Abstract
Biomineralization is the process by which corals build their exoskeletons, and ultimately the reef ecosystem framework. The current model of biomineralization in these animals involves the endocytosis of calcifying medium by the calcifying cells lining the exoskeleton, followed by the intracellular formation of amorphous calcium carbonate precursors. However, the intracellular trafficking pathways inside these cells are poorly described. In this study, we investigated the intracellular trafficking of calcifying medium at the growing edge of laterally extending Stylophora pistillata colonies. Using a combination of fluorescent probes and immunofluorescence, along with confocal microscopy in living and fixed samples, we observed that a portion of the endocytosed calcifying medium is directed towards the Rab11-dependent endosomal recycling system, and we suggest that this pathway can be involved in the process of biomineral formation.
Similar content being viewed by others
Introduction
Biomineralization is the process of mineral fabrication by living organisms. Biominerals are present in all kingdoms of life, including prokaryotes1, plants2, protists3, fungi4, and a variety of metazoans5. They come in a wide diversity of compositions, shapes and functions, e.g., calcium phosphate (hydroxyapatite) in vertebrate bones and teeth6,7, calcium carbonate in the form of calcite, aragonite or vaterite in mollusk shells, coral skeletons, avian eggshells or tunicate spicules8,9,10,11. Biomineral formation follows non-classical crystallization processes, which are highly controlled by mineral-producing organisms12,13
Regarding biomineral production at the global scale, coral reef ecosystems hold a primary importance and are major players of biogeochemical carbon and calcium cycles. Among the vast diversity of organisms dwelling in coral reef ecosystems, which represent ca. 25% of all marine species14, hermatypic scleractinian corals, also termed stony corals or reef-building corals, are keystone species. By constructing aragonite exoskeletons, they are the key architects of the three-dimensional structure of the reef11,15.
Scleractinian corals are constituted of two tissue layers, the oral tissue facing seawater and the aboral tissue facing the exoskeleton. These tissues comprise two epithelial layers: an outer cell layer named ectoderm (or epidermis) and an inner cell layer termed endoderm (or gastrodermis) lining the gastrovascular system11 (Supplementary Fig. 1). In the past decades, research into many aspects of coral physiology has provided a comprehensive picture of the biomineralization process. The site of calcification is the extracellular Calcifying Medium (CM), lying between the skeleton and the tissues. The calcifying epithelium, also termed calicodermis, is the aboral ectoderm lining the extracellular CM. It is composed of calicoblastic cells, or calcifying cells, and desmocytes responsible for anchoring coral tissues to the skeleton16,17. The composition of the CM in calcium, protons and dissolved inorganic carbon, is modified compared to external seawater. Physiological and molecular approaches have shown that the calicoblastic cells are responsible for modifying the concentration of these ions through transmembrane proteins such as the calcium ATPase PMCA, the bicarbonate transporter SLC4γ or the ammonium transporter AMT1d18,19,20,21. The composition of the extracellular CM thus shows an aragonite saturation state (Ωara) that favors aragonite precipitation22. Furthermore, a complex assemblage of proteins, polysaccharides, and lipids, termed skeletal organic matrix (SOM)23,24 is present in coral skeletons. It has been shown that the SOM is secreted by the calicoblastic cells and can play various roles in mineral formation, from mineral nucleation and stabilization to crystal growth/inhibition of growth25. Additionally, it was observed that calicoblastic cells are constantly engulfing extracellular CM through macropinocytosis26, an evolutionary conserved, non-specific, fluid phase endocytic process allowing for the internalization of large gulps of extracellular milieu in endocytic vesicles ranging from 0.2 to 5 µm, termed macropinosomes27,28,29.
Recently, it was proposed that mineral formation also involves intracellular steps, with amorphous calcium carbonate (ACC) nanoparticles nucleating within macropinocytotic vesicles inside calicoblastic cells before being deposited on the growing skeleton surface5,30. While this intracellular biomineralization model is supported by experimental observations of ACC particles in corals30,31, there is a lack of information regarding the intracellular traffic and the fate of the vesicles resulting from engulfment of extracellular CM through macropinocytosis, and their recycling towards the plasma membrane. Different modes of calcifying fluid transport have been previously described in marine calcifiers, e.g., urchins, coccolithophores and foraminifera32,33, but have not been explored yet in corals, therefore it is of primary importance to decipher these processes in these organisms.
Several endocytic mechanisms exist in eukaryotes, which can be classified as clathrin-dependent and clathrin-independent pathways, macropinocytosis belonging to the latter34,35. After being internalized by endocytosis, molecules and extracellular milieu are sorted and either delivered to late endosomes and lysosomes for degradation, transported to the trans-Golgi network, or exported back to the plasma membrane through the endosomal recycling system36. This intracellular trafficking takes place in the endomembrane system, composed of membrane-bound organelles where biochemical reactions and subcellular processes are compartmentalized. The endomembrane system is orchestrated by Rab (Ras-related in brain) GTPases, a large family of evolutionary conserved small GTPases. As central determinants of membrane and organelle identity, Rab GTPases indeed perform essential functions, namely the formation of vesicles at the plasma membrane or at the surface of organelles, their transport between subcellular compartments, e.g., the Golgi complex or the endosomal system, and subsequently their tethering and fusion with their targets37,38,39. A major component of the endomembrane system is the endosomal machinery, which is involved in the uptake, degradation and recycling of molecules and fluids taken up from the extracellular milieu40.
The endosomal recycling is essential for the redirection of endocytosed membrane lipids and proteins towards the plasma membrane. This allows maintaining the integrity of the plasma membrane, which otherwise would be rapidly depleted by endocytic processes engulfing lipid bilayers inside the cell. Additionally, it contributes to maintaining cell polarity in epithelia41,42. Two recycling systems have been characterized in eukaryotic cells42: the fast and the slow recycling pathways. The fast recycling pathway, regulated by Rab4, allows for direct and rapid recycling from the early endosome to the plasma membrane, with a t1/2 < 5 minutes and sometimes as fast as 90 seconds in mammalian CHO cells43. The slow recycling pathway, under the control of Rab11, involves a transition from the early endosome to the recycling endosome before addressing vesicles to the plasma membrane and displays a t1/2 of 10-20 min41,44,45.
In the context of the coral intracellular biomineralization model proposed by30, endosomal recycling could provide a way for vesicles containing endocytosed CM (to return to the apical plasma membrane). However, the endomembrane system and the intracellular traffic pathways in coral calicoblastic cells are poorly characterized. Ultrastructural studies using transmission electron microscopy in the coral Pocillopora damicornis have revealed that calicoblastic cells contain large vesicles (ca. 250 nm in diameter), some of them containing a granular material being exocytosed at the apical plasma membrane46. Observations by scanning electron microscopy in Galaxea fascicularis have also described large membrane-bound vesicles (ca. 370 nm) within calicoblastic cells, although fusion with plasma membrane was not observed47. These large vesicles could correspond to macropinosomes observed by26 using confocal microscopy. However, electron microscopy in fixed and/or frozen coral sample, while providing exquisite subcellular details, provides little information on the protein content of intracellular compartments and on the dynamics of intracellular traffic.
The observation of coral calicoblastic cells in living samples is technically challenging : the convoluted geometry of coral skeletons, the opacity of overlying cell layers, the autofluorescence of coral tissues which naturally express fluorescent proteins48 and the strong chlorophyll fluorescence from endosymbiotic dinoflagellate microalgae render observation difficult on mature colonies. However, observations at the growing edge (GE) of laterally extending coral colonies allow for direct observation of actively calcifying calicoblastic cells in living coral samples using confocal microscopy49. Furthermore, this kind of sample can be processed for immunolabeling and subsequent imaging with minimal processing. Studies at the GE have provided unprecedented details on some aspects of coral calicoblastic cells and their physiology, e.g., pH regulation19,50,51, paracellular transport52, calcifying medium chemistry22 presence of primary cilia and lipid droplets53,54.
