Proteomic Dissection of a Giant Cell

Many individual proteins have been identified as having defined positions relative to cell polarity axes, raising the question of what fraction of all proteins may have polarized localizations. We took advantage of the giant ciliate Stentor coeruleus to quantify the extent of polarized localization proteome-wide. This trumpet-shaped unicellular organism shows a clear morphological anterior-posterior axis defined by a circular array of cilia known as a membranellar band at one end, and a holdfast at the other end. Because individual Stentor cells are over a millimeter in length, we were able to cut the cells into three pieces along the anterior-posterior axis, followed by proteomic analysis of proteins enriched in each piece. We find that approximately 30% of all detected proteins show a polarized location relative to the anterior-posterior cell axis. Proteins with polarized enrichment include centrin-like proteins, calcium-regulated kinases, orthologs of SFI1 and GAS2, and proteases. At the organelle level, nuclear and mitochondrial proteins are enriched in the anterior half of the cell body, but not in the membranellar band itself, while ribosome related proteins are apparently uniformly distributed. RNAi of signaling proteins enriched in the membranellar band, which is the anterior-most structure in the cell, revealed a protein phosphatase 2 subunit b ortholog required for closure of the membranellar band into the ring shape characteristic of Stentor. These results suggest that a large fraction of the Stentor proteome has a polarized localization, and provide a protein-level framework for future analysis of pattern formation and regeneration in Stentor as well as defining a general strategy for subcellular spatial proteomics based on physical dissection of cells.

Regeneration in Stentor coeruleus

We often think about regeneration in terms of replacing missing structures, such as organs or tissues, with new structures generated via cell proliferation and differentiation. But at a smaller scale, single cells, themselves, are capable of regenerating when part of the cell has been removed. A classic model organism that facilitates the study of cellular regeneration in the giant ciliate Stentor coeruleus. These cells, which can grow to more than a millimeter in size, have the ability to survive after extensive wounding of their surface, and are able to regenerate missing structures. Even a small piece of a cell can regenerate a whole cell with normal geometry, in a matter of hours. Such regeneration requires cells to be able to trigger organelle biogenesis in response to loss of structures. But subcellular regeneration also relies on intracellular mechanisms to create and maintain global patterning within the cell. These mechanisms are not understood, but at a conceptual level they involve processes that resemble those seen in animal development and regeneration. Here we discuss single-celled regeneration in Stentor from the viewpoint of standard regeneration paradigms in animals. For example, there is evidence that regeneration of the oral apparatus in Stentor follows a sender-receiver model similar to crustacean eyestalk regeneration. By drawing these analogies, we find that many of the concepts already known from the study of animal-scale regeneration and development can be applied to the study of regeneration at the cellular level, such as the concepts of determination, induction, mosaic vs. regulative development, and epimorphosis vs. morphallaxis. We propose that the similarities may go beyond analogy, and that some aspects of animal development and regeneration may have evolved by exploiting pre-existing subcellular developmental strategies from unicellular ancestors.

The nuclear transport factor CSE1 drives macronuclear volume increase and macronuclear node condensation in Stentor coeruleus

The giant ciliate, Stentor coeruleus, provides a unique opportunity to study nuclear shape because its macronucleus undergoes a rapid, dramatic, and developmentally regulated shape change. During a 2 hour time period within cell division and regeneration, the 400 um long moniliform macronucleus condenses into a single mass, elongates into a vermiform shape, and then renodulates, returning to its original beads-on-a-string morphology (Tartar 1961). Previous work from the 1960s - 1980s demonstrated that the macronuclear shape change is a highly regulated part of cell division and regeneration, but there were no molecular studies into this process (De Terra 1964; De Terra 1983). With the recent availability of a sequenced Stentor genome, a transcriptome during regeneration, and molecular tools like RNAi, it is now possible to investigate the molecular mechanisms that drive macronuclear shape change (Slabodnick et al. 2014; Slabodnick et al. 2017; Sood et al. 2021). We found that the volume of the macronucleus increases during condensation. When the nuclear transport factor, CSE1, is knocked down by RNAi, this volume increase is reduced, and the nodes are unable to fuse. This affects the final morphology of the macronucleus: 24 hours after regeneration the macronucleus is misshapen. We found that CSE1 is mainly cytoplasmic during interphase and in early regeneration, and then becomes mainly macronuclear during condensation. At the end of regeneration CSE1 is degraded while the macronucleus returns to its pre-condensation volume. We propose a model in which nuclear transport via CSE1 increases the volume of the macronucleus, driving the condensation of the many nodes into a single mass.

Modular, Cascade-like Transcriptional Program of Regeneration in Stentor

The giant ciliate Stentor coeruleus is a classical model system for studying regeneration and morphogenesis at the level of a single cell. The anterior of the cell is marked by an array of cilia, known as the oral apparatus, which can be induced to shed and regenerate in a series of reproducible morphological steps, previously shown to require transcription. If a cell is cut in half, each half will regenerate an intact cell, including a new oral apparatus in the posterior half. We used RNAseq to assay the dynamic changes in Stentors transcriptome during regeneration, after both oral apparatus shedding and bisection, allowing us to identify distinct temporal waves of gene expression including kinases, RNA binding proteins, centriole biogenesis factors, and orthologs of human ciliopathy genes implicated in Meckel and Joubert syndromes. By comparing transcriptional profiles of different regeneration events in the same species, we were able to identify distinct modules of gene expression corresponding to oral apparatus regeneration, posterior holdfast regeneration, and recovery after wounding. By measuring gene expression in cells in which translation is blocked, we show that the sequential waves of gene expression involve a cascade mechanism in which later waves of expression are triggered by translation products of early-expressed genes. Among the early-expressed genes, we identified an E2F transcription factor and the conserved RNA binding protein Pumilio as potential regulators of regeneration based on the expression pattern of their predicted target genes. This work allows us to classify regeneration genes into groups based on their potential role for regeneration in distinct cell regeneration paradigms, and provides new insight into how a single cell can coordinate complex morphogenetic pathways to regenerate missing structures.

