Multicellular Colonies Research Articles

Growth-mediated autochemotactic pattern formation in self-propelling bacteria.

Bacteria, while developing a multicellular colony or biofilm, can undergo pattern formation by diverse intricate mechanisms. One such route is directional movement or chemotaxis toward or away from self-secreted or externally employed chemicals. In some bacteria, the self-produced signaling chemicals or autoinducers themselves act as chemoattractants or chemorepellents and thereby regulate the directional movements of the cells in the colony. In addition, bacteria follow a certain growth kinetics which is integrated in the process of colony development. Here, we study the interplay of bacterial growth dynamics, cell motility, and autochemotactic motion with respect to the self-secreted diffusive signaling chemicals in spatial pattern formation. Using a continuum model of motile bacteria, we show growth can act as a crucial tuning parameter in determining the spatiotemporal dynamics of a colony. In action of growth dynamics, while chemoattraction toward autoinducers creates arrested phase separation, pattern transitions and suppression can occur for a fixed chemorepulsive strength.

Analysis of motility in multicellular Chlamydomonas reinhardtii evolved under predation.

The advent of multicellularity was a watershed event in the history of life, yet the transition from unicellularity to multicellularity is not well understood. Multicellularity opens up opportunities for innovations in intercellular communication, cooperation, and specialization, which can provide selective advantages under certain ecological conditions. The unicellular alga Chlamydomonas reinhardtii has never had a multicellular ancestor yet it is closely related to the volvocine algae, a clade containing taxa that range from simple unicells to large, specialized multicellular colonies. Simple multicellular structures have been observed to evolve in C. reinhardtii in response to predation or to settling rate-based selection. Structures formed in response to predation consist of individual cells confined within a shared transparent extracellular matrix. Evolved isolates form such structures obligately under culture conditions in which their wild type ancestors do not, indicating that newly-evolved multicellularity is heritable. C. reinhardtii is capable of photosynthesis, and possesses an eyespot and two flagella with which it moves towards or away from light in order to optimize input of radiant energy. Motility contributes to C. reinhardtii fitness because it allows cells or colonies to achieve this optimum. Utilizing phototaxis to assay motility, we determined that newly evolved multicellular strains do not exhibit significant directional movement, even though the flagellae of their constituent unicells are present and active. In C. reinhardtii the first steps towards multicellularity in response to predation appear to result in a trade-off between motility and differential survivorship, a trade-off that must be overcome by further genetic change to ensure long-term success of the new multicellular organism.

Role of tumbling in bacterial swarming.

Typical wild-type bacteria swimming in sparse suspensions exhibit a movement pattern called "run and tumble," characterized by straight trajectories (runs) interspersed by shorter, random reorientation (tumbles). This is achieved by rotating their flagella counterclockwise, or clockwise, respectively. The chemotaxis signaling network operates in controlling the frequency of tumbles, enabling navigation toward or away from desired regions in the medium. In contrast, while in dense populations, flagellated bacteria exhibit collective motion and form large dynamic clusters, whirls, and jets, with intricate dynamics that is fundamentally different than trajectories of sparsely swimming cells. Although collectively swarming cells do change direction at the level of the individual cell, often exhibiting reversals, it has been suggested that chemotaxis does not play a role in multicellular colony expansion, but the change in direction stems from clockwise flagellar rotation. In this paper, the effects of cell rotor switching (i.e., the ability to tumble) and chemotaxis on the collective statistics of swarming bacteria are studied experimentally in wild-type Bacillus subtilis and two mutants-one that does not tumble and one that tumbles independently of the chemotaxis system. We show that while several of the parameters examined are similar between the strains, other collective and individual characteristics are significantly different. The results demonstrate that tumbling and/or flagellar directional rotor switching has an important role on the dynamics of swarming, and imply that swarming models of self-propelled rods that do not take tumbling and/or rotor switching into account may be oversimplified.

Evolution of Individuality: A Case Study in the Volvocine Green Algae

While numerous criteria have been proposed in definitions of biological individuality, it is not clear whether these criteria reflect the evolutionary processes that underlie transitions in individuality. We consider the evolution of individuality during the transition from unicellular to multicellular life. We assume that “individuality” (however it is defined) has changed in the volvocine green algae lineage during the transition from single cells, to simple multicellular colonies with four to one hundred cells, to more complex multicellular organisms with thousands of differentiated cells. We map traits associated with the various proposed individuality criteria onto volvocine algae species thought to be similar to ancestral forms arising during this transition in individuality. We find that the fulfillment of some criteria, such as genetic homogeneity and genetic uniqueness, do not change across species, while traits underpinning other aspects of individuality, including degrees of integration, group-level fitness and adaptation, and group indivisibility, change dramatically. We observe that different kinds of individuals likely exist at different levels of organization (cell and group) in the same species of algae. Future research should focus on the causes and consequences of variation in individuality.

