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PubMed1996

Tyrosine phosphorylation in Myxococcus xanthus, a multicellular prokaryote.

Tyrosine phosphorylation is an extremely rare event in prokaryotes, occurring almost exclusively in multicellular eukaryotes. We have identified, for the first time, by the use of antiphosphotyrosine monoclonal antibody and Western blot (immunoblot) analysis, two tyrosine-phosphorylated membrane proteins in the multicellular prokaryote Myxococcus xanthus. The pattern of tyrosine phosphorylation was shown to change during development, indicating a possible role for this regulatory modification during two stages of development, i.e., aggregation and sporulation. Furthermore, the altered pattern of tyrosine phosphorylation observed in a variety of signaling mutants was shown to differ from that observed in the wild type, suggesting further the possible involvement of tyrosine phosphorylation during the development program.

Antibodies, Monoclonal↗
PubMed1998

Multicellular stalk-like structures in Saccharomyces cerevisiae.

Stalk formation is a novel pattern of multicellular organization. Yeast cells which survive UV irradiation form colonies that grow vertically to form very long (0.5 to 3.0 cm) and thin (0.5 to 4 mm in diameter) multicellular structures. We describe the conditions required to obtain these stalk-like structures reproducibly in large numbers. Yeast mutants, mutated for control of cell polarity, developmental processes, UV response, and signal transduction cascades were tested and found capable of forming stalk-like structures. We suggest a model that explains the mechanism of stalk formation by mechanical environmental forces. We show that other microorganisms (Candida albicans, Schizosaccharomyces pombe, and Escherichia coli) also form stalks, suggesting that the ability to produce stalks may be a general property of microorganisms. Diploid yeast stalks sporulate at an elevated frequency, raising the possibility that the physiological role of stalks might be disseminating spores.

Candida albicans↗
PubMed1999

A novel membrane protein influencing cell shape and multicellular swarming of Proteus mirabilis.

Swarming in Proteus mirabilis is characterized by the coordinated surface migration of multicellular rafts of highly elongated, hyperflagellated swarm cells. We describe a transposon mutant, MNS185, that was unable to swarm even though vegetative cells retained normal motility and the ability to differentiate into swarm cells. However, these elongated cells were irregularly curved and had variable diameters, suggesting that the migration defect results from the inability of these deformed swarm cells to align into multicellular rafts. The transposon was inserted at codon 196 of a 228-codon gene that lacks recognizable homologs. Multiple copies of the wild-type gene, called ccmA, for curved cell morphology, restored swarming to the mutant. The 25-kDa CcmA protein is predicted to span the inner membrane twice, with its C-terminal major domain being present in the cytoplasm. Membrane localization was confirmed both by immunoblotting and by electron microscopy of immunogold-labelled sections. Two forms of CcmA were identified for wild-type P. mirabilis; they were full-length integral membrane CcmA1 and N-terminally truncated peripheral membrane CcmA2, both present at approximately 20-fold higher concentrations in swarm cells. Differentiated MNS185 mutant cells contained wild-type levels of the C-terminally truncated versions of both proteins. Elongated cells of a ccmA null mutant were less misshapen than those of MNS185 and were able to swarm, albeit more slowly than wild-type cells. The truncated CcmA proteins may therefore interfere with normal morphogenesis, while the wild-type proteins, which are not essential for swarming, may enhance migration by maintaining the linearity of highly elongated cells. Consistent with this view, overexpression of the ccmA gene caused cells of both Escherichia coli and P. mirabilis to become enlarged and ellipsoidal.

Amino Acid Sequence↗
PubMed2004

Calcium inhibits bap-dependent multicellular behavior in Staphylococcus aureus.

