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Ubiquitous macropinocytosis in anthozoans

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Cite this article as: eLife 2020;9:e50022 doi: 10.7554/eLife.50022

Abstract

Transport of fluids, molecules, nutrients or nanoparticles through coral tissues are poorly documented. Here, we followed the flow of various tracers from the external seawater to within the cells of all tissues in living animals. After entering the general coelenteric cavity, we show that nanoparticles disperse throughout the tissues via the paracellular pathway. Then, the ubiquitous entry gate to within the cells’ cytoplasm is macropinocytosis. Most cells form large vesicles of 350–600 nm in diameter at their apical side, continuously internalizing their surrounding medium. Macropinocytosis was confirmed using specific inhibitors of PI3K and actin polymerization. Nanoparticle internalization dynamics is size dependent and differs between tissues. Furthermore, we reveal that macropinocytosis is likely a major endocytic pathway in other anthozoan species. The fact that nearly all cells of an animal are continuously soaking in the environment challenges many aspects of the classical physiology viewpoints acquired from the study of bilaterians.

Introduction

Unicellular eukaryotes use endocytosis, in particular phagocytosis, to probe their surrounding medium and obtain their next meal. In higher metazoans, that is bilaterians, phagocytic cells mostly represent an essential branch of the immune system, whereas feeding is mediated by digestive organs which degrade complex molecules into nutrients further absorbed by individual cell (Desjardins et al., 2005; Goodman, 2010; Buchon et al., 2014; Cosson and Soldati, 2008). In early branching metazoans such as sponges or anthozoans (corals and sea anemones), comparatively little is known in regard to the cellular processes by which the surrounding medium with its nutritive, signaling or infectious contents, is processed.

From a histological and anatomical point of view (Allemand et al., 2011; Tambutté et al., 2011), anthozoans (included in the phylum Cnidaria) are simple organisms: their mouth is surrounded by a crown of tentacles and serves both for feeding and excreting waste, their body cavity hosts digestive and reproductive organs. The tissues are made up of two cell layers, the ectoderm and the endoderm separated by an acellular layer of mesoglea. Stony corals (scleractinians) represent the branch of anthozoans that synthetize a skeleton. They are colonial, meaning that the individuals called polyps are linked together by the cœnosarc, a portion of continuous tissue that connects polyps together (Figure 1). Their tissues are qualified as oral or aboral depending on whether their ectoderm is facing seawater or the skeleton, respectively. Between the oral and aboral tissues lies the common central cavity of the colony, called the coelenteric cavity. Most reef-building corals are symbiotic, hosting in their endodermal cells photosynthetic dinoflagellates (Symbiodiniaceae) from which they derive a large part of their organic nutrients (Muscatine and Porter, 1977). At the interface between the calcifying ectodermal layer (also called the calicoblastic epithelium) and the skeleton lies the extracellular calcifying medium (ECM) (Tambutté et al., 2011). Despite their relatively simple tissular organization, many questions remain concerning the biology/physiology of corals. Firstly, the process of cell to cell communication and exchange is unknown; secondly, these animals depend on both heterotrophic and autotrophic feeding but the cellular pathways involved in these two feeding modes are unknown; thirdly, these animals build a calcareous skeleton but the path by which ions and organic molecules are supplied to the site of calcification remains debated. Importantly, although cell-cell communication, feeding and calcification seem to refer to different aspects of biology, these processes all involve the properties of the coral’s epithelial cell layers. Indeed, as is the case for any multicellular organism, the coral epithelial cell layers (i) serve as functional barriers, forming selectively permeable interfaces between compartments of different chemical composition and regulating the transport of ions and molecules, (ii) act as sensors of the extracellular environment, triggering the cellular response to extracellular variations and (iii) are involved in cell-cell communication.

Coenosarc anatomy.

The tissue connecting individual polyps is composed of two tissues. The oral tissue is composed of the oral ectoderm (in contact with the sea water) and the oral endoderm (which contains Symbiodiniaceae in most cells) separated by the acellular mesoglea. The aboral tissue is composed of the aboral endoderm (which contains Symbiodiniaceae in some cells) and the calicoblastic ectoderm (responsible for the formation of the skeleton) separated by the mesoglea. Tissue layers have their basal side contacting the mesoglea whereas motile cilia and septate junctions mark their apical side. In between the two endodermal layers lies the coelenteric cavity which carries most of the internal fluids.

Transport of ions/molecules from one cell layer to another cell layer and between the two tissues has been shown to occur through two different pathways: a paracellular and a transcellular pathway. The paracellular pathway (i.e. passing in between cells) occurs through septate junctions (Davy et al., 2012; Tambutté et al., 2012; Barott et al., 2015), the properties of which reflect the cell layer permeability. This paracellular pathway is selective and depends on the size and charge of each transported entity (Tambutté et al., 2012). Based on physiological experiments, it has been shown that the permeability to ions is different between the oral and aboral tissues (Bénazet-Tambutté et al., 1996b). Based on ultrastructural and molecular data, this could be due to constituent differences between the septate junctions of the oral and the aboral cell layers (Ganot et al., 2015). In regard to coral anatomy and fluid circulation, it must be noted that 1) only the oral ectoderm is covered by a layer of mucus which can affect the velocity of ion/molecule/fluid direct uptake from seawater; 2) both oral and aboral endoderms are bathed by the coelenteric fluid the renewal rate of which depends on mouth closure/aperture and on fluid diffusion through the paracellular pathway; 3) the calcifying aboral ectoderm is neither in direct contact with seawater nor with the coelenteric fluid. Therefore, both paracellular diffusion and internal fluid renewal should be considered when studying the transport of fluid/molecules/ions through coral epithelial cell layers. The supply of ions to the ECM involves a transcellular pathway with channel/carriers located on the apical and basal membranes of the polarized calicoblastic cells. For example, bicarbonate and calcium have been proposed to be transcellularly transported through these carriers/channels (Barott et al., 2015; Zoccola et al., 2015; Zoccola et al., 2004; Zoccola et al., 1999). Transcellular and paracellular transport can only help explain how ions and specific molecules are transported from seawater to the different cell layers and to the ECM (Allemand et al., 2011). In fact, molecules and fluids presumably also enter and exit coral cells through endocytosis and exocytosis, respectively. These processes have been poorly characterized in corals but some authors suggest that trans-cellular transport of vesicles occurs during the calcification process (Mass et al., 2017). In these models, it has been proposed that seawater is endocytosed into vesicles and enriched in carbonate ions in the calcifying cells. These vesicles would then be transported and their content exocytosed in the ECM.

Endocytosis includes different cellular pathways which can be classified according to vesicle size. Clathrin- and caveolin- dependent/independent internalization are endocytic processes using small (less than 100 nm) vesicles (Hansen and Nichols, 2009; McMahon and Boucrot, 2011). On the other hand, endocytic processes operating via large vesicles (200 nm – 5 µm) are typical of phagocytosis and macropinocytosis (Bloomfield and Kay, 2016; Kerr et al., 2009; Mayor and Pagano, 2007). Macropinocytosis is a highly conserved process found from unicellular eukaryotes to metazoans, similar to that of phagocytosis, although direct contact with the internalized material is not required as for phagocytosis (McNeil, 1981; Levin et al., 2015; Doherty and McMahon, 2009). Macropinocytosis is an actin-driven process: plasma membrane ruffling and closing takes up surrounding extracellular fluids and its content into large vesicles, which may further fuse intracellularly with endosomal vesicles (Swanson, 2008). In humans, macropinocytosis can be constitutive or induced and has been involved in various cellular functions including antigen presentation, cell metabolism, as well as in cancer (increased nutrient uptake), pathogen entries (from viruses to protozoans), and therapeutics (drug entry gate) (Bloomfield and Kay, 2016; Levin et al., 2015; Recouvreux and Commisso, 2017; Canton et al., 2016; Canton, 2018; Yoshida et al., 2018; Zhang et al., 2011; Lim and Gleeson, 2011; Commisso et al., 2013). Hence, macropinocytosis can be considered as the endocytic pathway allowing non-specific endocytosis of large nanoparticles, up to 5 µm in diameter (Swanson, 2008).

In anthozoans (which include sea anemones, gorgonians and corals), macropinocytosis has never been characterized despite its potential role in fluid uptake. Of note, pioneering studies concerning the uptake of India ink (Olano and Bigger, 2000) or dissolved organic matter (DOM) by epithelial cell layers (Apte et al., 1996; Schlichter, 1982) might be worth being revisited with regard to more recent macropinocytosis data. In the present study, we used several type/size of nanoparticles, that is gold nanoparticles, latex beads and dextrans to characterize the transport of fluids and associated molecules/nanoparticles in the coral Stylophora pistillata for which many physiological, biochemical and molecular data are available (Tambutté et al., 2011). Using mostly confocal microscopy approaches, we followed nanoparticle movement from the surrounding seawater to the coelenteron, then to the different tissue layers via the paracellular pathway and further into the cells’ cytoplasm. We show that the large majority of cells continuously take up large volumes of their surrounding medium using macropinocytosis. Macropinocytosis was confirmed both with transmission electronic microscopy and specific inhibitor experiments. In addition, we show that macropinocytosis is polarized from the apical to the basal side of cells in all tissues. Thus, the oral ectoderm facing the seawater directly absorbs the media, more precisely what is trapped in the mucus covering the animal; the two endoderms lining the coelenteron directly absorb the coelenteric fluid, whereas the calicoblastic ectoderm samples the ECM. In terms of dynamics, the mucus apparently represents a mesh slowing down large particles from being immediately taken up by the oral ectoderm. The coelenteric cavity is filled up within ca. 5 min, whereas only nanoparticles below 20 nm width reach the ECM with an additional ca. 10 min delay, likely due to the passage through the septate junctions filtering the paracellular diffusion to the ECM. Finally, we also described macropinocytosis in the sea anemone Anemonia viridis and in the octocorallian Corallium rubrum, thereby revealing macropinocytosis as a major endocytic process in anthozoans.

Results

Dextran uptake by Stylophora pistillata occurs through vesicles

To investigate the endocytic route in the coral Stylophora pistillata, we first analysed tracer molecule uptake within the whole organism down to the cell level by incubating microcolonies in sea water containing gold dextran-coated nanoparticles of two sizes (3 nm and 10 nm). Using transmission electron microscopy (Figure 2) we detected gold nanoparticles within large intracellular vesicles bigger than 200 nm in diameter, in both the ectodermal and the endodermal layers. Dextran particles were never observed free in the cytoplasm. This result shows that nanoparticles follow an endocytotic pathway with the aspect of vesicles varying from simple to multivesicular body endosomes. Such large vesicles could be characteristic of phagocytosis, macropinocytosis, or even late endosomes resulting from the intracellular fusion of smaller early endosomes. However, when osmium post-fixation was performed (Figure 2—figure supplement 1), electron micrographs showed large vesicle formation right below the apical membrane of both ectodermal and endodermal cells which is typical of macropinocytotic processes.

Figure 2 with 1 supplement see all
Endocytosis of gold dextran-coated nanoparticle by large vesicles.

Microcolonies were incubated in sea water complemented with Gold dextran coated nanoparticles of 3 nm (a,b,d,e) or 10 nm (c) in diameter and imaged with transmission electron microscopy in the ectoderm (a–b) or the endoderm (c–e). Samples here were not contrasted with OsO4. In (a) gold nanoparticles are detected in 3 out of the four vesicles (finger pointing) present in the tissue layer, but not in other parts of the cytoplasm. Higher magnifications (b–e) clearly delineate the large vesicles containing the nanoparticles. Of note, multivesicular body endosomes containing the nanoparticles (d, vesicle on the right, and e) were also observed. Ext, external sea water medium; nu, nucleus.

