To confirm if endocytosis in G. lemaneiformis can internalize extracellular fluid, the cell slices were treated with 0.4% trypan blue. As a dye used to identify cell activity, trypan blue could be effectively blocked by the PM of living cells but could enter the living cell through endocytosis. Therefore, trypan blue has been used to visualize pinocytosis in viable cells (Kerschbaum et al., 2021). In the present study, dead cells in the slices were stained dark blue, but the living cells were observed to internalize trypan blue into endocytic vesicles within 10 min ( Figure 1A ). After 20 min, number of vesicles coated with trypan blue further increased. Of note, the diameter of the dye-coated vesicles was larger than 0.2 μm; 1.94 ± 0.51 μm ( Figure 1B ). According to the size of endocytotic vesicles, we speculated that macropinocytosis exists in G. lemaneiformis. Therefore, G. lemaneiformis cells were treated with fluorescein-dextran, a well-established marker for detecting macropinocytosis (Hewlett et al., 1994; Commisso et al., 2013). When cells were incubated with 4 kDa FITC-dextran, FITC-dextran was internalized in the vesicles with diameters of approximately 2 μm, whereas the FITC signals (round or oval particles) were missing in the negative control ( Figure 1C ). Amiloride and its derivative EIPA can effectively inhibit macropinocytosis in animals and amoeba (West et al., 1989; Ivanov, 2008). When G. lemaneiformis was treated with different concentrations of EIPA, the number of endocytic vesicles decreased with increasing EIPA in a dose-dependent manner ( Figure 1D ).

To further prove the presence of macropinocytosis in G. lemaneiformis, cell ruffles were observed, and the relationship between ruffles and F-actin was detected. To detect the PM cell ruffles, slices of G. lemaneiformis was observed using electron microscopy ( Figure 2 ). Ruffles were formed by the cells and seemed to grow into cups, contacting and closing successfully into the cell body ( Figure 2A ). Cell ruffles were also observed on the cell surface by confocal microscopy ( Figure 2B ). F-Actin is one of the main regulating molecules for ruffle formation and closure. Therefore, cells were stained with phalloidin to observe F-actin. F-Actin was concentrated where ruffles occurred, and it aggregated at the edge of the cup ruffles, showing an “F-actin ring” ( Figure 2B ). When the cells were treated with latrunculin B, an inhibitor of F-actin polymerization, the endocytic activity of the cells significantly decreased with increasing dosage of this inhibitor ( Figure 2C ).

F-Actin polymerization cannot be achieved without the action of its key factors that regulate F-actin polymerization. The key proteins associated with F-actin aggregation were blasted in algal genomes. Arp2 and Arp3 are nucleating factors that can form the Arp2/3 complex to bind to microfilaments and promote the generation of new branched microfilaments (Goley and Welch, 2006). The Arp2/3 complex acts downstream of the WASP/SCAR/WAVE signal, forming nucleating branches on both sides of the existing parent filaments to generate a dense actin network (Seastone et al., 2001; Pollitt and Insall, 2009; Veltman et al., 2016; Davidson et al., 2018). However, Arp2, Arp3, and the WASP/SCAR/WAVE are generally lacking in red algal genomes ( Figure 2D ). Formin is another nucleating factor of F-actin that mediates the formation of unbranched actin filaments (Breitsprecher and Goode, 2013). Formins were found in G. lemaneiformis and other red algal genomes ( Figure 2D ), which suggested that F-actin polymerization was mediated by Formins.

When macropinosome forms from ruffles, it is acidified via trafficking to the endosome/lysosome pathway (Racoosin and Swanson, 1993). To detect the acidification of endocytic vesicles, LysoTracker, a fluorescent dye that labels acidic organelles in living cells, was incubated with G. lemaneiformis cells. After 2 h, the vesicles marked by FITC-dextran were observed to be acidified ( Figure 3A ). Meanwhile, cells that internalized FITC-dextran were cultured and observed at different time points. The results showed that FITC-dextran signals gradually weakened and disappeared with time ( Supplementary Figure S1 ). After 48 h, the FITC-dextran signals had almost disappeared. In contrast, the positive control (fixed cells) still had FITC-dextran signals even after 48 h ( Supplementary Figure S1 ). FITC fluorescent signal is sensitive to pH changes, and fluorescence of FITC-dextran will be progressively quenched with decreasing pH (Geisow, 1984). This suggested that the endocytic vesicles of G. lemaneiformis were acidified. When macropinosome is acidified, it acquires the digestive function, which can degrade cargos to provide nutrients for cells (Kerr and Teasdale, 2009). Floridean starch is one of the main carbon sinks in Gracilaia/Gracilariopsis species (Viola et al., 2001; Chen et al., 2022). In the current study, the floridean starch content of cells was examined after inhibition of endocytosis in G. lemaneiformis. The results indicated that the floridean starch content of the cells experienced a significant reduction subsequent to a 1-week incubation period with EIPA ( Figures 3B, C ). This suggested that inhibition of endocytosis in G. lemaneiformis led to a decrease in the presence of energy storage substances within the cells.

