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SummaryMany 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.

Although learning is often viewed as a unique feature of organisms with complex nervous systems, single-celled organisms also demonstrate basic forms of learning. The giant ciliate Stentor coeruleus responds to mechanical stimuli by contracting into a compact shape, presumably as a defense mechanism. When a Stentor cell is repeatedly stimulated at a constant level of force, it will learn to ignore that stimulus but will still respond to stronger stimuli. Prior studies of habituation in Stentor reported a graded response, suggesting that cells transition through a continuous range of response probabilities. By analyzing single cells using an automated apparatus to deliver calibrated stimuli, we find that habituation occurs via a single step-like switch in contraction probability within each cell, with the graded response in a population arising from the random distribution of switching times in individual cells. This step-like response allows Stentor behavior to be represented by a simple two-state model whose parameters can be estimated from experimental measurements. We find that transition rates depend on stimulus force and also on the time between stimuli. The ability to measure the behavior of the same cell to the same stimulus allowed us to quantify the functional heterogeneity among single cells. Together, our results suggest that the behavior of Stentor is governed by a two-state stochastic machine whose transition rates are sensitive to the time series properties of the input stimuli.

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 Stentor’s 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. RNAi mediated knockdown experiments indicate that Pumilio is required for regenerating oral structures of the correct size. We show that E2F is involved in the completion of regeneration but is dispensable for earlier steps. This work allows us to classify regeneration genes into groups based on their potential role for regeneration in distinct cell regeneration paradigms, and provides insight into how a single cell can coordinate complex morphogenetic pathways to regenerate missing structures.

The cytoskeleton is essential for the maintenance of cell morphology in eukaryotes. In fission yeast for example, polarized growth sites are organized by actin whereas microtubules (MT) acting upstream control where growth occurs (La Carbona et al, 2006). Growth is limited to the cell poles when MTs undergo catastrophes there and not elsewhere on the cortex (Brunner and Nurse, 2000). Here we report that the modulation of MT dynamics by forces as observed in vitro (Dogterom and Yurke, 1997; Janson et al, 2003) can quantitatively explain the localization of MT catastro-phes in S. pombe. However, we found that it is necessary to add length-dependent catastrophe rates to make the model fully consistent with other measured traits of MTs. This result demonstrates the possibility that MTs together with associated proteins such as kinesins having a depolymerization activity can reliably mark the tips of the cell., 25 pages, 3 figures

Cellular components are non-randomly arranged with respect to the shape and polarity of the whole cell.1-4 Patterning within cells can extend down to the level of individual proteins and mRNA.5,6 But how much of the proteome is actually localized with respect to cell polarity axes? Proteomics combined with cellular fractionation7-11 has shown that most proteins localize to one or more organelles but does not tell us how many proteins have a polarized localization with respect to the large-scale polarity axes of the intact cell. Genome-wide localization studies in yeast12-15 found that only a few percent of proteins have a localized position relative to the cell polarity axis defined by sites of polarized cell growth. Here, we describe an approach for analyzing protein distribution within a cell with a visibly obvious global patterning-the giant ciliate Stentor coeruleus.16,17 Ciliates, including Stentor, have highly polarized cell shapes with visible surface patterning.1,18 A Stentor cell is roughly 2mm long, allowing a "proteomic dissection" in which microsurgery is used to separate cellular fragments along the anterior-posterior axis, followed by comparative proteomic analysis. In our analysis, 25% of the proteome, including signaling proteins, centrin/SFI proteins, and GAS2 orthologs, shows a polarized location along the cell's anterior-posterior axis. We conclude that a large proportion of all proteins are polarized with respect to global cell polarity axes and that proteomic dissection provides a simple and effective approach for spatial proteomics.

The phenomenon of ciliary coordination has garnered increasing attention in recent decades and multiple theories have been proposed to explain its occurrence in different biological systems. While hydrodynamic interactions are thought to dictate the large-scale coordinated activity of epithelial cilia for fluid transport, it is rather basal coupling that accounts for synchronous swimming gaits in model microeukaryotes such asChlamydomonas.Unicellular ciliates present a fascinating yet understudied context in which coordination is found to persist in ciliary arrays positioned across millimetre scales on the same cell. Here, we focus on the ciliateStentor coeruleus, chosen for its large size, complex ciliary organization, and capacity for cellular regeneration. These large protists exhibit ciliary differentiation between cortical rows of short body cilia used for swimming, and an anterior ring of longer, fused cilia called the membranellar band (MB). The oral cilia in the MB beat metachronously to produce strong feeding currents. Remarkably, upon injury, the MB can be shed and regenerated de novo. Here, we follow and track this developmental sequence in its entirety to elucidate the emergence of coordinated ciliary beating: from band formation, elongation, curling and final migration towards the cell anterior. We reveal a complex interplay between hydrodynamics and ciliary restructuring inStentor, and highlight for the first time the importance of a ring-like topology for achieving long-range metachronism in ciliated structures.This article is part of the Theo Murphy meeting issue ‘Unity and diversity of cilia in locomotion and transport’.