Dynamics of dextran uptake differs between cell layers and dextran size

To get further insight into coral endocytosis, we next sought to qualitatively characterize fluid/molecule dynamics within whole S. pistillata animals using common fluorescent dextran as endocytic markers (Kerr et al., 2009; Clarke et al., 2002; Wang et al., 2014; Li et al., 2015; Chen et al., 2018). Furthermore, we asked whether dextran uptake by the different cell layers was particle size dependent. Therefore, we performed a time course experiment using two sizes of dextrans, 3 kDa and 10 kDa (D3K and D10K) (Figure 3 and Figure 3—figure supplements 13). The control at T0 shows no fluorescent labeling (ie. no autofluorescence) whatever the epithelial layer analyzed. After 5 min, dextran molecules were visible at the apical surface of all epithelial layers except for the aboral calicoblastic ectoderm where the labeling appeared 10–20 min later. With incremental pulse duration (from 10 to 240 min incubations), dextran labeling increasingly invaded every cell layer in an apparent apical to basal manner. Importantly, the pattern of dextran uptake in the oral ectoderm appeared to be size-dependent, with preferential internalization of D3K versus D10K. This was not the case within both endoderms where dextran fluorescence in the cells was similar whatever the size of the dextran (Video 1). Of note, D3K and D10K appeared to co-localize only in a few vesicles (Figure 3). However, close inspection of the separate dextran confocal emission channels revealed that both dextrans co-localized in most vesicles in all cell layers, albeit with seemingly variable relative concentrations (ratio). Altogether, this data suggests that the dynamics of molecule internalization is tissue specific and size-dependent and suggests that internalization occurs at the apical membrane in all tissues. To test whether the uptake of dextatran into large vesicles could be extend to other distant anthozoans, we incubated the octocorallian Corallium rubrum in a similar manner (Figure 3—figure supplement 4). Apparently all cells showed D3K and D10K uptake into large vesicles.

Figure 3 with 4 supplements see all
Kinetic of dextran uptake by Stylophora pistillata.

Incremental time length of Dextran 3K and 10K incubation shows gradual penetration of dextrans inside the tissues. Note that this figure only shows 5, 20, and 240 min’ incubation times, see Figure 3—figure supplement 3 for complementary time points. All images correspond to y-projections of Z stacks acquired through the oral (top panel) and the aboral tissues (bottom panel). The different tissue layers, depicted besides the photos as a,b,c,d, correspond to the oral ectoderm, the oral endoderm, the aboral endoderm, and the calicoblastic ectoderm respectively (SW: sea water; coel.: coelenteron; ECM: Extracellular Calcifying Medium). Dextran 3K and 10K individual channel acquisitions are shown in black and white, merged are shown in color: Dextran 10 KDa (D10K) in blue, Dextran 3 KDa (D3K) in green, DAPI (nuclei) in red and chlorophyll autofluorescence from the Symbiodiniaceae present in the endodermal tissue layers in gray. Each photograph represents 144 µm wide and 9 µm depth tissues.

Video 1
Macropinosomes.

A branch of Stylophora pistillata Coral was incubated for 30 min with D3K and D10K, then fixed. The video shows a 3D reconstruction (LAS-AF) of a Z-stack encompassing the aboral endoderm. Symbiodiniaceae (10 µm diameter) are in gray, nuclei (3 µm diameter) in red, texasRed-D3K in green and TRITC-D10K in blue. Note the macropinosomes labeled with D3K and D10K which appear as merged color.

Dextran vesicle progression inside the cells

Next, we further characterized vesicular intracellular progression in all S. pistillata tissues through a series of pulse-chase experiments using three dextran sizes (3, 10 and 70 kDa). From the previous time course experiment, we determined that 15 min was the time necessary to visualize dextran within cells of all S. pistillata epithelia. Indeed, after 15 min incubation, most D3K and D10K signals were, as expected, concentrated at the apical side of the different cell layers (Figure 4A). However, after a 4 hr chase period preceded by a wash (ie. dextran was no longer present in the incubation medium), the dextran signal was more centrally positioned inside the cells (compare with nuclei position), confirming an apical to basal progression in all tissues. Again (see above), in the oral epithelium, penetration of the D10K was delayed as compared with D3K (Figure 4B). This was even more obvious when comparing 3 KDa and 70 KDa dextrans (Figure 4C). These results indicate that endocytic vesicles of dextran follow an apical to basal progression within all S. pistillata tissues reminiscent of endocytic processes.

Cellular progression of the dextran uptake.

Dextran pulse of 15 min followed by 4 hr chase. Microcolonies were incubated for 15 min in sea water supplemented with D3K and D10K and fixed (A), or pulsed with D3K and D10K (B) or D3K and D70K (C) and left for an additional 4 hr in sea water before fixation. Tissues and labelling legends are as in Figure 2. Note that in all tissues, dextrans appear to follow an apical to basal progression inside the cells. Contrary to the other cell layers, the oral ectoderm have preferential endocytosis toward smaller dextran molecules (D3K >> D10K>D70K). Each photograph represents 144 µm wide and 9 µm depth tissues.

Endocytosis occurs at the apical side of the cells

In order to firmly conclude that the uptake of dextran occurs apically, we performed double dextran pulse experiments (Figure 5). After a first 15 min pulse and a 4 hr chase, another 15 min pulse was applied using only D3K so that dextran size did not come into play. Combinations of different fluorochomes were used in pulse 1 and 2 to make sure that the choice of the fluorochome had no effect on the results, which it did not (not shown). Whichever the epithelial cell layer considered, we show that after the pulse1-chase-pulse2 experiment, dextran fluorescence from the first pulse was observable within the cells whereas the fluorescence of the dextran from the second pulse was only found at the apical side of the cells. Combined with the results of Figure 4, these results clearly demonstrate that dextran endocytosis occurs on the apical side of all cell layers.

Figure 5 with 2 supplements see all
Apical to basal endocytosis: two 15 min pulses separated by a 4 hr chase.

Microcolonies were first incubated for 15 min with D3K either coupled with Texas-red (left panel) or Alexa 488 (right panel), chased in sea water for 4 hr to let the dextran progress through the epithelial cells’ cytoplasms, and then incubated for another 15 min with D3K coupled with TRITC. Regardless of the cell layer, the second pulsed dextran was always found at the apical face of the tissues whereas the first pulsed dextran was visible throughout the cells’ cytoplasms. Each photograph represents 144 µm wide and 9 µm depth tissues.

In the case of the endoderms, the results accordingly show that the up-taken fluids/molecules came from the coelenteron and not from fluids absorbed basally from the mesoglea, as the apical sides of both cell layers face the coelenteron. This was corroborated with experiments performed on the sea anemone Anemonia viridis whereby tentacles were filled with D3K and D10K (Figure 5—figure supplement 1). In this latter set-up, only the endodermal cells (and not the ectodermal cells) were bathed on their apical sides with the dextran solution. Similar to what is observed in corals, dextran uptake gradually increased in the endodermal cells from one to 15 min (Figure 5—figure supplement 2), implying that endodermal cells apically endocytose dextrans from the coelenteron.

In corals, since the apical side of the oral ectoderm lines seawater and since the apical side of the aboral calicoblastic ectoderm lines the extracellular calcifying medium (ECM), we reasoned that dextran uptake by these two layers had to come from the seawater and the ECM, respectively (note that seawater is separated from the apical side of the oral ectoderm by the mucus layer). Therefore, apical endocytosis of dextran by the calicoblastic ectoderm implies that dextran first follows the paracellular pathway from the coelenteron to the ECM.

Paracellular diffusion through the aboral cell layers

To verify Dextran paracellular diffusion from the coelenteron to the ECM, we recorded movement of fluorescent dextran D3K between the cells composing the aboral tissue with live imaging of microcolonies grown on coverslips at the edge of the microcolony (where there are gaps in between the growing crystals). As was already observed with the tracer calcein in Tambutté et al. (2012), dextran diffuses along the paracellular pathway through septate junctions (Video 2). Septate junctions are 15–20 nm in width and control the perm-selectivity of molecules. Therefore, in order to further characterize the paracellular diffusion through the septate junctions, we followed the flow of latex beads of 20 and 200 nm in diameter (particles ten to hundreds of times bigger than dextrans; Table 1, Figure 6). Confocal observation of the different tissues showed clear intra-cellular localization of the beads in the oral ectoderm as well as the oral and aboral endoderms, regardless of their size. However, in the aboral calicoblastic ectoderm, no particle uptake was detected. This result clearly shows that the oral ectoderm and the two endoderms can absorb the beads (and not only dextran-based molecules) from the seawater and the coelenteron, respectively. However, barriers, likely represented by the septate junctions of the two aboral cell layers (plus potentially the mesoglea), prevent the paracellular diffusion of latex beads over 20 nm in diameter towards the ECM and thus, prevent their access to the calicoblastic ectoderm.

Latex beads paracellular diffusion and macropinocytosis.

microcolonies were incubated with 20 nm and 200 nm latex beads in order to challenge (i) the paracellular barrier and (ii) dextran specific endocytosis. Incubations of Fluorescent latex beads for 2 hr followed by a chase of 1 day show that the three tissue layers (A, oral ectoderm; B, oral endoderm; C, aboral endoderm; D, calicoblastic ectoderm) in contact with the medium could engulf particles of 20 nm (A,B) and 200 nm (C,D) in diameter. Thus, after passing via the polyps’ mouths (data not shown), beads enter the coelenteric cavity and are endocytosed by the endoderm tissue layers, although with less efficiency than dextran molecules. Alternatively, they are directly acquired from the sea water by the oral ectoderm. However, in the calicoblastic cells, neither 20 nm nor 200 nm beads were detected. The paracellular barrier made by septate junctions prevented the access to the extra calcifying medium, and thus the calicoblastic cells. Scale bar = 10 µm.

Video 2
Paracellular diffusion.

The video correspond to screencasts of the LasAM program graphical interface (Leica) displaying the time-laps acquisition (xzyt) in the video mode. The laterally grown microcolony was set in an incubation chamber and analyzed by inverted confocal microscopy from beneath, at the edge of the microcolony where there are gaps in-between the growing crystals. Time laps imaging was recorded (single Z section, 5sec/image). After 3 min 30 s of recording, texasRed-D3K was added to the medium. D3K is in green (TexasRed detected 615–625 nm); Symbiodiniaceae (10 µm diameter) are in gray. Note the timer display at the bottom right corner of the screen. Within tens of seconds, the paracellular labeling is apparent.

Table 1
Tracers’ endocytosis.
TracerSize
(nm)
Charge*Oral ecto.Oral endo.Aboral endo.CalicoPara-cellular
dextran 3 KDa Alexa4881.3(-)+++++++++
dextran 3 KDa TexRed1.3(+/-)+++++++++
dextran 10 KDa TRITC2.3(-)++++++++
dextran 70 KDa TRITC6(-)-+++++++
gold-dextan-coated-3nm3(?)+++nana
gold-dextan-coated-10nm10(?)-++nana
microsphere-20nm20(--)+++--
microsphere-200nm200(--)+++---
  1. *charge: (?) unknown, (+/-) zwitterionic; (-) anionic; (--) highly negatively charged.

Taken together, the data with dextran, gold nanoparticles and latex beads show that particles of different sizes and nature enter the cells into large vesicles analogous to the macropinosomes described in other taxa. Particles are engulfed from the medium lining the apical side of the cells. With regard to the calicoblastic ectoderm, although small molecules such as dextran can pass through the paracellular pathway, latex beads of bigger sizes (see Table 1 for particle sizes) are blocked by the septate junctions.

