Chlamydomonas as a model system to study cilia and flagella using genetics, biochemistry, and microscopy.

The unicellular green alga, Chlamydomonas reinhardtii, has played a central role in discovering much of what is currently known about the composition, assembly, and function of cilia and flagella. Chlamydomonas combines excellent genetics, such as the ability to grow cells as haploids or diploids and to perform tetrad analysis, with an unparalleled ability to detach and isolate flagella in a single step without cell lysis. The combination of genetics and biochemistry that is possible in Chlamydomonas has allowed many of the key components of the cilium to be identified by looking for proteins that are missing in a defined mutant. Few if any other model organisms allow such a seamless combination of genetic and biochemical approaches. Other major advantages of Chlamydomonas compared to other systems include the ability to induce flagella to regenerate in a highly synchronous manner, allowing the kinetics of flagellar growth to be measured, and the ability of Chlamydomonas flagella to adhere to glass coverslips allowing Intraflagellar Transport to be easily imaged inside the flagella of living cells, with quantitative precision and single-molecule resolution. These advantages continue to work in favor of Chlamydomonas as a model system going forward, and are now augmented by extensive genomic resources, a knockout strain collection, and efficient CRISPR gene editing. While Chlamydomonas has obvious limitations for studying ciliary functions related to animal development or organ physiology, when it comes to studying the fundamental biology of cilia and flagella, Chlamydomonas is simply unmatched in terms of speed, efficiency, cost, and the variety of approaches that can be brought to bear on a question.

Physical Forces in Regeneration of Cells and Tissues.

The ability to regenerate after the loss of a part is a hallmark of living systems and occurs at both the tissue and organ scales, but also within individual cells. Regeneration entails many processes that are physical and mechanical in nature, including the closure of wounds, the repositioning of material from one place to another, and the restoration of symmetry following perturbations. However, we currently know far more about the genetics and molecular signaling pathways involved in regeneration, and there is a need to investigate the role of physical forces in the process. Here, we will provide an overview of how physical forces may play a role in wound healing and regeneration, in which we compare and contrast regenerative processes at the tissue and cell scales.

Modeling homologous chromosome recognition via nonspecific interactions.

In many organisms, most notably Drosophila, homologous chromosomes associate in somatic cells, a phenomenon known as somatic pairing, which takes place without double strand breaks or strand invasion, thus requiring some other mechanism for homologs to recognize each other. Several studies have suggested a "specific button" model, in which a series of distinct regions in the genome, known as buttons, can associate with each other, mediated by different proteins that bind to these different regions. Here, we use computational modeling to evaluate an alternative "button barcode" model, in which there is only one type of recognition site or adhesion button, present in many copies in the genome, each of which can associate with any of the others with equal affinity. In this model, buttons are nonuniformly distributed, such that alignment of a chromosome with its correct homolog, compared with a nonhomolog, is energetically favored; since to achieve nonhomologous alignment, chromosomes would be required to mechanically deform in order to bring their buttons into mutual register. By simulating randomly generated nonuniform button distributions, many highly effective button barcodes can be easily found, some of which achieve virtually perfect pairing fidelity. This model is consistent with existing literature on the effect of translocations of different sizes on homolog pairing. We conclude that a button barcode model can attain highly specific homolog recognition, comparable to that seen in actual cells undergoing somatic homolog pairing, without the need for specific interactions. This model may have implications for how meiotic pairing is achieved.

Cell motility: Bioelectrical control of behavior without neurons.

Single cells are capable of remarkably sophisticated, sometimes animal-like, behaviors. New work demonstrates bioelectric control of motility through the differential regulation of appendage movements in a unicellular organism that walks across surfaces using leg-like bundles of cilia.

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

Stentor coeruleus provides a unique opportunity to study how cells regulate nuclear shape because its macronucleus undergoes a rapid, dramatic, and developmentally regulated shape change. We found that the volume of the macronucleus increases during coalescence, suggesting an inflation-based mechanism. When the nuclear transport factor, CSE1, is knocked down by RNAi, the shape and volume changes of the macronucleus are attenuated, and nuclear morphology is altered. CSE1 protein undergoes a dynamic relocalization correlated with nuclear shape changes, being mainly cytoplasmic prior to nuclear coalescence, and accumulating inside the macronucleus during coalescence. At the end of regeneration, CSE1 protein levels are reduced as the macronucleus returns to its pre-coalescence volume. We propose a model in which nuclear transport via CSE1 is required to increase the volume of the macronucleus, thereby decreasing the surface-to-volume ratio and driving coalescence of the nodes into a single mass.

