Abstract
Sponges are capable of rebuilding entire functional organisms from dissociated somatic cells. This feat demands radical cytoskeletal reprogramming whose molecular logic remains obscure. Using the Arctic demosponge Halisarca dujardinii, we mapped molecular signatures across free-swimming larvae, sessile adults, and early cell aggregates. Integrating RNA-seq, single-cell deconvolution, DIA-LC-MS proteomics, native complex fractionation, live-cell proteasome imaging, and immunofluorescence, we identified stage-associated profiles of cytoskeletal proteins and post-translational modifications (PTMs). Larvae enrich non-classical myosins (Myosin-9/15) and dynamic microtubules for motility; adults deploy spectrin scaffolds and deacetylated microtubules for stability; cell aggregates shift toward microtubule-based transport with targeted proteolysis and stage-specific PTMs on divergent actin HdA6 and ferritin. Live imaging shows ubiquitin-independent 20S proteasome activity at cell contacts in forming aggregates, while mass spectrometry reveals coordinated methionine oxidation and ubiquitination events coinciding with cytoskeletal remodeling. Although bulk data cannot fully separate cell-type composition from intracellular reprogramming, the multi-layered signatures suggest a tightly regulated, stage-specific program. Together, these findings establish a correlative framework linking cytoskeletal architecture, redox-sensitive PTMs, and proteostasis within sponge development and early reaggregation stage. We propose that conserved structural networks are chemically tuned to enable morphological plasticity in early-branching metazoans - a foundation for future functional studies of multicellular morphogenesis.
Introduction
Cytoskeletal reorganization accompanies regeneration in a wide variety of animals, but its mechanisms can differ fundamentally. In planarians (Platyhelminthes), microtubules regulate the expression of key factors in the muscles used to form the blastema during regeneration, but actin-mediated motility is not completely suppressed (; ). In Hydra (Cnidaria), remodeling of the actin cortex occurs during budding and regeneration, and microtubules are involved in maintaining polarity determined by signaling pathways (; ; ). During limb regeneration in the axolotl (Chordata), blastema cell migration is carried out mainly by actin-dependent mechanisms, although microtubules are involved in the transport within the growing limb bud, they do not completely replace actin-dependent motility (; ; ).
Sponges (Porifera), one of the earliest-branching metazoan lineages, have captivated biologists for over a century with their extraordinary capacity to rebuild new functional organisms from dissociated cells - a phenomenon first demonstrated in Wilson’s reaggregation experiments (). This whole-body regenerative process is far from monolithic. Crucially, not all sponge species can reassemble new functional bodies from cellular aggregates, suggesting that this morphological flexibility extends well beyond injury repair, and regeneration competence is an adaptive trait shaped by ecological pressures (). Branching demosponges like Raspailia ramosa sculpt forms within hydrodynamics, simplifying architecture in heavily silted habitats rather than elaborating structures for sediment shedding (). The tropical sponge Anthosigmella varians dynamically modulates spicule density that fortifying its skeleton under predation threat while reducing mineral investment in safer waters to conserve energy (). During Hymeniacidon heliophila regeneration, pinacocytes and archaeocytes migrate with purposeful coordination while differentiated cells undergo transdifferentiation to rebuild tissue boundaries (). Oscarella lobularis (cl. Homoscleromorpha) is capable to form the clonal buds in vitro, which regenerate after damage and complete dissociation into the cells (). In calcareous sponge Sycon ciliatum, regeneration partially recapitulates post-larval development, reactivating conserved molecular pathways to reestablish body axes and tissue architecture (). Beneath these visible transformations lies cellular reprogramming of remarkable sophistication. Transcriptomic surveys across Porifera reveal deep conservation of developmental toolkits (Wnt, TGF-β, Notch, Hedgehog) alongside striking lineage-specific innovations in cytoskeletal regulation (; ; ; ). Particularly striking is the facultative microtubule organization in certain demosponges: while most eukaryotes maintain stable interphase microtubule arrays, Halisarca dujardinii (cl. Demospongiae) dynamically assembles cytoplasmic microtubules only during regeneration and, keeping them largely absent in sessile adults (). This contrasts sharply with calcareous sponges where microtubule networks remain constitutively present, highlighting how cytoskeletal strategies reflect adaptive solutions to distinct ecological niches ().