The size of dextran vesicles is in the 350–1500 nm range

In order to further characterize the endocytotic vesicles in the coral S. pistillata, we performed a quantitative analysis (size and number) of dextran labeling in all epithelial layers at two time points, after a pulse of 15 min or after a 15 min pulse/4 hr chase. Representative images of dextran labeling within the different cell layers are shown in Figure 7A. As aforementioned, the absence of color merging reflects the ratio variability in terms of dextran concentrations within vesicles, whichever the tissue. In the oral ectoderm, most vesicles were 350 to 800 nm in width, with or without the chase period (Figure 7B). As previously observed, the number of vesicles was greater with smaller dextran. In the oral endoderm, D3K was rapidly endocytosed (P15C0), the diameter of the first observable vesicles being in the 350–800 nm range. However, after 4 hr of chasing, the size distribution shifted towards much larger vesicles (up to 1.5 µm in width), which possibly implies vesicle fusion with time and/or cytoplasm progression. The same holds true for the aboral endoderm except that the number of vesicles was significantly higher (maybe due to lower steric hindrance since Symbiodiniaceae are much less numerous than in the oral endoderm) and that there was no preference for dextran size at 15 min pulse. Finally, in the calicoblastic aboral ectoderm, only a few vesicles were counted after the 15 min pulse, likely because of the delay inherent to the passage via the paracellular pathway (D3K passing faster than D10K). After 4 hr of chasing, the vesicle number was higher although vesicle size remained in the 350–800 nm diameter range regardless of dextran particle size. Hence, vesicle quantification reflects what was observed previously, i.e. 1) the vesicles in the ectodermal cells are smaller than those in the endodermal cells (350–800 nm versus 350–1500 nm), potentially due to vesicular fusion; 2) the oral ectoderm shows dextran uptake size dependence; 3) the calicoblastic cells have a delayed kinetic of dextran uptake as compared to the other cell layers.

Quantitative analysis of the dextran endocytic vesicles.

High magnification Z stack sections encompassing the different individual tissue layers (from top to bottom: oral ectoderm, oral endoderm, aboral endoderm, calicoblastic ectoderm) were acquired after the same pulse-chase experiments than in Figure 3A) Representative Z projections for each tissue after D3K/D10K 15 min pulse/4 hr chase (legends as in Figure 3). Note the variable relative content of the co-localizing dextrans from one vesicle to another. (B) For each experiment, the diameters (nm) of the dextran vesicles were measured then ranked according to their size distribution and their counts were normalized per square units (detailed in Appendix 1). Note that the very large vesicles (D > 2 µm) observed in the aboral ectoderm with D3K-D70K were in most cases due to the piling of smaller vesicles that were recognized by the imageJ program as one single artefactual vesicle after Z-stack projection.

The endocytotic pathway of dextran is macropinocytosis

Combining all the data described above, we hypothesize that the endocytic pathway described here in S.pistillata is macropinocytosis. Macropinocytosis has been shown to be specifically inhibited by EIPA (that blocks the NHE exchanger), by PI3K (Phosphoinositide 3-kinases) inhibitors such as wortmannin (Clague et al., 1995; Araki et al., 1996), and by actin inhibitors such as latrunculin (Cosson et al., 1989; Koivusalo et al., 2010; Gold et al., 2010). This is the case in mammalian cells as well as in the protist Dictyostelium discoideum (Neuhaus et al., 2002; Williams and Kay, 2018), implying highly conserved cellular pathways among Eukaryota. To ascertain that macropinocytosis is indeed the endocytic pathway observed in corals, we monitored the uptake of dextran in the presence of these inhibitors. Contrary to the DMSO control experiment (Figure 8A), incubation with EIPA efficiently impaired the formation of macropinosomes (Figure 8B) in all cell layers. When incubated with 1 µM wortmannin, dextran uptake was impaired both quantitatively and qualitatively (Figure 8C) in every cell layer except for the aboral epitheliums. Indeed, Dextran (D3K) uptake in the aboral ectoderm was faint and in the aboral endoderm, the signal was stretched or horse-shoe like, and did not resemble the typical large and round vesicles seen in the control experiments.

EIPA and Wortmannin inhibits macropinocytosis in corals.

Microcolonies were first incubated in the presence of DMSO (control) (A), 100 µM EIPA (B) or 1 µM Wortmannin (C) for 45 min. Then D3K was added to the incubations and pulsed for an additional 30 min before being fixed and processed for confocal analysis. Top (a,b, a’,b’) and bottom (c,d,c’,d’) panels corresponds to oral and aboral tissue analyses, respectively. In each panel, the top picture is a y projection of a Z-stack through the tissue layers as in Figure 3, and the two bottom pictures correspond to a Z projection of a stack embracing individual tissue layers at higher magnification as in Figure 7A. Although DMSO has no effect on dextran endocytosis, EIPA and Wortmannin strongly impaired dextran internalization. Note that for the Wortmannin experiment, PMTs were increased to pick up the low signals of dextran intake. Scale bars = 10 µm.

Vesicle formation was also inhibited by Latrunculin (LatA) and inhibition was reversible (Figure 9). Similar to the previous experiments, dextran uptake was inhibited in the two oral cell layers as well as in the calicoblastic ectoderm in the presence of 500 nM LatA. In the aboral endoderm, although endocytosis was modestly compromised at 500 nM (Figure 9A), it was completely abolished at 1000 nM LatA (Figure 9—figure supplement 1). F-Actin inhibition by latrunculin has been shown to be reversible in vertebrate cultured cells (Spector et al., 1983). To evidence such reversibility in corals, after the inhibitory step previously described (step1), only half of the colonies was fixed while the other half was set back into regular seawater for 4 hr in order to washout the LatA. Then these (semi-)colonies were assayed for dextran uptake. After this second step, endocytosis was restored in all tissues treated with LatA (500 nM and 1000 nM), except for the calicoblastic ectoderm after the 1000 nM LatA treatment as integrity of this particular tissue was compromised at such a concentration. Thus, vesicle formation in S. pistillata was sensitive to EIPA, Wortmannin, and LatA, corroborating our previous results suggesting that vesicle formation follows the macropinocytosis route.

Figure 9 with 1 supplement see all
Actin is required for coral macropinocytosis: at T = 0, microcolonies were incubated in the presence of 500 (panels A and B) Latrunculin or control DMSO (panel C).

At T = 45 min., D3K-TRITC was added to the incubation. At T = 75 min. colonies were cut into two halves, one half was fixed (step1) the other half was set to normal seawater. At T = 315 min., the second halves of the colonies were incubated with D3K-TexRed for another 30 min. before fixation (step2). Panels and legends are as in Figure 7. At 500 nM, micropinocytosis (intake of D3K-TRITC) is clearly inhibited in the oral and calicoblastic cell layers whereas only slightly impaired in the aboral endoderm (step1, Panel A) (note that macropinocytosis in the aboral endoderm is completely inhibitied at 1000 nM LatA, Figure 9—figure supplement 1). However, after 4 hr washing, inhibition was removed as macropinocytic activity (intake of D3K-TexRed) was restored (PanelB). Scale bars = 10 µm.

Finally, in order to get further insight into the mechanism of vesicle formation, we next analyzed the cellular F-actin network during macropinosome formation. To do so, we had to minimize the time of decalcification prior to phalloidin labeling. Hence, we used microcolonies grown on coverslips, which were only partially decalcified before Phalloidin staining. We also used a SP8 confocal equipped with hybrid detectors to increase the detection sensitivity. In this experiment, only the aboral tissues (ie. those in contact with the coverslip) could be visualized (Figure 10, Videos 35). After incubation with D3K for 15 min, we clearly observed an F-actin dent, that we termed ‘cap’, associated with the forming macropinosomes at the apical side of the endodermal and calicoblastic cells. Importantly, these actin caps did not surround the dextran vesicles, at least in the initial stage of formation. Our interpretation of the 3D reconstruction image stacks is that the formation of an intracellular actin cap could create a depression at the surface of the plasma membrane provoking the engulfment of the extracellular liquid.

F-actin is linked to macropinosome formation.

A microcolony grown on coverslip was incubated for 15 min. with D3K, fixed and labeled with phalloidin. Representative confocal Z-stacks (a, c) and 3D reconstruction snapshots (b, d) of the aboral endodermal (a, b) and calicoblastic (c, d) cell layers are shown; Phalloidin (F-actin) in cyan and D3K in red (see also Videos 35). For the vast majority of the forming macropinosomes, as visualized by D3K labeling, an intracellular ‘cap’ of actin is associated (e), illustrating the participation of F-Actin to macropinosome formation.

Video 3
Actin and macropinosomes, large view.

A laterally grown microcolony was incubated in Dextran for 15 min, fixed and shortly decalcified before F-actin labeling with phalloidin. The video corresponds to a 3D reconstruction (LAS-X) of a Z-stack encompassing the aboral endoderm. The video describes a large view of the cellular F-actin network and the process of macropinocytosis (D3K uptake) in the endoderm. TexasRed-D3K in red (macropinosomes), F-actin in cyan and nuclei in blue.

Video 4
Actin and macropinosomes, zoom into the endoderm.

Same as in Video 3 (endoderm), showing a closer view, without nuclei. Note the cap of actin (cyan) always associated with the forming macropinosome (red).

Video 5
Actin and macropinosomes, zoom into the calicoblastic cells.

The video corresponds to a 3D reconstruction (LAS-X) of a Z-stack encompassing 3 cells of the calicoblastic ectoderm. Like for the endoderm, note the cap of actin (cyan) always associated with the forming macropinosome (red).

These results combined with the results concerning vesicle size observed during uptake of dextrans and beads and the localisation of vesicle formation observed at the apical membrane of the cells clearly show that the endocytotic process observed here is macropinocytosis and that these vesicles can be named macropinosomes.

Our data suggest that macropinocytosis is ubiquitous to most cells since every field we observed with the microscope showed a labeling pattern similar to that shown in Video 1. Such a process is not specific to the scleractinian coral Stylophora pistillata since we also observed dextran internalisation in the sea anemone Anemonia viridis and in the red coral, Corallium rubrum. Macropinocytosis thus appears as an ubiquitous process found in different anthozoan species.

Discussion

Here, we show that macropinocytosis is a ubiquitous endocytic pathway found in all the cell layers of the coral S. pistillata (Hexacorallia/Scleractinia). Furthermore, we show that this pathway is conserved in anthozoans since we also observed similar vesicles in the sea-anemone Anemonia viridis (Hexacorallia/Actiniaria) and the red coral Corallium rubrum (Octocorallia/Scleraxonia); Hexacorallia and Octocorallia are the two major subclasses of Anthozoa, which likely diverged more than 600 Mya (Kayal et al., 2018; Guzman et al., 2018). Such a ubiquitous use of macropinocytosis was largely unexpected as this process is restricted to specialized cells in the sister group of Bilateria.

Characterization of macropinocytosis in anthozoans

Macropinocytosis is one of the endocytotic pathways that exists in eukaryotic cells, along with others such as clathrin-mediated endocytosis or phagocytosis (for more details see reviews Kerr et al., 2009; Canton, 2018; Lim and Gleeson, 2011). A budding structure from the plasma membrane is a prerequisite for any endocytotic pathway. In the case of macropinocytosis, it usually occurs from highly ruffled regions of the plasma membrane (flat sheet-like protrusions; Swanson, 2008) that extend from the cell surface, constrict and close, with membrane fusion producing a large intracellular vesicle containing a droplet of medium (Doherty and McMahon, 2009). Using transmission electron microscopy, we could not show outward extensions of the plasma membrane. However, we could clearly picture the vesicles as part of the apical plasma membrane, with apparent subsequent internalization of the extracellular medium, which is typical of macropinocytosis (Figure 2). Macropinocytosis requires a dynamic actin skeleton essential for contractile motility. In corals, the forming vesicles were associated with what appears as an inward actin ‘cap’ (Figure 10 and Videos 35). Furthermore, dextran uptake was reversibly inhibited by latrunculin, a potent actin inhibitor (Figure 9).

