crenactin (figure 4). Images showed a spiral like protein that spanned the cell providing support that Crenactin does indeed form a backbone-like structure in the cells. It was also clearly stated that despite the lack of genetic similarity between MreB and Crenactin they do share a great range of functional similarity. Extensive genetic analysis however shows that eukaryotic actin is Crenactin’s closest evolutionary relative.

The dynamics of Crenactin were also studied by exposing exponentially growing P. calidifontis cells to actin and MreB inhibitors. Contrary to possible expected results, Crenactin was not highly affected by these inhibitors, therefore showing that Crenactin may fulfill the same purpose as MreB. MreB may share evolutionary similarities with actin, but function in a different form that the other two proteins.

       The Arcade gene cluster was also of great importance to the study, and the gene’s roles in the cell cytoskeleton were also studied. It was found that the location of the genes in this cluster varied among species of archaea, where certain species had the genes spread out through their whole genome while others had all of the genes clustered except for the first gene, rkd-1.

Figure 6. Immunofluorescence microscopy of gene products from the Arcade cluster in P. calidifontis cells. A-C. Representation of the three proteins that aid Crenactin in the cytoskeleton, rkd-1, -3, and -4 are seen forming helical shapes within the cell. D.Double staining of Crenactin (red) and rkd-1(green).

Most of the gene products from the Arcade cluster did not show any structural similarity to other known cytoskeletal protein in eukaryotic or bacterial species, however one protein rkd-4 may share some evolutionary similarity with a chromosome structure maintenance protein found in other archaea and some bacteria. This cluster was studied in species that contained Crenactin, and interestingly enough, almost all of the species of archaea that contained Crenactin also contained most of the genes in the Arcade cluster. The gene products were tagged with appropriate antibodies and stained for imaging in order to determine whether they also played an integral part in the cell cytoskeleton (figure 4). Imagining analysis revealed very thought provoking results. Apparently rkd-1, -3, and -4 all play roles in helping support cell shape with Crenactin. Rkd-1 appears to work closest with Crenactin while the other two proteins are supplementary. The gene products of all three genes appear to span the cell in a similar helical shape. Rkd-1 was seen to overlap Crenactin in the overlay, (figure 5) and both gene products appeared to localize to the same area. This was very important, as it gave scientist the support in their conclusion that both gene products may be interacting directly with each other. Rkd-3 and -4 on the other hand varied in their intensity and pronunciation compared to Crenactin and Rkd-1, therefore hinting to the idea that these gene products may be supplementary in cytoskeletal function. Rkd-2 on the other hand may potentially play a role in cell division, as seen by double staining images with DAPI chromosomal staining. The archaea being studied does not contain any genes for bacterial proteins that would aid in cell division, such as the constricting Ftsz ring. The Rkd-2 gene is located in close proximity of the Rkd-3 and -4, genes that are thought to aid in cell shape (as seen in Figure 4). The close proximity of these genes may be a form of organization for the archaeal organism, in order to link cell division to cell structure. The DAPI images (not shown) appear to display the cells during cell division, with the DNA on opposite poles while the gene product of Rkd-2 remains in the relative center. It is this distribution of the Rkd-2 gene product that may point to its possible role in cell division or constriction.   

            All of these findings pose a new possible hypothesis in the field of Eukaryogenesis. One theory that stems from the well-known endosymbiotic hypothesis states that it was actually a hybrid between the prokaryote and eukaryote cell (“proto-eukaryote”), what engulfed other small organisms (our current mitochondria organelle) very long ago. This close relatedness between Crenactin and actin support a hypothesis stating that eukaryotic organisms actually evolved from a lineage within the archaea instead than parallel to the kingdom, and is in accordance with the idea of a proto-eukaryote. Actin is essential for phagocytosis and formation of appendages; therefore obtaining these cytoskeletal proteins was of upmost importance to developing eukaryotic organisms. This apparently simple characteristic may very likely have been vital for the phagocytosis of ancient mitochondrion and as a result, emergence of the eukarya as a new kingdom of life. As stated by the investigators themselves, more research must be done on other archaeal organisms in order to be able to draw more definite conclusions regarding the possible ancestral relationship between these and eukarya. Due to the lack of fully sequenced archaeal genomes compared to the vast variety of archaeal organisms currently known to exist, there is still much left to discover regarding the genomes of archaea. Nevertheless, this study can definitely serve as a jumping off point for future researchers who may be interested in searching for a possible correlation between the evolutionary histories of the two domains. These promising results hold hope for the role of archaea in eukaryogenesis; the Arcade games are not over yet!

If you are interested in additional information regarding this study please visit: http://www.sciencedirect.com/science/article/pii/S0014579314000581

*All information provided by:

Ettema, Thijs J. G., Ann-Christin Lindås, and Rolf Bernander. 2011."An Actin-based Cytoskeleton in Archaea." Molecular Microbiology 80: 1052-061.