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Dynamics of formation and secretion of heterococcoliths by Coccolithus pelagicus ssp. Braarudii
Abstract and Figures
The formation and secretion of heterococcoliths by the non-motile life phase of the coccolithophore Coccolithus pelagicus was investigated using electron microscopy and time-lapse bright field imaging. Coccolithogenesis in C. pelagicus exhibited sequential mineralization of single coccoliths in Golgi-derived and nuclear-associated vesicles, a pattern similar to the formation of heterococcoliths in Emiliania huxleyi. Our TEM data show that only on maturation does the single coccolith vesicle migrate away from the nucleus before secretion. A reticular body, distinct from the Golgi body, was also clearly visible at the distal surface of the developing coccolith vesicle, suggesting this is a common structural feature in placolith cells that mineralize and secrete coccoliths one at a time. Time-lapse imaging revealed that the coccolith secretion process is rapid, taking 60–190 seconds, and involves considerable contractile activity to eject and position the coccolith on the surface of the cell. An intact flagellar root apparatus was discovered at the anterior pole of this non-motile cell from which polarized secretion of coccoliths occurs, which may indicate a novel role for such cytoskeletal structures. Freeze-fracture preparations revealed columnar deposits and adhesions linking the scales and coccolith baseplates to the cell, across the periplasmic space providing points of attachment for cellular movement. Rotatory movements of the cell relative to external coccoliths were exhibited by all actively calcifying cells. These movements enable the cell, while exhibiting morphologically polarized secretion, to locate and secrete a mature coccolith in a spatially well-defined manner. Finally, the time-lapse imaging approach described here provides an opportunity to quantify the regulation of coccolith production in single cells with high temporal resolution allowing responses of calcification to rapidly fluctuating environmental conditions such as light–dark transitions to be examined in detail, which has not been possible with bulk calcification studies.
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... For example, the coccolith vesicles in E. huxleyi and Coccolithus pelagicus ssp. braarudii are associated with a complex membrane structure known as the reticular body (Wilbur and Watabe 1963;Taylor et al. 2007), while coccolith vesicles of Pleurochrysis (Crysotila) carterae are associated with small vesicles termed 'coccolithosomes' (van der Wal et al. 1983). To add to the complexity, some species (such as P. carterae) produce unmineralized organic scales which pass through the Golgi system in parallel to the coccoliths (van der Wal et al. 1983), and other species produce more than one type of coccolith on the same cell (Young et al. 2009;Drescher et al. 2012). ...
... The principle that the cytosol is not homogenous but consists of distinct domains has gained traction in recent years (Boeynaems et al. 2018), so it is not impossible that regions of the cytosol have biophysical properties that allow Ca 2+ to accumulate to higher than normal concentrations. Multiple studies have noted that in some species the coccolith vesicle is close to the nuclear envelope for most of the calcification process (Westbroek et al. 1984;Taylor et al. 2007;Drescher et al. 2012;Yin et al. 2018), leading to speculation that Ca 2+ could be transported across the narrow intermembrane space with locally raised Ca 2+ concentrations. There have also been suggestions that calcium accumulating in acidocalcisome-like organelles, which are found in a wide variety of mineralizing and non-mineralizing algae (Gal et al. 2018), may have been co-opted as part of the calcium transport pathway in coccolithophores (Sviben et al. 2016). ...
... Calcification in coccolithophores is subject to multiple layers of regulation which differ from species to species, but we are completely ignorant of the underlying molecular mechanisms. For example, E. huxleyi and C. pelagicus only produce one coccolith at a time (Taylor et al. 2007), so what control mechanisms operate to prevent the formation of multiple coccoliths simultaneously in a cell, or even in the same vesicle? What controls the final size of a coccolith, and how does a cell sense that a coccolith is complete and ready for exocytosis? ...
Microbial organisms are responsible for the synthesis of some of the most elaborate of biominerals. In particular, diatoms make intricate silica frustules and coccolithophores make morphologically complex CaCO3 scales. The significance of these algae and their biominerals in the natural environment, as well as the potential to use them in biotechnology, makes understanding the molecular genetic underpinnings of biomineralization in these organisms an important research area. In this chapter I discuss our current understanding of the molecular genetics of biomineralization in diatoms and coccolithophores while highlighting gaps in our knowledge. I also compare these intracellular biomineralization processes with those in magnetotactic bacteria, to emphasize commonalities and differences in the genetic blueprints of these biominerals. After considering the regulation of coccolithophore and diatom biomineralization, outstanding question, methodological opportunities, and future directions of research are discussed.
