Search

The axoneme-the conserved core of eukaryotic cilia and flagella-contains highly specialized doublet microtubules (DMTs). A long-standing question is what protein(s) compose the junctions between two tubules in DMT. Here we identify a highly conserved flage

Morphogenesis and pattern formation are vital processes in any organism, whether unicellular or multicellular. But in contrast to the developmental biology of plants and animals, the principles of morphogenesis and pattern formation in single cells remain

The dynamics by which homologous chromosomes pair is currently unknown. Here, we use fluorescence in situ hybridization in combination with three-dimensional optical microscopy to show that homologous pairing of the somatic chromosome arm 2L in Drosophila occurs by independent initiation of pairing at discrete loci rather than by a processive zippering of sites along the length of chromosome. By evaluating the pairing frequencies of 11 loci on chromosome arm 2L over several timepoints during Drosophila embryonic development, we show that all 11 loci are paired very early in Drosophila development, within 13 h after egg deposition. To elucidate whether such pairing occurs by directed or undirected motion, we analyzed the pairing kinetics of histone loci during nuclear cycle 14. By measuring changes of nuclear length and correlating these changes with progression of time during cycle 14, we were able to express the pairing frequency and distance between homologous loci as a function of time. Comparing the experimentally determined dynamics of pairing to simulations based on previously proposed models of pairing motion, we show that the observed pairing kinetics are most consistent with a constrained random walk model and not consistent with a directed motion model. Thus, we conclude that simple random contacts through diffusion could suffice to allow pairing of homologous sites.

We have analyzed the progressive changes in the spatial distribution of telomeres during meiosis using three-dimensional, high resolution fluorescence microscopy. Fixed meiotic cells of maize (Zea mays L.) were subjected to in situ hybridization under conditions that preserved chromosome structure, allowing identification of stage-dependent changes in telomere arrangements. We found that nuclei at the last somatic prophase before meiosis exhibit a nonrandom, polarized chromosome organization resulting in a loose grouping of telomeres. Quantitative measurements on the spatial arrangements of telomeres revealed that, as cells passed through premeiotic interphase and into leptotene, there was an increase in the frequency of large telomere-to-telomere distances and a decrease in the bias toward peripheral localization of telomeres. By leptotene, there was no obvious evidence of telomere grouping, and the large, singular nucleolus was internally located, nearly concentric with the nucleus. At the end of leptotene, telomeres clustered de novo at the nuclear periphery, coincident with a displacement of the nucleolus to one side. The telomere cluster persisted throughout zygotene and into early pachytene. The nucleolus was adjacent to the cluster at zygotene. At the pachytene stage, telomeres rearranged again by dispersing throughout the nuclear periphery. The stage-dependent changes in telomere arrangements are suggestive of specific, active telomere-associated motility processes with meiotic functions. Thus, the formation of the cluster itself is an early event in the nuclear reorganizations associated with meiosis and may reflect a control point in the initiation of synapsis or crossing over.

We developed a computerized medical informatics tool to identify patients who had a culture performed on a sterile body site specimen during their hospitalization that subsequently turned positive after hospital dismissal. During a 13-month period, 533 patients had a positive culture identified by our Computer-Based Antimicrobial Monitoring (CBAM) program after hospital dismissal, and 112 (21%) of these culture results necessitated an intervention and communication with the primary health care professional. Thirty-two (29%) of positive cultures were from the blood. Thirty-eight (34%) of the CBAM interventions with available outcome data resulted in initiation of, change in, or prolongation of outpatient antimicrobial therapy. The CBAM program serves an important role in optimizing patient care and communication with the health care professional during the transition from inpatient to outpatient management. [ABSTRACT FROM AUTHOR]