Microfluidic guillotine reveals multiple timescales and mechanical modes of wound response in Stentor coeruleus

Background Wound healing is one of the defining features of life and is seen not only in tissues but also within individual cells. Understanding wound response at the single-cell level is critical for determining fundamental cellular functions needed for cell repair and survival. This understanding could also enable the engineering of single-cell wound repair strategies in emerging synthetic cell research. One approach is to examine and adapt self-repair mechanisms from a living system that already demonstrates robust capacity to heal from large wounds. Towards this end, Stentor coeruleus, a single-celled free-living ciliate protozoan, is a unique model because of its robust wound healing capacity. This capacity allows one to perturb the wounding conditions and measure their effect on the repair process without immediately causing cell death, thereby providing a robust platform for probing the self-repair mechanism. Results Here we used a microfluidic guillotine and a fluorescence-based assay to probe the timescales of wound repair and of mechanical modes of wound response in Stentor. We found that Stentor requires ~ 100–1000 s to close bisection wounds, depending on the severity of the wound. This corresponds to a healing rate of ~ 8–80 μm2/s, faster than most other single cells reported in the literature. Further, we characterized three distinct mechanical modes of wound response in Stentor: contraction, cytoplasm retrieval, and twisting/pulling. Using chemical perturbations, active cilia were found to be important for only the twisting/pulling mode. Contraction of myonemes, a major contractile fiber in Stentor, was surprisingly not important for the contraction mode and was of low importance for the others. Conclusions While events local to the wound site have been the focus of many single-cell wound repair studies, our results suggest that large-scale mechanical behaviors may be of greater importance to single-cell wound repair than previously thought. The work here advances our understanding of the wound response in Stentor and will lay the foundation for further investigations into the underlying components and molecular mechanisms involved.

RNA Interference by Cyanobacterial Feeding Demonstrates the SCSG1 Gene Is Essential for Ciliogenesis during Oral Apparatus Regeneration in Stentor

In the giant ciliate Stentor coeruleus, oral apparatus (OA) regeneration is an experimentally tractable regeneration paradigm that occurs via a series of morphological steps. OA regeneration is thought to be driven by a complex regulatory system that orchestrates the temporal expression of conserved and specific genes. We previously identified a S. coeruleus-specific gene (named SCSG1) that was significantly upregulated during the ciliogenesis stages of OA regeneration, with an expression peak at the stage of the first OA cilia appearance. We established a novel RNA interference (RNAi) method through cyanobacteria Synechocystis sp. PCC6803 feeding in S. coeruleus. The expression of SCSG1 gene was significantly knocked down by using this method and induced abnormal ciliogenesis of OA regeneration in S. coeruleus, suggesting that SCSG1 is essential for OA regeneration in S. coeruleus. This novel RNAi method by cyanobacterial feeding has potential utility for studying other ciliates.

Microfluidic guillotine reveals multiple timescales and mechanical modes of wound response in Stentor coeruleus

BackgroundWound healing is one of the defining features of life and is seen not only in tissues but also within individual cells. Understanding wound response at the single-cell level is critical for determining fundamental cellular functions needed for cell repair and survival. This understanding could also enable the engineering of single-cell wound repair strategies in emerging synthetic cell research. One approach is to examine and adapt self-repair mechanisms from a living system that already demonstrates robust capacity to heal from large wounds. Towards this end, Stentor coeruleus, a single-celled free-living ciliate protozoan, is a unique model because of its robust wound healing capacity. This capacity allows one to perturb the wounding conditions and measure their effect on the repair process without immediately causing cell death, thereby providing a robust platform for probing the self-repair mechanism.ResultsHere we used a microfluidic guillotine and a fluorescence-based assay to probe the timescales and mechanisms of wound repair in Stentor. We found that Stentor requires ∼100 – 1000 s to close bisection wounds, depending on the severity of the wound. This corresponds to a healing rate of ∼8 – 80 μm2/s, faster than most other single cells reported in the literature. Further, we observed and characterized three distinct mechanical modes of wound repair in Stentor: contraction, cytoplasm retrieval, and twisting/pulling. Using chemical perturbations, active cilia were found to be important for only the twisting/pulling mode. Contraction of myonemes, a major contractile fiber in Stentor, was surprisingly not important for the contraction mode and was of low importance for the others.ConclusionsWhile events local to the wound site have been the focus of many single-cell wound repair studies, our results suggest that large-scale mechanical behaviors may be of greater importance to single-cell wound repair than previously thought. The work here advances our understanding of the wound response in Stentor, and will lay the foundation for further investigations into the underlying components and molecular mechanisms involved.