... Firstly, coccolith size is, at least partly, controlled by cell size (working hypothesis 1; e.g. Taylor et al., 2007). Secondly, coccoliths are produced during a growth phase of the cell, specifically G1 interphase (working hypothesis 2; Müller et al., 2008). ...
... The cell diameter Ȼ Ø is considered to be directly linked to the coccosphere diameter Cs Ø (i.e., the cell wall thickness is constant; Fig. 1A). This working hypothesis is coherent with observations in Taylor et al. (2007; based on C. braarudii (PLY182g) growth observations). However, this working hypothesis also quite significantly constrains the growth model. ...
... Conversely to the model depicted hereafter, in natural conditions, cells are rarely naked and host coccoliths from a previous phase growth. Here, we simplified the model starting with no coccoliths, as is sometimes conducted in coccolithophore laboratory experiments (e.g., Taylor et al., 2007). Firstly, the observations confirm that coccolith size is at least partly controlled by cell size (working hypothesis 1). ...
Heterococcoliths are calcite platelets produced inside diploid coccolithophore cells and extruded to form a covering on the cell surface called a coccosphere. The size of coccoliths is an important parameter sometimes used to identify species, and it is observed to be influenced in extant species by abiotic parameters (e.g., CO2, light). However, the variable distribution of coccolith sizes occurring within a single coccosphere questions the mechanisms controlling coccolith size. A relationship between cell/coccosphere size and mean coccolith size was previously identified, called the “coccolithophore size rules”. In this study, we query the mechanisms controlling the size of a coccolith during coccolithogenesis. A culture experiment on Gephyrocapsa huxleyi strain RCC1216 shows that coccolithogenesis occurs during the cell growth G1 interphase and newly produced coccoliths get bigger as the cell grows. These observations provide parameters for the development of two numerical models used to simulate the coccolith size distribution within a coccolithophore population. Neither model can accurately reproduce an empirical monoclonal coccolith size distribution, indicating that additional factors influence coccolith size. According to our results, coccolith size is only clearly related to cell size at the time of its formation. We confirm that application of the coccolithophore size rules model should be limited to inferring average cell dimensions from (fossil) coccolith biometry, and that comparisons are valid only in multipopulational studies. The coccolith size rule model – the constraining effect of coccolith production during G1 interphase cell growth on coccolith size – proposed here is applicable only for some placolith-forming species.
... 6 Pg year −1 of calcium carbonate production in the pelagic ocean and collectively ballast and facilitate~83% of the particulate organic carbon flux from the surface to the deep ocean. They are produced intracellularly in a Golgi-derived coccolith deposition vesicle (CDV), within which an organic baseplate scaffolds the nucleation of calcium carbonate onto a protococcolith ring (1). During coccolith maturation, an array of organic matter constituents (proteins, lipids, and polysaccharides) facilitates CaCO 3 precipitation, patterning, transport, and adherence to the cell (2)(3)(4)(5). ...
Marine coccolithophores are globally distributed, unicellular phytoplankton that produce nanopatterned, calcite biominerals (coccoliths). These biominerals are synthesized internally, deposited into an extracellular coccosphere, and routinely released into the external medium, where they profoundly affect the global carbon cycle. The cellular costs and benefits of calcification remain unresolved. Here, we show observational and experimental evidence, supported by biophysical modeling, that free coccoliths are highly adsorptive bio-minerals that readily interact with cells to form chimeric coccospheres and with viruses to form "viroliths," which facilitate infection. Adsorption to cells is mediated by organic matter associated with the coccolith base plate and varies with biomineral morphology. Biomineral hitchhiking increases host-virus encounters by nearly an order of magnitude and can be the dominant mode of infection under stormy conditions, fundamentally altering how we view biomineral-cell-virus interactions in the environment.
... Both heterococcoliths and holococcoliths are primarily produced in the light phase of the diel cycle (Paasche, 1969). Production of each single large heterococcolith in the HET phase takes approximately 3 hours, whereas holococcolith production is much more rapid (Taylor et al., 2007;Langer et al., 2021). It seems likely that the extensive rearrangement of the cytoskeleton required for cell division would interfere with its essential role in heterococcolith formation (Langer et al., 2010;Durak et al., 2017) and intracellular heterococcoliths are not observed in dividing HET cells (Walker et al., 2018). ...