Here, our aim was to determine whether the intracellular macropinocytotic vesicles, resulting from the engulfment of extracellular CM, can be recycled towards the apical plasma membrane before releasing their content towards the site of calcification. Using confocal microscopy, we investigated the vesicles containing newly endocytosed CM, with a special focus on the slow endosomal recycling pathway. We chose to study the slow recycling pathway for two reasons. First, it partially overlaps with the secretory pathway42,55,56, which could potentially allow the delivery of newly synthetized skeletal organic matrix (SOM) proteins in CM-containing vesicles. Second, the temporal resolution of the pulse-chase assay we used here is limited by the relatively slow renewal of extracellular CM in corals: it was determined that the half-time of calcein influx in the extracellular CM of S. pistillata microcolonies is ca. 7 min52, making technically challenging the use of very short pulses of fluorescent dyes followed by immediate washing to study the fast recycling pathway. However, CM renewal is fast enough to allow for the exploration of the slow recycling pathway whose dynamics are lengthier.
We first performed data mining in the genome of Stylophora pistillata to search for a homolog of the small GTPase Rab11, the major regulator of slow endosomal recycling, Then, we used a combination of fluorescent endocytosis probes, Rab11 immunolabelling, and observations at the growing edge of S. pistillata colonies, to determine the fate of macropinocytotic vesicles. Our results show that a part of the extracellular calcifying medium, which is engulfed by calicoblastic cells through macropinocytosis, is directed towards the slow endosomal recycling pathway and thus the apical membrane. These results provide new information about the cell biology of the calcifying cells of corals, paving the way towards a better understanding of the mechanisms linked to biomineralization.
Results & Discussion
S. pistillata possesses a homolog of the slow endosomal regulating protein Rab11
To gain insight on the intracellular traffic of the calcifying medium after its entry in calicoblastic cells, we chose to investigate the slow endosomal recycling pathway, which is regulated by the small GTPase Rab1142. We performed data mining based on a BLAST approach in molecular databases, followed by phylogenetic analysis (Supplementary Fig. 2A), and determined that the genome of S. pistillata contains the homologs of major Rab GTPases involved in endocytic and recycling processes: Rab4 (regulator of fast endosomal recycling), Rab5 (involved in endocytic processes, including macropinocytosis) and Rab7 (regulator of late endosome maturation and fusion with lysosomes)38. Notably, we found that S. pistillata possesses a clear Rab11 homolog, namely SpiRab11, with 89.7% similarity with human HsaRab11 (Supplementary Fig. 2B). This is not surprising as all eukaryotic organisms possess an endosomal recycling system, from plants and yeasts to insects, nematodes and vertebrates57,58,59. Moreover, the function of a Rab11 homolog, EdiRab11, was studied in the context of cnidarian-dinoflagellate endosymbiosis in the sea anemone Exaiptasia diaphana60. Functional assays using wild-type and mutant EdiRab11 heterologous expression in primate cells revealed EdiRab11 is involved in endosomal recycling, showing that its function is conserved between vertebrates and cnidarians. The Exaiptasia diaphana and Stylophora pistillata homologs share 93.09% identity. Thus, it is likely that Rab11 recycling function is also conserved between E. diaphana and S. pistillata (both belong to the Hexacorallian subclass of anthozoan cnidarians).
Then, to explore the endosomal recycling pathway in S. pistillata calicoblastic cells, we evaluated the ability of a monoclonal antibody raised against human Rab11 (anti-HsaRab11) to specifically detect the Rab11 homolog SpiRab11 in S. pistillata. This antibody was previously used by60 to label intracellular Rab-11 bearing vesicles in the sea anemone E. diaphana, which belongs to the Hexacorallia subclass of anthozoans, similarly to S. pistillata. Western-blot analysis showed that the antibody labelled a protein band in S. pistillata tissue lysate (Supplementary Fig. 3), with a measured molecular weight (MW) of 24.5 kDa closely matching the computed MW of SpiRab11 (24.3 kDa). Immunostaining of S. pistillata aboral epithelia revealed that anti-HsaRab11 specifically labels 221 ± 89 nm (mean ± S.D.) intracellular Rab11 vesicles in the cytosol of the calicoblastic cells (Fig. 1C). The endosomal recycling system is often described as a pericentriolar or perinuclear cluster of vesicles61; here, we could not determine a preferential subcellular localization for Rab11-positive vesicles, which appear randomly distributed throughout the cell cytoplasm. Such absence of perinuclear clustering was also observed in phagocytic cells of E. diaphana60 : the organization of the endosomal recycling system is probably variable between different cell types and organisms. The density of Rab11-positive vesicles appeared significantly lower in the endodermal cell layer compared to the calicodermis (Fig. 1D). This could reflect a lower activity of the slow recycling pathway, which may be compensated in endodermal cells by the fast recycling pathway. In control samples immunostained without the primary anti-HsaRab11 antibody (Fig. 1 A-B), no signal was observed (except rare fluorescent objects in the endoderm only, which may correspond to endocytosed microalgae debris or fluorescent protein granules, Fig. 1B) confirming the specificity of the labeling.
S. pistillata microcolonies growing on glass coverslips were fixed and processed for immunostaining. A, B control sample (incubated with secondary antibody alone); C, D Sample incubated with the anti-HsaRab11 antibody and the secondary antibody. A, C calicoblastic cells; B, D aboral endoderm. Maximum intensity projections of Z-stacks (160 nm sections) acquired through the calicoblastic cells. Grey : Hoechst (nuclei); Magenta: SpiRab11. Maximum intensity projections of Z-stacks acquired through the aboral epithelia. Scale bar 5 µm. Brightfield images of the aboral epithelia are displayed in Supplementary Fig. 4.