The Arctic demosponge H. dujardinii, lacking mineralized spicules and exhibiting exceptional reaggregation capacity, provides a compelling model to a unique opportunity to trace the cytoskeletal rearrangements during the transitions. Its life cycle spans free-swimming larvae (developing in June) deploying dynamic microtubule networks for locomotion, sessile adults exist throughout the annual cycle (). The in vitro reaggregation of dissociated somatic cells from H. dujardinii captures the earliest phase of a broader regenerative process, because the early cell aggregates retain the capacity to develop into a juvenile sponge, and offer a straightforward and reproducible model for studying the molecular triggers of body assembly (). Despite decades of fascination with sponge regeneration, the molecular mechanisms driving cytoskeletal remodeling remain poorly understood. In particular, how PTMs repurpose existing protein scaffolds without de novo synthesis is still unknown. Moreover, while sponge genomes have been extensively characterized, comprehensive proteomic comparisons across life stages are largely absent. Here, we integrate transcriptomics, proteomics, single-cell RNA-seq deconvolution, and live imaging to trace how H. dujardinii achieves remarkable morphological plasticity. This work establishes a correlative framework linking transcriptional programs, protein abundance, and PTM dynamics to stage-specific cellular architecture. Together, these patterns suggest that chemical tuning of conserved cytoskeletal networks may have provided an energy-efficient strategy for early metazoans to navigate the transition toward complex multicellularity.
Materials and methods
The specimens of Arctic sponge H. dujardinii were collected in the sublittoral zone of the White sea (2–5 m depth) near the Polar Circle tourist Center (66° 30′N, 33° 08 E) during their reproductive period in late June 2025 (Supplementary Figures S1A–E). The sponges were collected together with their substrate (macroalgae) to preserve the natural microenvironment, and maintained as described earlier (). The adult sponges with larvae were placed in an aquarium with seawater, and larvae that left the parent and swam freely were collected. Dissociation of sponge body and collection of cell aggregates were performed as described in detail in the Supplementary methods.
The in vivo imaging of aggregating cells of sponge specimens was performed by Zeiss LSM880 confocal microscope, equipped with Zeiss Plan-APOCHROMAT 63х/1.4 Oil DIC objective lens (Zeiss, Oberkochen, Germany) as described previously (). Active proteasomes were detected using 1 nM Me4BodipyFL-Ahx3Leu3VS (UbiQbio, Amsterdam, Netherlands) added immediately after the sponge body dissociation at +6 to +8 °C. The aggregating sponge cells were recorded 1 hour after the dissociation by confocal laser scanning microscope Carl Zeiss LSM 880 with excitation and emission parameters 511 and 533 nm, respectively.
To test whether proteasome activity is required for cell reaggregation, the dissociated H. dujardinii cells were treated with selective proteasome inhibitor, bortezomib (2.5, 5, and 10 nM) during reaggregation and then assessed the morphological parameters of 24 hpd aggregates, as described in the Supplementary methods.
Electrophoresis of sponge proteins in a native polyacrylamide gel followed by detection of chymotrypsin-like proteasome activity (ChLA) in the gel was performed as described earlier (). Extended electrophoresis allowed protein complexes to reach positions in the polyacrylamide gradient according to their masses. The electrophoresis was terminated when two colored and differently charged thyroglobulin markers labelled with Cy3.5 (Lumiprobe, Hunt Valley, MD, United States) reached nearly identical positions in the gel. To visualize proteasome proteolytic activities, 200 mM Na-HEPES buffer, pH 7.5, containing 30 μM fluorogenic substrate N-succinyl-leu-leu-val-tyr-amido-4-methyl coumarin (Suc-LLVY-AMC) (1/20 of gradient gel volume) was applied to the gel surface. The gel was covered by cellulose acetate film and incubated at 37 °C for 20 min. Fluorescence bands in the gel were photographed in a dark room under illumination at 365 nm. To reveal the specificity of signal of proteasome activity the selective proteasome inhibitor Bortezomib at the dose of 5 nM was added to the sample 30 min before the proteasome substrate Suc-LLVY-AMC. The inhibitor concentration was selected based on the survival of cells within its presence (Supplementary Table S1).
To identify the cytoskeletal protein contents and post-translation features of adult sponges, cell aggregates, and larvae were analyzed by LC–MS/MS. The native protein complexes (ferritin complex - 550 kDa band) of H. dujardinii body, larvae, and 24 hpd aggregates were isolated from the native gel and analyzed by LC–MS/MS as described previously (). The H. dujardinii body, larvae, 24 hpd cell aggregate lysates were also separated by sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis, followed by Coomassie blue staining and the bands corresponding to (35–40 and 55 kDa) were analyzed by LC–MS/MS as described earlier (). Each group of proteomic samples was represented by at least three biological replicates. To study post-translational modifications of proteins, we used DIA-NN analysis, as described in the Supplementary methods.