Coccolithophores feature a haplo-diplontic life cycle comprised of diploid cells producing heterococcoliths and haploid cells producing morphologically different holococcoliths. These life cycle phases of each species appear to have distinct spatial and temporal distributions in the oceans, with the heavily calcified heterococcolithophores (HET) often more prevalent in winter and at greater depths, whilst the lightly calcified holococcolithophores (HOL) are more abundant in summer and in shallower waters. The haplo-diplontic life cycle may therefore allow coccolithophores to expand their ecological niche, switching between life cycle phases to exploit conditions that are more favourable. However, coccolithophore life cycles remain poorly understood and fundamental information on the physiological differences between life cycle phases is required if we are to better understand the ecophysiology of coccolithophores. In this study, we have examined the physiology of HET and HOL phases of the coccolithophore Coccolithus braarudii in response to changes in light and nutrient availability. We found that the HOL phase was more tolerant to high light than the HET phase, which exhibited defects in calcification at high irradiances. The HET phase exhibited defects in coccolith formation under both nitrate (N) and phosphate (P) limitation, whilst no defects in calcification were detected in the HOL phase. The HOL phase grew to a higher cell density under P-limitation than N-limitation, whereas no difference was observed in the maximum cell density reached by the HET phase at these nutrient concentrations. HET cells grown under a light:dark cycle divided primarily in the dark and early part of the light phase, whereas HOL cells continued to divide throughout the 24 h period. The physiological differences may contribute to the distinct biogeographic distributions observed between life cycle phases, with the HOL phase potentially better adapted to high light, low nutrient regimes, such as those found in seasonally stratified surface waters.
HIGHLIGHTS
• Coccolithus braarudii life cycle phases exhibit different physiological responses.
• The heavily calcified heterococcolithophores (HET) life cycle phase is more sensitive to high light.
• The lightly calcified holococcolithophores (HOL) life cycle phase may be better suited to growth under low phosphate availability.
The coccolithophore algae (Calcihaptophycidae) produce an extracellular covering of calcium carbonate plates (coccoliths) and are responsible for up to 10% of global carbon fixation through photosynthesis and biomineralization. Therefore, any alteration in cell functioning and calcification may strongly affect the ocean-atmosphere system. In this regard, the identification of any variation of the element pathways to the cell and their incorporation in the coccolith is important to understand the response of these organisms to changes in (past) environmental conditions and to identify (paleo)ecological proxies. Here, we focus on manganese which is an essential element for coccolithophore functioning: understanding whether manganese is also incorporated in coccolith calcite and if its distribution is species-specific is of seminal importance. In order to obtain ultra-high resolution (50 x 50 nm) spatial elemental distribution maps of Early to mid-Cretaceous (ca. 122 to 110 Ma) coccoliths we used synchrotron-based XRF analyses at the nano scale. The studied nannofossils belong to different taxa preferring low to higher surface-water fertility and were extracted from samples of different ages (from early Aptian to early Albian) collected in distant oceanic settings including the western Tethys, the Pacific and proto-North Atlantic Ocean. The analyses showed a high correlation in the spatial distribution of calcium and manganese in the coccoliths, suggesting that manganese is present in the coccolith calcite, and it was likely incorporated during coccolith formation. Moreover, the studied species show different Mn/Ca ratios suggesting that there is a species-specific control on the manganese uptake. Secondary calcite was also identified but it displays higher Mn/Ca ratios compared to biogenic calcite. Only the specimens collected from a black shale displayed secondary calcite crusts probably formed at the sediment-water interface after coccolith precipitation to the seafloor. Suboxic/anoxic conditions promoted manganese remobilization from Mn-oxides and Mn-oxyhydroxides which was incorporated in the secondary calcite. The Mn/Ca ratio in coccolith calcite crusts may therefore be indicator of (past) oxygen variations in bottom waters.