Macropinocytosis differs from other types of endocytosis by its unique susceptibility to Na+/H+ exchanger inhibitors (Cosson et al., 1989; Koivusalo et al., 2010). Here we show that EIPA, a specific blocker of this exchanger blocks macropinosome formation. Macropinocytosis is also a Phosphoinositide 3-kinase (PI3K) dependent process in eukaryotes (Araki et al., 1996; Williams and Kay, 2018), and blocking PI3K with wortmannin inhibited macropinocytosis (Figure 8). Thus, although the initial shape of the macropinosome-forming membrane may differ in corals versus vertebrate cells or Dictyostelium, the pharmacological approach used here suggests that the molecular processes leading to macropinocytosis are likely to be conserved throughout evolution. On the other hand, although macropinocytosis strongly supports our observations of nanoparticle uptake, we cannot rule out the existence of other types of Clathrin-independent endocytosis (CIE) (Ferreira and Boucrot, 2018). Among CIE, the CLIC/GEEC (Clathrin-independent carrier/GPI anchored protein enriched endosomal compartments) pathway rapidly uptakes bulk fluid into early endosomes (Howes et al., 2010). The CLIC/GEEC pathway is driven by actin and is PI3K-dependent like macropinocytosis (Hemalatha et al., 2016), however it forms small tubular intermediates that we did not observe.

The size of the vesicles that we observed with the fluorescent fluid phase marker dextran is between 350 and 1500 nm, which is typical of macropinocytosis (Levin et al., 2015; Swanson, 2008). This large vesicle size, orders of magnitude larger than the molecules they capture, enables the uptake of solutes that are excluded from other endocytotic processes. Studies in mammalian cells have shown that macropinocytosis can be induced in a variety of cell types by growth factors and other stimuli whereas dendritic cells and macrophages perform macropinocytosis constitutively (Canton et al., 2016; Canton, 2018). Currently, from our observations, we cannot conclude whether macropinocytosis in corals is induced or constitutive and no data is available in the literature. However, we never performed an experiment without observing macropinocytosis in all cells, suggesting that the process occurs at all times.

Particle diffusion and cell progression of macropinosomes

In corals, particles from the seawater medium are ingested into the coelenteric cavity via the mouth and can diffuse between the cells via the paracellular pathway (gated by 20 nm wide septate junctions; Tambutté et al., 2012; Figure 11). Our time course study shows that within 5 min, particles are detectable in the coelenteric cavity (Figure 4). Then to reach the calicoblastic layer, particles follow the paracellular pathway down to the ECM through the endodermal septate junctions, the mesoglea and the calicoblastic septate junctions, separating the coelenteron from the ECM. On the other side of the animal, particles can reach the oral ectodermal cells directly from the seawater medium. However, in this particular tissue layer, we observed a size dependent particle uptake. The oral ectoderm is covered by mucus which forms a diffusion barrier at the apical side of the cells (Brown and Bythell, 2005). This mucus layer is a gel-forming layer composed of polysaccharides that are constantly released by all coral species (Bythell and Wild, 2011). One possibility to explain the preferential endocytosis of smaller particles is that bigger particles would be trapped or delayed by this mucus layer. This is supported by the Transmission Electron Microscope observation of the content of the ectodermal versus endodermal macropinosomes (TEM observations): the former contain a heterogeneous medium with a fibrillary network (mucus-like) and the latter a homogeneous medium (Figure 2—figure supplement 1). Alternatively, other micropinocytic processes could ensure fluid phase uptake in addition to macropinocytosis. In the slime mold Dyctiostelium, concurrently to macropinocytosis, clathrin-mediated endocytosis is also able to capture Dextran fluorescent tracers into small vesicles that further fuse into large late endosomes (Neuhaus et al., 2002). Multiple endocytotic pathways presumably exist in anthozoans. Endocytosis of small size dextrans (e.g. D3K vs D70K) in small vesicles which would later fuse as larger endosomes may account for some selective aspects of fluid phase uptake. However, the endocytotic process in the different tissue layers is rapid. In A. viridis, macropinosome formation in the aboral endoderm takes 1 to 4 min. In S. pistillata, given the time necessary for the dextran to reach the cellular apical membrane, whichever the cell layer we can estimate to less than 5 min the formation of macropinosomes. Hence, given the kinetics of vesicle formation as well as their size and number, macropinocytosis likely accounts for most of the fluid phase uptake in coral tissues, including the oral ectoderm.

Fluids/particles flow from the sea water to the cell cytoplasm.

Schematic representation of the movement of particles in the different tissue layers of a coral (see text). Stars represent particles of two different sizes (inferior and superior to 20 nm); mp, macropinosome; mr, membrane ruffling; nu; nucleus; sj, septate junction (apical side); zoox, Symbiodiniaceae.

Macropinocytosis occurs in apparently the vast majority of coral cells. Particles are engulfed into large vesicles originating from the apical membrane of the cells. These macropinosomes then move towards the inner part of the cells, down to the basal side. Macropinosomes in the endodermal cells are notably bigger than those of the ectodermal cells. A likely explanation is that in the endodermal cells, macropinosomes fuse during their progression. In vertebrate models, macropinosome fate after internalization varies depending on the cell type, as they can be recycled to the cell membrane or they can adopt degradative properties by fusing with lysosomes and undergoing a lysosome-dependent acidification (Recouvreux and Commisso, 2017). Further investigation would be required to decipher the fate of macropinosomes after internalization in anthozoans.

Macropinocytosis and coral physiology

Macropinocytosis has general implications for coral physiology. With regard to feeding, symbiotic corals not only feed on autotrophic nutrition (symbiosis with Symbiodiniaceae) but also rely on two modes of heterotrophic nutrition, predation and assimilation of DOM (Houlbrèque and Ferrier-Pagès, 2009). The observation that in corals, diverse particles ranging in size from 1 to 200 nm are engulfed by most cell types suggests that exogenous particles such as DOM or particulate organic matter may enter these cells by macropinocytosis. The oral ectodermal cell layer was shown to directly absorb DOM sources from the surrounding sea water by its apical membrane (Schlichter, 1982; Sorokin, 1973), which corresponds to the side of macropinocytosis, and thus corroborates the fact that macropinocytosis could be the DOM feeding pathway in the oral ectoderm. Yet, nutrition in corals involves the production of photosynthates by the Symbiodiniceae which is mostly hosted in the oral endodermal cells. How these photosynthates are further transported to the other tissues remains largely unknown. Bulk fluid absorption via macropinocytocis could form part of the mechanism for the uptake of photosynthates released in the coelenteric cavity. Hence, individual coral cells, and more generally anthozoan cells, would have the capacity to use macropinocytosis for both auto-and heterotrophic feeding.

With regard to calcification, recently Mass and collaborators observed mineral particles within the calicoblastic cells, with sizes varying from 400 nm to 9.4 μm in diameter, 400 nm being the most frequent (Mass et al., 2017). There was no direct evidence that these particles were localized inside vesicles but by analogy between the size of the mineral particles and the vesicles (380 nm) observed by Clode and Marshall (2002), they deduced that the particles and the vesicles referred to the same object. In the present study, sizes of the macropinosomes in the calicoblastic cells were in the same size range as in Clode and Marshall (2002), that is 350 to 800 nm. However, we cannot conclude whether the macropinosomes that we observed correspond to the same particles observed by Mass et al. (2017) and whether or not they contain mineral matter. Of major interest, apical to basal macropinocytosis is commonly observed during endocytotic events including micropinocytosis (Shivas et al., 2010; Apodaca, 2001) and is the prevalent endocytic pathway in all coral tissues, including calicoblastic cells. Thus, calicoblastic cells capture the extracellular medium that is present at the interface between the cells and the skeleton (i.e. the ECM). Taking this into account and the studies showing that the composition of the ECM is different from seawater (higher pH, Ca and carbonate [Sevilgen et al., 2019; Cai et al., 2016]), it is clear that ‘calicoblastic’ macropinosomes do not capture seawater from the medium but rather modified seawater. This brings novel insight into the recent model of calcification/biomineralization that implies transcytosis across the different cell layers where vesicular transport of ions occurs from the surrounding sea water down to the site of calcification (Mass et al., 2017). Of note, we never observed dextran labeled vesicles within the mesoglea, suggesting that vesicle passage from one tissue layer to the other does not occur. Overall, the role of macropinocytosis in the calicoblastic cells and its potential link with calcification awaits deeper investigations.

Finally with regard to corals and environmental stressors (Hoegh-Guldberg et al., 2007; Hughes et al., 2017), pollution by plastics is of primary concern for the global marine environment. Plastics do not dissolve in seawater but gradually fragment into microparticulate matter due to continuous physical, mechanical and chemical attack. With time, microsized plastics become nanosized particles that accumulate in the marine environment (Cole et al., 2011; Law, 2017). In the present study, we show that latex beads as large as 200 nm are able to enter the general coelenteric cavity, likely via the polyps’ mouth, as we observed their accumulation at the oral opening (not shown). Then, they are found within the cells, although large latex beads were apparently internalized with less efficiency than smaller nanoparticles such as dextrans (see Figures 4 and 6). Thus, it seems likely that the size of plastic fragments (micrometer vs nanometer) also affects their internalization efficiency both at the coelenteric entry level and within the cells. However, current studies on corals have so far only addressed the toxicological impact of micrometer sized (entry inside the organism) and not nanometer sized (entry into the cells) plastics (Reichert et al., 2018; Allen et al., 2017; Hankins et al., 2018; Rotjan et al., 2019), as it has been done in other organisms (e.g. Della Torre et al., 2014). Since macropinocytosis allows internalization of nanoparticles of different sorts in all coral cells, this endocytic pathway should now certainly be taken into account in future eco-toxicology studies.

Materials and methods

Culture of corals and microcolonies

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Stylophora pistillata mother colonies were grown in the culture facilities at the Centre Scientifique de Monaco where aquaria were supplied with flowing seawater from the Mediterranean sea (exchange rate 2% h−1), at a salinity of 38, under an irradiance of 175 µmol photons m−2 s−1 of photosynthetically active radiation (PAR) (400–700 nm) on a 12 hr light: dark cycle. Microcolonies were prepared from mother colonies by sectioning samples of 2–3 cm at the apex of a branch. Microcolonies were attached to a monofilament thread in the culture tanks and were used only after complete healing (3 weeks) (Tambutté et al., 2012; Al-Moghrabi et al., 1993). In the case of phalloidin staining and live imaging, another type of microcolony was used that grew laterally on coverslips (Tambutté et al., 2012). These types of microcolonies allow the direct visualization of the aboral tissues (Tambutté et al., 2012). Anemonia viridis sea anemone and Corallium rubrum red coral specimens were collected in Monaco and Marseille and grown in the culture facilities at the Centre Scientifique de Monaco as in Bénazet-Tambutté et al. (1996a); Le Goff et al. (2017). Corals were fed daily with frozen rotifers and twice weekly with live Artemia salina nauplii.

Incubation of microcolonies with nanoparticles

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Incubations of the coral Stylophora pistillata were all performed under similar conditions as cultured corals and experimental time always started between 13:00 PM and 14:00PM. Typical incubation experiments were carried out in 50 ml beakers with a magnetic stirrer. Incubations were all performed in sterile-filtered seawater (SFSW) freshly supplemented with 1 mg/L of lyophilized Rotifer (Ocean nutrition) per liter and further filtrated at 0.2 microns (supplemented sterile filtered seawater = S SFSW). Of note, initial experiments with dextran incubations of less than 60 min in SFSW alone gave similar results, but we preferred supplemented SFSW to avoid potential nutritional stress. Samples were incubated in 20 ml S-SFSW containing the nanoparticle for the desired pulse time and then optionally transferred to a new S-SFSW for the desired chase time, before fixation and further processing. Parallel control experiments without nanoparticles were always carried out. The time of the different pulses and chases are given in the text for each experiment.

