Take a look at any pop culture article about the most important things in romantic relationships, and you’ll see “communication” listed right there at the top. This makes sense, right? In any relationship, you need to be able to talk to each other to figure things out. But us humans aren’t the only creatures that rely on communication; the importance of communication extends throughout the wide world of multicellular organisms. Here, we’re going to dive deep into an ancient method of communication of one such organism – the cyanobacteria Anabaena spp. – but let's first establish a basic understanding of what it means to be multicellular.
First off – who even counts as a multicellular organism? Your average science textbook posits eukaryotes as the hallmark of cell-cell communication, but a closer look into the world of prokaryotic organisms by Arévalo et al. shows that there is more to this story. Indeed, we see examples such as cable bacteria that facilitate division of labor through electrically connected systems, the cave bacterium HS-3 that can form multicellular “bodies,” and even ~3.42 billion-year-old filamentous microfossils that are thought to be some of the oldest known occurrences of multicellularity.
Many other examples of multicellular microbes exist, and most can be sorted into two general categories; aggregative multicellularity and clonal multicellularity (Figure 1). Aggregative multicellularity involves social aggregation, in which single cells come together to form some sort of fruiting body or swarm. Clonal multicellularity involves serial cell division, which just means that cells stay together even after division. Examples of this include filamentation, biofilm formation, patterned multicellularity, or multicellular magnetotactic prokaryotes.
Figure 1: A comprehensive look at the types of prokaryotic multicellularity. Credit: Mizuno et al. 2022
According to Lyons and Kolter (2015), filamentous microbes are thought to be both the first multicellular organisms on Earth and the first known instance of cellular differentiation. In 2021, Cavalazzi et al. presented more evidence with their discovery of 3.42 billion-year-old microbial fossils that inhabited a paleo-subseafloor hydrothermal vein system. These microfossils were filamentous in nature, suggesting an ancient origin of the filamentous bacteria that exist today.
Figure 2: Extended depth of field transmitted optical micrograph showing microfossil filament details. Chert is the type of rock that the filaments were found in. Credit: Cavalazzi et al. 2021.
This category of filamentous bacteria includes our titular organism: Anabaena spp! Anabaena spp. are a type of cyanobacteria, known commonly as blue-green algae. As Demoulin et al. 2019 explains, cyanobacteria are responsible for the oxygenation of the atmosphere and oceans, are major primary producers in past and present oceans, and are ancestors of the chloroplast. Cyanobacteria like Anabaena spp. provide an informative window into the mechanisms of early multicellularity and cell-cell communication.
One key characteristic of Anabaena spp. is its nitrogen fixation ability. Anabaena spp. often form symbiotic relationships with plants, who are not capable of getting nitrogen on their own. According to NOAA, N2 makes up 78% of the Earth’s atmosphere. There is plenty of nitrogen in the atmosphere, but it exists in a form that plants can’t use. Nitrogen fixation broadly refers to the natural processes by which atmospheric nitrogen reacts and becomes nitrogenous compounds like nitrate (NO2) or ammonium (NH4+) (Figure 3). Diazotrophs can siphon nitrogen out of thin air, turning it into something useful, and sometimes sharing it with other organisms. Thanks to diazotrophs, who can perform nitrogen fixation, plants are able to access the otherwise unavailable nitrogen they crave. Thus, diazotrophs like Anabaena spp. are absolutely crucial to the ecosystem of the Earth.
Figure 3: The biotic nitrogen cycle. Credit: Nitrogen Cycle
Anabaena spp. perform nitrogen fixation in their heterocysts, which are simply differentiated vegetative cells that are specialized for the fixation of nitrogen. In a single filament of Anabaena, there are generally lots of vegetative cells interrupted by the occasional heterocyst (Figure 4). The existence of vegetative cells and heterocysts in a single filament raises the question of how, if at all, these cells might be communicating with each other to modulate intercellular functions and processes, such as the acquisition and dispersal of nitrogen throughout the filament. Arévalo et al. explores just this. Using a variety of methods, they examine the ways that this filamentous cyanobacteria facilitates intracellular molecular exchange.
Figure 4: A filament of Anabaena spp. The largest cell is the heterocyst, with the rest being vegetative cells. Credit: PhycoKey Image Database
One method that Arévalo et al. uses to explore the mediation of intercellular molecular exchange in Anabaena filaments is called FRAP, otherwise known as Fluorescence Recovery After Photobleaching. In their experiment, FRAP is used to detect molecules moving between cells. FRAP begins by attaching a fluorescent molecule to another molecule of interest. This method could be used to make a fluorescent version of a particular molecule or a particular population of molecules.
If there is a molecule under suspicion of movement, we want to know whether that molecule is moving around, and if so, where it’s going. Tracking its movement involves fixing a fluorescent molecule known as a fluorophore to it. When something is fluorescent, that molecule can be detected by fluorescence microscopy. The fluorophore absorbs a specific wavelength of light, then emits light of a different wavelength, making the location of the molecule(s) of interest detectable.