Larvae of the Arctic sponge H. dujardinii appear only once a year, that gives us a naturally synchronized, developmentally coherent stage that can be cleanly compared to adults sampled at the same time. The proteomic analysis of adult sponge and larvae (three biological replicates per stage) was performed using DIA-LC-MS on an Ultimate 3000 Nano LC system coupled to an Orbitrap Tribrid Lumos mass spectrometer (Thermo Fisher Scientific) as described in detail in the Supplementary methods and Supplementary Table S2. Identification and quantification were performed in DIA-NN v.2.2.0 using a protein database constructed from H. dujardinii genome assembly (GCA_054858975.1). Quantitative analysis of protein abundance was performed in Perseus v.2.0.10 (for more details see Supplementary methods). The cell aggregates are formed in vitro by dissociating adult sponges, whose physiological state and consequently their molecular composition varies within the seasons. The informative H. dujardinii aggregate proteome would require sampling across multiple seasons, which was beyond the scope of this work.
Transcriptome profiling was performed using RNA-seq (BioProject: PRJNA1418124) for three sponge stages: adult sponge tissue, free-swimming larvae, and 24 hpd cell aggregates (three biological replicates per stage) as described in detail in the supplementary methods.
Functional enrichment analysis was performed in STRING (https://string-db.org/) using default parameters as described in the supplementary methods.
Single-cell RNA sequencing (scRNA-seq) data of the cold-water demosponge H. dujardinii (NCBI SRA database under BioSample accessions SAMN42483027 and SAMN42483028) were generated in our previous study (). Data processing and analysis were performed using Seurat v5.0.1, which included read count normalization, selection of variable features, scaling, dimensionality reduction, and cell clustering. Cluster relationships were visualized using Uniform Manifold Approximation and Projection (UMAP). Marker genes for each cluster were identified with the FindAllMarkers function and used to annotate cell identities.
To validate the tubulin localizations in adult (sponge body and 24 hpd cell aggregates) and larval stages, immunofluorescence microscopy was performed with mouse monoclonal against α-tubulin DM-1A (T9026, Sigma-Aldrich, Saint Louis, MO, USA) as described in details in the Supplementary methods. For imaging, a Zeiss LSM900 confocal microscope equipped with Zeiss Plan-APOCHROMAT 63×/1.4 Oil DIC objective lens (Zeiss, Oberkochen, Germany) was used (provided by the Moscow State University Development Program).
Statistical analysis is described in the Supplementary methods. To study the oxidation of methionine, we used Python codes that were created using the Google Colab service and are available on the website (https://colab.research.google.com/drive/1ljzWHx3ypQKZ2tLXPJ-tFFJZ4LARDVs9#scrollTo=qJ93wLyYXlcG).
Results
To understand how Arctic demosponge H. dujardinii coordinates cytoskeletal reorganization across its life stages, we integrated transcriptomic and proteomic profiling of adults, larvae, and 24 hpd cell aggregates, asking whether stage-specific morphological transitions are driven primarily by transcriptional reprogramming, post-transcriptional tuning, or a combination of both? The proteomic DIA-LC-MS analysis of naturally synchronized sampling of sponge H. dujardinii identified a total of 10,733 proteins (Supplementary Table S3) across larval and adult samples (2,918–10,087 proteins per sample). Principal component analysis (PCA) demonstrated separation of adult and larval samples (Supplementary Figure S2A). Among the 25 most abundant proteins across the developmental stages examined, significant enrichment was observed for the conserved actins HdA1/2/3 and ferritins (HdF1a/b), the 20S proteasome α4 subunit (PSMA4), and several sponge-specific proteins (Figure 1A). Analysis of the 500 most abundant proteins in each proteome (Figure 1B) revealed a core of highly abundant factors involved in fundamental cellular processes, including cytoskeletal organization, the ubiquitin-proteasome system, membrane-associated complexes, histones and their modifying enzymes, and proteins mediating mRNA synthesis, splicing, translation, and cell-cell interactions. In adults, the 500 most abundant proteins were relatively evenly distributed in abundance, consistent with functionally stabilized tissue architecture. In contrast, larval proteomes exhibited a more skewed distribution, dominated by a subset of highly abundant proteins (Figure 1B). Comparative proteomic analysis identified 1,213 proteins with differential expression between larvae and adults (|log2FC| > 1, FDR <0.05), of which 272 were found exclusively in adults and only 86 in larvae (Figure 1C; Supplementary Table S4). Of the 855 proteins common to both stages, 352 were elevated in adults and 503 were downregulated compared to larvae. Gene ontology (GO) enrichment analysis of differentially expressed proteins revealed that adults had elevated levels of regulators of actin cytoskeleton dynamics and tubulin deacetylation (Figure 1D), but reduced levels of proteins involved in microtubule cytoskeleton organization and microtubule transport (Figure 1E). Stage-specific proteins were also subjected to GO enrichment analysis (Supplementary Figure S3). The limited number of larval-specific proteins (Supplementary Table S4) precluded robust functional enrichment; however, the RBR-type E3 ligase RNF217 was identified among the unique larval proteins. In contrast, adult-specific proteins were significantly enriched in several biological processes, including negative regulation of phosphorylation (GO:0042326, FDR = 0.02), sensory organ morphogenesis (GO:0090596, FDR <3.4E-4), and embryonic development (GO:0009790, FDR = 0.04). This group also included the HECT-type SMURF E3 ubiquitin ligases associated with the regulation of cell proliferation and DNA repair (Supplementary Figure S3).