Homologous chromosome recognition via nonspecific interactions.

In many organisms, most notably Drosophila, homologous chromosomes in somatic cells associate with each other, a phenomenon known as somatic homolog pairing. Unlike in meiosis, where homology is read out at the level of DNA sequence complementarity, somatic homolog pairing takes place without double strand breaks or strand invasion, thus requiring some other mechanism for homologs to recognize each other. Several studies have suggested a "specific button" model, in which a series of distinct regions in the genome, known as buttons, can associate with each other, presumably mediated by different proteins that bind to these different regions. Here we consider an alternative model, which we term the "button barcode" model, in which there is only one type of recognition site or adhesion button, present in many copies in the genome, each of which can associate with any of the others with equal affinity. An important component of this model is that the buttons are non-uniformly distributed, such that alignment of a chromosome with its correct homolog, compared with a non-homolog, is energetically favored; since to achieve nonhomologous alignment, chromosomes would be required to mechanically deform in order to bring their buttons into mutual register. We investigated several types of barcodes and examined their effect on pairing fidelity. We found that high fidelity homolog recognition can be achieved by arranging chromosome pairing buttons according to an actual industrial barcode used for warehouse sorting. By simulating randomly generated non-uniform button distributions, many highly effective button barcodes can be easily found, some of which achieve virtually perfect pairing fidelity. This model is consistent with existing literature on the effect of translocations of different sizes on homolog pairing. We conclude that a button barcode model can attain highly specific homolog recognition, comparable to that seen in actual cells undergoing somatic homolog pairing, without the need for specific interactions. This model may have implications for how meiotic pairing is achieved.

Mitochondrial networks through the lens of mathematics.

Mitochondria serve a wide range of functions within cells, most notably via their production of ATP. Although their morphology is commonly described as bean-like, mitochondria often form interconnected networks within cells that exhibit dynamic restructuring through a variety of physical changes. Further, though relationships between form and function in biology are well established, the extant toolkit for understanding mitochondrial morphology is limited. Here, we emphasize new and established methods for quantitatively describing mitochondrial networks, ranging from unweighted graph-theoretic representations to multi-scale approaches from applied topology, in particular persistent homology. We also show fundamental relationships between mitochondrial networks, mathematics, and physics, using ideas of graph planarity and statistical mechanics to better understand the full possible morphological space of mitochondrial network structures. Lastly, we provide suggestions for how examination of mitochondrial network form through the language of mathematics can inform biological understanding, and vice versa.

The flagellar length control system: exploring the physical biology of organelle size.

How cells build and maintain dynamic structures of defined size is currently an important unsolved problem in quantitative cell biology. The flagella of the unicellular green algaChlamydomonasprovide a highly tractable model system to investigate this general question, but while the powerful genetics of this organism have revealed numerous genes required for proper flagellar length, in most cases we do not understand their mechanistic role in length control. Flagellar length can be viewed as the steady state solution of a dynamical system involving assembly and disassembly of axonemal microtubules, with assembly depending on an active transport process known as intraflagellar transport (IFT). The inherent length dependence of IFT gives rise to a family of simple models for length regulation that can account for many previously described phenomena such as the ability of flagella to maintain equal lengths. But these models requires that the cell has a way to measure flagellar length in order to adjust IFT rates accordingly. Several models for length sensing have been modeled theoretically and evaluated experimentally, allowing them to be ruled out. Current data support a model in which the diffusive return of the kinesin motor driving IFT provides a length dependence that ultimately is the basis for length regulation. By combining models of length sensing with a more detailed representation of cargo transport and availability, it is now becoming possible to formulate concrete hypotheses to explain length altering mutants.

Cooperative hydrodynamics accompany multicellular-like colonial organization in the unicellular ciliate Stentor.