In FRAP, once a population of fluorescent molecules has been established, scientists take a specific region and photobleach it with a laser, disabling the fluorescence of these molecules. This looks like a dark region where molecules in a specific location of the population are “burned” by the laser. After “burning”, scientists are able to examine if intercellular molecular transfers occur. Over time, scientists see how that “burn scar” heals. Does it blend out, or does it stay put? If the dark region stays dark over a timed period of observation, then those molecules can be distinguished as non-mobile or non-communicating. If the borders of the photobleached area bend and blur with time, it is likely due to the movement of molecules away from where the laser originally removed their fluorescent qualities.
This really cool, visually pleasing experiment was performed to track the movement of a fluorescent marker, calcein, in the filamentous, heterocyst-forming cyanobacterium Anabaena species strain PCC 7120. Here, researchers observed the movement of these calcein from one cell to another. Individual cells of Anabaena are joined together by intercellular doorways called septal junctions. Septal junctions mediate intercellular communication and traverse septal peptidoglycan, the thick material comprising bacterial cell walls, through small holes called nanopores. By attaching calcein to cells inside a filament of Anabaena, photobleaching them, and then observing the healing process of the “burn scars”, they were able to see this fluorophore pass along through nanopores from cell to cell (Figure 5). From this, the authors measured the rates of intercellular exchange by watching these fluorescent calcein molecules through the lenses of their fluorescence microscopes.
Figure 5: Calcein FRAP analysis of two filaments from Arévalo et al. 2021.
In Figure 5, part A shows an example of recovery (or lack thereof) from photobleaching in two different filaments of Anabaena PCC 7120. The yellow arrow points to a filament which did recover from photobleaching, and the white arrow points to a filament that did not recover from photobleaching. Part B graphically displays the percentage of recovery rate constants (R) for vegetative cells grown in a medium containing sodium nitrate (BG11, blue) or in a medium lacking sodium nitrate (BG110, orange). Any cells exhibiting an R value of less than 0.01 were considered non-communicating, since higher R values are associated with cells whose cytoplasm might not be fully divided from daughter or neighbor cells. From these graphics, it is clear that recovered cells grown in a more nitrogen rich environment (BG11) had a higher percentage of non-communicating cells than cells grown in an environment with less available nitrogen (BG110). Indeed, the authors statistical analyses revealed that for cells grown in BG11 medium, non-communicating cells composed 58% of all cells, while cells grown in BG110 had a non-communicating cell percentage of about 16%.
Since septal junctions are known to mediate intercellular experimentation, this experiment suggests that septal junctions can largely coordinate, regulate, and cooperate with the needs of the cells to communicate, especially when in diazotrophic conditions. To re-summarize the results, when deprived of nitrogen, intercellular communication increased. This is likely due to more septal junctions opening, since vegetative cells didn’t have enough stored nitrogen and became dependent on the heterocysts for their nitrogen. Conversely, when cells were grown in a more nitrogen rich environment, intercellular exchange decreased and there was a more random occurrence of communicating versus non-communicating cells. This more random occurrence was likely due to some vegetative cells not being dependent on the heterocysts to receive adequate nitrogen.
These findings suggest that depending on the metabolic status of individual cells in a filament (i.e carbon to nitrogen ratios), septal junction activity, and thus intercellular communication, is altered. In general, it seems that the activity of septal junctions (the determining factor when considering if a cell is communicating or non-communicating) is mediated on an individual cell level. Depending on the needs of the vegetative cells, septal junction activity and communication status changed. These results also reveal that non-communicating and communicating cells can exist in the same filament!
Understanding the mechanisms that allow multicellular communication helps us to learn more about the functions of some of the most important organisms on Earth. Communication between cells in Anabaena filaments, although decidedly complex, actually represents one of the simplest and oldest models of intercellular communication known. Studies like Arévalo et al. 2021 are essential to unpack how these cyanobacteria are able to exist and regulate some of the most necessary processes on Earth. Perhaps in the future, the knowledge presented in their research will enable us to discover additional information about the past, or even future, of multicellular life. Maybe you can even take a leaf out of Anabaena’s book – how you communicate can be directly related to your environmental conditions, and so it’s important to make sure you’re getting what you need to thrive!
About the Authors:
Ceren Çıtak '23 and Maddie Burke '23 study in the Biological Sciences department at Mount Holyoke College. Ceren does independent research in the Woodard Lab studying molecular eukaryotic genetics and hormone signaling transduction pathways in Drosophila melanogaster. This research studies larval fat body remodeling, a process analogous to tumor metastasis. Maddie does their research in the Bacon Lab. When they are not Gram-staining bacteria or identifying unknown microbes isolated from soil samples, this duo can be found in the forest searching for the fruiting bodies of fungal networks or visiting wildlife sanctuaries.
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