The single-celled ciliate Stentor coeruleus demonstrates habituation to mechanical stimuli, showing that even single cells can manifest a basic form of learning. Although the ability of Stentor to habituate has been extensively documented, the mechanism of learning is currently not known. Here we take a bottom-up approach and investigate a simple biochemistry-based model based on prior electrophysiological measurements in Stentor along with general properties of receptor molecules. In this model, a mechanoreceptor senses the stimulus and leads to channel opening to change membrane potential, with a sufficient change in polarization triggering an action potential that drives contraction. Receptors that are activated can become internalized, after which they can either be degraded or recycled back to the cell surface. This activity-dependent internalization provides a potential means for the cell to learn. Stochastic simulations of this model confirm that it is capable of showing habituation similar to what is seen in actual Stentor cells, including the lack of dishabituation by strong stimuli and the apparently step-like response of individual cells during habituation. The model also can account for several habituation hallmarks that a previous two-state Markov model could not, namely, the dependence of habituation rate on stimulus magnitude, which had to be added onto the two state model but arises naturally in the receptor inactivation model; the rate of response recovery after cessation of stimulation; the ability of high frequency stimulus sequences to drive faster habituation that results in a lower response probability, and the potentiation of habituation by repeated rounds of training and recovery. The model makes the prediction that application of high force stimuli that do not normally habituate should drive habituation to weaker stimuli due to decrease in the receptor number, which serves as an internal hidden variable. We confirmed this prediction using two new sets of experiments involving alternation of weak and strong stimuli. Furthermore, the model predicts that training with high force stimuli delays response recovery to low force stimuli, which aligns with our new experimental data. The model also predicts subliminal accumulation, wherein continuation of training even after habituation has reached asymptotic levels should lead to delayed response recovery, which was also confirmed by new experiments. The model is unable to account for the phenomenon of rate sensitivity, in which habituation caused by higher frequency stimuli is more easily reversed leading to a frequency dependence of response recovery. Such rate sensitivity has not been reported in Stentor . Here we carried out a new set of experiments which are consistent with the model's prediction of the lack of rate sensitivity. This work demonstrates how a simple model can suggest new ways to probe single-cell learning at an experimental level. Finally, we interpret the model in terms of a kernel estimator that the cell may use to guide its decisions about how to response to new stimuli as they arise based on information, or the lack thereof, from past stimuli.

Spirostomum is a unicellular ciliate capable of contracting to a quarter of its body length in less than five milliseconds. When measured as fractional shortening, this is an order of magnitude faster than motion powered by actomyosin. Myonemes, which are protein networks found near the cortex of many protists, are believed to power Spirostomum contraction. Fast contraction, slow elongation, and calcium-triggering are hallmarks of myoneme-based motion. The biochemical basis of this motion and the molecular mechanism that supports such fast speeds are not well understood. Previous work suggests that myoneme structures in some protists are rich in centrin and Sfi1 homologs, two proteins that may underlie contraction. Centrin undergoes a significant conformational change in the presence of calcium, allowing it to bind to other centrin molecules. To understand Spirostomum contraction, we measure changes in cortical structures and model contraction of the whole cell and of the underlying protein complexes. We provide evidence that centrin/Sfi1 structures are responsible for contraction, which we propose is powered by a modulated entropic spring. Using this model, we recapitulate organismal-scale contraction in mesh simulation experiments and demonstrate the importance of structural organization of myoneme in a fishnet-like structure. These results provide a cohesive, multiscale model for the contraction of Spirostomum . Deeper understanding of how single cells can execute extreme shape changes holds potential for advancing cell biophysics, synthetically engineering contractile machinery, and cellular-inspired engineering designs.

Soft X-ray tomography (SXT) is an invaluable tool for quantitatively analyzing cellular structures at sub-optical isotropic resolution. However, it has traditionally depended on manual segmentation, limiting its scalability for large datasets. Here, we leverage a deep learning-based auto-segmentation pipeline to segment and label cellular structures in hundreds of cells across three Saccharomyces cerevisiae strains. This task-based pipeline employs manual iterative refinement to improve segmentation accuracy for key structures, including the cell body, nucleus, vacuole, and lipid droplets, enabling high-throughput and precise phenotypic analysis. Using this approach, we quantitatively compared the 3D whole-cell morphometric characteristics of wild-type, VPH1-GFP, and vac14 strains, uncovering detailed strain-specific cell and organelle size and shape variations. We show the utility of SXT data for precise 3D curvature analysis of entire organelles and cells and detection of fine morphological features using surface meshes. Our approach facilitates comparative analyses with high spatial precision and statistical throughput, uncovering subtle morphological features at the single cell and population level. This workflow significantly enhances our ability to characterize cell anatomy and supports scalable studies on the mesoscale, with applications in investigating cellular architecture, organelle biology, and genetic research across diverse biological contexts., Significance Statement: Soft X-ray tomography offers many powerful features for whole-cell multi-organelle imaging, but, like other high resolution volumetric imaging modalities, is typically limited by low throughput due to laborious segmentation.Auto-segmentation for soft X-ray tomography overcomes this limitation, enabling statistical 3D morphometric analysis of multiple organelles in whole cells across cell populations. The combination of high 3D resolution of SXT data with statistically useful throughput represents an avenue for more thorough characterizations of cells in toto and opens new mesoscale biological questions and statistical whole-cell modeling of organelle and cell morphology, interactions, and responses to perturbations.

