Effect of mycalolides isolated from a marine sponge Mycale aff. nullarosette on actin in living cells

Discovery of novel bioactive compounds is important not only for therapeutic purposes but also for understanding the mechanisms of biological processes. To screen bioactive compounds that affect nuclear morphology in marine organism extracts, we employed a microscopy-based assay using DNA staining of human cancer cells. A crude extract from a marine sponge Mycale aff. nullarosette, collected from the east coast of Japan, induced cellular binucleation. Fractionation of the extract led to the isolation of mycalolides A and B, and 38-hydroxymycalolide B as the active components. Mycalolides have been identified as marine toxins that induce depolymerization of the actin filament. Live cell imaging revealed that low concentrations of mycalolide A produce binucleated cells by inhibiting the completion of cytokinesis. At higher concentrations, however, mycalolide A causes immediate disruption of actin filaments and changes in cell morphology, yielding rounded cells. These results suggest that the completion of cytokinesis is a process requiring high actin polymerization activity. Furthermore, luciferase reporter assays with mycalolide A treatments support the view that the level of globular actin can affect transcription of a serum response gene.

induction of binucleated cells by the crude marine sponge extract. During fractionation of the active components, we found that the fractions that induced binucleation were also associated with actin depolymerizing activity, as revealed by F-actin staining using rhodamine-conjugated phalloidin. Three fractions that produced binucleation and actin depolymerization in cells were found to contain mycalolides A, B, and 38-hydroxymycalolide B. We then employed live-cell imaging to directly monitor the effect on actin filaments and cell morphology of the fraction containing mycalolide A. When cells were incubated with the fraction at low concentrations, cells entered into mitosis, but harbored two daughter nuclei. At higher concentrations, actin fibers were immediately disrupted, causing the loss of cellular tension. These results are consistent with the biochemical property observed in vitro for mycalolides that bind to F-actin to promote depolymerization. In addition, the luciferase reporter assay suggests that mycalolide A can bind to nuclear actin and affect transcription from a serum response gene.

Materials and Methods
Animal material. Mycale  Extraction and isolation. Mycalolides from Miyagi Mycale were isolated as described below. The frozen material (232 g) was extracted with methanol (MeOH) at room temperature. The crude methanolic extract was concentrated and partitioned between H 2 O and CHCl 3 . The aqueous phase was extracted with n-BuOH and the n-BuOH phase was combined with the CHCl 3 phase. The combined extract was subjected to the modified Kupchan's procedure 13 . The sample was partitioned between n-hexane and MeOH/H 2 O (9:1), and water was added to the aqueous MeOH layer to adjust the water content to 40%, which then was extracted with CHCl 3 .
Mycalolides were also isolated from Kagoshima Mycale (1180 g) using essentially the same procedure as above, except that a reversed phase ODS HPLC fraction containing mycalolides was further fractionated through a reversed phase HPLC (CAPCELL PAK UG120; 70% MeOH) to yield mycalolide A (13.4 mg).
To compare the rate of binuclear cell formation, cells were plated at different densities in 12-well plates, and 4 hr later, purified mycalolide A, cytochalasin D (Wako Pure Chemical Industries) and latrunculin B (Wako Pure Chemical Industries) were added. Cells were cultured for 20 hr, before fixation and staining with Hoechst 33342 and rhodamine-phalloidin.
Fluorescence images of cells in individual wells were collected using an inverted microscope (Ti-E, Nikon) equipped with a motorized XY-stage (Nikon) with a PlanApo 20× dry objective lens (NA = 0.6), combined with an electron multiplying charge coupled device (EM-CCD; iXon+; Andor; normal mode; gain ×5.1), under the operation of NIS Element ver 3.0 (Nikon).