Evolution of multicellularity from early unicellular ancestors is arguably one of the most important transitions since the origin of life1,2. Multicellularity is often associated with higher nutrient uptake3, better defense against predation, cell specialization and better division of labor4. While many single-celled organisms exhibit both solitary and colonial existence3,5,6, the organizing principles governing the transition and the benefits endowed are less clear. Using the suspension-feeding unicellular protist Stentor coeruleus, we show that hydrodynamic coupling between proximal neighbors results in faster feeding flows that depend on the separation between individuals. Moreover, we find that the accrued benefits in feeding current enhancement are typically asymmetric- individuals with slower solitary currents gain more from partnering than those with faster currents. We find that colony-formation is ephemeral in Stentor and individuals in colonies are highly dynamic unlike other colony-forming organisms like Volvox carteri 3. Our results demonstrate benefits endowed by the colonial organization in a simple unicellular organism and can potentially provide fundamental insights into the selective forces favoring early evolution of multicellular organization.

Genome-wide analysis of anterior-posterior mRNA localization in Stentor coeruleus reveals a role for the microtubule cytoskeleton.

Cells have complex and beautiful structures that are important for their function, but understanding the molecular mechanisms that produce these structures is a challenging problem due to the gap in size scales between molecular interactions and cellular structures. The giant ciliate Stentor coeruleus is a unicellular model organism whose large size, reproducible structure, and ability to heal wounds and regenerate has historically allowed the formation of structure in a single cell to be addressed using methods of experimental embryology. Such studies have shown that specific cellular structures, such as the oral apparatus, always form in specific regions of the cell, which raises the question: what is the source of positional information within this organism? By analogy with embryonic development, in which localized mRNA is often used to mark position, we asked whether position along the anterior-posterior axis of Stentor might be marked by specific regionalized mRNAs. By physically bisecting cells and conducting half-cell RNA sequencing, we were able to identify sets of messages enriched in either the anterior or posterior half. We repeated this analysis in cells in which a set of longitudinal microtubule bundles running down the whole length of the cell, known as KM-fibers, were disrupted by RNAi of b-tubulin. We found that many messages either lost their regionalized distribution or switched to an opposite distribution, such that anterior-enriched messages in control became posterior-enriched in the RNAi cells, or vice versa. This study indicates that mRNA can be regionalized within a single giant cell and that microtubules may play a role, possibly by serving as tracks for the movement of the messages.

Testing the ion-current model for flagellar length sensing and IFT regulation.

Eukaryotic cilia and flagella are microtubule-based organelles whose relatively simple shape makes them ideal for investigating the fundamental question of organelle size regulation. Most of the flagellar materials are transported from the cell body via an active transport process called intraflagellar transport (IFT). The rate of IFT entry into flagella, known as IFT injection, has been shown to negatively correlate with flagellar length. However, it remains unknown how the cell measures the length of its flagella and controls IFT injection. One of the most-discussed theoretical models for length sensing to control IFT is the ion-current model, which posits that there is a uniform distribution of Ca2+ channels along the flagellum and that the Ca2+ current from the flagellum into the cell body increases linearly with flagellar length. In this model, the cell uses the Ca2+ current to negatively regulate IFT injection. The recent discovery that IFT entry into flagella is regulated by the phosphorylation of kinesin through a calcium-dependent protein kinase has provided further impetus for the ion-current model. To test this model, we measured and manipulated the levels of Ca2+ inside of Chlamydomonas flagella and quantified IFT injection. Although the concentration of Ca2+ inside of flagella was weakly correlated with the length of flagella, we found that IFT injection was reduced in calcium-deficient flagella, rather than increased as the model predicted, and that variation in IFT injection was uncorrelated with the occurrence of flagellar Ca2+ spikes. Thus, Ca2+ does not appear to function as a negative regulator of IFT injection, hence it cannot form the basis of a stable length control system.

Studying Habituation in Stentor coeruleus.