In another experiment, a branch of the Mediterranean red coral Corallium rubrum was incubated with D3K and D10K for 4 hr. The branch was then fixed and thin slices of tissues were dissected, then mounted on a microscope slide.

Nanoparticles used

Fluorescent dextrans

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Five type of dextran conjugates have been used (ThermoFisher D-34682, D-3328, D-3308, D-1817, D-1818). D3K (Dextran-Alexa488, 3000 MW, Anionic, Ex/Em = 495/519), D3K (Dextran-Texas Red, 3000 MW, lysine fixable, Zwitterionic, Ex/Em = 595/615), D3K (Dextran- tetramethylrhodamine, 3000 MW, lysine fixable, anionic, Ex/Em = 555/580), D10K (Dextran-tetramethylrhodamine, 10,000 MW, lysine fixable, Anionic, Ex/Em = 555/580), D70K (Dextran, tetramethylrhodamine, 70,000 MW, lysine fixable, Anionic, Ex/Em = 555/580) stock solutions were prepared from powder resuspended in SFSW at 10, 10, 10, 25 and 25 mg/ml, respectively, centrifuged at 12000 g, aliquoted and stored frozen at −20°C. D3K, D10K, and D70K working concentrations were 100, 250, and 500 ng/ml, respectively, to keep equivalent molarity.

Fluospheres

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Fluorescent latex beads of 20 and 200 nm diameter (ThermoFisher F-8887, red fluorescent carboxylate-modified Microspheres, Ex/Em = 580/605) were used at 2% final concentration in S-SFSW.

Gold dextran-coated nanoparticles

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5 ml of 3 nm or 10 nm Dextran coated Gold Nanoparticles (0.1 mg/ml H2O, Interchim Uptima 1P6270 and 1P3660) were complemented with 5 ml 2X artificial sea water (ASW, see below) and added to 120 ml S-SFSW in a beaker. Microcolonies were incubated in this solution for 6 hr before being processed for transmission electron microscopy.

Transmission electronic microscopy of gold dextran-coated nanoparticles containing vesicles

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Sample preparation and electron micrographs obtained with a JEOL transmission microscope were described in Tambutté et al. (2007), except that no Osmium was added to avoid artifact contrast due to precipitation with aldehyde residues. Image contrast and brightness were adjusted with the Photoshop levels tool.

Inhibitor experiments

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Latrunculin A (Sigma), EIPA (5-(N-Ethyl-N-isopropyl) amiloride (Sigma), and Wortmannin (Selleckchem), were dissolved in DMSO at 5 mM, 100 mM, and 10 mM, stock solutions, respectively, and diluted in S-SFSW to 500 nM (or 1000 nM), 100 µM [Laurent et al., 2014], and 1 µM, working concentrations, respectively. A control experiment with 1 µl/ml DMSO was carried out in parallel. For EIPA, and Wortmannin inhibitor experiments, microcolonies were first incubated in S-SFSW supplemented with the inhibitor for 45 min to allow the drug to act, and then transferred into S-SFSW supplemented with the inhibitor and Dextrans (D3K-TRITC) for 30 min, before fixation. For the Latrunculin experiment, microcolonies were also pre-incubated in the presence of the drug (500 nM, 1000 nM, or control DMSO) for 45 min before addition of D3K-TRITC and further incubated for 30 min. At the end of this first step, microcolonies were cut into two equal parts, one half being fixed and the other half being set back to regular seawater for 4 hr (washing). Then these second halves were subjected to another 30 min dextran labeling in S-SFSW with D3K-TexRed before fixation (step2), to assess the reversibility of the inhibition.

General preparation of tissue sections for confocal microscopy

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The procedure for sample fixation, decalcification, microdissection and labeling was previously described in Ganot et al. (2015). Briefly (Figure 1), each sample was fixed in 50 ml chilled artificial-sea-water/paraformaldehyde (PAF) fixation buffer [425 mM NaCl, 9 mM KCl, 9.3 mM CaCl2, 25.5 mM MgSO4, 23 mM MgCl2, 2 mM NaHCO3, 100 mM HEPES pH = 7.9, 4.5% PAF] for 2 hr on ice. The skeleton part of the samples were decalcified in 50 ml [100 mM HEPES pH = 7.9, 500 mM NaCl, 250 mM EDTA pH = 8.0, 0.4% PAF, 0.1% Tween 20] at 4°C until skeleton complete dissolution (3–5 days). The remaining tissues were transferred into PBS for dissection under a binocular where polyps were removed using micro-scissors (Vannas), leaving only the cœnosarc (inter-polyp tissues). A/ For all dextran experiments, the dissected cœnosarc (~5×5 mm) was cut into two equivalent pieces, rinsed 3 times 10 min in PBS; with DAPI in the second rinse for nuclear counter-staining. Then, the two cœnosarc pieces were mounted, one with the calicoblastic (aboral) ectoderm up and the other one with the oral ectoderm up, in anti-fading medium (Slow Fade Gold antifade reagent, Molecular Probes) between microscope slide and coverslip separated by a frame layer (Gene Frame Thermo scientific) to compensate for the thickness of the cœnosarc. B/ For FluoSphere experiments, sagittal slices as thin as possible were manually cut using a scalpel through the cœnosarc. Slices were rinsed, counterstained with DAPI and mounted in anti-fading medium (Slow Fade Gold antifade reagent, Molecular Probes) between microscope slide and coverslip.

Imaging of fluorescent nanoparticles on fixed tissue sections

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Confocal imaging was performed using TCS SP5 DMI 6000 CS (Leica Microsystems) monitored by LasAF software platform with an HC PL APO 40x/1.3 oil CS2 objective. DAPI, Symbiodiniaceae, Green Fluorescent Proteins, FluoSpheres, or fluorescent dextran imaging were acquired sequentially (see Table 2 for tuning). Experiments including dextran were acquired using only two settings: i) high resolution 1024 × 1024 frame size, 0.8 µm Z step size, and 5.2X digital zoom; ii) medium resolution 512 × 32 frame size, 0.5 µm Z step size, and 2.6X digital zoom corresponding to 9 × 144 μm of tissue field. Medium resolution stacks covered the entire ectoderm and endoderm layer of either oral or aboral side. Z-stacks were projected along the y axis (3D projection with X viewing set to 90°) resulting in a pseudo transversal cross section of the tissues (see also Figure 3—figure supplement 2). For FluoSpheres, only high resolution z-stacks were acquired.

Table 2
Dye detection.
FluoLaser ex (nm)Em max (nm)Detection range (nm)
DAPI405461430–470
Symbiodiniaceae*405680–740
GFP*488500–530
Alexa 488488495500–530
TRITC543577557–585
TexRed543613607–630
FluoSphere543605600–640
  1. *autofluorescence.

Imaging of phalloidin and dextran

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Laterally grown microcolonies were incubated for 15 min in S-SFSW supplemented D3K-TRITC. Fixation and decalcification were as before except that decalcification was stopped after 16 hr. Then, the microcolonies grown on their coverslip were rinsed in PBS 3 times and incubated in PBS/3%BSA with Phalloidin-Alexa488 (ThermoFisher A12379) for 4 hr before rinsing in PBS (counterstained with DAPI) and imaged using TCS SP8 inverted microscope with hybrid detectors. Individual channels were acquired sequentially. Three dimensional reconstruction images were computed using the 3D application included in the Leica LasX (Leica) software platform.

Automated counting of fluorescent nanoparticles containing vesicles

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For the dextran D3K/D10K/D70K pulse chase experiment, high resolution imaging of the different tissues layers were processed with imageJ using the methods developed in Wang et al. (2014). The macro that we used is supplied in the Appendix 1.

Live imaging of fluorescent dextran

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Confocal imaging was performed using TCS SP5 DMI 6000 CS (Leica Microsystems) with a PL FLUOTAR 16x/0.5 oil objective. For paracellular live imaging experiments, we used microcolonies grown laterally on glass coverslips as in Muscatine et al. (1997) and Venn et al. (2011). Briefly, the microcolony was set in an incubation chamber and analyzed by inverted confocal microscopy from beneath, at the edge of the microcolony where there are gaps in between the growing crystals. Time laps imaging was recorded (one image every 5 s) using appropriate channels. After 3 min 30 s of recording, texasRed-D3K was added to the medium. Videos correspond to screencasts of the LasAM program graphical interface (Leica) displaying the timelaps acquisition (xzyt) in the video mode.

Appendix 1

Similar to Wang et al. (2014) imageJ macro for counting of the vesicles:

  • 1) confocal high resolution Z-stack series were acquired through individual tissue layer.

  • 2) individual stacks were processed using imageJ:

    • *import lif file with/plugins/BioFormats/Bioformats_importer

    • *Split channels with/Image/Colors/Split_channels

    • *On the channel of interest, project the Z-stacks corresponding to the tissue of interest with/Image/Stacks/Zprojects (maximum intensity)

    • *run the following macro with/plugins/macros/

    • run(‘8-bit’);

    • run(‘Subtract Background...‘, ‘rolling = 15’);

    • run(‘Auto Threshold’, ‘method = MaxEntropy white’);

    • setOption(‘BlackBackground’, false);

    • run(‘Make Binary’); run(‘Watershed’);

    • run(‘Analyze Particles...‘, ‘size = 0.1–20 display summarize’);

  • 3) This imageJ macro gives individual areas in µm2. Given the formula Area(A)=πr2,  the diameter (D, in nm) of each individual vesicle was deduced in Excel using the formula.

    • D=2*(A/π)*1000
  • 4) Diameter values for individual Z-stack were processed into frequencies and normalized per 1000 µm2 using excel.

Results are shown in Figure 7.

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Decision letter

  1. María Isabel Geli
    Reviewing Editor; Institut de Biología Molecular de Barcelona (IBMB), Spain
  2. Suzanne R Pfeffer
    Senior Editor; Stanford University School of Medicine, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

The cell biology of early metazoans is massively understudied. This work supports the view that the major pinocytic process in anthozoans might be macopinocytosis.

Decision letter after peer review:

Thank you for submitting your article "Massive ubiquitous macropinocytosis in anthozoans" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Suzanne Pfeffer as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Even though the manuscript is descriptive, two reviewers with experience in anthozoans agreed that this is a careful study, showing for the first time that basal methazoans take up fluids mainly from the apical side of the oral and aboral cell layers by macropinocytosis. Further, these reviewers felt that this information is of general interest and importance to understand the physiology of this ecologically relevant group and to learn on how they are facing ocean acidification. Based on these comments, it is my pleasure to inform you that the manuscript could be accepted in eLife provided you are able to address some of the major concerns to better characterize the endocytic pathway used by corals.

We summarize here the most important issues to be addressed (and at the bottom include the detailed reviews) but we ask that you focus on these summarized comments to guide your next steps.

1) One of the criteria to state that anthozoans take up fluid mainly by macropinocytosis is the use of "specific" inhibitors. There is a general concern related to the claimed specificity of such inhibitors, because, first, their action has not been properly characterized or reported in these organisms, and second, inhibition of their supposed targets might have many other effects besides inhibiting macropinocytosis. In this context, it would be essential to show that the drugs do not compromise the viability of the corals under the experimental conditions used, by showing that their effects are reversible. Further, it would be important to demonstrate that the drugs act on their expected targets. This should not be difficult for inhibitors of actin and tubulin polymerization, where reversible disruption of the cytoskeleton could be demonstrated by fluorescence microscopy. On the other hand, the inhibition with Latrunculin A and EIAPA might affect many other cellular processes, which indirectly impact on endocytic traffic. One of the reviewers, who is an expert in macropinocytosis, suggested the use of PI3K inhibitors to convincingly show that corals use macropinocytosis for fluid uptake. Finally, I think that it would be important to show that the primary endocytic profiles transiently acquired the machinery involved in macropinosome formation (at least actin).