The calcite platelets of coccolithophores (Haptophyta), the coccoliths, are among the most elaborate biomineral structures. How these unicellular algae accomplish the complex morphogenesis of coccoliths is still largely unknown. It has long been proposed that the cytoskeleton plays a central role in shaping the growing coccoliths. Previous studies have indicated that disruption of the microtubule network led to defects in coccolith morphogenesis in Emiliania huxleyi and Coccolithus braarudii. Disruption of the actin network also led to defects in coccolith morphology in E. huxleyi, but its impact on coccolith morphology in C. braarudii was unclear, as coccolith secretion was largely inhibited under the conditions used. A more detailed examination of the role of actin and microtubule networks is therefore required to address the wider role of the cytoskeleton in coccolith morphogenesis. In this study, we have examined coccolith morphology in C. braarudii and Scyphosphaera apsteinii following treatment with the microtubule inhibitors vinblastine and colchicine (S. apsteinii only) and the actin inhibitor cytochalasin B. We found that all cytoskeleton inhibitors induced coccolith malformations, strongly suggesting that both microtubules and actin filaments are instrumental in morphogenesis. By demonstrating the requirement for the microtubule and actin networks in coccolith morphogenesis in diverse species, our results suggest that both of these cytoskeletal elements are likely to play conserved roles in defining coccolith morphology.
Directing crystal growth into complex morphologies is challenging, as crystals tend to adopt thermodynamically stable morphologies. However, many organisms form crystals with intricate morphologies, as exemplified by coccoliths, microscopic calcite crystal arrays produced by unicellular algae. The complex morphologies of the coccolith crystals were hypothesized to materialize from numerous crystallographic facets, stabilized by fine-tuned interactions between organic molecules and the growing crystals. Using electron tomography, we examined multiple stages of coccolith development in three dimensions. We found that the crystals express only one set of symmetry-related crystallographic facets, which grow differentially to yield highly anisotropic shapes. Morphological chirality arises from positioning the crystals along specific edges of these same facets. Our findings suggest that growth rate manipulations are sufficient to yield complex crystalline morphologies.
We report single-entity measurements of the degree of calcification of individual phytoplankton cells. Electrogenerated acid is used to dissolve the calcium carbonate (CaCO3) shell (coccosphere) of individual coccolithophores and the...
Coccoliths are plates of biogenic calcium carbonate secreted by calcifying marine phytoplankton; annually these phytoplankton are responsible for exporting >1 billion tonnes (10 15 g) of calcite to the deep ocean. Rapid and reliable methods for assessing the degree of calcification are technically challenging because the coccoliths are micron sized and contain picograms (pg) of calcite. Here we pioneer an opto‐eletrochemical acid titration of individual coccoliths which allows 3D reconstruction of each individual coccolith via in situ optical imaging enabling direct inference of the coccolith mass. Coccolith mass ranging from 2 to 400 pg are reported herein, evidencing both inter‐ and intra‐species variation over four different species. We foresee this scientific breakthrough, which is independent of knowledge regarding the species and calibration‐free, will allow continuous monitoring and reporting of the degree of coccolith calcification in the changing marine environment.
In the past few years rapid progress has been made regarding our understanding of one of the dominant phytoplankton groups of the world's oceans: the coccolithophores. Among other initiatives, the EU-funded TMR network CODENET (Coccolithophores Evolutionary Biodiversity and Ecology Network) has provided new results and insights. These and many other findings are reviewed and synthesized in a number of individual chapters which focus on coccolithophore biology (molecular to cellular), ecology (experimental and in the ocean), evolutionary phylogeny and its impact on current and past global changes. Coccolithophores are emerging as a prime model for interdisciplinary global change research due to their great abundance, wide distribution and exemplary geological record. The results presented in this book address the fundamental question of the interaction between the biota and the environment at various temporal and spatial scales.
Calcite crystals grown by organisms can be elaborate and enigmatic. One of the best examples is the tiny calcite shields known as coccoliths that are produced by unicellular algae. Coccoliths consist of interlocking single crystals of highly modified morphology, and complex organic molecules called CAPs (coccolith associated polysaccharides) are known to be intimately associated with their formation. Here, we show how a CAP can regulate crystal morphology to enhance precipitation of specific faces, a crucial aspect of the biomineralization process. Using atomic force microscopy (AFM), we investigated the interaction of CAP from the species Emiliania huxleyi with the calcite surface during dissolution, precipitation, and dynamic equilibrium. We were able to see the polysaccharide adsorbed to the surface and observe its impact on mineral behavior. These experiments demonstrate that CAP preferentially interacts with surface sites defined by acute, rather than obtuse, angles and blocks acute sites during dissolution and growth. Therefore, CAP makes the calcite face that is most stable in the pure system, 101̄4, extend preferentially on the obtuse edges, promoting development of faces with lower angles to c-axis. AFM images of E. huxleyi at micrometer and atomic scales established that this is precisely the type of faces that define the morphology of the coccolith crystals. Therefore, we propose that crystal shape regulation by CAP is a fundamental aspect of coccolith biomineralization, and that preferential growth inhibition by site-specific functional groups is the mechanism causing CAP functionality.