FIGURE 1
Do the stage-specific proteomic signatures of tubulin deacetylation and cytoskeletal regulators we identified lead to differences in cellular architecture? To answer this question, we visualized microtubule organization of adult and larval H. dujardinii stages using the anti-tubulin antibody DM1A, which efficiently labels both cytoplasmic microtubules and axonemes (the 20-microtubule core of flagella; ). Immunofluorescence staining reveals striking differences in microtubule organization between adult sponge H. dujardinii and larval stages. In adults, tubulin signal is concentrated almost exclusively in the axonemes of choanocytes (red arrows, Figure 2A), with only sparse and poorly developed cytoplasmic microtubule networks visible in rare cells (yellow arrow, Figure 2B). The 24 hpd cell aggregates do not reveal choanocyte flagella, while cytoplasmic microtubules (shown by yellow arrows, Figure 2C) are present in many cells, in contrast to adult sponge tissue (Figure 2F). By the contrast, larvae exhibit a well-developed cytoplasmic microtubule network in nearly all cells (yellow arrows, Figures 2D–F), irrespective of their flagellar content. Notably, two distinct larval morphotypes were evident: one nearly devoid of flagella (Figure 2D) and another densely packed with them (Figures 2DG) yet both maintained extensive cytoplasmic microtubule arrays.
FIGURE 2
To determine whether transcriptional shifts or post-transcriptional mechanisms drive H. dujardinii’s developmental transitions, we performed RNA-seq analysis of adult sponges, their 24 hpd cell aggregates, and larvae and identified 15,132 out of 28,634 genes with expression levels ≥3 CPM in at least one of these sample groups. PCA demonstrated clear separation of adult, larval, and cell aggregate samples, indicating distinct transcriptional profiles among these conditions (Supplementary Figure S2B). Comparative transcriptomic analysis identified 7,509 differentially expressed genes (DEGs; adjusted p < 0.05, |log2FC| > 1) in larvae and only 106 DEGs in 24-h cell aggregates when compared to adult sponges (Supplementary Table S5). We examined in more detail whether the transcription levels of key genes associated with the actin- and tubulin-based cytoskeletal functions differ between larvae, adult sponges, and cell aggregates (Supplementary Figure S4; Supplementary Table S6). The larva exhibits profilin and filamin at minimal levels (mRNA and protein), but has high levels of non-classical myosins. Myosin-15, which attains its highest expression level at the larval stage, is canonically involved in the organization of microvilli and the positioning of primary cilia - structures critical for navigation. Myosin-9, whose expression level also peaks at the larval stage, functions as a “molecular brake,” inactivating Rho-GTPases (RhoA, Rac, Cdc42) and allowing for the immediate halt of local actin remodeling processes. At the protein level, the amount of bicaudal, the key adaptor for dynein and kinesin is reduced in larvae compared to the adults (Supplementary Table S6). The adult radically changes the strategy: profilin, filamin, and spectrin (Supplementary Figure S4; Supplementary Table S6) are expressed at their highest mRNA and protein levels. Interestingly, actin-dependent cell motility is suppressed in cellular aggregates: profilin, filamin, and spectrin mRNA levels reach their minimum levels. The amount of α-tubulin mRNA in cell aggregates is about half that of the adult sponges and even less in the larvae, while the β-tubulin mRNA level does not differ significantly (Supplementary Figure S4; Supplementary Table S6). Mitochondrial proteins LACTB and ABHD10, which form a complex regulating lipid metabolism, were identified in larvae, as well as the E3 ubiquitin ligase RNF217 (RBR family), which is important for mitochondrial homeostasis and the control of reactive oxygen species (Supplementary Figure S3). Taken together, these findings may point to stage-specific rewiring of the cytoskeletal apparatus tuning larvae for active dispersal while equipping adults for the structural stability required by their sessile, filter-feeding lifestyle.
Are the proteomic differences due to changes in sponge stage cellular composition? To link observed transcriptional transitions to underlying cell molecular regulation, we analyzed bulk RNA Seq (adult, larva and cell aggregates) and H. dujardinii single-cell RNA-Seq data from our previous study which are publicly accessible via Zenodo (doi.org/10.5281/zenodo.14981466) ().