Regeneration is the remarkable phenomenon through which an organism can regrow lost or damaged parts with fully functional replacements, including complex anatomical structures, such as limbs. In 2019, Development launched its 'Model systems for regeneration' collection, a series of articles introducing some of the most popular model organisms for studying regeneration in vivo. To expand this topic further, this Perspective conveys the voices of five expert biologists from the field of regenerative biology, each of whom showcases some less well-known, but equally extraordinary, species for studying regeneration., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Cells have complex and beautiful structures that are important for their function. However, 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 have 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 membranellar band, always form in particular 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 regionalized mRNA is often used to mark position, we asked whether specific regionalized mRNAs might mark position along the anterior-posterior axis of Stentor . By physically bisecting cells and conducting bulk RNA sequencing, we were able to identify sets of messages enriched in either the anterior or posterior half. We then conducted half-cell RNA-sequencing in paired anteriors and posteriors of cells in which the microtubule cytoskeleton was disrupted by RNAi of β-tubulin or dynein intermediate chains. 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., Competing Interests: Declaration of Competing Interests The authors declare that they have no competing interests.

The study of evolution at the cellular level traditionally has focused on the evolution of metabolic pathways, endomembrane systems, and genomes, but there has been increasing interest in evolution of more complex cellular structures and behaviors, particularly in the eukaryotes. Ciliates have major advantages for such studies due to their easily visible surface patterning and their dramatic and complex behaviors that can be easily analyzed. Among the ciliates, the genus Stentor epitomizes the features that are useful for studying evolution: they are widespread in freshwater environments, easy to visualize because of their large size, and capable of complex behaviors such as learning, decision-making, and phototaxis. Here, we introduce the discovery of a new species within this genus: Stentor stipatus , so named for their distinctive dark brown aggregates. We present morphological, phylogenetic, ecological, and behavioral characterizations of these cells. The S. stipatus clade has a bootstrap value of 93 and is phylogenetically distinct from S. amethystinus , the closest related species which shares a sequence similarity of 98.9%. S. stipatus is capable of phototaxis and can also habituate more quickly than S. coeruleus , the Stentor species in which most habituation studies have previously been conducted. These findings expand our understanding of Stentor species diversity, natural history, and demonstrate common principles of complex behavior that are present in single-celled organisms.

The unicellular green alga, Chlamydomonas reinhardti i, 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., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Marshall.)

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., (Copyright © 2024 Cold Spring Harbor Laboratory Press; all rights reserved.)

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., Competing Interests: Competing interests statement:The authors declare no competing interest.

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., (Creative Commons Attribution license.)

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., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

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.

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 alga Chlamydomonas provide 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., (Creative Commons Attribution license.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 Ca 2+ channels along the flagellum and that the Ca 2+ current from the flagellum into the cell body increases linearly with flagellar length. In this model, the cell uses the Ca 2+ 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 Ca 2+ inside of Chlamydomonas flagella and quantified IFT injection. Although the concentration of Ca 2+ 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 Ca 2+ spikes. Thus, Ca 2+ does not appear to function as a negative regulator of IFT injection, hence it cannot form the basis of a stable length control system., Competing Interests: HI, JM, DD, MD, WM No competing interests declared, (© 2023, Ishikawa et al.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: PS, AL, CY, RM, UD, TM, AN, ST, WM No competing interests declared, (© 2022, Sood et al.)

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., Competing Interests: The authors have declared that no competing interests exist.

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 fractionation 7-11 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 yeast 12-15 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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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 × 10 5 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.

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., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Marshall.)

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds of genes-many not associated with the oral apparatus (OA)-that are differentially regulated in phases throughout the regeneration process. It was hypothesized that this systemic reorganization and mobilization of cellular resources towards growth of a new OA will lead to observable changes in movement and behavior corresponding in time to the phases of differential gene expression. However, the morphological complexity of S. coeruleus necessitated the development of an assay to capture the statistics and timescale. A custom script was used to track cells in short videos, and statistics were compiled over a large population (N ~100). Upon loss of the OA, S. coeruleus initially loses the ability for directed motion; then starting at ~4 h, it exhibits a significant drop in speed until ~8 h. This assay provides a useful tool for the screening of motility phenotypes and can be adapted for the investigation of other organisms.

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 μm 2 /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.