Small fluorescent probes are internalized by calicoblastic cells through macropinocytosis
To investigate the dynamics of the calcifying medium in S. pistillata calicoblastic cells, we needed a fluorescent probe capable of labelling the extracellular medium in coral samples and to be endocytosed by calicoblastic cells. Calcein has been widely used in biomineralization studies owing to its properties: this fluorescent, non-toxic and cell impermeant molecule can be used as a fluid phase tracer to follow seawater and calcifying medium transport. Furthermore, since it strongly binds calcium, it is incorporated into newly formed calcium carbonate minerals. This valuable fluorescent probe was used in a variety of calcifying organisms, including coccolithophores62, foraminifera32, mollusks63 or sea urchins64. In corals, it has been extensively used to investigate tissue permeability and paracellular transport52,65 or crystal growth66,67,68,69. However, despite its intrinsic qualities, calcein comes with a major drawback: it is non fixable, and hence is lost in samples subjected to the necessary fixation and permeabilization steps prior to immunolabeling in immunofluorescence experiments. Therefore, we investigated the ability of another fluorescent, fixable probe, to be used as an endocytic tracer in S. pistillata calicoblastic cells. Lucifer Yellow carbohydrazide (LY) is a fixable derivative of the fluorescent probe Lucifer Yellow, with a maximum excitation peak at 430 nm and an emission peak at 540 nm. Lucifer yellow has been used as a fluid phase endocytosis tracer in yeasts70, plants71 and vertebrates72, or in neurons to investigate intra-axonal transport73,74. Similarly to calcein, it displays a net negative electrical charge which renders it membrane impermeant. Both are small, soluble molecules with similar molecular weights (LY: 457.25 g.mol-1; calcein: 622.53 g.mol-1) and therefore are unlikely to be separated by size-dependent sorting in the endosomal system after internalization by cells75. Importantly, LY is aldehyde-fixable, allowing its use in immunofluorescence experiments to investigate the protein content of endosomes labelled by LY76
We first evaluated the ability of fluorescent dyes to be used as an endocytic tracer in the calicoblastic cells of living coral samples. First, we observed, as in previous studies, that calcein accesses the extracellular CM from the surrounding sea water through the paracellular pathway, similar to other soluble fluorescent probes19,26,52. Similarly, the LY probe rapidly diffused between cells through the paracellular pathway and stained the extracellular CM in the same way as calcein. We observed on living microcolonies that, similarly to calcein (Fig. 2 A1-A3), LY is endocytosed at the apical pole of calicoblastic cells and accumulates in large endosomes whose diameter (0.2–1.5 µm) corresponds to macropinosomes (Fig. 2 B1-B3) based on their size previously characterized by26. When delivered simultaneously, both probes are present in endocytic vesicles, indicating a similar entry mechanism (Fig. 2 C1-C3). It was previously established, using pharmacological inhibitors, that the entry route of fluorescent dextrans from 3 kDa to 70 kda in the calicoblastic cells of S. pistillata samples is macropinocytosis26. To confirm that the LY and/or calcein small fluorescent molecules were also internalized through macropinocytosis, we used EIPA, a potent macropinocytosis inhibitor. The uptake of calcein was markedly reduced in the calicoblastic cells of microcolonies treated with EIPA (Fig. 3B) compared to control with DMSO (Fig. 3A). We measured a 71% reduction of the fluorescence intensity of calcein inside the calicoblastic cells (Fig. 3C), along with a 62% reduction in the volume of calcein-labeled endosomes compared to the control experiment with DMSO (Fig. 3D), confirming that macropinocytosis is a major endocytic route for internalization of extracellular CM containing calcein or LY by calicoblastic cells in S. pistillata. This is not surprising as macropinocytosis is a non-selective endocytic mechanism which indiscriminately engulfs extracellular milieu along with the soluble molecules it contains35. The strong fluorescence of EIPA under UV excitation at 405 nm (Supplementary Fig. 5D) prevented us to directly quantify the uptake of LY (which is also excited at 405 nm) by calicoblastic cells. However since calcein and LY are both present in the extracellular CM and are found in the same vesicles after endocytosis, and macropinocytosis being a receptor-independent, non-selective, fluid-phase endocytosis process, we can thus reasonably assume that LY is endocytosed by the same mechanism than calcein, i.e., macropinocytosis.
S. pistillata Microcolonies growing on glass coverslips were incubated with S-FSW containing calcein (A1-A3), Lucifer yellow (B1-B3) or both probes (C1-C3) for 10 min, then chased for 5 min in S-FSW before live confocal imaging. Images are maximum intensity projections of Z-stacks (160 nm sections) acquired from the apical pole to the basal pole of the the calicoblastic cells layer. The absence of crosstalk of Lucifer yellow in the calcein channel (cyan, A2-C2) and of calcein in the Lucifer yellow channel (glow, A1-C1) was verified. A3-C3 : overlay of calcein and LY fluorescence. Cyan: Calcein; Glow: Lucifer yellow. Cr: aragonite crystal. S-FSW = supplemented filtered seawater. Scale bars 10 µm.
S. pistillata microcolonies growing on glass coverslips were pre-incubated for 10 min in S-FSW containing either 0.01% DMSO (control) or 10 µM EIPA, and 40 µM Hoechst for nuclear counterstaining. Then, incubation was performed for 10 min in S-FSW containing either calcein and 0.01% DMSO (control) (A) or calcein and 10 µM EIPA (B). Colonies were then chased for 5 min in S-FSW containing either 0.01% DMSO (control) or 10 µM EIPA before live confocal imaging. A, B : Maximum intensity projections of Z-stacks (160 nm sections) acquired through the calicoblastic cells. Cyan : calcein. Cr : aragonite crystal. C total calcein fluorescence intensity (i.e., sum of fluorescence intensities from calcein vesicles) in the calicoblastic cells of microcolonies incubated with calcein or calcein + 10 µm EIPA. D : sum of volumes of calcein-positive vesicles in the calicoblastic cells of microcolonies incubated with calcein or calcein + 10 µm EIPA. Bars represent mean ± S.D., n = 3 (independent experiments, distinct samples). C Two tailed unpaired T-test preceded by Shapiro-Wilk test, t = 3.3167, df=4 *p = 0.0295. D Two tailed unpaired T-test preceded by Shapiro-Wilk test, t = 4.2363, df=4 * p = 0.0133. Scale bars 5 µm.
A portion of the endocytosed CM undergoes slow endosomal recycling
To determine whether CM-containing macropinosomes (also termed endosomes once they enter the trafficking pathway) are directed towards the recycling endosomes, we used LY to label endocytosed calcifying medium in the calicoblastic cells of S. pistillata microcolonies in a pulse-chase experiment, followed by double immunostaining of samples with the anti-HsaRab11 antibody and the anti-SOM antibody. The LY pulse allowed the labelling of macropinosomes formed through engulfment of ECM by calicoblastic cells, while chasing of different durations allowed these macropinsomes to mature and potentially associate to Rab11. We observed that LY is retained inside endosomes within calicoblastic cells after fixation and permeabilization (Fig. 4, A1-A5), confirming the usefulness of this fixable probe to investigate CM traffic in coral calicoblastic cells. Close examination indicates that a portion of the endosomes containing LY also contain Rab11 and SOM (Supplementary Fig. 6).
S. pistillata microcolonies growing on glass coverslips were incubated with S-FSW containing Lucifer yellow for 10 min (control: S-FSW alone: A1-D1), then chased for various times (0 min: A2-D2; 5 min: A3-D3; 20 min: A4-D4; 50 min: A5-D5) in S-FSW before fixation and subsequent immunostaining for confocal microscopy. Immunostaining was performed with the anti-HsaRab11 antibody and the anti-SOM antibody (control: secondary antibodies alone) and nuclei counterstained with Syto Deep Red. Maximum intensity projections of Z-stacks (160 nm sections) acquired through the calicoblastic cells. A1-A5, Glow: Lucifer Yellow; B1-B5, Grey: SOM (small vesicles) and nuclei (large oval objects); C1-C5, Magenta: SpiRab11; D1-D5: overlay. S-FSW = supplemented filtered seawater; SOM = skeletal organic matrix. Scale bars 5 µm.
To quantify the degree of association of SpiRab11 with the LY-containing endosomes, we performed 3D object-based image analysis on confocal images of calicoblastic cells. Fig. 5C shows that the LY-containing macropinosomes rapidly acquire Rab11 after macropinocytosis, with a peak after 5 min of chase (24% of LY endosomes harboring Rab11). The intensity of Rab11 fluorescent signal in the LY-containing endosomes also significantly increased after the LY pulse, then progressively decreased back to the basal level after 50 min of chase (Fig. 5A). This indicates that a portion of the endosomes containing LY (i.e., containing calcifying medium) enter the slow endosomal pathway. Note that the presence of Rab11 immediately after the 10 min LY pulse is probably due to the limited temporal resolution of our pulse-chase assay: at the moment the coral microcolonies were aldehyde-fixed, some endosomes were already formed (10 minutes before fixation) and had probably already undergone association with Rab11 and endosomal recycling.
S. pistillata microcolonies were pulsed for 10 min with S-FSW containing 0.5 mg/mL Lucifer Yellow and chased in S-FSW. Colonies were subsequently fixed and immunostained for SOM and Rab11. A mean fluorescence intensity of Rab11 in Lucifer yellow-containing endosomes; B mean fluorescence intensity of SOM in Lucifer yellow-containing endosomes. C proportion of Lucifer yellow-containing endosomes labelled with Rab11. Bars represent mean ± S.D., n = 3 (independent experiments, distinct samples). ANOVA preceded by Levene’s test, followed by Tukey’s post-hoc test, F = 11.84, df = 11; p = 0.0026, * p < 0.05.