Live cell microscopy. For live cell imaging, HeLa human cervical cancer cells were plated on a glass-bottom dish (Mat-Tek) and the medium was replaced by phenol red-free DMEM (Nacalai Tesque) supplemented with antibiotics and 10% FCS. Rhodamine-conjugated actin (1 mg/ml; 3 μl; Cytoskeleton) was introduced into cells using glass beads 17 . Phase contrast and fluorescence images were captured using an inverted microscope (Ti-E; Nikon), featuring a culture system (Tokai Hit) at 37 °C under 5% CO 2 , with a PlanApo VC 100× (NA = 1.4) oil-immersion objective lens, using an EM-CCD (iXon+; Andor; normal mode; gain × 5.1) with filter sets (Semrock; DAPI-1160 for Hoechst 33342 and LF561-A for rhodamine) and the exposure period set to 200 ms.
www.nature.com/scientificreports www.nature.com/scientificreports/ A 75 W Xenon lamp was used as a fluorescence light source and attenuated through neutral-density and 440 nm long-pass filters to achieve a light intensity of 6-10 μW at the specimen. Phase-contrast images were collected (200 ms) using an external phase ring.

Results and Discussion
Purification of mycalolides in cell based assays. A cell-based assay monitored by a motorized fluorescence microscope equipped with an EM-CCD was used to screen bioactive compounds from marine organisms. MDA-MB-231 breast cancer cells were grown in 96-well glass-bottom plates and cultured for ~20 hr with the organic compounds extracted from the marine organisms. Cells were fixed and incubated with a DNA-staining dye, Hoechst 33342, to evaluate the effects of the compounds on the nuclear morphology (Fig. 1A). An extract from Mycale aff. nullarosette, collected at Miyagi prefecture, the North-Eastern coast of Japan ( Supplementary  Fig. S1A), was found to promote the production of cells harboring two nuclei, or binucleated cells (Fig. 1B).
To purify the active components that cause binucleation, the extract was fractionated by solvent partitioning and column chromatography (Fig. 2). Since cellular binucleation is typically observed in agents that affect actin-filament turnover 18-20 , F-actin was also stained with rhodamine-conjugated phalloidin for subsequent assays. The F-actin formation was indeed disturbed by the active fractions (Fig. 3). For example, F-actin disappeared when cells were incubated at higher concentrations of a fraction (>222 ng/ml of 11-3(1)) ( Fig. 3A). At lower concentrations (25 or 75 ng/ml of 11-3(1)), binucleated cells were produced and only small patches of actin filaments were observed (Fig. 3B). LC-MS and NMR analyses revealed that the fraction 11-3(1) contained mycalolide A (82% purity) ( Supplementary Fig. S1B,C). Mycalolide B and 38-hydroxymycalolide B were also identified in other chromatography fractions, 9-6 and 11-2(1), respectively ( Fig. 3 and Supplementary Fig. S1C). These mycalolides have been shown to bind to actin and induce depolymerization in vitro 10,11,14 , suggesting that the mycalolides are the major compounds that induce the cellular phenotype. We initially used the fraction 11-3(1) as mycalolide A for live cell imaging (Figs 4-6). However, as the purity of mycalolide A in 11-3(1) fraction was only 82% measured using LC-MS, we prepared a more purified mycalolide A (92% purity) from another Mycale sample collected at Kagoshima (Supplementary Fig. S2), by which similar cell phenotypes were observed as for 11-3(1) (Supplementary Fig. S3), and used this for quantitative comparisons with other actin polymerization inhibitors (Figs 7 and 8).
Visualizing F-actin disrupting activity by live-cell imaging. We investigated the effects of compounds in the fraction 11-3(1) (82% mycalolide A) by live cell microscopy using HeLa cells, which are less motile than MDA-MB-231 cells and suitable for live imaging. Cells were grown on a heated stage in an inverted microscope, and phase contrast images were acquired every 10 min. The fraction 11-3(1) was added to the medium to give a final concentration of 10 ng/ml. Within 10 min of its addition, the morphology of the plasma membrane was altered (Fig. 4A,B), consistent with the inhibition of actin polymerization in lamellipodia. When cells entered www.nature.com/scientificreports www.nature.com/scientificreports/ into mitosis, chromosome segregation occurred normally and a contractile ring apparently formed. However, most (~90%; 17 out of 19) cells that had entered into mitosis during ~9 hr failed to complete cytokinesis and the outcome was two sister nuclei sharing a single cytoplasm (Fig. 4C,D). This result suggests that mycalolide A in the fraction 11-3(1) inhibits F-actin formation during mitosis, resulting in failure of the completion of cytokinesis.
