Proteome Dynamics Across the Blastogenic Cycle of Botryllus schlosseri Reveals Targets for Cell Immortalization

Background The colonial tunicate Botryllus schlosseri undergoes a weekly blastogenic cycle in which old zooids regress while new buds proliferate. Despite this species’ advantages for studying coordinated proliferation and degeneration, proteome-level regulation across blastogenic stages and actionable targets for advancing proliferation of cell culture models remain undefined. Results DIA proteomics enabled quantitation of 15,156 unique peptides mapping to 3,155 unambiguous protein groups across zooids from four blastogenic cycle stages and takeover primary buds (TOB), with 1,432 proteins (45%) changing significantly across these stages. Principal component analysis (PCA) and network analyses revealed most distinct proteomes in TOB versus regressing takeover zooids (TOZ). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that TOB were enriched in DNA replication, cell cycle progression, ribosome biogenesis, and translation pathways, reflecting a strong proliferative and biosynthetic program. Analysis of differentially abundant proteins and enriched pathways across different blastogenic stages revealed that the regulation of cyclin-dependent kinase 1 (CDK1), CDK2, replication licensing, chromatin remodeling, proteostasis, and enhancing translation are central to the TOB proliferation program. In contrast, TOZ were enriched for pathways associated with stress responses, proteolysis, metabolic remodeling, and catabolism, indicating a proteomic signature of programmed degradation and macromolecular integrity control during zooid regression. Conclusions The pro- and anti-proliferative proteomic signatures identified CDK1, CDK2, histone deacetylase 2 (HDAC2), S-phase kinase-associated protein 1 (SKP1), and proliferating cell nuclear antigen (PCNA) as the major nodes to be targeted for manipulation of cell proliferation in vitro to overcome crisis/senescence and achieve reliable immortalization of tunicate cell lines.