Finding patches in a heterogeneous aquatic environment: pH‐taxis by the dispersal stage of choanoflagellates

AbstractMicrobial eukaryotes that feed on bacteria are critical links in aquatic food webs. We used the choanoflagellate Salpingoeca rosetta, whose life history includes fast‐ and slow‐swimming unicellular forms and multicellular colonies, to study performance of these different forms at finding and accumulating in patches of prey. Video microscopy of behavior in microfluidic experiments showed that only unicellular fast swimmers exhibited taxis into patches of attractant. Of the chemical cues tested, only pH affected behavior: fast swimmers moved toward patches of water that had pH in the range of 6–7 from water with higher or lower pH. Since bacteria can lower the pH of the water in their vicinity, we suggest that seeking regions with a moderately more acidic pH than average seawater helps the fast‐swimming dispersal stage of choanoflagellates find particles or surfaces rich in prey.

Multiscale microenvironmental perturbation of pluripotent stem cell fate and self-organization

The combination of microfluidics with engineered three-dimensional (3D) matrices can bring new insights into the fate regulation of stem cells and their self-organization into organoids. Although there has been progress in 3D stem cell culturing, most existing in vitro methodologies do not allow for mimicking of the spatiotemporal heterogeneity of stimuli that drive morphogenetic processes in vivo. To address this, we present a perfusion-free microchip concept for the in vitro 3D perturbation of stem cell fate. Stem cells are encapsulated in a hydrogel compartment that is flanked by open reservoirs for the diffusion-driven generation of biomolecule gradients. Juxtaposing additional compartments bearing supportive cells enables investigating the influence of long range cell-cell communication. We explore the utility of the microchips in manipulating early fate choices and self-organizing characteristics of 3D-cultured mouse embryonic stem cells (mESCs) under neural differentiation conditions and exposure to gradients of leukemia inhibitory factor (LIF). mESCs respond to LIF gradients in a spatially dependent manner. At higher LIF concentrations, multicellular colonies maintain pluripotency in contrast, at lower concentrations, mESCs develop into apicobasally polarized epithelial cysts. This versatile system can help to systematically explore the role of multifactorial microenvironments in promoting self-patterning of various stem cell types.

In vivo modified organic matrix for testing biomineralization-related protein functions in differentiated Dictyostelium on calcite.

This work reports an in vivo approach for identifying the function of biomineralization-related proteins. Synthetic sequences of n16N, OC-17 and perlucin with signal peptides are produced in a novel Gateway expression system for Dictyostelium under the control of the [ecmB] promoter. A fast and easy scanning electron microscopic screening method was used to differentiate on the colony level between interplay effects of the proteins expressed in the extracellular matrix (ECM). Transformed Dictyostelium, which migrated as multicellular colonies on calcite crystals and left their ECM remnants on the surface were investigated also by energy-dispersive X-ray spectroscopy (EDX). Calcium minerals with and without phosphorous accumulated very frequently within the matrix of the Dictyostelium colonies when grown on calcite. Magnesium containing phosphorous granules were observed when colonies were exposed on silica. The absence of calcium EDX signals in these cases suggests that the external calcite crystals but not living cells represent the major source of calcium in the ECM. Several features of the system provide first evidence that each protein influences the properties of the matrix in a characteristic mode. Colonies transformed with perlucin produced a matrix with cracks on the length scale of a few microns throughout the matrix patch. For colonies with OC-17, almost no cracks were observed, regardless of the length scale. The non-transformed Dictyostelium (Ax3-Orf+) produced larger cracks. The strategy presented here develops the first step toward an efficient eukaryotic screening system for the combinatorial functionalization of materials by bioengineering in close analogy to natural biomineralization concepts.