Deconvolution analysis revealed that the studied sponge stages involve some molecular changes and reorganization of cellular composition (Supplementary Figures S5A, B). Importantly, cell populations of cluster 0_2 (corresponds to choanocytes and archaeocytes) are present at all stages, while individual pinacocyte clusters are enriched or depleted. In adults, more than 50% of cells belong to cluster 0_2 along with cluster 7 (corresponds to neuroid pinacocytes). The cluster 7 represents a specialized pinacocyte population enriched in tenascin-like proteins, tyrosine-protein phosphatase Lar, and the T-box transcription factor TBX19 genes likely involved in extracellular matrix formation and maintain the complex tissue architecture of adult sponges. In cell aggregates, the minor cluster 15 with representation of cluster 5 (corresponds to transition choanocytes to pinacocytes) dominates, while larvae consist mainly of cells of cluster 8 (corresponds to transition pinacocytes). The cluster 15 (present predominantly in aggregates) expresses neurocalcin homologues and receptor tyrosine kinases (including a RET proto-oncogene homologue) and exhibits enrichment of collagen-related genes and unique coexpression of receptor signaling genes with peroxidase activity and heme-binding components. Notably, the proportion of cells in clusters 5 and 8 (pinacocytes expressing HdA6 actin) increases in both larvae and cell aggregates (Supplementary Figure S5C). Interestingly, Myosin 15 is predominantly expressed in cluster 13 associated with pinacocytes in larvae, while Rho GDP dissociation inhibitor (ARHGDI) exhibits increased expression in cluster 15 of cell aggregates (Supplementary Figure S5C) ().
Thus, specific changes in the expression of cytoskeleton-related proteins in certain cell types may influence the functional reorganization required for free-swimming larval forms, cellular aggregates, and filter-feeding adults.
Despite the transcriptional rearrangements we observed, proteomic analysis revealed that some core components remained consistently abundant across all developmental stages. This apparent paradox between dynamic transcriptional regulation and conserved protein abundance suggests that rapid cellular reorganization during sponge metamorphosis may rely not so much on de novo protein synthesis as on post-translational mechanisms that dynamically repurpose preexisting cytoskeletal components. The consistent presence of actin HdA1/2/3 and 20S proteasome throughout all developmental stages (Figure 1A), despite significant functional differences between free-swimming larvae and filter-feeding, attached adults, indicated a complex regulatory system operating at the level of protein modification (for example, protein phosphorylation, tubulin deacetylation), not just transcriptional control (Figure 1E; Supplementary Figure S3A). We hypothesized that the rapid reaggregation of dissociated cells can be associated not with the de novo synthesis of new components, but with spatiotemporal rearrangement of existing actin networks, and proteasome activity for targeted protein turnover and adhesion. To test this hypothesis, we performed vital confocal microscopy of reaggregating H. dujardinii cells using the Me4BodipyFL-Ahx3Leu3VS probe, which detects active forms of the proteasome. This approach was designed to determine whether proteasome activity is localized with filopodia, actin-rich protrusions, and forming cell-cell contacts during the first hours of reaggregation-a time interval when transcriptome analysis revealed minimal changes in gene expression (RNA-seq NCBI Bioprojects PRJNA594150) and highlights the need for chemical rewiring or post-translational regulation of existing cytoskeletal proteins.
Reaggregation of sponge cells emerged as a highly dynamic process involving active cell migration, frequent intercellular contacts, and progressive aggregate growth. Aggregating cells extended elongated pseudopodia containing active proteasomes (Figure 3), while time-lapse sequences captured sequential attachment of single cells to aggregates via thin filamentous contacts (Figures 3A1–A10; Supplementary Figure S6). A strong fluorescence signal confirmed proteasome activity in the attaching cells (Figures 3A9), at 21.49 s, with cyclic fluctuations in signal intensity during the attachment (Figures 3A5–A10), suggesting dynamic regulation of proteolytic activity during cell–cell adhesion. The intensity of Me4BodipyFL-Ahx3Leu3VS fluorescence in aggregating cells within aggregates after 1.5 h of aggregation was significantly higher than in individual cells (Figure 3B). Thus, monitoring the pulsating and changing fluorescent signals of Me4BodipyFL-Ahx3Leu3VS in real time of aggregation revealed that targeted proteasome proteolysis can acts as an immediate, spatially precise regulator of cytoskeleton remodeling. Concurrently, native PAGE analysis of proteasome complexes from adults, 24 hpd aggregates, and larvae revealed that ChLA was predominantly associated with the core proteasome 20S particle in cell aggregate extracts (Figure 3C). The ChLA was not observed in cell aggregates at the selective proteasome inhibitor bortezomib presence (Supplementary Figure S6C). Moreover, proteasome inhibition by the bortezomib (2.5, 5, and 10 nM), significantly impaired aggregate development in a dose-dependent manner: unit aggregate area increased progressively with increasing bortezomib concentration (p < 0.001 for all comparisons) and circularity and roundness parameters were decreased (p < 0.001, 0.029, and 0.021, respectively) (Supplementary Figure S7). These aggregates fail to progress to later primmorph or juvenile stages (Supplementary Figure S1H-J). These findings demonstrate that ubiquitin-independent 20S proteasome activity is essential for proper cell-cell adhesion, aggregate assembling during early reaggregation in H. dujardinii.