Endosomes contain Skeletal Organic Matrix
As the slow endosomal recycling pathway partially overlaps with the protein secretory pathway42,55,56, we sought to determine whether CM-containing endosomes acquire skeletal organic matrix (SOM) during their intracellular journey. SOM is a complex mixture of proteins, polysaccharides and lipids, synthesized by coral calicoblastic cells and is essential in skeleton formation11,23,77. Therefore, we used a polyclonal antibody raised in rabbit against S. pistillata SOM24 in our experiment to label SOM components inside the calicoblastic cells of S.pistillata. We then used 3D object-based analysis to evaluate the SOM content of LY-labelled endosomes (Fig. 5B). While we observed that this endosome population indeed contained SOM immediately after the LY pulse and at all chase times, we could not detect a significant increase in the SOM fluorescent signal in the LY containing endosome population during their intracellular journey. The presence of SOM in endosomes immediately after the pulse suggests two possible origins : (i) endosomes are rapidly enriched in SOM after their internalization by calicoblastic cells, through overlap of the secretory pathway with the slow endosomal recycling pathway; (ii) part of the SOM, which is delivered in the extracellular CM through the secretory pathway, is engulfed back along with CM through nonspecific apical macropinocytosis. Indeed, organic material which probably corresponds to SOM, was observed at the interface between the calcifying cells and the skeleton in the coral Galaxea fascicularis by47. Thus, it is not surprising that macropinosomes contain SOM which can be endocytosed along with fluorescent probes in our experiment. Of note, we also observed SOM vesicles which do not contain LY nor RAB11, and we hypothesize that these are secretory vesicles budding from the Golgi complex where SOM is synthetized.
Implications for biomineralization in corals
We acknowledge that while the CM-containing endosomes labelled with SpiRab11 and antiSOM may represent the compartment where ACC particles nucleation occurs in the coral biomineralization model30, the observation of the first phases of mineral formation is not achievable using confocal microscopy. Furthermore, we cannot exclude that a Rab4-dependent fast endosomal recycling pathway, out of the temporal resolution of our pulse-chase assay, also allows for the redirection of macropinosomes back to the plasma membrane. Indeed, we identified a Rab4 homolog in the genome of S. pistillata (Supplementary Fig. 2). However, our results clearly indicate that S. pistillata calicoblastic cells engulf CM containing skeletal organic matrix in large endosomes, and that a portion of these endosomes rapidly associates with SpiRab11. This suggests that a portion of the engulfed CM is redirected towards the plasma membrane through the endosomal recycling pathway. Fig. 6 summarizes the intracellular traffic of the CM with the following steps that have been identified in our study: (1) Seawater containing small fluorescent probes (calcein and LY in this study) gain access to the extracellular calcifying medium through the paracellular pathway; (2) After endocytosis of extracellular CM at the apical membrane of the calicoblastic cells, macropinosomes containing calcifying medium undergo intracellular traffic and can be qualified as endosomes; (3) a fraction of the endosomes transiently acquires SpiRab11, which promotes their recycling towards the plasma membrane, (4) The SOM synthesized in the endoplasmic reticulum and Golgi complex by calicoblastic cells is either delivered directly to endosomes through an overlap of the secretory pathway with the slow endosomal recycling pathway, either secreted in the extracellular CM and engulfed in endosomes through macropinocytosis. There are other steps in the process which have not been shown directly but which can be logically proposed in relation to our results: the endosomes tagged with SpiRab11 and containing organic matrix are sorted to the apical cell membrane, since Rab11 is known to promote their recycling towards the plasma membrane. Other questions are still pending such as which part of the endocytosed calcifying medium is diverted towards late endosomes or lysosomes? Do the SOM-containing vesicles come exclusively from acquisition from the extracellular CM, or can they also result from fusion of the secretory pathway with the recycling pathway? Is the Rab4-dependent fast endosomal recycling system also involved in CM intracellular traffic? These questions open new avenues of research that will help improve our understanding of the cellular coral biomineralization pathway.
pistillata. (1) Ion and molecules from seawater and small fluorescent probes (red) gain access to the extracellular calcifying medium, sandwiched between the skeleton and the apical pole of calicoblastic cells, through the paracellular pathway26,52. (2) the extracellular calcifying medium enters the calicoblastic cells via macropinocytosis26. After being endocytosed at the apical membrane, macropinosomes containing calcifying medium undergo intracellular traffic and sorting as endosomes (proposed in current study). (3) A fraction of the endosomes transiently acquires SpiRab11 (magenta), which promotes their recycling towards the plasma membrane (proposed in current study). (4) The skeletal organic matrix proteins (SOM) (orange) synthetized in the endoplasmic reticulum and the Golgi complex is delivered in the extracellular CM through the secretory pathway and partly endocytosed back by the calicoblastic cells, along with CM, through macropinocytosis (proposed in current study). A fusion of recycling endosomes with secretory vesicles budding from the trans-Golgi network (TGN) may exist but we have not observed it. Note that a fraction of the endocytosed CM might undergo fast recycling through the Rab4-dependent pathway, or be directed towards the late endosomal/lysosomal compartments (proposed in current study).
Concluding remarks
In conclusion, our findings shed a new light on the biology of the coral calicoblastic cells which is, to this date, poorly described. More broadly, these results underline the importance of investigating the intracellular trafficking pathways in calcifying organisms. Being major regulators of vesicular transport, Rab GTPases are likely to play a crucial role in these organisms. Several Rabs, including Rab11, have been identified in extracellular matrix vesicles excreted by cultured osteoblast-like Saos-2 human cells78. In sea urchins, knockdown of the fast endosomal recycling regulator Rab35 impairs gastrulation and skeletogenesis through perturbations of actin polymerization and vesicular traffic79. In foraminifera, the expression of Rab5 (involved in endocytosis), Rab7 (marker of late endosome and lysosome) and Rab8 (involved in traffic from the trans-Golgi network to the plasma membrane) is increased during calcification, which is probably linked to the biogenesis of vesicles involved in calcium transport and storage80.
Along with its potential involvement in intracellular biomineral formation, the Rab11-dependent endosomal recycling pathway is essential to maintain cell homeostasis and plasma membrane integrity, especially in the polarized epithelia of corals performing constitutive endocytosis26, and thus is likely to sustain, at least indirectly, the calcification process. As an example, macropinocytosis and subsequent endosomal recycling may also be involved in nutrient acquisition from other tissues of the coral colony81,82, necessary to fuel the metabolic processes linked to calcification in these highly active calicoblastic cells, which represent 30% of energy expenditure in corals83. Increasing knowledge of the intracellular trafficking pathways in coral cells will undoubtedly illuminate biological processes which are, directly or indirectly, linked to biomineralization. In the future, genomic edition84,85 will probably allow the fluorescent tagging of Rab GTPases, ion transporters or skeletal matrix proteins, which will provide avenues to investigate the intimate subcellular processes of biomineralization in living coral samples.
Materials and methods
Biological material culture and maintenance
Coral colonies of Stylophora pistillata were grown in the culture facilities at the Centre Scientifique de Monaco. Aquaria were supplied with seawater from the Mediterranean Sea (exchange rate 50% h-1), at 25 °C and under an irradiance of 100 µmol photons m-2 s-1 of photosynthetically active radiation (PAR) on a 12 h: 12 h day: night light-cycle. Corals were fed 5 days a week with frozen rotifers, and twice a week with live Artemia salina nauplii. Microcolonies were prepared following the lateral skeleton preparative assay16,86,87. Briefly, flat portions (0.5 - 1 cm²) of S. pistillata mother colonies grown on glass supports were fixed with Devcon® epoxy resin on circular glass coverslips and left to grow across coverslip to a size of 1-2 cm² over 3 weeks in the culture conditions described above. Coverslips were cleaned every week with a razorblade to remove algae growing around the microcolony.