Learning is usually associated with a complex nervous system, but there is increasing evidence that life at all levels, down to single cells, can display intelligent behaviors. In both natural and artificial systems, learning is the adaptive updating of system parameters based on new information, and intelligence is a measure of the computational process that facilitates learning. Stentor coeruleus is a unicellular pond-dwelling organism that exhibits habituation, a form of learning in which a behavioral response decreases following a repeated stimulus. Stentor contracts in response to mechanical stimulation, which is an apparent escape response from aquatic predators. However, repeated low-force perturbations induce habituation, demonstrated by a progressive reduction in contraction probability. Here, we introduce a method for quantifying Stentor habituation using a microcontroller board-linked apparatus that can deliver mechanical pulses at a specified force and frequency, including methods for building the apparatus and setting up the experiment in a way that minimizes external perturbations. In contrast to the previously described approaches for mechanically stimulating Stentor, this device allows the force of stimulation to be varied under computer control during the course of a single experiment, thus greatly increasing the variety of input sequences that can be applied. Understanding habituation at the level of a single cell will help characterize learning paradigms that are independent of complex circuitry.

Single-cell analysis of habituation in Stentor coeruleus.

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.

Role of intraflagellar transport in transcriptional control during flagellar regeneration in Chlamydomonas.

Biosynthesis of organelle precursors is a central part of the organelle size control problem, but what systems are required to control precursor production? Genes encoding flagellar proteins are up-regulated during flagellar regeneration in Chlamydomonas, and this up-regulation is critical for flagella to reach their final length, but it not known how the cell triggers these genes during regeneration. We present two models based on transcriptional repressor that is produced either in the flagellum or in the cell body and sequestered in the growing flagellum. Both models lead to stable flagellar length control and can reproduce the observed dynamics of gene expression. The two models make opposite predictions regarding the effect of mutations that block intraflagellar transport (IFT). Using quantitative measurements of gene expression, we show that gene expression during flagellar regeneration is greatly reduced in mutations of the heterotrimeric kinesin-2 that drives IFT. This result is consistent with the predictions of the model in which a repressor is sequestered in the flagellum by IFT. Inhibiting axonemal assembly has a much smaller effect on gene expression. The repressor sequestration model allows precursor production to occur when flagella are growing rapidly, representing a form of derivative control.

Modular, cascade-like transcriptional program of regeneration in Stentor.

The giant ciliate Stentor coeruleus is a classical model system for studying regeneration and morphogenesis in 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 regenerates an intact cell. We used RNA sequencing (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. By comparing transcriptional profiles of different regeneration events, we identified distinct modules of gene expression corresponding to oral apparatus regeneration, posterior holdfast regeneration, and recovery after wounding. By measuring gene expression after blocking translation, 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 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. 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.

A unicellular walker controlled by a microtubule-based finite-state machine.

Cells are complex biochemical systems whose behaviors emerge from interactions among myriad molecular components. Computation is often invoked as a general framework for navigating this cellular complexity. However, it is unclear how cells might embody computational processes such that the theories of computation, including finite-state machine models, could be productively applied. Here, we demonstrate finite-state-machine-like processing embodied in cells using the walking behavior of Euplotes eurystomus, a ciliate that walks across surfaces using fourteen motile appendages (cirri). We found that cellular walking entails regulated transitions among a discrete set of gait states. The set of observed transitions decomposes into a small group of high-probability, temporally irreversible transitions and a large group of low-probability, time-symmetric transitions, thus revealing stereotypy in the sequential patterns of state transitions. Simulations and experiments suggest that the sequential logic of the gait is functionally important. Taken together, these findings implicate a finite-state-machine-like process. Cirri are connected by microtubule bundles (fibers), and we found that the dynamics of cirri involved in different state transitions are associated with the structure of the fiber system. Perturbative experiments revealed that the fibers mediate gait coordination, suggesting a mechanical basis of gait control.

Modeling cell biological features of meiotic chromosome pairing to study interlock resolution.