Bap (biofilm-associated protein) is a 254-kDa staphylococcal surface protein implicated in formation of biofilms by staphylococci isolated from chronic mastitis infections. The presence of potential EF-hand motifs in the amino acid sequence of Bap prompted us to investigate the effect of calcium on the multicellular behavior of Bap-expressing staphylococci. We found that addition of millimolar amounts of calcium to the growth media inhibited intercellular adhesion of and biofilm formation by Bap-positive strain V329. Addition of manganese, but not addition of magnesium, also inhibited biofilm formation, whereas bacterial aggregation in liquid media was greatly enhanced by metal-chelating agents. In contrast, calcium or chelating agents had virtually no effect on the aggregation of Bap-deficient strain M556. The biofilm elicited by insertion of bap into the chromosome of a biofilm-negative strain exhibited a similar dependence on the calcium concentration, indicating that the observed calcium inhibition was an inherent property of the Bap-mediated biofilms. Site-directed mutagenesis of two of the putative EF-hand domains resulted in a mutant strain that was capable of forming a biofilm but whose biofilm was not inhibited by calcium. Our results indicate that Bap binds Ca2+ with low affinity and that Ca2+ binding renders the protein noncompetent for biofilm formation and for intercellular adhesion. The fact that calcium inhibition of Bap-mediated multicellular behavior takes place in vitro at concentrations similar to those found in milk serum supports the possibility that this inhibition is relevant to the pathogenesis and/or epidemiology of the bacteria in the mastitis process.

Amino Acid Sequence↗
PubMed2005

The Erwinia chrysanthemi type III secretion system is required for multicellular behavior.

Enterobacterial animal pathogens exhibit aggregative multicellular behavior, which is manifested as pellicles on the culture surface and biofilms at the surface-liquid-air interface. Pellicle formation behavior requires production of extracellular polysaccharide, cellulose, and protein filaments, known as curli. Protein filaments analogous to curli are formed by many protein secretion systems, including the type III secretion system (TTSS). Here, we demonstrate that Erwinia chrysanthemi, which does not carry curli genes, requires the TTSS for pellicle formation. These data support a model where cellulose and generic protein filaments, which consist of either curli or TTSS-secreted proteins, are required for enterobacterial aggregative multicellular behavior. Using this assay, we found that hrpY, which encodes a two-component system response regulator homolog, is required for activity of hrpS, which encodes a sigma54-dependent enhancer-binding protein homolog. In turn, hrpS is required for activity of the sigma factor homolog hrpL, which activates genes encoding TTSS structural and secreted proteins. Pellicle formation was temperature dependent and pellicles did not form at 36 degrees C, even though TTSS genes were expressed at this temperature. We found that cellulose is a component of the E. chrysanthemi pellicle but that pellicle formation still occurs in a strain with an insertion in a cellulose synthase subunit homolog. Since the TTSS, but not the cellulose synthase subunit, is required for E. chrysanthemi pellicle formation, this inexpensive assay can be used as a high throughput screen for TTSS mutants or inhibitors.

Adaptation, Physiological↗
PubMed2004

Gene expression analysis of tumor spheroids reveals a role for suppressed DNA mismatch repair in multicellular resistance to alkylating agents.

Drug resistance is a major obstacle in the successful treatment of cancer. Thus, elucidation of the mechanisms responsible is a critical first step in trying to prevent or delay such manifestations of resistance. In this regard, three-dimensional multicellular tumor cell spheroids are intrinsically more resistant to virtually all anticancer cytotoxic drugs than conventional monolayer cultures. We have employed the EMT-6 subline PC5T, which forms highly compact spheroids, and differential display to identify candidate genes whose expression differs between monolayer and spheroids. Approximately 5,000 bands were analyzed, revealing 26 to be differentially expressed. Analysis of EMT-6 tumor variants selected in vivo for acquired resistance to alkylating agents identified eight genes whose expression correlated with drug resistance in tumor spheroids. Four genes (encoding Nop56, the NADH SDAP subunit, and two novel sequences) were found to be down-regulated in EMT-6 spheroids and four (encoding 2-oxoglutarate carrier protein, JTV-1, and two novel sequences) were up-regulated. Analysis of the DNA mismatch repair-associated PMS2 gene, which overlaps at the genomic level with the JTV-1 gene, revealed PMS2 mRNA to be down-regulated in tumor spheroids, which was confirmed at the protein level. Analysis of PMS2(-/-) mouse embryo fibroblasts confirmed a role for PMS2 in sensitivity to cisplatin, and DNA mismatch repair activity was found to be reduced in EMT-6 spheroids compared to monolayers. Dominant negative PMS2 transfection caused increased resistance to cisplatin in EMT-6 and CHO cells. Our results implicate reduced DNA mismatch repair as a determinant factor of reversible multicellular resistance of tumor cells to alkylating agents.