SummaryThe phenomenon of ciliary coordination has garnered increasing attention in recent decades, with multiple theories accounting for its emergence in different contexts. The heterotrich ciliateStentor coeruleusis a unicellular organism which boasts a number of features which present unrivalled opportunities for biophysical studies of cilia coordination. With their cerulean colour and distinctive morphology, these large protists possess a characteristic differentiation between cortical rows of short body cilia used for swimming, and an anterior ring structure of fused oral cilia forming a membranellar band. The oral cilia beat metachronously to produce strong feeding currents. In addition to this complex body plan,Stentorhave remarkable regenerative capabilities. Minute fragments of single cells can over the period of hours or days, regenerate independently into new, proportional individuals. Certain environmental perturbations elicit a unique programmed response known as oral regeneration wherein only the membranellar band is shed and a new, ciliated oral primordium formed on the side of the body. Here, we target oral regeneration induced by sucrose-shock to reveal the complex interplay between ciliary restructuring and hydrodynamics inStentor, which accompanies the complete developmental sequence from band formation, elongation, curling, and migration toward the cell anterior.“When the anterior part is open, one may perceive about its Edges a very lively Motion; and when the Polyps presents itself in a certain manner, it discovers, on either side of these edges of its anterior part, somewhat very much resembling the wheels of a little Mill, that move with great velocity.”A. Trembley F.R.S describing the membranellar band of Stentor, Phil. Soc. Trans. Royal Society (London), 1744.

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Cell polarization is crucial for the functioning of all organisms. The cytoskeleton is central to the process but its role in symmetry breaking is poorly understood. We study cell polarization when fission yeast cells exit starvation. We show that the basis of polarity generation is de novo sterol biosynthesis, cell surface delivery of sterols, and their recruitment to the cell poles. This involves four phases occurring independent of the polarity factor cdc42p. Initially, multiple, randomly distributed sterol-rich membrane (SRM) domains form at the plasma membrane, independent of the cytoskeleton and cell growth. These domains provide platforms on which the growth and polarity machinery assembles. SRM domains are then polarized by the microtubule-dependent polarity factor tea1p, which prepares for monopolar growth initiation and later switching to bipolar growth. SRM polarization requires F-actin but not the F-actin organizing polarity factors for3p and bud6p. We conclude that SRMs are key to cell polarization.

SummaryThe cylindrical rod shape of the fission yeast Schizosaccharomyces pombe is organized and maintained by interactions between the microtubule, cell membrane, and actin cytoskeleton [1]. Mutations affecting any components in this pathway lead to bent, branched, or round cells [2]. In this context, the cytoskeleton controls cell polarity and thus dictates cell shape. Here, we use soft-lithography techniques to construct microfluidic channels to control cell shape. We show that when wild-type rod-shaped cells are physically forced to grow in a bent fashion, they will reorganize their cytoskeleton and redirect cell polarity to make new ectopic cell tips. Moreover, when bent or round mutant cells are physically forced to conform to the wild-type rod-shape, they will reverse their mutational phenotypes by reorganizing their cytoskeleton to maintain proper wild-type-like localization of microtubules, cell-membrane proteins, and actin. Our study provides direct evidence that the cytoskeleton controls cell polarity and cell shape and demonstrates that cell shape also controls the organization of the cytoskeleton in a feedback loop. We present a model of the feedback loop to explain how fission yeast maintain a rod shape and how perturbation of specific parameters of the loop can lead to different cell shapes.

The field of fluorescent proteins (FPs) is constantly developing. The use of FPs changed the field of life sciences completely, starting a new era of direct observation and quantification of cellular processes. The broad spectrum of FPs (see Fig. 1) with a wide range of characteristics allows their use in many different experiments. This review discusses the use of FPs for imaging in budding yeast (Saccharomyces cerevisiae) and fission yeast Schizosaccharomyces pombe). The information included in this review is relevant for both species unless stated otherwise.

The field of fluorescent proteins (FPs) is constantly developing. The use of FPs changed the field of life sciences completely, starting a new era of direct observation and quantification of cellular processes. The broad spectrum of FPs (see Fig. 1) with a wide range of characteristics allows their use in many different experiments. This review discusses the use of FPs for imaging in budding yeast (Saccharomyces cerevisiae) and fission yeast Schizosaccharomyces pombe). The information included in this review is relevant for both species unless stated otherwise.

To study virus propagation, we have developed a method by which the propagation of the Lambda bacteriophage can be observed and quantified. This is done by creating a fusion protein of the capsid protein gpD and the enhanced yellow fluorescent protein (EYFP). We show that this fusion allows capsid formation and that the modified viruses propagate on a surface covered with host bacteria thus forming fluorescent plaques. The intensity of fluorescence in a growing plaque determines the distribution of phages. This provides a new tool to study the propagation of infection at the microscopic level.