FIGURE 3
Next, we turned to mass spectrometry of native and denatured fractions, mapping post-translational modifications (PTMs) of actin, tubulin, and their regulatory partners in larvae, adults, and regenerating 24 hpd cell aggregates to determine whether H. dujardinii achieves its remarkable plasticity through chemical reconfiguration of preexisting protein scaffolds. LC–MS/MS of native and denatured protein fractions revealed stage-specific cytoskeletal organization in sponge H. dujardinii (Supplementary Table S7 A-B). The α-tubulin-6 and α-tubulin-7 incorporate into native ferritin complexes exclusively in adults (α-tubulin-6 oxidized at M398). While all α-tubulins appear in SDS fractions across all stages, phosphorylation signatures differ: adults primarily modify S379, whereas larvae and aggregates exclusively target Y312. β-Tubulin shows sequential “oxidative profile” shifts: adults display oxidation at M73 with carbamidomethylation at C12; larvae exhibit deamidation at N337; aggregates transition to oxidation at M330/363 and deamidation at N347 (Supplementary Table S7B; Supplementary Figure S8). Regulatory protein PTMs demonstrate switching: RhoGDI undergoes stage-specific modifications - carbamidomethylation at C106 (adults), deamidation at Q157 (larvae), oxidation at M160 (aggregates). Clathrin (CLTC) appears in native complexes only in larvae and aggregates, acquiring triple modifications (oxidation M1121, deamidation N240, carbamidomethylation C738) specifically in aggregates. Ferritin-actin-tubulin colocalization showed that complexes contain conserved actins (HdA1/2/3) and β-tubulin with overlapping oxidative PTMs (actin: M45/48/228/306/326; tubulin: M73/164/257/293/330) (Supplementary Table S7A-B; Supplementary Figure. S8). Divergent actin HdA6 exhibits stage-specific PTM patterns in cell aggregates: M48 undergoes both oxidation and carbamidomethylation; in larvae it displays maximal PTM diversity including oxidation (M48/51/86/329), carbamidomethylation (C37/56/289), deamidation (Q45), methylation (R43), and ubiquitination (K88/T93) (Supplementary Table S7B). Moreover, the specific markers were detected exclusively in 24 hpd cell aggregates: oxidized ARPC2 (M148/178) and ubiquitinated Ferritin HdF1a/b at S92/C127/T150/K153. Bortezomib exposure (5 µM) during reaggregation shifted ferritin HdF1a/b ubiquitination to residues S13/K19/S118/S120/C127/T150. These data demonstrate that larvae and cell aggregates employ distinct, PTM-driven mechanisms to remodel cytoskeletal proteins, enabling morphological plasticity during development and reaggregation processes.