Alkylating Agents↗
PubMed1975

The development and structure of thick-walled, multicellular, aerial spores in Diheterospora chlamydosporia (=Verticillium chlamydosporium).

The aerial, thick-walled spores in Diheterospara chlamydosporia arose as terminal swellings on erect hyphae. Repeated septation of the continuously swelling spore resulted in a multicellular structure. Immediately after the onset of septation secondary wall material was laid down between the two-layered primary wall and the plasmalemma. The presence of secondary wall material indicates that the multicellular spore is a dictyochlamydospore and not an aleuriospore. The relationship between chlamydospores and aleuriospores in other fungi is discussed.

Cell Membrane↗
PubMed1997

Mitochondrial function in oncogene-transfected rat fibroblasts isolated from multicellular spheroids.

Two mitochondrion-specific fluorochromes, 10-N-nonyl acridine orange (NAO) and rhodamine 123 (Rh123), were used to determine the mechanism responsible for alterations in energy metabolism of transformed rat embryo fibroblast cells isolated from different locations within multicellular spheroids. Accumulation of Rh123 depends on intact mitochondrial membrane potential, whereas NAO is taken up by mitochondria independently of their function and thus represents mitochondrial distribution only. A reproducible selective dissociation procedure was used to isolate cells from different locations within the spheroids. After isolation, cells were simultaneously stained with one mitochondrial stain and the DNA dye Hoechst 33342, and several parameters, including cell volume, were monitored via multilaser-multiparameter flow cytometry. Our data clearly show a decrease in the uptake of Rh123 in cells from the periphery to the inner regions of the tumor spheroids, reflecting a persistent alteration in mitochondrial function. However, NAO staining experiments showed no reduction in the total mitochondrial mass per unit cell volume. Because cells were exposed to stain under uniform conditions after isolation from the spheroid, these data indicate the downregulation of mitochondrial function is associated with cell quiescence rather than a transient effect of reduced nutrient availability. This result, which is in accordance with data from two other cell lines (EMT6 and 9L), might reflect a general phenomenon in multicellular spheroids, supporting the hypothesis that quiescent cells in the innermost viable spheroid layer stably reduce their mitochondrial function, presumably to compensate for lower nutrient supply and/or decreased energy demand.

Acridine Orange↗
PubMed2000

Complex organization in multicellularity as a necessity in evolution.

By introducing a dynamical system model of a multicellular system, it is shown that an organism with a variety of differentiated cell types and a complex pattern emerges through cell-cell interactions even without postulating any elaborate control mechanism. Such an organism is found to maintain a larger growth speed as an ensemble, by achieving a cooperative use of resources, than do simple homogeneous cells, which behave "selfishly." This suggests that the emergence of multicellular organisms with complex organization is a necessity in evolution. According to our theoretical model, there initially appear multipotent stem cells, which undergo stochastic differentiation to other cell types. With development and differentiation, both the chemical diversity and the complexity of intra-cellular dynamics are decreased, as a general consequence of our system. Robustness of the developmental process is also confirmed.

Biological Evolution↗
PubMed2004

The use of 3-D cultures for high-throughput screening: the multicellular spheroid model.