Generalized squirming motion of a sphere

A number of swimming microorganisms, such as ciliates (Opalina) and multicellular colonies of flagellates (Volvox), are approximately spherical in shape and swim using beating arrays of cilia or short flagella covering their surfaces. Their physical actuation on the fluid may be mathematically modeled as the generation of surface velocities on a continuous spherical surface—a model known in the literature as squirming, which has been used to address various aspects of the biological physics of locomotion. Previous analyses of squirming assumed axisymmetric fluid motion and hence required all swimming kinematics to take place along a line. In this paper we generalize squirming to three spatial dimensions. We derive analytically the flow field surrounding a spherical squirmer with arbitrary surface motion and use it to derive its three-dimensional translational and rotational swimming kinematics. We then use our results to physically interpret the flow field induced by the swimmer in terms of fundamental flow singularities up to terms decaying spatially as \({\sim } 1/r^3\). Our results will make it possible to develop new models in biological physics, in particular in the area of hydrodynamic interactions and collective locomotion.

A massively parallel microfluidic device for long-term visualization of isolated motile cells

Visualizing the natural behavior of motile cells over many hours is a challenge, as cells can leave the field of view of a microscope in a matter of minutes. Many interesting cell behaviors—such as cell division, motility phenotype, cell–cell interactions, and multicellular colony formation—require hours of observation to characterize. We present a microfluidic device that traps hundreds of single motile cells in isolated chambers, thereby allowing observation over several days. This polydimethylsiloxane device features 400 circular chambers, connected to a central serpentine channel. Motile cells are loaded into these chambers through the serpentine channel. The channel is then purged with air, fluidically isolating the chambers from each other and effectively trapping the cells. We applied the device to observe the behavior of the choanoflagellate Salpingoeca rosetta. Because of its ability to live in both solitary and colonial forms, S. rosetta is a useful model organism for the study of the evolutionary origins of multicellularity. In particular, S. rosetta can take on two distinct colonial forms: chain colonies and rosette colonies. With our device, we are able to observe the formation of these colonies from single cells more easily and with higher throughput than ever before. This device has the potential to be a powerful tool for studying the long-term behavior of motile cells.

Aging and differentiation in yeast populations: elders with different properties and functions

Over the past decade, it has become evident that similarly to cells forming metazoan tissues, yeast cells have the ability to differentiate and form specialized cell types. Examples of yeast cellular differentiation have been identified both in yeast liquid cultures and within multicellular structures occupying solid surfaces. Most current knowledge on different cell types comes from studies of the spatiotemporal internal architecture of colonies developing on various media. With a few exceptions, yeast cell differentiation often concerns nongrowing, stationary-phase cells and leads to the formation of cell subpopulations differing in stress resistance, cell metabolism, respiration, ROS production, and others. These differences can affect longevity of particular subpopulations. In contrast to liquid cultures, where various cell types are dispersed within stationary-phase populations, cellular differentiation depends on the specific position of particular cells within multicellular colonies. Differentiated colonies, thus, resemble primitive multicellular organisms, in which the gradients of certain compounds and the position of cells within the structure affect cellular differentiation. In this review, we summarize and compare the properties of diverse types of differentiated chronologically aging yeast cells that have been identified in colonies growing on different media, as well as of those found in liquid cultures.

The antibiotic planosporicin coordinates its own production in the actinomycete Planomonospora alba

Planosporicin is a ribosomally synthesized, posttranslationally modified peptide lantibiotic produced by the actinomycete Planomonospora alba. It contains one methyl-lanthionine and four lanthionine bridges and inhibits cell wall biosynthesis in other Gram-positive bacteria probably by binding to lipid II, the immediate precursor for cell wall biosynthesis. Planosporicin production, which is encoded by a cluster of 15 genes, is confined to stationary phase in liquid culture and to the onset of morphological differentiation when P. alba is grown on agar. This growth phase-dependent gene expression is controlled transcriptionally by three pathway-specific regulatory proteins: an extracytoplasmic function σ factor (PspX), its cognate anti-σ factor (PspW), and a transcriptional activator (PspR) with a C-terminal helix-turn-helix DNA-binding domain. Using mutational analysis, S1 nuclease mapping, quantitative RT-PCR, and transcriptional fusions, we have determined the direct regulatory dependencies within the planosporicin gene cluster and present a model in which subinhibitory concentrations of the lantibiotic function in a feed-forward mechanism to elicit high levels of planosporicin production. We show that in addition to acting as an antibiotic, planosporicin can function as an extracellular signaling molecule to elicit precocious production of the lantibiotic, presumably ensuring synchronous and concerted lantibiotic biosynthesis in the wider population and, thus, the production of ecologically effective concentrations of the antibiotic.