2) A second important criteria used to conclude that anthozoans use macropinocytosis is the size of vesicles accumulating dextran. Besides the technical and conceptual issues raised by two of the reviewers, which strongly suggest to use vesicle volumes and not areas to describe their size, the measures are taken by confocal microscopy earliest upon 15 minutes exposure to the dextran, plus 15 minutes chase. Within this time window, most Transferrin taken up by clathrin-mediated endocytosis in mammals would be found in sorting and recycling endosomes (not in clathrin coated vesicles) and therefore, the timing is not really suitable to define the primary endocytic profiles. Also, as the authors mention, piling up of small vesicles would be scored as big vacuoles with the technique used. Ideally, if the authors want to identify the primary endocytic vesicles derived from the plasma membrane, the experiment should be performed by electron microscopy upon much shorter (5-10 minutes max) exposure to the dextran, followed by a wash out step to remove the surface-exposed dextran. Otherwise, the authors could use a membrane dye such as FM4-64 combined with super-resolution fluorescence microscopy.

3) In the context of defining whether corals macropinocytose constitutively, it would be important to define or clarify if they do so in the absence of rotifer extract. Also, there seems to be an agreement on the lack of conclusion regarding the dextran size sorting issue. I would suggest that you discuss the results in light of the article (Neuhaus et al., 2002); addressing the issue experimentally seems difficult at this point and probably out of the scope of the manuscript. In this context thought, you claim that the delay in the uptake in the D10K and D70K as compared to the D3K in the oral ectoderm is due to the mucus. If you can enzymatically remove the mucus to directly test this hypothesis, the point could be made. Otherwise, please discuss other possibilities such as the existence of different uptake pathways in different cell types.

4) In general terms, all reviewers agreed that the text needs extensive editing (I attach specific comments bellow) and I would certainly encourage you to shorten the Discussion and reduce speculation. The evidence for the existence of other endocytic pathways should be discussed and properly referenced (see specific comments below). The word massive should be removed from the title.

Specific comments from the reviewers:

Reviewer 1:

1) The amount of Materials and methods-like sentences has to be reduced in the Results section. For example, in the subsections “Dynamics of dextran uptake differs between cell layers and size of dextran”, and “Endocytosis occurs at the apical side of the cells”, and many other places.

Reviewer 3:

1) Introduction: Wording a little hard to follow. Thorough editing needed for verb tenses and singular vs. plural.

2) Introduction: second paragraph needs citations throughout.

3) Introduction, third paragraph: should cite primary literature by these authors and others showing coral septate jxn, the Davy review only presupposes their existence (e.g. Barott et al., 2015).

4) Introduction, third paragraph: Barott et al., 2015 also shows evidence of bicarbonate transporters, as well as Na and K ions.

5) Important to mention in the Introduction that the anthozoans studied here have endosymbiotic algae they derive organic nutrients from. These should be referred to as "Symbiodiniaceae" and not zooxanthellae (a nonspecific term that includes many different families of unrelated algae).

6) Just because macropinocytosis is shown here to be utilized by anthozoans for the first time, the phrase 'at the forefront' seems like an overstatement. We know anthozoans do phagocytosis, that's how they take up their symbionts. It also seems safe to assume that have clathrin and caveolae-mediated endocytosis, as many researchers have seen small vesicles that would be consistent with these modes of uptake. Either clarify what macropinocytosis as forefront of or remove.

7) Results should be in past tense.

8) Subsection “Dextran uptake by Stylophora pistillata occurs through vesicles”: change 'shows' to 'suggests'.

9) Discussion needs to introduce how closely related these different anthozoans are for readers unfamiliar with cnidaria.

10) Subsection “Macropinosomes and coral calcifying cells”: Other examples of more recent studies showing vesicles in the CE: https://doi.org/10.1371/journal.pone.0209734 and Barott et al., 2015.

11) Subsection “Macropinocytosis and coral health”: wild corals injest plastic microparticles: Rotjan et al., 2019.

12) Subsection “Macropinocytosis and coral health”, second to last sentence: confusingly worded.

13) "fixed on ice" – with what? For how long?

14) Subsection “Incubation of microcolonies with nanoparticles”, last sentence: fixed how? Was it decalcified?

15) It would be helpful to refer to different sized corals with different names. Microcolonies seems more appropriate for the corals growing on coverslips (size not indicated but certainly <2cm) than the 2-3 cm coral fragments hanging from a thread. The coral literature typically refers to corals this size as fragments or nubbins. Minicolonies would be fine as well.

Reviewer #1:

This article reveals some interesting aspects of the massively understudied cell biology of early metazoans. Most of the studies on anthozoans have focused on their symbiotic/mutualistic association with dinoflagellates, but despite their essential place in the Earth ecosystem, little is known about how they communicate with and extract food from their environment. This article is an elegant and thorough, though a bit preliminary, dissection of the major pinocytic process in anthozoans, which appear to be of macropinocytic nature. Overall, the article is very well written and reads like a detective novel, but some parts are overly detailed, with un-necessary technical presentations of otherwise relatively standard procedures (except in the field of anthozoans).

1) The methods used to visualise and describe the endocytic mechanisms and compartments are standard, but applied with extreme care and thoroughness. What is missing is a form of quantification of the progression of the tracers in the various cells and layers. It is likely possible to monitor, quantitate and represent the (differential, see point 2 below) advance of the tracers in the form of a "gradient" through the tissue, similarly as is performed when researchers measure and model the gradients of morphogens progressing through a tissue such as the Drosophila imaginal disk.

2) The authors present some interesting data about "size selection" of two different dextran molecules used as fluid-phase tracer. First, such a phenomenon has been reported earlier (for example in Neuhaus et al., 2002) and the authors could compare/contrast their findings with those. Second, because the "molecular sieving" probably does not occur during the ingestion, subsequent macropinosome fragmentation, tubulation and cargo sorting, or other size-discriminating phenomena have to take place. The authors could try to visualise such subsequent phenomena by live microscopy, measuring the fluorescence ratio as a read-out of the selection.

3) Again, the manuscript is well written, but the amount of Materials and methods-like sentences has to be reduced in the Results section. For example, in the subsections “Dynamics of dextran uptake differs between cell layers and size of dextran” and “Endocytosis occurs at the apical side of the cells”, and many other places.

4) The macropinocytic process revealed in this study, as well as the progression through tissue layers are well documented (but see point 1-). Now, in terms of mechanistic insight, beside the size of the compartments, which classify plausibly the process as macropinocytic, the authors should relativise their conclusions based on the use of "specific" drugs to block the pathway, because this is based on experiments performed in evolutionarily distant organisms and the molecular targets and mechanisms of action of the drugs might not be conserved.

5) The authors speculate about the fact that fluid-phase tracers are taken up in a "constant" manner and might become concentrated during the uptake process (subsection “Macropinocytosis and signaling”). The former sounds plausible, but there is no real proof of that. The latter can be substantiated by an experiment (it is easy to measure the intensity of fluorescence per volume units during the process), or the authors have to cut short their speculations, especially "It is thus tantalizing to extrapolate a role for macropinocytosis in cell to cell signaling pathways". Keep this to write a review once the article is published.

Reviewer #2:

This paper potentially adds any interesting piece of biology to the macropinocytosis field, although a lot of it is also very descriptive for eLife. The core is the observation that particles that are too big to be taken up through transporters are being endocytosed via macropinocytosis. That point is clearly made and may be very important for the field. The finding that the macropinocytosis comes from a particular surface seems clear and may be important – a reviewer with expertise in anthozoa will have to comment.

Others are less sound, for one reason or another:

- The focus on the different sizes of dextrans is currently unfocused – there are no conclusions drawn, and "the reason for this is unknown". This party of the story is not ready for eLife yet – it's a set of results that don't yet lead to understanding. Interestingly, the authors may find some clues in the work of Thierry Soldati, who has published on the differential sorting of different dextran sizes. I think the relevant paper was about eight years ago, but can't remember precisely.

I would like to see this work led to a justified scientific conclusion.

- The inhibitors are called "specific" in the Abstract (it is incidentally odd to say that without saying what they're specific for) – but they aren't. Blocking Nhe1 for 45' could really harm cells; the authors need to show that the rest of cellular physiology is unaffected and that the organisms can recover. The latrunculin is possibly worse – single cells that have been incubated that long in high concentrations are slaughtered; it blocks single-cell macropinocytosis assays within minutes, but also blocks other vesicle traffic, makes the membranes go floppy and blebby, and more generally breaks down cell architecture over tens of minutes. It's conceivable that it takes that long for the drug to diffuse in to the cells of interest (have the authors tested this?) but in any case they should consider secondary effects from actin disruption, including but not only changes to cell viability.

I would like to see careful controls that these inhibitors were behaving specifically, and the appropriate part of the Abstract rewritten. Also, why not examine π 3-kinase inhibitors? Those are (somewhat) less likely to kill the cells.

Measuring vesicles and expressing the size as an "area" is not sensible. Vesicles are spherical; the images presumably slice through them at different parts; this needs to be considered and turned into an informed estimate of a volume. And saying " size of the vesicles that we have observed.… is between 350 to 1500 nm" is also unclear – does it mean "the diameter of the slices in EM" or "the diameter of slices in the confocal" or is it a measure of the true diameter like widefield?

The references for dextran in imaging are very late and miss the real innovators – suggest citing for example Clarke et al., 2002.

Reviewer #3:

This study describes for the first time a mechanism, macropinocytosis, for the uptake and intracellular transport of dissolved compounds and nanoparticles within corals and two other anthozoans. The use of fluorescence and electron microscopy, coupled with pharmacological inhibitors of macropinocytosis, provide convincing support for this mechanism. It would be nice to see F-actin staining of the tissues across the pulse/chase time series to help confirm macropinocytosis (and that the 'ruffles' are not just cilia and/or microvilli), something that given the experience of these authors would not be hard to do. Directional transport from the apical side of both endo- and ectodermal cells is convincingly demonstrated for the oral tissues, however I am not convinced they have shown this pattern of uptake in the calicoblastic epithelium. Instead, it looks like the dextrans are moving through the aboral endodermal cells (taken up apically as indicated) and then transported throughout those cells and across via the basolateral side of the CE cells. I was also surprised to read in the Materials and methods that the corals are given a rotifer extract (feeding stimulant) during all of the incubations, but this is never mentioned in the Discussion. This is an important point to bring up and I would like to know if the authors first tried their experiments without the extract and saw no/less macropinocytosis activity.

Overall this study is an important step forward in our understanding of coral biology that has important implications for cnidarian physiology, particularly as it relates to coral exposure to water soluble pollutants but also for regulation of coral uptake of inorganic and organic nutrients. The authors also argue that corals may be a useful model system for the study of macropinocytosis as it relates to human health and drug delivery, a valuable point that was not articulated as clearly as it should be. Overall, it does not seem surprising that anthozoans use macropinocytosis given that this mechanism is conserved with the most basal eukaryotes, and the authors overstate this novelty. That said, to have described this mechanism for the first time in a class of basal metazoans is an important and interesting advance in our understanding of the physiology of this ecologically and evolutionarily significant group of taxa. It also is a step towards describing the mechanisms of intra- and inter-cellular transport necessary for coral calcification, a timely topic of study for understanding how corals facing ocean acidification may fare.