To directly visualize the effect of the compound(s) in the fraction 11-3(1) on actin filaments in real-time, cells were loaded with rhodamine-actin, and the fluorescence and phase-contrast images were collected every 5 sec. Rhodamine-actin was enriched in cytoskeleton filaments as well as in filopodia and lamellipodia, which were also identified by phase-contrast images. In the normal medium, the actin cytoskeleton appeared quite stable; the steady-state structure can be maintained by the rapid turnover of actin polymerization and depolymerization [21][22][23] . In contrast, filopodia and lamellipodia moved rapidly during the imaging period for several minutes (Movie 1; The disruption of actin filaments was more clearly observed at a higher concentration (300 ng/ml) of the fraction 11-3(1) (Movie 2; Fig. 5B). The disappearance of actin filaments was first observed within a few minutes after addition ( Fig. 5B; 0:30 and 1:50). Actin filaments were often disrupted (2:00, closed arrowhead; 2:20, open arrowhead) and retracted with the passage of time. Actin molecules that were released from the cytoskeleton filaments appeared to be mostly diffused throughout the cytoplasm (Fig. 5A; 10:30; and 5B; 10:00) rather than forming aggregations. These observations are consistent with the actin depolymerizing activity of mycalolides 10 , and differ from the effects of actin filament stabilizing agents like amphidinolide H, which has been shown to induce actin aggregations after retraction of the cytoskeleton 20 . Even though the cells became rounder after F-actin disruption, they remained attached to the dish probably through focal adhesion complexes. While the original filopodia disappeared quickly after the addition of the fraction 11-3(1), many spikes reappeared later ( Fig. 5A; 10:30; and 5B; 10:00). To examine how such spikes are formed when F-actin is disrupted, we visualized tubulin and F-actin in fixed cells using the specific antibody and phalloidin, respectively. In cells treated with the fraction 11-3(1), www.nature.com/scientificreports www.nature.com/scientificreports/ spikes contained tubulin but not F-actin (Fig. 6), implying that they are not identical to normal filopodia. We repeated this experiment using mycalolide A that was purified at a higher level (92% purity) from another Mycale sp. source, and a similar result was obtained ( Supplementary Fig. S3), supporting the view that the active compound that disrupts F-actin in the fraction 11-3(1) is mycalolide A. In addition, when cells were pretreated with nocodazole (to inhibit microtubule polymerization) just before mycalolide A administration, such spikes did not appear (Supplementary Fig. S3). These results suggest that the microtubule filaments can grow to create spikes by pushing out the plasma membrane when the actin cytoskeleton is disrupted, since the extension of microtubules is normally prevented by the actin cytoskeleton.  www.nature.com/scientificreports www.nature.com/scientificreports/ Effects of mycalolide A on binucleation. Using the higher (92%)-purified mycalolide A, we compared its effects on cell phenotypes (i.e., binucleation and transcription) with other actin polymerization inhibitors, such as latrunculin B (binding to a nearby ATP binding site of monomeric actin) and cytochalasin D (capping F-actin). To analyze the activity which induces binucleation, two human cell lines, HeLa and MDA-MB-231, were seeded at various densities and treated with the inhibitors for 20 hr. After fixation and staining with Hoechst and phalloidin, the rates of the binucleated cells were counted (Fig. 7A). At the lowest cell density, binucleated cells were produced at high percentages (70-80%) by mycalolide A at 3.6 and 11 nM for HeLa and MDA-MB-231, respectively (Fig. 7B). In the presence of higher concentrations of mycalolide A at the same cell density, few attached cells were observed (indicated by # in Fig. 7B), suggesting that cells had been detached by massive disruption of actin filaments under the influence of excess mycalolide A. When the cell density was increased, the optimized concentration for generating binucleated cells was also increased. Thus, the effects of mycalolide A depended both on the concentration and cell density, probably due to its F-actin severing activity and high-affinity