Any proposed mechanism for organelle size control should be able to account not only for average size but also for the variation in size. We analyzed cell-to-cell variation and within-cell variation of length for the two flagella in Chlamydomonas , finding that cell-to-cell variation is dominated by cell size, whereas within-cell variation results from dynamic fluctuations. Fluctuation analysis suggests tubulin assembly is not directly coupled with intraflagellar transport (IFT) and that the observed length fluctuations reflect tubulin assembly and disassembly events involving large numbers of tubulin dimers. Length variation is increased in long-flagella mutants, an effect consistent with theoretical models for flagellar length regulation. Cells with unequal flagellar lengths show impaired swimming but improved gliding, raising the possibility that cells have evolved mechanisms to tune biological noise in flagellar length. Analysis of noise at the level of organelle size provides a way to probe the mechanisms determining cell geometry., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Cells in culture display diverse motility behaviors that may reflect differences in cell state and function, providing motivation to discriminate between different motility behaviors. Current methods to do so rely upon manual feature engineering. However, the types of features necessary to distinguish between motility behaviors can vary greatly depending on the biological context, and it is not always clear which features may be most predictive in each setting for distinguishing particular cell types or disease states. Convolutional neural networks (CNNs) are machine learning models allowing for relevant features to be learned directly from spatial data. Similarly, recurrent neural networks (RNNs) are a class of models capable of learning long term temporal dependencies. Given that cell motility is inherently spacio-temporal data, we present an approach utilizing both convolutional and long- short-term memory (LSTM) recurrent neural network units to analyze cell motility data. These RNN models provide accurate classification of simulated motility and experimentally measured motility from multiple cell types, comparable to results achieved with hand-engineered features. The variety of cell motility differences we can detect suggests that the algorithm is generally applicable to additional cell types not analyzed here. RNN autoencoders based on the same architecture are capable of learning motility features in an unsupervised manner and capturing variation between myogenic cells in the latent space. Adapting these RNN models to motility prediction, RNNs are capable of predicting muscle stem cell motility from past tracking data with performance superior to standard motion prediction models. This advance in cell motility prediction may be of practical utility in cell tracking applications.

Objective: To report the Mayo Clinic experience with coronavirus disease 2019 (COVID-19) related to patient outcomes., Methods: We conducted a retrospective chart review of patients with COVID-19 diagnosed between March 1, 2020, and July 31, 2020, at any of the Mayo Clinic sites. We abstracted pertinent comorbid conditions such as age, sex, body mass index, Charlson Comorbidity Index variables, and treatments received. Factors associated with hospitalization and mortality were assessed in univariate and multivariate models., Results: A total of 7891 patients with confirmed COVID-19 infection with research authorization on file received care across the Mayo Clinic sites during the study period. Of these, 7217 patients were adults 18 years or older who were analyzed further. A total of 897 (11.4%) patients required hospitalization, and 354 (4.9%) received care in the intensive care unit (ICU). All hospitalized patients were reviewed by a COVID-19 Treatment Review Panel, and 77.5% (695 of 897) of inpatients received a COVID-19-directed therapy. Overall mortality was 1.2% (94 of 7891), with 7.1% (64 of 897) mortality in hospitalized patients and 11.3% (40 of 354) in patients requiring ICU care., Conclusion: Mayo Clinic outcomes of patients with COVID-19 infection in the ICU, hospital, and community compare favorably with those reported nationally. This likely reflects the impact of interprofessional multidisciplinary team evaluation, effective leveraging of clinical trials and available treatments, deployment of remote monitoring tools, and maintenance of adequate operating capacity to not require surge adjustments. These best practices can help guide other health care systems with the continuing response to the COVID-19 pandemic., (Copyright © 2020 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.)

As cells grow, the size and number of their internal organelles increase in order to keep up with increased metabolic requirements. Abnormal size of organelles is a hallmark of cancer and an important aspect of diagnosis in cytopathology. Most organelles vary in either size or number, or both, as a function of cell size, but the mechanisms that create this variation remain unclear. In some cases, organelle size appears to scale with cell size through processes of relative growth, but in others the size may be set by either active measurement systems or genetic programs that instruct organelle biosynthetic activities to create organelles of a size appropriate to a given cell type.

Flagellar length control in Chlamydomonas is a tractable model system for studying the general question of organelle size regulation. We have previously proposed that the diffusive return of the kinesin motor that powers intraflagellar transport can play a key role in length regulation. Here, we explore how the motor speed and diffusion coefficient for the return of kinesin-2 affect flagellar growth kinetics. We find that the system can exist in two distinct regimes, one dominated by motor speed and one by diffusion coefficient. Depending on length, a flagellum can switch between these regimes. Our results indicate that mutations can affect the length in distinct ways. We discuss our theory's implication for flagellar growth influenced by beating and provide possible explanations for the experimental observation that a beating flagellum is usually longer than its immotile mutant. These results demonstrate how our simple model can suggest explanations for mutant phenotypes., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