The text of the manuscript needs to be thoroughly edited for flow and clarity.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Congratulations, we are pleased to inform you that your article, "Ubiquitous macropinocytosis in anthozoans", has been accepted for publication in eLife. However, before full acceptance you would need to address a few points by rewriting some sections:

This article reveals some interesting aspects of the massively understudied cell biology of early metazoans and supports the view that the major pinocytic process in anthozoans might be macopinocytosis. There is a general agreement that you have done a major effort:

1) To demonstrate that the Latrunculin A effect is reversible;

2) To image actin in association with the dextran loaded structures;

3) To demonstrate that inhibition of PI3K inhibit dextran uptake; and,

4) To more properly quantify the vesicle-vacuole size.

The reviewers also agree though that the new results further support the view that macropinocytosis might be the prevalent endocytic pathway in corals but they do not conclusively prove it because:

1) The actin associated with the dextran vesicles-vacuoles might still correspond to endosomal actin rather than actin structures associated with forming macropinosomes; and,

2) Because other endocytic mechanisms such as the GEEC pathway are also sensitive to PI3K inhibitors, EIPA and Latrunculin A. In any case, since precise dissection of the endocytic pathways in corals is out of the scope of the manuscript and experimentally very challenging, we are willing to accept the manuscript with no further experimental work, but we ask you to please discuss the fact that other endocytic mechanisms such as the GEEC pathway cannot be excluded.

Some other points need to be addressed in addition:

1) The reviewers accept that you did not really probe that the drugs target the expected proteins, but discuss instead the likelihood that this is the case in evolutionary terms. This is fine for the experiments where you observe an effect of the drug. However, it would be important to show that colchicine indeed depolymerizes the microtubules in your system, since you claim that microtubule depolymerization does not affect dextran uptake. If you cannot do this, this negative result should be omitted.

2) All reviewers also agree that the Discussion is still too long and re-describes some of the results in more detail than needed. The comparison to early TEM work in corals showing the presence of vesicles seems particularly long, as is the discussion of the possible importance of coral macropinocytosis with regard to microplastics. Those last three sections could be combined and condensed into a 'Macropinocytosis and coral physiology' section. Please condense the Discussion to no more than 5 pages double spaced in your draft manuscript.

https://doi.org/10.7554/eLife.50022.sa1

Author response

Several very important concerns were raised and complementary experiments were required in order to improve our demonstration of macropinocytosis in the anthozoan coral Stylophora pistillata (Cnidaria). These major points included i) the use of more specific inhibitors such as PI3K inhibitors, ii) showing that the effect of the inhibitors were reversible, and iii) trying to visualize the interplay between the movement of the plasma membrane and/or iv) the cortical actin network associated with macropinocytosis. Indeed, in other eukaryotic models such as vertebrate cells or the unicellular protist Dictyostelium where the mechanisms of macropinocytosis have been carefully studied, plasma membrane ruffling guided by F-actin reorganization was demonstrated and could be inhibited.

We have tried our best to comply with these very justified demands using different experimental set-ups and we believe that we managed to fill many of the gaps raised by our initial submission. However, working with a whole organism, moreover a coral, is certainly not as straightforward as working on cells (or even other organisms), and some of our experimental attempts were unsuccessful, likely due to the complexity associated with coral biology.

A) General answers and major modifications

1) Membrane visualization: we tried to visualize the cellular membranes of the different tissues using 3 different membrane dyes, i.e. FM4-64 (thermofisher), lipilight (idylle) and Vybrant DiO (thermofisher), without success. Whether using live or fixed samples, we never managed to label any plasma or intracellular membrane. The reason of this is unknown, and would probably require further investigation that falls outside the frame of our present manuscript. We will explore this intriguing “non-result” in the future.

2) Actin visualization: on the one hand, Sir-actin probes (Cytoskeleton) on live animals did not work. On the other hand, we used fluorescent-conjugated Phalloidin on fixed samples to visualize the F-actin network after a short pulse (15 minutes) of dextran, and obtained pretty good results. We had to modify the experimental set-up in the sense that we used laterally grown coral colonies instead of coral fragments: laterally grown coral colonies are grown on coverslips producing a rather thin layer of skeleton. After fixation, they can be (partially) decalcified in a relatively short period of time, i.e. over-night instead of 3-5 days in the regular protocol. Hence, we experienced a much better preservation of the F-Actin network. Also, we moved to a more sensitive confocal equipped with hybrid detectors (Leica SP8). In fine, we were able to clearly image the actin sheet associated with the forming macropinosome (Dextran labeling). Using 3D reconstructions (Leica LasX), we were able to visualize a strong signal of actin (which we called a “cap”) associated with the majority of dextran vesicles. This is now Figure 10 and Videos 3-5 of the manuscript.

3) PI3K inhibitors: we first used LY294002 (cayman chemical) as it is a reversible PI3K inhibitor. However, this drug was inadequate as it had another very strong and unexpected side effect on the general autofluorescence of the cells. We had never observed such a phenotype previously. Author response image 1 shows an example of imaging after LY294002 treatment. We also included a phylogenetic analysis the PI3K family in Stylophora, showing that there is only one PI3K homolog, against 4 in human.

Author response image 1
LY294002 treatment triggers unexpected secondary effect and phylogenetic comparison of the PI3K family in Stylophora pistillata vs human.

(A and B) Confocal Z-stack of Stylophora incubated in LY294002. Red=Dapi; Green=dextra; gray=Zoox, and Cyan is the GFP chanel. A and B correspond to the oral ectoderm and the aboral endoderm, respectively. Note that in the oral ectoderm, the cell layer is filled with GFP-like signal that is never seen under other conditions. In B, the GFP-like signal is less strong (although different than controls), and the vesicles of dextran are abnormally large (4-5 μm in diameter). However, based on the side effect of LY294002, this assay was not validated. (C) PhyML phylogeny of the PI3K families in human (names in black) and in Stylophora (names in blue). In Stylophora, there is only one cognate PI3K homolog (4 in human). The other sequences are PI3K-related members (i.e. PI3K-C2, PI3K-type3 and PI4K).

On the other hand, we successfully used Wortmannin (Selleckchem) another PI3K inhibitor, although irreversible. Like EIPA, Wortmannin blocked Dextran uptake, showing that macropinocytosis in anthozoans is PI3K dependent as in other eukaryotes. This is now Figure 8.

4) Inhibitor reversibility: in the first version of our manuscript, we had shown that the actin inhibitor Latrunculin inhibited dextran uptake at 500 nM (not effective on the aboral endoderm) and 1000 nM (effective on the aboral endoderm). In order to verify that latrunculin (LatA) was not destroying the cellular integrity of the animals’ tissues (which would also end up as an apparent inhibition), we tested reversibility. Corals are colonial organisms. This means that a fragment (corresponding to approx. 100 individuals or polyps) can be cut into 2 pieces without much harm to the colony. In order to assess the reversibility of the drug, we used a similar set-up than in the first version, albeit this time, after the LatA inhibition and dextran pulse, we sampled only one half of the colony. The second half was then submitted to a second step with another dextran pulse after a 4 hour wash without the drug. At 500 nM LatA, we again experienced a strong impairment of the dextran uptake (30 minutes pulse), except in the aboral endoderm (step1). However, after a 4 hours wash in regular seawater, the same colony had recovered the ability to uptake dextran (step2). Concurrently, in the aboral endoderm (as well as in the oral cell layers), inhibition of dextran uptake was achieved at 1000 nM, and was reversibly restored after washing. However, at this concentration, we experienced that the cells composing the thin calicoblastic cell layer were rounded and not forming a true epithelium. Nevertheless, we conclusively showed that latrunculin specifically (since reversible) blocked actin in the process of macropinocytosis, with tissue layers responding differently depending on drug dosage. The 500 nM experiment is now Figure 8 (and figure supplements).

We believe that within the frame of a technically challenging investigation of cell biology in corals, this novel set of experiments should provide sufficient additional proof to conclude that macropinocytosis is indeed the process that we show in the present manuscript. We have developed several innovative imaging approaches for the field of coral biology that we hope should satisfy the general and specific demands of the reviewers, as summed up by the editor.

Other figure changes:

i) Figure supplements have to complement a main text figure. We created a new Figure 1 (which was previously in our supplementary data) to schematize the anatomy and histology of the coral. This helps the comprehension of the following figures for scientist non-familiar to scleractinian corals. Consequently, all previous figures have their number incremented by +1 (ex-Figure 1 is now Figure 2 etc.)

ii) We totally agree that describing vesicles with surface areas in µm2 was inappropriate. We went back to the measurement files and converted every area (which corresponds to Z-stack projections) into diameters (A=πr2). We then reconstructed the distributions of the different diameter ranges, using 200 nm steps. Figure 7 shows vesicles diameters in nm. There was no change to the conclusions of this part.

iii) We changed one picture in the Figure 2—figure supplement 1C which was showing transmission electronic microscope picture of the aboral endoderm. The previous picture was misleading with regard to extracellular microvilli versus intracellular macropinosomes. The new picture clearly shows that endodermal cells contain (forming) macropinosomes (mp) as part of the apical plasma membrane, like in the ectoderm (b). Microvilli (Mv) are now indicated with arrows to avoid confusion. To our understanding, the positioning of these vesicles is not likely to represent large late endosomes resulting from the fusion of smaller early endosomes. The text describing the macropinosome formation as part of the apical membrane has been changed accordingly.

B) Detailed answers to the issues summarized by the editors

[…] We summarize here the most important issues to be addressed (and at the bottom include the detailed reviews) but we ask that you focus on these summarized comments to guide your next steps.

1) One of the criteria to state that anthozoans take up fluid mainly by macropinocytosis is the use of "specific" inhibitors. There is a general concern related to the claimed specificity of such inhibitors, because, first, their action has not been properly characterized or reported in these organisms, and second, inhibition of their supposed targets might have many other effects besides inhibiting macropinocytosis. In this context, it would be essential to show that the drugs do not compromise the viability of the corals under the experimental conditions used, by showing that their effects are reversible. Further, it would be important to demonstrate that the drugs act on their expected targets. This should not be difficult for inhibitors of actin and tubulin polymerization, where reversible disruption of the cytoskeleton could be demonstrated by fluorescence microscopy. On the other hand, the inhibition with Latrunculin A and EIAPA might affect many other cellular processes, which indirectly impact on endocytic traffic. One of the reviewers, who is an expert in macropinocytosis, suggested the use of PI3K inhibitors to convincingly show that corals use macropinocytosis for fluid uptake. Finally, I think that it would be important to show that the primary endocytic profiles transiently acquired the machinery involved in macropinosome formation (at least actin).

See answers in A.

2) A second important criteria used to conclude that anthozoans use macropinocytosis is the size of vesicles accumulating dextran. Besides the technical and conceptual issues raised by two of the reviewers, which strongly suggest to use vesicle volumes and not areas to describe their size, the measures are taken by confocal microscopy earliest upon 15 minutes exposure to the dextran, plus 15 minutes chase. Within this time window, most Transferrin taken up by clathrin-mediated endocytosis in mammals would be found in sorting and recycling endosomes (not in clathrin coated vesicles) and therefore, the timing is not really suitable to define the primary endocytic profiles. Also, as the authors mention, piling up of small vesicles would be scored as big vacuoles with the technique used. Ideally, if the authors want to identify the primary endocytic vesicles derived from the plasma membrane, the experiment should be performed by electron microscopy upon much shorter (5-10 minutes max) exposure to the dextran, followed by a wash out step to remove the surface-exposed dextran. Otherwise, the authors could use a membrane dye such as FM4-64 combined with super-resolution fluorescence microscopy.