The domains of PI3Ks were searched in genome of G. lemaneiformis using HMMER ( Supplementary Table S1 ). Protein containing the Ras binding domain (RBD) appeared to be lacking ( Supplementary Table S1 ). Additionally, proteins containing the p85B domain and C2 domain were also lacking. Meanwhile, a set of protein sequences of phosphatidylinositol kinase, which contained class I PI3K, VPS34, PI4K, and PIP5K, were used to blast the protein database of G. lemaneiformis. A total of 10 proteins were found in G. lemaneiformis and the sequence sets ( Supplementary File 1 ) containing these proteins were used to construct a phylogenetic tree. The results showed that none of the proteins of G. lemaneiformis were clustered into the branches of class I PI3K and VPS34 ( Figure 4A ). LXC001784.1 and LXC007569.1 were clustered with PI4Ks, and these proteins both contained the domain of PI3_PI4_kinase ( Supplementary Table S1 ; Figure 4A ). Although LXC001784.1 contained a PI3Ka domain related to substrate presentation in PI3Ks ( Supplementary Table S1 ; Figure 4B ), conserved domain analyses showed that LXC001784.1 and LXC007569.1 had a catalytic domain of PI4K ( Figure 4B ). In addition, LXC006155.1 was clustered with the PI4K of plants and had a Pkc_like superfamily catalytic domain, a domain that has the catalytic activity of phosphokinase ( Figures 4A, B ). In addition, LXC006370.1 was clustered with PIP5K and had a PIPKc domain, which is a conserved core region in the PIP5K family ( Figures 4A, B ). Wortmannin, an inhibitor of phosphatidylinositol kinases, was incubated with G. lemaneiformis to investigate whether phosphatidylinositol kinases participate in G. lemaneiformis endocytosis. After treatment with 20 μM wortmannin, the endocytic activity of cells was significantly decreased, while 10 μM had no significant effect ( Figure 4C ).

In this study, the hidden Markov model of the Ras family (PF00071) and a sequence set of Ras superfamily members were used to blast the genome of G. lemaneiformis and to construct a phylogenetic tree using the sequence set containing the protein sequences being blasted ( Supplemental File 2 ). The results showed that LXC002888.1 was clustered into one branch with Rho, Rac1, and CDC42, which has a Rho domain ( Figures 5A, B ). Meanwhile, Rab7, a marker of a mature macropinosomes, was also clustered with the protein of G. lemaneiformis, and the protein has the Rab7 domain. LXC000339.1 and LXC004497.1 were clustered into one branch with the Ras family (bootstrap=99), and both of them has a conserved domain of P-loop NTPase superfamily. Although Rab5 that was enriched in the early macropinosomes was clustered together with seven proteins of G. lemaneiformis (bootstrap=94), the proteins do not contain the typical domain of Rab5 but contain the domain of other members in Rab family such as Rab11-like or Rab18.

Gracilariopsis lemaneiformis were collected from aquaculture areas of Rongcheng, Shandong, China, and cultured in Provasoli’s medium (Pflugmacher et al., 1999) at 20°C under a 12-h/12-h light/dark cycle and illumination intensity of 30 μmol photons m⁻² s⁻¹.

The shoot apices of the algae branches (~3 cm in length) were harvested and sliced using razor blades. To detect the non-selective internalized extracellular fluid by endocytosis, approximately 50 slices were incubated with 500 μL of 0.04% trypan blue (Solarbio, Cat. C0040) for 10 and 20 min, and the vesicles containing trypan blue in living cells were determined. Approximately 50 slices were also incubated with 500 μL of 0.1 mg/mL FITC-dextran (Xi’an ruixi Biological Technology Co., Ltd., Cat. R-FD-001) to determine whether they internalized dextran. For determining the inhibition of endocytosis, different inhibitors [5-(N-ethyl-N-isopropyl)] amiloride (EIPA) (MedChemExpress, Cat. HY-101840), lacunculin B (Aladdin, Cat. L275457), and wortmannin (MedChemExpress, Cat. HY-10197) were incubated with slices for 1 h, and then, the slices were incubated with the mixture of inhibitors and FITC-dextran for 50 min. After fixing in 4% paraformaldehyde (Solarbio, Cat. P1110) at 4°C for 2 h, the slices were observed using a laser confocal microscope (Nikon A1R HD25, Japan). For F-actin staining, the slices were fixed overnight in 1 mL of 4% paraformaldehyde at 4°C and washed three times using 0.1% Triton X 100 (1× PBS). The slices were then stained with 100 μL of 0.67 µM Alexa Fluor Plus 405 Phalloidin (Invitrogen, Cat. A30104) for 5 h. For detecting vesicle acidification, the slices were incubated with 500 μL of 0.1 mg/mL FITC-dextran for 50 min and then with 200 μL of 1 μM Lysotracker Blue DND 22 (Invitrogen, Cat. L7525) for 1 h. After washing, the slices were observed using laser confocal microscopy.

Protein sequences were download from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/), and the sequence sets were searched in the G. lemaneiformis genome using Blastp. Hidden Markov models of domain sequences were downloaded from Pfam (http://pfam-legacy.xfam.org/), and HMMER was used to search the gene family members from the alga genome. The conserved domain was predicted by the CD search of the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) and Smart (http://smart.embl-heidelberg.de/). Multiple sequence alignment was performed with Mafft (Katoh and Standley, 2013), and a phylogenetic tree was constructed using Iqtree (Minh et al., 2020).

Shoot apices (3 mm) were harvested for ultrastructural observation. The electron microscopy samples were prepared and observed as described by Chen et al. (2022). In brief, the samples were fixed in 1% osmium tetroxide after being fixed overnight in 2.5% glutaraldehyde. Then, samples were dehydrated with ethanol treatments and embedded in Epon 812. After being cured, the samples were cut with a Reichert-Jung Ultracut E Ultramicrotome (Germany) and were observed under a JEOL JEM-1230 transmission electron microscope (Japan). The number of starch granules and particle diameters of cells were measured using Image J.

Statistical analysis of the obtained data was performed using SPSS. Statistical differences were evaluated by Student’s t-test for two-group comparison.