Cooperatively Generated Stresslet Flows Supply Fresh Fluid to Multicellular Choanoflagellate Colonies

The flagellated protozoan Salpingoeca rosetta is one of the closest relatives of multicellular animals. Unicellular S. rosetta can be induced to form multicellular colonies, but colonies swim more slowly than individual cells so the advantages conferred by colony formation are uncertain. Here we use theoretical models to show that hydrodynamic cooperation between cells can increase the fluid supply to the colony, an important predictor of feeding rate. Our results suggest that hydrodynamic benefits may have been an important selective factor in the evolution of early multicellular animals.

A General Allometric and Life-History Model for Cellular Differentiation in the Transition to Multicellularity

The transition from unicellular, to colonial, to larger multicellular organisms has benefits, costs, and requirements. Here we present a model inspired by the volvocine green algae that explains the dynamics involved in the unicellular-multicellular transition using life-history theory and allometry. We model the two fitness components (fecundity and viability) and compare the fitness of hypothetical colonies of different sizes with varying degrees of cellular differentiation to understand the general principles that underlie the evolution of multicellularity. We argue that germ-soma separation may have evolved to counteract the increasing costs and requirements of larger multicellular colonies. The model shows that the cost of investing in soma decreases with size. For lineages such as the Volvocales, as reproduction costs increase with size for undifferentiated colonies, soma specialization benefits the colony indirectly by decreasing such costs and directly by helping reproductive cells acquire resources for their metabolic needs. Germ specialization is favored once soma evolves and takes care of vegetative functions. To illustrate the model, we use some allometric relationships measured in Volvocales. Our analysis shows that the cost of reproducing an increasingly larger group has likely played an important role in the transition to multicellularity and cellular differentiation.

Aging and longevity of yeast colony populations: metabolic adaptation and differentiation

Yeast multicellular colonies possess several traits that are absent from individual yeasts. These include the ability to synchronize colony population development and adapt its metabolism to different environmental changes, such as nutrient depletion. This, together with cell diversification to cell variants with distinct metabolic and other properties, contributes to the main goal of the colony population: to achieve longevity. In this respect, a benefit to individual cells is subordinated to the benefit to the whole population, exhibiting a kind of altruistic behaviour. For example, some colony cells located at particular positions undergo regulated cell dying and provide components to other cells located in more propitious areas. The enhancement of techniques that enable the in vivo investigation of three-dimensional spatiotemporal colony development may lead to new discoveries on metabolic differentiation and regulation in the near future.

Multicellular development in a choanoflagellate

Summary Little is known about how the first animals evolved from their single-celled ancestors. Over 120 years ago, Ernst Haeckel proposed that animals evolved through repeated self-division of [a] primary cell, [1] an idea supported by the observation that all animals develop from a single cell (the zygote) through successive rounds of cell division [2]. Nonetheless, there are multiple alternative hypotheses [3], including the formal possibility that multicellularity in the progenitor of animals occurred through cell aggregation, with embryogenesis by cell division being secondarily derived. The closest known relatives of animals, choanoflagellates, are emerging as a model system for testing specific hypotheses about animal origins [4–6]. Studying colony formation in choanoflagellates may provide a context for reconstructing the evolution of animal multicellularity. Here, we find that the transition from single cells to multicellular colonies in the choanoflagellate Salpingoeca rosetta (previously known as Proterospongia sp.) occurs by cell division, with sister cells remaining stably attached.

Modeling metastasis biology and therapy in real time in the mouse lung

Pulmonary metastasis remains the leading ca use of death for cancer patients. Opportunities to improve treatment outcomes for patients require new methods to study and view the biology of metastatic progression. Here, we describe an ex vivo pulmonary metastasis assay (PuMA) in which the metastatic progression of GFP-expressing cancer cells, from a single cell to the formation of multicellular colonies, in the mouse lung microenvironment was assessed in real time for up to 21 days. The biological validity of this assay was confirmed by its prediction of the in vivo behavior of a variety of high- and low-metastatic human and mouse cancer cell lines and the discrimination of tumor microenvironments in the lung that were most permissive to metastasis. Using this approach, we provide what we believe to be new insights into the importance of tumor cell interactions with the stromal components of the lung microenvironment. Finally, the translational utility of this assay was demonstrated through its use in the evaluation of therapeutics at discrete time points during metastatic progression. We believe that this assay system is uniquely capable of advancing our understanding of both metastasis biology and therapeutic strategies.