Reagents
Calcein (Sigma Aldrich, Saint-Louis, USA) stock solution was prepared at a concentration of 30 mM in distilled water and pH adjusted to 8 with NaOH to facilitate dissolution (adapted from65. Lucifer yellow CH (carbohydrazide) dilithium salt (Sigma Aldrich, Saint-Louis, USA) stock solution was prepared in artificial seawater (NaCl 425 mM, KCl 9 mM, CaCl2 9.3 mM, MgSO4 25.5 mM, MgCl2 23 mM) at a concentration of 5 mg/mL. 5-(N-ethyl-N-isopropyl)amiloride (EIPA) (Sigma Aldrich, Saint-Louis, USA) stock solution was prepared in DMSO at a concentration of 100 mM. Syto Deep Red Nucleic Acid Stain (Thermo Fisher, Waltham, USA) stock solution was prepared in DMSO at a concentration of 2 mM. Anti-Rab11 (clone 47) antibody raised against human Rab11 in mouse, which was previously used by60 for the immunodetection of Rab11 in the cnidarian modem system Exaiptasia diaphana, was purchased from BD Bioscience, Franklin Lakes, USA. Custom anti-SOM antibody directed against S. pistillata skeletal organic matrix was raised in rabbit (Eurogentec, Seraing, Belgium) following24 modified by88. Fluorescent secondary antibodies (Goat anti-Mouse IgG (H + L) coupled to Alexa Fluor 555 and Goat anti-Rabbit IgG (H + L) coupled to Alexa Fluor 647) were purchased from Thermo Fisher, Waltham, USA. Prolong Glass Antifade Mountant and nuclear counterstain Hoechst 33342 solution (20 mM) were obtained from Thermo Fisher, Waltham, USA. Donkey Serum and Bovine Serum Albumin were purchased from Sigma Aldrich, Saint-Louis, USA.
S. pistillata protein extraction
Tissue lysate of Stylophora pistillata was prepared from apexes (5 cm) ground on ice in 10 mL Laemmli extraction buffer (62,5 mM tris HCl pH 6,8, 2% SDS, PIC Roche protease inhibitor) (adapted from89. After grinding, the tissue slurry was agitated at 4 °C for 1 hour. A first centrifugation was performed for 10 minutes at 1000xg, 4 °C to pellet skeletal fragments. The supernatant was centrifuged for 15 minutes at 3 500xg, 4 °C to pellet insoluble fractions. Protein concentration in S. pistillata tissue lysate was determined using the BCA Protein Assay Kit (Interchim Uptima, Montluçon, France) according to the manufacturer’s instructions. Samples were deposited in a microplate in triplicate and the absorbance was determined using an Epoch Microplate Spectrophotometer (BioTek Instruments, Winooski, USA).
Western Blotting
Samples were denatured at 95 °C for 5 min in Laemmli buffer containing β-mercaptoethanol (Biorad, Hercules, USA) before deposition of 40 µg protein per lane on a 8-16% polyacrylamide Criterion polyacrylamide gel (Biorad, Hercules, USA). Migration was performed for 30 min at 200 V in electrophoresis TGS (Tris/glycine/SDS) buffer (Biorad, Hercules, USA).
The electrotransfer of proteins from electrophoresis gel to polyvinylidene difluoride (PVDF) membrane was performed with a Trans-Blot Turbo transfer system and the Trans-Blot Turbo RTA Transfer kit (Biorad, Hercules, USA) (7 min, 25 V, 2.5 A). The blotting membranes were incubated with a blocking solution (TBS, Tween-20 0,1%, powdered milk 5%) for 1 hour. Membranes were incubated overnight at 4 °C with the primary anti-Rab11 antibody at 1:1000 dilution in (Tris-Borate saline, Tween 20 0,1%, powdered milk 1%). The membranes were rinsed (10 ×10 min) at room temperature under gentle agitation in rinsing buffer (Tween-20 0,1%, TBS 1X, and 10% blocking solution). The membranes were then incubated with a goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) at 1:10.000 dilution (Zymed Laboratories, San Francisco, USA) overnight at 4 °C. Membranes were then rinsed (10 ×10 min) in rinsing solution, at room temperature under gentle agitation. Western Blot was revealed with the Enhanced Chemo Luminescence reagent (GE Healthcare, Chicago, USA) and luminescence detected by a Chemidoc device (Biorad, Hercules, USA). Images were analyzed and molecular weight was determined using Image lab software. Theoretical molecular weight of spiRab11 protein was computed using the Expasy online Compute pI/Mw tool (https://web.expasy.org/compute_pi/).
Rab GTPases phylogeny
Aminoacid sequences of the human small GTPases Rab4, Rab5, Rab7 and Rab11 were retrieved from Uniprot (https://www.uniprot.org). These sequences were used as baits in a reciprocal BLAST strategy using BLASTp (blast.ncbi.nlm.nih.gov/Blast.cgi) against reference sequence (refseq) databases of the following organism : the cnidarians Stylophora pistillata, Acropora digitifera, Dendronephtya gigantea, Exaiptasia diaphana, Nematostella vectensis, the tunicate Ciona intestinalis and the vertebrates Homo sapiens and Mus musculus. Protein sequences (listed in the Supplementary figure 2C) were aligned using the MEGA software (version 11.0.13) and ClustalW algorithm. Maximum likelihood tree was constructed using the JTT + G model.
Incubations of the colonies with fluorescent probes
Experiments on S. pistillata microcolonies were performed under an irradiance 100 µmol photons m-2 s-1 of photosynthetically active radiation (PAR) at 25 °C. Incubations were performed in filtered seawater (FSW) supplemented with 1 mg/mL of lyophilized rotifer powder (Ocean nutrition) and filtered at 0,45 µm to avoid nutritional stress (supplemented filtered seawater: S-FSW). To label the endosomal compartment, Lucifer Yellow was used at a concentration of 0.5 mg/mL (i.e., 1.09 mM)90 and calcein at a concentration of 160 µM65. Fluorescent probes were dissolved in S-FSW and pH adjusted to 8.0 with NaOH if needed. Samples were first pulsed for 10 min in 10 ml S-FSW containing probes or S-FSW alone (control). Then, samples were transferred into beakers containing 50 mL S-FSW under agitation for various chase times: 0, 5, 20, or 50 min, before performing either live confocal imaging, or fixation and further processing for immunostaining. For experiments with the macropinocytosis inhibitor EIPA, S. pistillata microcolonies were pre-incubated for 10 min in S-FSW containing either 0.01% DMSO (control) or 10 µM EIPA, and 40 µM Hoechst 33342 for nuclear counterstaining. Then, incubation was performed for 10 min in in S-FSW containing either 160 µM calcein and 0.01% DMSO (control) (A-E), or 160 µM calcein and 10 µM EIPA (B-F). Colonies were then chased for 5 min in S-FSW containing either 0.01% DMSO (control) or 10 µM EIPA before live confocal imaging.