G-actin binding 10 , causing irreversible disruption of the actin filaments. In contrast, effects of cytochalasin D and latrunculin B on binucleation were largely dependent on their concentration and the effective ranges were wider (Fig. 7B). As it has been reported that the effects of cytochalasin are reversible 24 , these molecules may dynamically bind to actin and be less sensitive to the cell density. www.nature.com/scientificreports www.nature.com/scientificreports/ Effect of mycalolide A on transcription. It has been reported that G-actin in the nucleus can control the expression of a subset of genes, partly through the interaction with the myocardin-related transcription factor (MRTF/MAL/MKL1), which is a coactivator of the serum response factor (SRF) 3,4 . The MRTF-SRF complex binds to the serum response elements (SREs) in promoter regions of target genes to facilitate transcription 25,26 . However, when G-actin binds to MRTF, the G-actin-MRTF complex is exported from the nucleus to the cytoplasm, resulting in inhibition of SRF activity 25 . Thus the level of nuclear G-actin influences MRTF-SRF-mediated  www.nature.com/scientificreports www.nature.com/scientificreports/ transcription activation. Consistently, it has been shown that cytochalasin D but not latrunculin B stimulates the SRF-mediated transcription 27 . We then investigated using the luciferase assay whether mycalolide A also affects the transcription from an SRE-containing promoter.
Cells were transiently transfected with the luciferase reporter gene under the influence of the SRE-containing promoter, and treated with different concentrations of actin inhibitors for 3 hr before lysing cells (Fig. 8). We chose a 3 hr time window for inhibitor treatments, because proteins may be synthesized within 3 hr after the induction of transcription 28 and a longer treatment may induce side effects. In the presence of 100 nM mycalolide A, the luciferase activity was increased (Fig. 8). With a dose of 300 nM mycalolide A, however, no increase in the activity was observed, which is consistent with the narrow optimal range, as seen in the binucleated cell formation (Fig. 4). Control experiments using cytochalasin D and latrunculin B essentially reproduced the previous data 27 . This result suggests that mycalolide A diffuses into the nucleus and outcompetes MRTF for G-actin binding, resulting in increasing G-actin-free MRTF in the nucleus to enhance SRF-dependent transcription. Indeed, MRTF and members of the macrolide family that mycalolide A belongs to (i.e., kabiramide C and jaspisamide A) were reported to bind the cleft between actin subdomains 1 and 3 29,30 . Therefore, mycalolide A may be useful for analyzing G-actin function not only in the cytoplasm but also in the nucleus. In addition to its binding to transcription factors including MRTF, nuclear G-actin is a critical component in multiple complexes for chromatin remodeling and histone modification 31 .

Conclusions
In this study, we isolated mycalolides from the marine sponge Mycale aff. nullarosette by using a cell-based assay with DNA and F-actin staining. Live cell imaging then revealed that actin depolymerization caused by the mycalolides results in incomplete cytokinesis that produced binucleated cells. At higher concentrations, mycalolides immediately disrupt actin filaments. Our microscopy-based screening and assay systems have proven useful in measuring biological activities of small molecules essential in cell morphology and molecular organizations, and will be beneficial in discovering future novel compounds. Figure 8. Effects of mycalolide A on SRF-responsive transcription by the luciferase reporter assay. SRFresponsive luciferase activity was measured in HeLa cells treated with the indicated concentrations of mycalolide A (92% purity), cytochalasin D, and latrunculin B for 3 hr. After normalizing the activities with cotransfected Renilla luciferase, the relative values to that in cells treated with DMSO (the solvent) were obtained. Data are expressed as means ± SD (from 3 independent experiments), where asterisks indicate a statistically significant difference (**p ≤ 0.01; *p ≤ 0.05). In 300 nM mycalolide A, many dead cells were observed and so the value at this condition (#) should not be directly compared with the others.