Knowledge of the mechanisms of calcification in different species of coccolithophores and the interactions between calcification and other cellular processes is required for further understanding the regulation of a key component of inorganic carbon flux in the oceans. In particular the functions of calcification in relation to photosynthesis and nutrient acquisition are still debated. Increased understanding of the cellular regulation of calcification is also required to accurately predict the responses to elevated atmospheric CO2 on a global scale. Moreover, transport processes in delivery of substrates for calcification will improve our ability to interpret isotopic fractionation in the fossil record that is increasingly being used as a proxy for past climatic conditions. Advances in single cell physiology and molecular biology are already contributing significantly to the study of calcification at the cellular level. The application of genomics approaches should, in the longer term, contribute further to the goal of understanding of how cellular functions contribute to processes at ecosystem and global levels.
Detailed descriptions are given of the structure and morphology of the heterococ-coliths of the six CODENET taxa; Gephyrocapsa oceanica, Coccolithus pelagicus, Calcidiscus leptoporus, Umbilicosphaera foliosa, Helicosphaera carteri and Syracosphaera pulchra. These descriptions highlight the quality of phylogenetic data which can be obtained from coccoliths, and the coccoliths of the closely related genera Coccolithus, Calcidiscus and Umbilicosphaera are compared to explore the interplay between evolutionarily conservative and non-conservative aspects of morphology. These case studies emphasize that identifying homologies between coccoliths is most meaningfully done at the crystal unit level rather than the overall morphology level. This way, focus is kept as close as possible to the process of biomineralisation itself, which is essential because a minor shift in process can cause a large morphological change — exemplified here particularly by development of bicyclicity in shield elements. These case studies are also used to develop our understanding of coccolith biomineralisation processes. In particular: (1) The role of crystal growth in controlling aspects of coccolith structure is highlighted. (2) The unique morphology of H. carteri coccoliths is related to a growth model. (3) The lath elements of S. pulchra are shown to have tangential c-axis orientations, resulting in an important modification of the V-/R-model.
We report the isolation of striated flagellar roots from the Prasinophycean green alga Tetraselmis striata using sedimentation in gradients of sucrose and flotation on gradients of colloidal silica. PAGE in the presence of 0.1% SDS demonstrates that striated flagellar roots are composed of a number of polypeptides, the most predominant one being a protein of 20,000 Mr. The 20,000 Mr protein band represents approximately 63% of the Coomassie Brilliant Blue staining of gels of isolated flagellar roots. Two-dimensional gel electrophoresis (isoelectric focusing and SDS PAGE) resolves the major 20,000 Mr flagellar root protein into two components of nearly identical Mr, but of differing isoelectric points (i.e., pl's of 4.9 and 4.8), which we have designated 20,000-Mr-alpha and 20,000-Mr-beta, respectively. Densitometric scans of two-dimensional gels of cell extracts indicate that the 20,000-Mr-alpha and -beta polypeptides vary, in their stoichiometry, between 2:1 and 1:1. This variability appears to be related to the state of contraction or extension of the striated flagellar roots at the time of cell lysis. Incubation of cells with 32PO4 followed by analysis of cell extracts by two-dimensional gel electrophoresis and autoradiography reveals that the more acidic 20,000-Mr-beta component is phosphorylated and the 20,000-Mr-alpha component contains no detectable label. These results suggest that the 20,000-Mr-alpha component is converted to the more acidic 20,000-Mr-beta form by phosphorylation. Both the 20,000-Mr-alpha and -beta flagellar root components exhibit a calcium-induced reduction in relative electrophoretic mobilities in two-dimensional alkaline urea gels. Antiserum raised in rabbits against the 20,000-Mr protein binds to both the 20,000-Mr-alpha and 20,000-Mr-beta forms of the flagellar root protein when analyzed by electrophoretic immunoblot techniques. Indirect immunofluorescence on vegetative or interphase cells demonstrate that the antibodies bind to two cyclindrical organelles located in the anterior region of the cell. Immunocytochemical investigations at ultrastructural resolution using this antiserum and a colloidal gold-conjugated antirabbit-IgG reveals immunospecific labeling of striated flagellar roots and their extensions. We conclude that striated flagellar roots are simple ion-sensitive contractile organelles composed predominantly of a 20,000 Mr calcium-binding phosphoprotein, and that this protein is largely responsible for the motile behavior of these organelles.