Immunostaining experiments
After incubation of the samples with Lucifer Yellow, micro-colonies were fixed overnight at 4 °C in artificial seawater containing paraformaldehyde (PAF) : (NaCl 425 mM, KCl 9 mM, CaCl2 9.3 mM, MgSO4 25.5 mM, MgCl2 23 mM, HEPES 100 mM, pH = 7.9, PAF 4%, Triton X-100 1%) (adapted from Ganot et al., 2015)91. Samples were then rinsed in PBS and incubated with a blocking solution for 2 hours (BSA 2%, Tween-20 0.05%, Donkey Serum 2%, in PBS). Microcolonies were incubated for 24 h at 4 °C in labelling solution (BSA 1%, Tween 20 0.05%, in PBS) containing the following primary antibodies: anti-SOM (1:10.000 dilution) and anti-Rab11 (1:50 dilution). Samples were rinsed (3 × 20 min) in rinsing solution (BSA 0.1%, Tween 20 0.05%, in PBS) under gentle agitation at room temperature. Immediately after, microcolonies were incubated in staining solution containing the following secondary anti-bodies: goat anti-rabbit IgG and goat anti-mouse IgG (both at 1:1000 dilution) for 24 h at 4 °C. Samples were rinsed in a PBST solution (Tween 20, 0.05%, PBS) for 20 min. Nuclei were counterstained using Hoechst (1:500 dilution) or Syto Deep Red (1:4 000 dilution) for 30 min in PBS. Colonies were rinsed with PBS performed for 20 min. Samples were covered with Prolong Glass Antifade Mountant and cured at room temperature for 72 h before visualization under a confocal microscope.
Confocal microscopy
Observations were carried out at the growing edge of S. pistillata microcolonies with a confocal laser scanning Leica SP8 inverted microscope monitored by the LAS X software (version 3.5.6.21594). Imaging of endocytosis probes in living S. pistillata microcolonies was conducted using a 63× water immersion objective with a numerical aperture of 1.2. The resolution was 1024 × 512 pixels and the z-step size 0.16 µm. High-speed stack acquisition was performed using a SuperZ galvanometric stage in GalvoFlow mode and the resonant scanner (8.000 Hz) in bidirectional mod. The pinhole size was 1 Airy Unit. Hoechst, Lucifer Yellow and Calcein were excited at 355 nm, 405 nm and 488 nm, respectively, and their emission signals were acquired at 420–470 nm, 500–550 nm and 500–550 nm, respectively. Signals were acquired sequentially to prevent dye crosstalk. Images were deconvoluted on-the-fly by the Las X-integrated Lightning tool using default parameters. Imaging of immunostained S. pistillata microcolonies was conducted using a 63× oil immersion objective with a numerical aperture of 1.4. The resolution was 512 × 512 pixels and the z-step size 0.11 µm. Z stack acquisition was performed with the galvanometric scanner (600 Hz) in unidirectional mode. The pinhole size was 1 Airy Unit. Lucifer yellow, AF555 and AF647 + Syto Deep Red were excited at 405 nm, 552 nm and 638 nm, respectively, and their emission signals were acquired at 500–540 nm, 560–600 nm, and 650–690 nm, respectively. Signals were acquired sequentially to prevent dye crosstalk. Signals from AF647 and Syto Deep Red were acquired in the same channel but were easily distinguished due to the difference in size of the objects they stain (3–5 µm for nuclei stained with Syto Deep red, 150–800 nm for vesicles containing OM and labeled with AF647. For each sample, 3 random regions were observed at the growing edge of the microcolony. Images were deconvoluted by the Las X-integrated Lightning tool.
3D image analysis
Image processing and analysis were conducted using the LAS X software (Leica) (version 3.7.6.25997) including a 3D-analysis module. Image stacks were cropped to delineate the calicodermis, using the calicoblastic cells nuclei and the mesoglea as visual controls to select images ranging from the apical to the basal pole of calicoblastic cells. For live imaging experiments, fluorescence intensities and vesicle dimensions were obtained as follows: background was removed (object size: 10 µm), threshold was set manually (2500), objects intersecting stack edges (including calcein-labeled primary aragonite crystals) were excluded and a morphological filter was applied (open & close, 1px). For imaging of fixed samples, fluorescence intensities and vesicle dimensions were obtained as follows: background was removed (object size: 2 µm), then each channel was thresholded separately, using a maximum entropy algorithm for Lucifer Yellow and Rab11 (deviation: 0 and -1500, respectively) and a manual threshold (6000) for SOM. Objects intersecting stack edges were excluded and a morphological filter was applied (split objects, 1px). A binary mask was created by combining Lucifer Yellow and Rab11 channels (operator OR), from which fluorescence intensities for each channel were derived.
Statistical Analysis
For experiments with the macropinocytosis inhibitor EIPA, statistical significance was assessed using Student’s two-tailed unpaired t-test. For the mean Rab11 fluorescence intensity in the LY-containing endosomes, the statistical significance was assessed by an ANOVA preceded by Levene’s test and followed by Tukey’s post-hoc test. Each experiment was repeated 3 times, and for each sample 3 random regions were observed at the growing edge of the microcolony. Tests were performed in Rstudio (version 2025.09.2, build 418 running R 4.5.2).
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
Numerical source data for graphs and charts can be found within the Supplementary Data files. Supplementary Data 1. data for supplementary Fig. 2; Supplementary Data 2. data for Fig. 3 graphs; Supplementary Data 3. data for Fig. 5 graphs. The unedited blot images for Supplementary Fig. 3A are provided as Supplementary Fig. 3B, C. Additional information and relevant data will be available from the corresponding author upon reasonable request.
References
Görgen, S. et al. The diversity of molecular mechanisms of carbonate biomineralization by bacteria. Discov. Mater. 1, 2 (2020).
Cuéllar-Cruz, M., Pérez, K. S., Mendoza, M. E. & Moreno, A. Biocrystals in plants: a short review on biomineralization processes and the role of phototropins into the uptake of calcium. Crystals 10, 591 (2020).
Matz, J. M. Plasmodium’s bottomless pit: properties and functions of the malaria parasite’s digestive vacuole. Trends Parasitol. https://doi.org/10.1016/j.pt.2022.02.010.(2022)
Whitney, K. D. Systems of Biomineralization in the Fungi. in Origin, Evolution, and Modern Aspects of Biomineralization in Plants and Animals (ed. Crick, R. E.) 433–441 (Springer US, Boston, MA). https://doi.org/10.1007/978-1-4757-6114-6_34, 1989.
Gilbert, P. U. P. A. et al. Biomineralization: Integrating mechanism and evolutionary history. Sci. Adv. 8, eabl9653 (2022).
Lacruz, R. S., Habelitz, S., Wright, J. T. & Paine, M. L. Dental enamel formation and implications for oral health and disease. Physiol. Rev. 97, 939–993 (2017).
Murshed, M. Mechanism of bone mineralization. Cold Spring Harb. Perspect. Med. 8, a031229 (2018).
Crichton, R. Chapter 19 - Biomineralization. in Biological Inorganic Chemistry (Third Edition) (ed. Crichton, R.) 517–544 (Academic Press). https://doi.org/10.1016/B978-0-12-811741-5.00019-9., 2019.
Gautron, J. et al. Avian eggshell biomineralization: an update on its structure, mineralogy and protein tool kit. BMC Mol. Cell Biol. 22, 11 (2021).
Pokroy, B. et al. Narrowly distributed crystal orientation in biomineral vaterite. Chem. Mater. 27, 6516–6523 (2015).
Tambutté, S. et al. Coral biomineralization: from the gene to the environment. J. Exp. Mar. Biol. Ecol. 408, 58–78 (2011).
Gower, L. Spiers Memorial Lecture: a retrospective view on the non-classical features revealed by advanced imaging of biominerals. Faraday Discuss. https://doi.org/10.1039/D5FD00054H (2025).
Karthika, S., Radhakrishnan, T. K. & Kalaichelvi, P. A review of classical and nonclassical nucleation theories. Cryst. Growth Des. 16, 6663–6681 (2016).
Raghukumar, S. The Coral Reef Ecosystem. in Fungi in Coastal and Oceanic Marine Ecosystems: Marine Fungi (ed. Raghukumar, S.) 143–161 (Springer International Publishing, Cham). https://doi.org/10.1007/978-3-319-54304-8_9, 2017.