In the context of animal or plant development, we tend to think of cells as small, simple, building blocks, such that complex patterns or shapes can only be constructed from large numbers of cells, with cells in different parts of the organism taking on different fates. However, cells themselves are far from simple, and often take on complex shapes with a remarkable degree of intracellular patterning. How do these patterns arise? As in embryogenesis, the development of structure inside a cell can be broken down into a number of basic processes. For each part of the cell, morphogenetic processes create internal structures such as organelles, which might correspond to organs at the level of a whole organism. Given that mechanisms exist to generate parts, patterning processes are required to ensure that the parts are distributed in the correct arrangement relative to the rest of the cell. Such patterning processes make reference to global polarity axes, requiring mechanisms for axiation which, in turn, require processes to break symmetry. These fundamental processes of symmetry breaking, axiation, patterning, and morphogenesis have been extensively studied in developmental biology but less so at the subcellular level. This review will focus on developmental processes that give eukaryotic cells their complex structures, with a focus on cytoskeletal organization in free-living cells, ciliates in particular, in which these processes are most readily apparent., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Murine muscle stem cells (MuSCs) experience a transition from quiescence to activation that is required for regeneration, but it remains unknown if the trajectory and dynamics of activation change with age. Here, we use time-lapse imaging and single cell RNA-seq to measure activation trajectories and rates in young and aged MuSCs. We find that the activation trajectory is conserved in aged cells, and we develop effective machine-learning classifiers for cell age. Using cell-behavior analysis and RNA velocity, we find that activation kinetics are delayed in aged MuSCs, suggesting that changes in stem cell dynamics may contribute to impaired stem cell function with age. Intriguingly, we also find that stem cell activation appears to be a random walk-like process, with frequent reversals, rather than a continuous linear progression. These results support a view of the aged stem cell phenotype as a combination of differences in the location of stable cell states and differences in transition rates between them., Competing Interests: Competing interestsJ.C.K. is now a paid employee of Calico Life Sciences. The authors have no other competing interests to disclose., (© 2020. Published by The Company of Biologists Ltd.)

Cells are complex machines with tremendous potential for applications in medicine and biotechnology. Although much effort has been devoted to engineering the metabolic, genetic, and signaling pathways of cells, methods for systematically engineering the physical structure of cells are less developed. Here we consider how coarse-grained models for cellular geometry at the organelle level can be used to build computer-aided design (CAD) tools for cellular structure.

Cilia and flagella are ideal model organelles in which to study the general question of organelle size control. Flagellar microtubules are steady-state structures whose size is set by the balance of assembly and disassembly. Assembly requires intraflagellar transport (IFT), and measurements of IFT have shown that the rate of entry of IFT particles into the flagellum is a decreasing function of length. It has been proposed that this length dependence of IFT may be the basis for flagellar length control. Here, we test this idea by showing that three different long-flagella mutations in Chlamydomonas all cause increased IFT injection, thus confirming that IFT can influence length control. However, quantitative comparisons with mathematical models suggest that the increase in injection is not sufficient to explain the full increase in length seen in these mutants; hence, some other mechanism may be at work. One alternative mechanism that has been proposed is length-regulated binding of tubulin to the IFT particles. However, we find that the apparent length dependence of tubulin loading that has previously been reported may actually reflect length-dependent organization of IFT trains. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.

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 as Chlamydomonas. 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 ciliate Stentor 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 in Stentor , 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'.

The giant unicellular ciliate Stentor coeruleus can be cut into pieces and each piece will regenerate into a healthy, full-sized individual. The molecular mechanism for how Stentor regenerates is a complete mystery, however, the process of regeneration shows striking similarities to the process of cell division. On a morphological level, the process of creating a second mouth in division or a new oral apparatus in regeneration have the same steps and occur in the same order. On the transcriptional level, genes encoding elements of the cell division and cell cycle regulatory machinery, including Aurora kinases, are differentially expressed during regeneration. This suggests that there may be some common regulatory mechanisms involved in both regeneration and cell division. If the cell cycle machinery really does play a role in regeneration, then inhibition of proteins that regulate the timing of cell division may also affect the timing of regeneration in Stentor. Here we show that two well-characterized Aurora kinase A+B inhibitors that affect the timing of regeneration. ZM447439 slows down regeneration by at least one hour. PF03814735 completely suppresses regeneration until the drug is removed. Here we provide the first direct experimental evidence that Stentor may harness the cell division machinery to regulate the sequential process of regeneration., Competing Interests: Conflict of interest The authors declare no conflicts of interest.