Generation and Characterization of Embryonic Stem‐Like Cell Lines Derived from In Vitro Fertilization Buffalo (Bubalus bubalis) Embryos

In the present study, buffalo embryonic stem-like (ES-like) cell lines were successfully isolated, cultured and characterized. From a total of 92 normal buffalo embryos obtained by in vitro fertilization, 18 were morulae, 33 were blastocyst and 41 were hatched blastocyst, the inside of morulae or inner cell masses of blastocysts were isolated mechanically and cultured onto mitomocin-C-inactivated buffalo embryonic fibroblasts as feeder layers. Alkaline phosphatase (AP) of ES-like cells, as well as the specific stage embryonic antigen SSEA-1, SSEA-3, SSEA-4 and transcription factor OCT-4, was used to evaluate the characterization of the cells. The spontaneous differentiation of ES-like cells was induced by culturing on leukaemia inhibitory factor-free medium for more than 2 weeks without passage. To evaluate mark gene expression, total RNA was extracted from cells, and specific primers were used for reverse transcriptase-polymerase chain reaction (RT-PCR). After 8-10 days of culture, primary ES-like cell colonies were formed in 0% (0/18) of morulae, 24.24% (8/33) of blastocysts and 60.98% (25/41) of hatched blastocysts, respectively. The forming rate of primary ES-like cells colonies in hatched blastocyst group was significantly (p < 0.05) higher than the obtained for other groups. Two ES-like cell lines could survive to eight passages at least by using the method of mechanical dissociation, but just three passages by using the method of enzymatic dissociation. The cells formed large, multicellular colonies with distinct boundaries, exhibited many important features of ES/ES-like cells, including positive AP, SSEA-1, SSEA-3 and SSEA-4 activity. Undifferentiated buffalo ES-like cells expressed Oct-4, Nanog, Sox2 gene mRNA. In vitro differentiation experiments had demonstrated that those cells were pluripotent.

Bacillus subtilis spreads by surfing on waves of surfactant

The bacterium Bacillus subtilis produces the molecule surfactin, which is known to enhance the spreading of multicellular colonies on nutrient substrates by lowering the surface tension of the surrounding fluid, and to aid in the formation of aerial structures. Here we present experiments and a mathematical model that demonstrate how the differential accumulation rates induced by the geometry of the bacterial film give rise to surfactant waves. The spreading flux increases with increasing biofilm viscosity. Community associations are known to protect bacterial populations from environmental challenges such as predation, heat, or chemical stresses, and enable digestion of a broader range of nutritive sources. This study provides evidence of enhanced dispersal through cooperative motility, and points to nonintuitive methods for controlling the spread of biofilms.

DNA-triggered innate immune responses are propagated by gap junction communication

Cells respond to infection by sensing pathogens and communicating danger signals to noninfected neighbors; however, little is known about this complex spatiotemporal process. Here we show that activation of the innate immune system by double-stranded DNA (dsDNA) triggers intercellular communication through a gap junction-dependent signaling pathway, recruiting colonies of cells to collectively secrete antiviral and inflammatory cytokines for the propagation of danger signals across the tissue at large. By using live-cell imaging of a stable IRF3-sensitive GFP reporter, we demonstrate that dsDNA sensing leads to multicellular colonies of IRF3-activated cells that express the majority of secreted cytokines, including IFNbeta and TNFalpha. Inhibiting gap junctions decreases dsDNA-induced IRF3 activation, cytokine production, and the resulting tissue-wide antiviral state, indicating that this immune response propagation pathway lies upstream of the paracrine action of secreted cytokines and may represent a host-derived mechanism for evading viral antiinterferon strategies.

LOOKING FOR EVIDENCE OF DIFFERENTIATION AND COOPERATION: NATURAL MEASURES FOR THE STUDY OF EVOLUTION OF MULTICELLULARITY

The understanding of the evolutionary transitions is a major area of research in artificial life and in biology. We follow an artificial life approach to investigate these phenomena, using a system inspired by Anabaena cyanobacteria (which exhibit rudimentary multicellular differentiation and cooperation) in order to look for evidence of emerging differentiation and multicellular cooperation in colonies of individual cells.We first evolve single free-living cells with the help of a Genetic Algorithm (GA). These cells are controlled with genetic regulatory networks. The single cells are evolved to each perform both of two tasks: an abstraction of house-keeping metabolism and a reproductive cycle. Once such a cell was evolved with the GA, the cell is used to seed the growth of a multicellular filamentous colony, whose constituent cells continue to reproduce and evolve. Two types of colonies generated from the seed cell are studied: one with intercellular communication ability and one without.We introduce and apply new measures for assessing the impact of multicellular interaction on individual reproduction and on life span.The conclusion of these studies shows that the colony with the ability to communicate shows, with the help of our new measures, behaviors that hint at the emergence of early cooperation.