Bozec, Y.-M., Alvarez-Filip, L. & Mumby, P. J. The dynamics of architectural complexity on coral reefs under climate change. Glob. Change Biol. 21, 223–235 (2015).
Muscatine, L., Tambutte, E. & Allemand, D. Morphology of coral desmocytes, cells that anchor the calicoblastic epithelium to the skeleton. Coral Reefs 16, 205–213 (1997).
Tambutté, E. et al. Observations of the tissue-skeleton interface in the scleractinian coral Stylophora pistillata. Coral Reefs 26, 517–529 (2007).
Capasso, L., Zoccola, D., Ganot, P., Aranda, M. & Tambutté, S. SpiAMT1d: molecular characterization, localization, and potential role in coral calcification of an ammonium transporter in Stylophora pistillata. Coral Reefs 41, 1187–1198 (2022).
Venn, A. A., Tambutté, E., Comeau, S. & Tambutté, S. Proton gradients across the coral calcifying cell layer: Effects of light, ocean acidification and carbonate chemistry. Front. Mar. Sci. 9, (2022).
Zoccola, D. et al. Molecular cloning and localization of a PMCA P-type calcium ATPase from the coral Stylophora pistillata. Biochim. Biophys. Acta 1663, 117–126 (2004).
Zoccola, D. et al. Bicarbonate transporters in corals point towards a key step in the evolution of cnidarian calcification. Sci. Rep. 5, 9983 (2015).
Sevilgen, D. S. et al. Full in vivo characterization of carbonate chemistry at the site of calcification in corals. Sci. Adv. 5, eaau7447 (2019).
Drake, J. L. et al. Proteomic analysis of skeletal organic matrix from the stony coral Stylophora pistillata. Proc. Natl. Acad. Sci. USA 110, 3788–3793 (2013).
Puverel, S. et al. Antibodies against the organic matrix in scleractinians: a new tool to study coral biomineralization. Coral Reefs 24, 149–156 (2005).
Falini, G., Fermani, S. & Goffredo, S. Coral biomineralization: a focus on intra-skeletal organic matrix and calcification. Semin. Cell Dev. Biol. 46, 17–26 (2015).
Ganot, P. et al. Ubiquitous macropinocytosis in anthozoans. eLife 9, e50022 (2020).
Kerr, M. C. & Teasdale, R. D. Defining macropinocytosis. Traffic 10, 364–371 (2009).
King, J. S. & Kay, R. R. The origins and evolution of macropinocytosis. Philos. Trans. R. Soc. B Biol. Sci. 374, 20180158 (2019).
Lim, J. P. & Gleeson, P. A. Macropinocytosis: an endocytic pathway for internalising large gulps. Immunol. Cell Biol. 89, 836–843 (2011).
Sun, C.-Y. et al. From particle attachment to space-filling coral skeletons. Proc. Natl. Acad. Sci. USA 117, 30159–30170 (2020).
Mass, T. et al. Amorphous calcium carbonate particles form coral skeletons. Proc. Natl. Acad. Sci. USA 114, E7670–E7678 (2017).
Bentov, S., Brownlee, C. & Erez, J. The role of seawater endocytosis in the biomineralization process in calcareous foraminifera. Proc. Natl. Acad. Sci. USA 106, 21500–21504 (2009).
Kahil, K., Weiner, S., Addadi, L. & Gal, A. Ion pathways in biomineralization: perspectives on uptake, transport, and deposition of calcium, carbonate, and phosphate. J. Am. Chem. Soc. https://doi.org/10.1021/jacs.1c09174 (2021).
Donaldson, J. G. Macropinosome formation, maturation and membrane recycling: lessons from clathrin-independent endosomal membrane systems. Philos. Trans. R. Soc. B Biol. Sci. 374, (2019).
Swanson, J. A. & King, J. S. The breadth of macropinocytosis research. Philos. Trans. R. Soc. B Biol. Sci. 374, 20180146 (2019).
Canton, I. & Battaglia, G. Endocytosis at the nanoscale. Chem. Soc. Rev. 41, 2718–2739 (2012).
Langemeyer, L., Fröhlich, F. & Ungermann, C. Rab GTPase function in endosome and lysosome biogenesis. Trends Cell Biol. 28, 957–970 (2018).
Stenmark, H. Rab GTPases as coordinators of vesicle traffic. Nat. Rev. Mol. Cell Biol. 10, 513–525 (2009).
Zerial, M. & McBride, H. Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol. 2, 107–117 (2001).
Brighouse, A., Dacks, J. B. & Field, M. C. Rab protein evolution and the history of the eukaryotic endomembrane system. Cell. Mol. Life Sci. 67, 3449–3465 (2010).
Grant, B. D. & Donaldson, J. G. Pathways and mechanisms of endocytic recycling. Nat. Rev. Mol. Cell Biol. 10, 597–608 (2009).
O’Sullivan, M. J. & Lindsay, A. J. The Endosomal Recycling Pathway—At the Crossroads of the Cell. Int. J. Mol. Sci. 21, 6074 (2020).
Hao, M. & Maxfield, F. R. Characterization of rapid membrane internalization and recycling. J. Biol. Chem. 275, 15279–15286 (2000).
Jonker, C. T. H. et al. Accurate measurement of fast endocytic recycling kinetics in real time. J. Cell Sci. 133, jcs231225 (2020).
Naslavsky, N. & Caplan, S. The enigmatic endosome – sorting the ins and outs of endocytic trafficking. J. Cell Sci. 131, jcs216499 (2018).
Johnston, I. S. The Ultrastructure of Skeletogenesis in Hermatypic Corals. in International Review of Cytology (eds Bourne, G. H. & Danielli, J. F.) vol. 67 171–214 (Academic Press, 1980).
Clode, P. L. & Marshall, A. T. Low temperature FESEM of the calcifying interface of a scleractinian coral. Tissue Cell 34, 187–198 (2002).
Alieva, N. O. et al. Diversity and evolution of coral fluorescent proteins. PLoS ONE 3, e2680 (2008).
Venn, A. A. et al. Coral calcification at the cellular scale: insight through the ‘window’ of the growing edge. in Front. Invertebrate Physiol. (2024).
Barott, K. L., Venn, A. A., Thies, A. B., Tambutté, S. & Tresguerres, M. Regulation of coral calcification by the acid-base sensing enzyme soluble adenylyl cyclase. Biochem. Biophys. Res. Commun. 525, 576–580 (2020).
Venn, A. A. et al. Effects of light and darkness on pH regulation in three coral species exposed to seawater acidification. Sci. Rep. 9, 2201 (2019).
Venn, A. A., Bernardet, C., Chabenat, A., Tambutté, E. & Tambutté, S. Paracellular transport to the coral calcifying medium: effects of environmental parameters. J. Exp. Biol. 223, (2020).
Chatin, B., Venn, A. A., Tambutté, É. & Tambutté, S. In vivo observation of lipid droplets in coral calcifying cells: fat stores to fuel the reef-building process? Coral Reefs https://doi.org/10.1007/s00338-023-02439-8 (2023).
Tambutté, E., Ganot, P., Venn, A. A. & Tambutté, S. A role for primary cilia in coral calcification?. Cell Tissue Res. 383, 1093–1102 (2021).
Saraste, J., Dale, H. A., Bazzocco, S. & Marie, M. Emerging new roles of the pre-Golgi intermediate compartment in biosynthetic-secretory trafficking. FEBS Lett. 583, 3804–3810 (2009).
Taguchi, T. Emerging roles of recycling endosomes. J. Biochem. (Tokyo) 153, 505–510 (2013).
Jedd, G., Mulholland, J. & Segev, N. Two new Ypt GTPases are required for exit from the yeast trans-golgi compartment. J. Cell Biol. 137, 563–580 (1997).
