UCSF

Regeneration and wound healing are essential biological processes that restore cellular and tissue integrity following injury from external perturbations. Central to these processes is the interpretation of positional cues which include chemical or mechanical signals that instruct cells on what to rebuild and where to place structures. While the mechanisms underlying tissue and organ regeneration have been extensively studied, the molecular and spatial logic of regeneration at the subcellular level remains less understood. The giant single-celled ciliate Stentor coeruleus offers a powerful model for uncovering how cells interpret positional information to reconstruct complex intracellular architecture. With a highly polarized body plan, anterior-posterior axis, and an oral apparatus critical for feeding, Stentor can regenerate entire structures from fragments, provided a part of the macronucleus is intact.Here, we explore how cytoskeletal patterning and cell cycle regulators support regeneration in Stentor. The oral apparatus regenerates at a stereotyped location along the anterior-posterior axis, guided by visible cortical landmarks such as pigmented stripes and organized arrays of cytoskeletal fibers. We find that Sfi1 family proteins, which scaffold centrin-based cytoskeletal assemblies, are upregulated during regeneration and are essential for both oral primordium formation and contractility. RNAi-mediated depletion of Sfi1 genes impairs regeneration and anterior-posterior centrin patterning, suggesting that Sfi1 proteins establish cytoskeletal polarity necessary for morphogenesis. These proteins are recruited in a temporally ordered manner to the regenerating oral primordium, linking gene expression timing with spatial organization.Moreover, we show that regeneration utilizes components of the canonical cell cycle. Using transcriptional and phosphoproteomic analyses, we identify upregulation of cell cycle regulators including E2F, CDK4, Rb, and cyclins during regeneration. Inhibition of CDK4 with Palbociclib disrupts this pathway and suppresses regeneration, indicating that CDK4-mediated phosphorylation of Rb and subsequent activation of E2F target genes is required. Interestingly, the morphological stages of regeneration mirror those seen during cell division, including macronuclear condensation and elongation, suggesting shared regulatory mechanisms. Our results raise the possibility that regeneration in Stentor reflects a partial redeployment of the developmental program associated with cell division.Together, our findings reveal that Stentor regeneration depends on the integration of cytoskeletal patterning with conserved cell cycle signaling pathways. This model system provides insight into how cells use positional cues and multifunctional molecular machinery to rebuild complex structures with spatial precision. Our work highlights the convergence of regeneration and cell cycle regulation as a general principle of morphogenesis, even at the level of a single cell.