We think that there was a misunderstanding with the timing of our dextran incubations (pulses). With the coral Stylophora pistillata, we have been incubating (without chase/wash) the fragments for as short as 5 minutes, as shown in the Figure 3—figure supplement 3 (even shorter pulses were tested; not shown). However, it is only after 10 minutes that the first vesicles were apparent in the aboral endoderm (Figure 3—figure supplement 3) or 15 minutes that they became apparent in most cells (Figures 4A, 5B, 10). Several parameters have to be considered for the dextran to arrive from the surrounding seawater to the 4 cell layers: for the oral ectoderm (in contact with the seawater), the mucus layer is a barrier. For the oral and aboral endoderms, the time for the renewal of the coelenteric cavity, which is the time for the dextran to first arrive in contact with the cellular membranes, is within the 5 minute range (discussed in the Discussion paragraph “Particle diffusion and cell progression of macropinosomes”). Of note, detection of a forming macropinosome requires a sufficient concentration of Dextran to have a detectable signal. Importantly, when we injected Dextran directly into the coelenteric cavity of the sea anemone Anemonia viridis (Figure 5—figure supplement 2), the first vesicles could be seen after only 1 minute, and were definitively evident after 4 minutes. Finally, in the calicoblastic ectoderm facing the skeleton, the dextran present in the coelenteric cavity needs to cross two septate junctions and the mesoglea before reaching the cells’ apical membranes, an additional time lag that we estimate to represent approx. 5 minutes. Thus, the time required for forming large (Diam. >350 nm) vesicles can be estimated to be less than 4 minutes. This is in agreement with experiments demonstrating macropinocytosis on cell lines (in direct contact with their media) from other organisms. It is less compatible with the accumulation of dextran in late endosomes.

3) In the context of defining whether corals macropinocytose constitutively, it would be important to define or clarify if they do so in the absence of rotifer extract. Also, there seems to be an agreement on the lack of conclusion regarding the dextran size sorting issue. I would suggest that you discuss the results in light of the article (Neuhaus et al., 2002); addressing the issue experimentally seems difficult at this point and probably out of the scope of the manuscript. In this context thought, you claim that the delay in the uptake in the D10K and D70K as compared to the D3K in the oral ectoderm is due to the mucus. If you can enzymatically remove the mucus to directly test this hypothesis, the point could be made. Otherwise, please discuss other possibilities such as the existence of different uptake pathways in different cell types.

The possibility of “inducing” macropinocytosis by supplying rotifer extract with the different tracers is a relevant point that we eluded in the first version of the manuscript. In the beginning of our investigation, we were using Sterile Filtered Seawater only to incubate the fragments. And we indeed observed macropinocytosis. Then, in order to avoid potential nutritional stress, we decided to conduct all our experiments with rotifer extracts, as there was no apparent changes in our observations. Thus, although we did not quantify the potential effect of the supplemented extracts, we can rule out the possibility that macropinocytosis is induced by rotifers. We added a comment in the relevant Materials and methods section.

With regard to the “selectivity” of the dextran size in the different tissues, we would rather prefer to avoid drawing definitive conclusions. It is an intriguing phenomenon that we observed (Figures 3 and 4) and measured (Figure 7) especially in the oral ectoderm. Several hypotheses could be drawn. The first and most easily accepted is size selectivity due to the mucus layer. A clear stack of Dextran is visible accumulating only after 5 min on the apical side of the oral ectoderm, where the mucus layer lies (see figures). This Surface Mucopolysaccharide Layer (mucus) is particularly abundant, representing a biological mesh and is part of the mechanisms for heterotrophic feeding from Dissolved Organic Material (DOM) to trapped bacteria (see review by Brown and Bythell, 2005 for extensive descriptions). Another possibility for nanoparticles selectivity could be the net charge of the particles (Table 2). The combination of a mucopolysaccharidic mesh layer with differently charged particles should also be taken into account. However, we certainly cannot exclude additional endocytic mechanisms that would contribute to the selective uptake of nanoparticles as was nicely demonstrated in Dictyostelium (Neuhaus et al., 2002). In this latter scenario, a “classical” endocytic pathway (e.g. clathrin-mediated) would selectively endocytose D3K in small vesicles that would later fuse as large late endosomes or with macropinosomes. This could explain why, after 15 minutes pulse and 4 hours chase (wash), there are still discrepancies between the different dextran size uptakes. However, short pulse experiments (e.g. Figure 4A) always show a rapid preferential uptake of D3K versus D10K, which would be better explained by mucus selective retention. Since we have yet no experimental evidence to conclude on the matter, we have modified the text to leave the field open and discussed the aforementioned hypotheses.

4) In general terms, all reviewers agreed that the text needs extensive editing (I attach specific comments bellow) and I would certainly encourage you to shorten the Discussion and reduce speculation. The evidence for the existence of other endocytic pathways should be discussed and properly referenced (see specific comments below). The word massive should be removed from the title.

We have reduced the Discussion and removed the speculative part on cell-cell signaling via macropinocytosis. The 15 following points (see below) were modified as requested. The entire manuscript has been revised by a professional text editor. We hope that this new version, which includes multiple text changes, will now be eligible for publication.

C) Specific answers to the reviewers

Reviewer #1:

1) The methods used to visualise and describe the endocytic mechanisms and compartments are standard, but applied with extreme care and thoroughness. What is missing is a form of quantification of the progression of the tracers in the various cells and layers. It is likely possible to monitor, quantitate and represent the (differential, see point 2 below) advance of the tracers in the form of a "gradient" through the tissue, similarly as is performed when researchers measure and model the gradients of morphogens progressing through a tissue such as the Drosophila imaginal disk.

2) The authors present some interesting data about "size selection" of two different dextran molecules used as fluid-phase tracer. First, such a phenomenon has been reported earlier (for example in Neuhaus et al., 2002) and the authors could compare/contrast their findings with those. Second, because the "molecular sieving" probably does not occur during the ingestion, subsequent macropinosome fragmentation, tubulation and cargo sorting, or other size-discriminating phenomena have to take place. The authors could try to visualise such subsequent phenomena by live microscopy, measuring the fluorescence ratio as a read-out of the selection.

Thank you for these two very constructive comments. We have the feeling however that they fall beyond the scope of the present study which is the journey of nanoparticles from the seawater to the cell uptake. Nevertheless, the actual study shall be continued; with the use of more advanced imaging facilities as well as combination of other tracers, in order to tentatively decipher the nanoparticle selectivity (size/charge/composition) as well as the cellular fate of the macropinosomes inside the cell. And live imaging using laterally grown micro-colonies is, among others, some of the tools we are currently developing.

3) Again, the manuscript is well written, but the amount of Materials and methods-like sentences has to be reduced in the Results section. For example, in the subsections “Dynamics of dextran uptake differs between cell layers and size of dextran” and “Endocytosis occurs at the apical side of the cells”, and many other places.

See section B for answer.

4) The macropinocytic process revealed in this study, as well as the progression through tissue layers are well documented (but see point 1-). Now, in terms of mechanistic insight, beside the size of the compartments, which classify plausibly the process as macropinocytic, the authors should relativise their conclusions based on the use of "specific" drugs to block the pathway, because this is based on experiments performed in evolutionarily distant organisms and the molecular targets and mechanisms of action of the drugs might not be conserved.

Macropinocytosis has been studied in eukaryotes as distant as the amoebae Dictyostelium and vertebrates using similar inhibitors. Cnidaria is the sister group to Bilateria, part of the Epitheliozoa. Moreover, the genomic sequencing efforts have shown that in most cases, gene sequences similarity was higher between cnidarian and vertebrate than between protostomian (e.g. Drosophila, C. elegans) and vertebrates. Actin is such a conserved molecule that there is no doubt about the inhibitory effects of Latrunculin. EIPA has been shown to be specific to NHE in Stylophora (Laurent et al., 2014). Although LY294002 had unexpected side effects that made its use invalid, we now show the effect of Wortmannin: the phylogenetic tree of the PI3K shows that the Stylophora homolog is positioned between the human homologs, which reflect the high level of sequence homology between the human and cnidarian sequences. Hence, we are confident that the action of the drugs used in our study is specific.

5) The authors speculate about the fact that fluid-phase tracers are taken up in a "constant" manner and might become concentrated during the uptake process (subsection “Macropinocytosis and signaling”). The former sounds plausible, but there is no real proof of that. The latter can be substantiated by an experiment (it is easy to measure the intensity of fluorescence per volume units during the process), or the authors have to cut short their speculations, especially "It is thus tantalizing to extrapolate a role for macropinocytosis in cell to cell signaling pathways". Keep this to write a review once the article is published.

We have removed this paragraph as it was too speculative.

Reviewer #2 and Reviewer #3

See sections A and B for responses.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

[…] Some other points need to be addressed in addition:

1) The reviewers accept that you did not really probe that the drugs target the expected proteins, but discuss instead the likelihood that this is the case in evolutionary terms. This is fine for the experiments where you observe an effect of the drug. However, it would be important to show that colchicine indeed depolymerizes the microtubules in your system, since you claim that microtubule depolymerization does not affect dextran uptake. If you cannot do this, this negative result should be omitted.

We removed the (negative) results from the colchicine experiment, i.e. the corresponding Figure 9—figure supplement 2 and related sentences in the Materials and methods, Results and figure legends sections.

2) All reviewers also agree that the Discussion is still way too long and re-describes some of the results in more detail than needed. The comparison to early TEM work in corals showing the presence of vesicles seems particularly long, as is the discussion of the possible importance of coral macropinocytosis with regard to microplastics. Those last three sections could be combined and condensed into a 'Macropinocytosis and coral physiology' section. Please condense the Discussion to no more than 5 pages double spaced in your draft manuscript.

We modified the Discussion section:

– We added a small paragraph on the CLIC/GEEC pathways and its putative participation in the formation of the vesicles that we observed.

– We shortened the Discussion down to 5 pages, which includes removing detailed re-description of some of the results and merging of the previous last 3 sections into one 'Macropinocytosis and coral physiology' section.

– We added an acknowledgment paragraph.

https://doi.org/10.7554/eLife.50022.sa2

Article and author information

Author details

  1. Philippe Ganot

    Marine Biology Department, Centre Scientifique de Monaco, Monaco, Monaco
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Investigation, Visualization, Methodology
    For correspondence
    pganot@centrescientifique.mc
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1743-9709
  2. Eric Tambutté

    Marine Biology Department, Centre Scientifique de Monaco, Monaco, Monaco
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Investigation
    Competing interests
    No competing interests declared
  3. Natacha Caminiti-Segonds

    Marine Biology Department, Centre Scientifique de Monaco, Monaco, Monaco
    Contribution
    Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared
  4. Gaëlle Toullec

    Marine Biology Department, Centre Scientifique de Monaco, Monaco, Monaco
    Present address
    EPFL Lausanne, Lausanne, Switzerland
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared
  5. Denis Allemand

    Marine Biology Department, Centre Scientifique de Monaco, Monaco, Monaco
    Contribution
    Resources, Supervision, Funding acquisition
    Competing interests
    No competing interests declared
  6. Sylvie Tambutté

    Marine Biology Department, Centre Scientifique de Monaco, Monaco, Monaco
    Contribution
    Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Visualization, Methodology, Project administration
    Competing interests
    No competing interests declared

Funding

Government of the Principality of Monaco

  • Philippe Ganot
  • Eric Tambutté
  • Natacha Caminiti-Segonds
  • Gaëlle Toullec
  • Denis Allemand
  • Sylvie Tambutté

This work was supported by the Centre Scientifique de Monaco research program, funded by the Government of the Principality of Monaco.

Acknowledgements

We thank Dominique Desgré for assistance with coral culture. We thank the editorial board and the three anonymous reviewers for their valuable comments which have helped us to improve our manuscript.

Senior Editor

  1. Suzanne R Pfeffer, Stanford University School of Medicine, United States

Reviewing Editor

  1. María Isabel Geli, Institut de Biología Molecular de Barcelona (IBMB), Spain

Publication history

  1. Received: July 8, 2019
  2. Accepted: February 8, 2020
  3. Accepted Manuscript published: February 10, 2020 (version 1)
  4. Version of Record published: February 20, 2020 (version 2)

Copyright

© 2020, Ganot et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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