Pereira-Leal, J. B. & Seabra, M. C. Evolution of the rab family of small GTP-binding proteins11. J. Thornton. J. Mol. Biol. 313, 889–901 (2001).
Takahashi, S. et al. Rab11 regulates exocytosis of recycling vesicles at the plasma membrane. J. Cell Sci. 125, 4049–4057 (2012).
Chen, M.-C. et al. ApRab11, a cnidarian homologue of the recycling regulatory protein Rab11, is involved in the establishment and maintenance of the Aiptasia-Symbiodinium endosymbiosis. Biochem. Biophys. Res. Commun. 338, 1607–1616 (2005).
Welz, T., Wellbourne-Wood, J. & Kerkhoff, E. Orchestration of cell surface proteins by Rab11. Trends Cell Biol. 24, 407–415 (2014).
Fox, E., Meyer, E., Panasiak, N. & Taylor, A. R. Calcein staining as a tool to investigate coccolithophore calcification. Front. Mar. Sci. 5, 326 (2018).
Ramesh, K., Hu, M. Y., Thomsen, J., Bleich, M. & Melzner, F. Mussel larvae modify calcifying fluid carbonate chemistry to promote calcification. Nat. Commun. 8, 1709 (2017).
Vidavsky, N. et al. Initial stages of calcium uptake and mineral deposition in sea urchin embryos. Proc. Natl. Acad. Sci. USA 111, 39–44 (2014).
Tambutté, E. et al. Calcein labelling and electrophysiology: insights on coral tissue permeability and calcification. Proc. R. Soc. B Biol. Sci. 279, 19–27 (2012).
Gagnon, A. C., Adkins, J. F. & Erez, J. Seawater transport during coral biomineralization. Earth Planet. Sci. Lett. 329, 150–161 (2012).
Holcomb, M., Cohen, A. L. & McCorkle, D. C. An evaluation of staining techniques for marking daily growth in scleractinian corals. J. Exp. Mar. Biol. Ecol. 440, 126–131 (2013).
Ohno, Y. et al. Calcification process dynamics in coral primary polyps as observed using a calcein incubation method. Biochem. Biophys. Rep. 9, 289–294 (2017).
Venn, A. A. et al. Impact of seawater acidification on pH at the tissue–skeleton interface and calcification in reef corals. Proc. Natl. Acad. Sci. USA 110, 1634–1639 (2013).
Basrai, M. A., Naider, F. & Becker, J. M. Internalization of lucifer yellow in Candida albicans by fluid phase endocytosis. J. Gen. Microbiol. 136, 1059–1065 (1990).
Cardoso-Gustavson, P., Robazzi, B. ignelli, Valente Aguiar, J. M., Ricardo Pansarin, E. & de Barros, F. A light in the shadow: the use of Lucifer Yellow technique to demonstrate nectar reabsorption. Plant Methods 9, 20 (2013).
Page, E., Goings, G. E., Upshaw-Earley, J. & Hanck, D. A. Endocytosis and uptake of lucifer yellow by cultured atrial myocytes and isolated intact atria from adult rats. Regulation and subcellular localization. Circ. Res. 75, 335–346 (1994).
Hanani, M. Lucifer yellow – an angel rather than the devil. J. Cell. Mol. Med. 16, 22–31 (2012).
Stewart, W. W. Lucifer dyes-highly fluorescent dyes for biological tracing. Nature 292, 17–21 (1981).
Berthiaume, E. P., Medina, C. & Swanson, J. A. Molecular size-fractionation during endocytosis in macrophages. J. Cell Biol. 129, 989–998 (1995).
Swanson, J. Chapter 9 fluorescent labeling of endocytic compartments. in Methods in Cell Biology (eds Wang, Y.-L., Taylor, D. L. & Jeon, K. W.) vol. 29 137–151 (Academic Press, 1988).
Wang, X. et al. The evolution of calcification in reef-building corals. Mol. Biol. Evol. https://doi.org/10.1093/molbev/msab103.(2021)
Thouverey, C. et al. Proteomic characterization of biogenesis and functions of matrix vesicles released from mineralizing human osteoblast-like cells. J. Proteom. 74, 1123–1134 (2011).
Remsburg, C., Testa, M. & Song, J. L. Rab35 regulates skeletogenesis and gastrulation by facilitating actin remodeling and vesicular trafficking. Cells Dev. 165, 203660 (2021).
Ujiié, Y. et al. Unique evolution of foraminiferal calcification to survive global changes. Sci. Adv. 9, eadd3584 (2023).
Pearse, V. B. & Muscatine, L. Role of symbiotic algae (zooxanthellae) in coral calcification. Biol. Bull. 141, 350–363 (1971).
Taylor, D. Intra-Colonial Transport Of Organic Compounds And Calcium In Some Atlantic Reef Corals. http://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=PASCAL7850225407 (1977).
Allemand, D., Tambutté, É., Zoccola, D. & Tambutté, S. Coral Calcification, Cells to Reefs. in Coral Reefs: An Ecosystem in Transition (eds Dubinsky, Z. & Stambler, N.) 119–150 (Springer Netherlands, Dordrecht). https://doi.org/10.1007/978-94-007-0114-4_9.2011
Cleves, P. A., Strader, M. E., Bay, L. K., Pringle, J. R. & Matz, M. V. CRISPR/Cas9-mediated genome editing in a reef-building coral. Proc. Natl. Acad. Sci. USA 115, 5235–5240 (2018).
Cleves, P. A. et al. Reduced thermal tolerance in a coral carrying CRISPR-induced mutations in the gene for a heat-shock transcription factor. Proc. Natl. Acad. Sci. USA 117, 28899–28905 (2020).
Raz-Bahat, M., Erez, J. & Rinkevich, B. In vivo light-microscopic documentation for primary calcification processes in the hermatypic coral Stylophora pistillata. Cell Tissue Res. 325, 361–368 (2006).
Venn, A., Tambutté, E., Holcomb, M., Allemand, D. & Tambutté, S. Live tissue imaging shows reef corals elevate pH under their calcifying tissue relative to seawater. PLoS ONE 6, e20013 (2011).
Debreuil, J. et al. Specific organic matrix characteristics in skeletons of Corallium species. Mar. Biol. 158, 2765–2774 (2011).
Zoccola, D. et al. Structural and functional analysis of coral Hypoxia Inducible Factor. PLoS ONE 12, e0186262 (2017).
Wright, K. M. & Oparka, K. J. Uptake of Lucifer Yellow CH into plant-cell protoplasts: a quantitative assessment of fluid-phase endocytosis. Planta 179, 257–264 (1989).
Ganot, P. et al. Structural molecular components of septate junctions in cnidarians point to the origin of epithelial junctions in eukaryotes. Mol. Biol. Evol. 32, 44–62 (2015).
Acknowledgements
We are grateful to Nathalie Técher, Dominique Desgré, Xavier Maccario, and Éric Elia for technical assistance and coral culture. This work was funded by the Government of the Principality of Monaco.
Author information
Authors and Affiliations
Contributions
B.C. and M.R. carried out research, contributed to the conception and design of the study and analyzed data. S.T. contributed to the conception and design of the study and secured funding. A.V., P.G., N.C.S., and E.T. carried out supporting research to the study. B.C., M.R., A.V., P.G., N.C.S, E.T., and S.T. wrote the manuscript. All authors contributed to the article and approved the submitted version
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Communications Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editors: Jin-Min Nam and Mengtan Xing.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Chatin, B., Robert, M., Venn, A.A. et al. Insights on the intracellular trafficking of calcifying medium in a reef-building coral. Commun Biol 9, 22 (2026). https://doi.org/10.1038/s42003-025-09275-2
Received:
Accepted:
Published:
Version of record:
DOI: https://doi.org/10.1038/s42003-025-09275-2