Discussion
In this study, we characterize for the first time the proteomes of adult and larval sponges H. dujardinii collected simultaneously from the same Arctic habitat, taking advantage of the natural synchronization of sampling. Our analysis reveals extensive cytoskeletal rewiring that underpins both developmental transitions and cell reaggregation in this cold-water demosponge. We demonstrate that larvae, adult sponge, and 24 hpd cell aggregates may employ distinct combinations of cytoskeletal regulators, PTMs, and proteostasis mechanisms to support stage-specific functions: from larval motility to adult structural stability to aggregate-mediated tissue assembly. The proteomic and transcriptomic analyses (Supplementary Table. S6) revealed that larvae exhibit elevated expression of non-classical myosins (myosin-15 and myosin-9), which support ciliary organization and rapid actin remodeling required for swimming (Supplementary Figure S4). Deconvolution of single-cell RNA-seq data localized these myosins primarily to pinacocyte-like cluster 13, which is abundant in aggregates and larvae, but not in adults (Supplementary Figure S5). Notably, myosin-15 expression decreases in cell aggregates (Supplementary Figure S4), suggesting that elevated larval expression reflects genuine stage-specific regulation rather than simply altered cell-type proportions. In contrast, adult sponges display maximal expression of profilin, filamin, and spectrin (Supplementary Figure S4) - proteins that form a dense submembrane scaffold supporting structural stability within the cellularly diverse mesohyl. Confocal immunofluorescence confirmed that adult sponges restrict microtubules largely to stable, acetylated axonemes within choanocytes, whereas larval cells maintain an extensive network of dynamic cytoplasmic microtubules (Figures 2A–D). This architectural shift aligns with the transition from active locomotion to sessile filter-feeding (; ). During cell reaggregation - initiating phase of a juvenile sponge assembling, cells downregulate actin-based motility regulators that can shifts cellular transport toward microtubule-dependent mechanisms. The coordinated molecular switch likely supports the intracellular trafficking demands of tissue reassembly that has been described for demosponge metamorphosis (; ). Beyond proteome and transcriptional regulation (Figure 1; Supplementary Figure S4; Supplementary Table S6), we identified extensive stage-specific PTMs on core cytoskeletal components (Supplementary Table S7 A, B). Despite the high expression of tyrosine kinase Src in adults (Supplementary Table S3), α-tubulins showed no tyrosine phosphorylation, suggesting that adults can actively suppress tyrosine phosphorylation (Figure 1D; Supplementary Figure S3A). The negatively charged phosphate group may prevent the incorporation of tubulin dimers into growing microtubules, thereby promoting their destabilization (). Notably, deamidation of asparagine 101 in α-tubulins was detected exclusively in cell aggregates, reminiscent of a “molecular clock” mechanism described in mammalian neurons that stabilizes senescent microtubules (; ). Similarly, oxidation of ARPC2 in aggregates (Supplementary Table S7) suggests localized activation of the Arp2/3 complex for membrane remodeling at nascent cell contacts. The divergent actin HdA6, β-tubulin, ARHGDI, and CLTC display stage-specific dynamic patterns of methionine oxidation, asparagine deamidation, cysteine carbamidomethylation, and ubiquitination (Supplementary Table S7A, B). The findings position HdA6 not merely as a structural element but as a redox-sensitive regulatory node linking oxidative status to cytoskeletal dynamics (; ; ; ). Interestingly, a population of pinacocytes expressing HdA6 (clusters 5 and 8) is present in both larvae and aggregates (Supplementary Figure S5), but HdA6 has distinctly different PTM signatures in each context (Supplementary Figure S8; Supplementary Table S7), suggesting that chemical “rewiring” can adapts the cytoskeleton to stage-specific processes (). Importantly, bulk transcriptomic and proteomic profiling cannot fully distinguish whether these molecular shifts reflect only intracellular chemical reprogramming or cell-type abundance changes. Future studies using higher-resolution approaches, such as spatial transcriptomics, single-cell proteomics, or lineage tracing will be needed to disentangle these contributions. Constitutively high abundance of actin, ferritin, and the α4 subunit of 20S proteasome (PSMA4) across stages (Figure 1A) supposes a conserved proteostasis framework that is differentially engaged during transitions. In humans, PSMA4 was reported to induce metabolic reprogramming by improving oxidative phosphorylation activity in a hypoxia state (). The shifts in proteasome activity during reaggregation coupled with the constitutive high abundance of both actin and 20S proteasome (Figure 3; Supplementary Figure S6) suggest a concerted cellular response, with the actin cytoskeleton providing the framework for cell reassociation, adhesion remodeling, and tissue assembly. This pattern is similar to that observed in mammalian neurons, where the membrane-associated 20S proteasome regulates adhesion without relying on canonical ubiquitin signaling (). In H. dujardinii, a burst of proteasome activity during cell reaggregation, along with cell aggregate-specific ubiquitination of ferritin HdF1a/b suggests that targeted protein turnover can manage redox balance within controlled cell remodeling. Bortezomib-induced redistribution of ferritin HdF1a/b ubiquitination sites parallels impaired aggregate compaction (Supplementary Figure S7), suggesting that precise spatiotemporal control of ferritin modification is required for proper morphogenetic progression. Interestingly, cell differentiation in the marine choanoflagellate Salpingoeca rosetta depends on utilization of insoluble ferric ions (). The redox-cytoskeletal coupling has been reported in regenerating planarians () and during embryonic development in basal metazoans, underscoring the evolutionary conservation of this strategy (; ). The stage-specific PTM patterns we describe extend this paradigm by demonstrating how chemical modifications of conserved scaffolds can rapidly reconfigure cellular architecture without requiring de novo protein synthesis - a principle increasingly recognized in stress responses across eukaryotes. Notably, the non-overlapping sets of E3 ligases expressed in larvae (RNF217) versus adults (HECT-type) propose distinct ubiquitination programs tailored to stage-specific demands. This observation resonates with recent reports of context-dependent ubiquitin signaling in developmental transitions (), though the precise substrates and regulatory networks in sponges remain to be elucidated.