During meiosis, homologous chromosomes become associated side by side in a process known as homologous chromosome pairing. Pairing requires long range chromosome motion through a nucleus that is full of other chromosomes. It remains unclear how the cell manages to align each pair of chromosomes quickly while mitigating and resolving interlocks. Here, we use a coarse-grained molecular dynamics model to investigate how specific features of meiosis, including motor-driven telomere motion, nuclear envelope interactions, and increased nuclear size, affect the rate of pairing and the mitigation/resolution of interlocks. By creating in silico versions of three yeast strains and comparing the results of our model to experimental data, we find that a more distributed placement of pairing sites along the chromosome is necessary to replicate experimental findings. Active motion of the telomeric ends speeds up pairing only if binding sites are spread along the chromosome length. Adding a meiotic bouquet significantly speeds up pairing but does not significantly change the number of interlocks. An increase in nuclear size slows down pairing while greatly reducing the number of interlocks. Interestingly, active forces increase the number of interlocks, which raises the question: How do these interlocks resolve? Our model gives us detailed movies of interlock resolution events which we then analyze to build a step-by-step recipe for interlock resolution. In our model, interlocks must first translocate to the ends, where they are held in a quasi-stable state by a large number of paired sites on one side. To completely resolve an interlock, the telomeres of the involved chromosomes must come in close proximity so that the cooperativity of pairing coupled with random motion causes the telomeres to unwind. Together our results indicate that computational modeling of homolog pairing provides insight into the specific cell biological changes that occur during meiosis.

Determining protein polarization proteome-wide using physical dissection of individual Stentor coeruleus cells.

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 2 mm 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.

Biosynthesis of Linear Protein Nanoarrays Using the Flagellar Axoneme.

Applications in biotechnology and synthetic biology often make use of soluble proteins, but there are many potential advantages of anchoring enzymes to a stable substrate, including stability and the possibility for substrate channeling. To avoid the necessity of protein purification and chemical immobilization, there has been growing interest in bio-assembly of protein-containing nanoparticles, exploiting the self-assembly of viral capsid proteins or other proteins that form polyhedral structures. However, these nanoparticles are limited in size, which constrains the packaging and the accessibility of the proteins. An axoneme, the insoluble protein core of the eukaryotic flagellum or cilium, is a highly ordered protein structure that can be several microns in length, orders of magnitude larger than other types of nanoparticles. We show that when proteins of interest are fused to specific axonemal proteins and expressed in living Chlamydomonas reinhardtii cells, they become incorporated into linear arrays, which have the advantages of high protein loading capacity and single-step purification with retention of biomass. The arrays can be isolated as membrane-enclosed vesicles or as exposed protein arrays. The approach is demonstrated for both a fluorescent protein and an enzyme (beta-lactamase), showing that incorporation into axonemes retains protein function in a stable, easily isolated array form.

A simple method to generate human airway epithelial organoids with externally orientated apical membranes.

Organoids, which are self-organizing three-dimensional cultures, provide models that replicate specific cellular components of native tissues or facets of organ complexity. We describe a simple method to generate organoid cultures using isolated human tracheobronchial epithelial cells grown in mixed matrix components and supplemented at day 14 with the Wnt pathway agonist R-spondin 2 (RSPO2) and the bone morphogenic protein antagonist Noggin. In contrast to previous reports, our method produces differentiated tracheobronchospheres with externally orientated apical membranes without pretreatments, providing an epithelial model to study cilia formation and function, disease pathogenesis, and interaction of pathogens with the respiratory mucosa. Starting from 3 × 105 cells, organoid yield at day 28 was 1,720 ± 302. Immunocytochemistry confirmed the cellular localization of airway epithelial markers, including CFTR, Na+/K+ ATPase, acetylated-α-tubulin, E-cadherin, and ZO-1. Compared to native tissues, expression of genes related to bronchial differentiation and ion transport were similar in organoid and air-liquid interface (ALI) cultures. In matched primary cultures, mean organoid cilia length was 6.1 ± 0.2 µm, similar to that of 5.7 ± 0.1 µm in ALI cultures, and ciliary beating was vigorous and coordinated with frequencies of 7.7 ± 0.3 Hz in organoid cultures and 5.3 ± 0.8 Hz in ALI cultures. Functional measurement of osmotically induced volume changes in organoids showed low water permeability. The generation of numerous single testable units from minimal starting material complements prior techniques. This culture system may be useful for studying airway biology and pathophysiology, aiding diagnosis of ciliopathies, and potentially for high-throughput drug screening.