We hypothesize how sponge H. dujardinii can achieve profound morphological changes through integrated transcriptional, post-translational, and proteostatic regulation across specific-stages (Figure 4).
FIGURE 4
While we identify stage-specific PTMs in H. dujardinii and propose their functional relevance, establishing causality will require targeted chemical proteomics in future studies. The biological variability linked to the reproductive cycle can mask subtler regulatory effects, underscoring the need for seasonal sampling to fully resolve cell aggregate proteomes. Furthermore, studying how sponge-associated bacteria modulate host cytoskeletal regulation through metabolite or signal exchange may reveal how the ubiquitin-proteasome system contributed to the evolutionary emergence of multicellularity.
Conclusion
In H. dujardinii, morphological plasticity emerges from the interplay between the stage-specific expression of pinacocyte-like cells and the dynamic post-translational tuning of conserved structural scaffolds. This “chemical rewiring”, which coordinates redox-sensitive PTMs, targeted proteolysis, and the selective expression of regulatory cytoskeletal proteins may represent an ancient strategy for cellular reorganization. Although direct functional validation is still required, this framework highlights the sophisticated regulatory networks operating at the base of the metazoan tree and suggests that post-translational control of preexisting protein networks likely played a fundamental role in the early evolution of multicellular morphogenesis.
Statements
Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.
Ethics statement
The animal study was approved by the Bioethics Commission of Koltzov Institute of Developmental Biology of Russian Academy of Sciences. The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
AK: Funding acquisition, Investigation, Visualization, Writing – original draft, Writing – review and editing, Conceptualization, Formal Analysis, Methodology, Software, Validation. AF: Funding acquisition, Investigation, Visualization, Writing – original draft. KA: Visualization, Writing – original draft, Data curation, Formal Analysis, Software, Validation, Methodology. VZ: Data curation, Formal Analysis, Funding acquisition, Investigation, Software, Validation, Visualization, Writing – original draft. AB: Conceptualization, Formal Analysis, Funding acquisition, Investigation, Visualization, Writing – original draft. MS: Data curation, Methodology, Software, Validation, Writing – original draft. KM: Formal Analysis, Software, Validation, Writing – original draft. RZ: Data curation, Formal Analysis, Investigation, Methodology, Resources, Software, Validation, Writing – original draft. NG: Writing – original draft, Investigation, Funding acquisition. PE: Investigation, Methodology, Visualization, Writing – original draft. ME: Writing – original draft, Investigation, Resources. ES: Investigation, Resources, Writing – original draft, Methodology. VM: Writing – original draft, Conceptualization, Supervision. OK: Funding acquisition, Investigation, Visualization, Writing – original draft, Writing – review and editing, Data curation. YL: Writing – review and editing, Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Visualization, Writing – original draft.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This research was funded by The Russian Science Foundation, grant number 25-14-00131.
Acknowledgments
The research was done using equipment of the Core Centrum of Institute of Developmental Biology RAS and Moscow State University Development Program. We would like to thank “The Arctic Circle Dive Centre and Lodge” and Nataliya Chervyakova for their assistance in collecting the sponges.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2026.1829393/full#supplementary-material
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Summary
Keywords
cytoskeleton, cytoskeleton remodeling, Halisarca dujardinii, Porifera, proteasome, proteome, redox signaling
Citation
Kotov AA, Finoshin AD, Adameyko KI, Zubarev VM, Burakov AV, Sabirov MS, Mikhailov KV, Ziganshin RH, Gornostayev NG, Erokhov PA, Ezhova MA, Shagimardanova EI, Mikhailov VS, Kravchuk OI and Lyupina YV (2026) Proteomic and transcriptomic signatures of cytoskeletal remodeling during morphogenesis in the basal metazoan Halisarca dujardinii (Porifera). Front. Cell Dev. Biol. 14:1829393. doi: 10.3389/fcell.2026.1829393
Received
12 March 2026
Revised
13 May 2026
Accepted
15 May 2026
Published
10 June 2026
Volume
14 - 2026
Edited by
Smadar Ben-Tabou De-Leon, University of Haifa, Israel
Reviewed by
Michaela Holzem, German Cancer Research Center (DKFZ), Germany
Suat Özbek, Heidelberg University, Germany
Updates
Copyright
© 2026 Kotov, Finoshin, Adameyko, Zubarev, Burakov, Sabirov, Mikhailov, Ziganshin, Gornostayev, Erokhov, Ezhova, Shagimardanova, Mikhailov, Kravchuk and Lyupina.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Oksana I. Kravchuk, kravchuk444@mail.ru
† These authors have contributed equally to this work
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.