Over the past few years, establishment and adaptation of cell-based assays for drug development and testing has become an important topic in high-throughput screening (HTS). Most new assays are designed to rapidly detect specific cellular effects reflecting action at various targets. However, although more complex than cell-free biochemical test systems, HTS assays using monolayer or suspension cultures still reflect a highly artificial cellular environment and may thus have limited predictive value for the clinical efficacy of a compound. Today's strategies for drug discovery and development, be they hypothesis free or mechanism based, require facile, HTS-amenable test systems that mimic the human tissue environment with increasing accuracy in order to optimize preclinical and preanimal selection of the most active molecules from a large pool of potential effectors, for example, against solid tumors. Indeed, it is recognized that 3-dimensional cell culture systems better reflect the in vivo behavior of most cell types. However, these 3-D test systems have not yet been incorporated into mainstream drug development operations. This article addresses the relevance and potential of 3-D in vitro systems for drug development, with a focus on screening for novel antitumor drugs. Examples of 3-D cell models used in cancer research are given, and the advantages and limitations of these systems of intermediate complexity are discussed in comparison with both 2-D culture and in vivo models. The most commonly used 3-D cell culture systems, multicellular spheroids, are emphasized due to their advantages and potential for rapid development as HTS systems. Thus, multicellular tumor spheroids are an ideal basis for the next step in creating HTS assays, which are predictive of in vivo antitumor efficacy.

Animals↗
PubMed1981

Simple adaptations to extend the range of flow cytometry five orders of magnitude for the DNA analysis of uni-and multicellular systems.

Procedures and instrumentation are described to extend the capability of a cytometry system to record samples that exhibit a wide range of fluorescence such as multicellular systems. The method employs a log amplifier in combination with a set of neutral density filters that reduces the incident light reaching the photomultiplier tube. With any given filter, signals within an intensity range of 200-fold can be measured; different filters can be used to obtain an extended overall range. Polystyrene fluorescent microspheres and a variety of mithramycin stained biological samples ranging from yeast cells to Paramecium were processed by the system. The relative DNA content of individual multicellular embryos was determined for a heterogeneous population of embryonic stages isolated from the nematode, Caenorhabditis elegans. As part of the evaluation of the procedure, the practical upper limit of range extension was determined. The most intense fluorescent signal was produced when untreated pecan pollen stained with ethidium bromide fluoresced with a factor (8.4 +/- 1.3) X 10(4) more than ethidium bromide stained E. coli cells.

Ambystoma↗
PubMed2004

A molecular timescale of eukaryote evolution and the rise of complex multicellular life.

BACKGROUND: The pattern and timing of the rise in complex multicellular life during Earth's history has not been established. Great disparity persists between the pattern suggested by the fossil record and that estimated by molecular clocks, especially for plants, animals, fungi, and the deepest branches of the eukaryote tree. Here, we used all available protein sequence data and molecular clock methods to place constraints on the increase in complexity through time. RESULTS: Our phylogenetic analyses revealed that (i) animals are more closely related to fungi than to plants, (ii) red algae are closer to plants than to animals or fungi, (iii) choanoflagellates are closer to animals than to fungi or plants, (iv) diplomonads, euglenozoans, and alveolates each are basal to plants+animals+fungi, and (v) diplomonads are basal to other eukaryotes (including alveolates and euglenozoans). Divergence times were estimated from global and local clock methods using 20-188 proteins per node, with data treated separately (multigene) and concatenated (supergene). Different time estimation methods yielded similar results (within 5%): vertebrate-arthropod (964 million years ago, Ma), Cnidaria-Bilateria (1,298 Ma), Porifera-Eumetozoa (1,351 Ma), Pyrenomycetes-Plectomycetes (551 Ma), Candida-Saccharomyces (723 Ma), Hemiascomycetes-filamentous Ascomycota (982 Ma), Basidiomycota-Ascomycota (968 Ma), Mucorales-Basidiomycota (947 Ma), Fungi-Animalia (1,513 Ma), mosses-vascular plants (707 Ma), Chlorophyta-Tracheophyta (968 Ma), Rhodophyta-Chlorophyta+Embryophyta (1,428 Ma), Plantae-Animalia (1,609 Ma), Alveolata-plants+animals+fungi (1,973 Ma), Euglenozoa-plants+animals+fungi (1,961 Ma), and Giardia-plants+animals+fungi (2,309 Ma). By extrapolation, mitochondria arose approximately 2300-1800 Ma and plastids arose 1600-1500 Ma. Estimates of the maximum number of cell types of common ancestors, combined with divergence times, showed an increase from two cell types at 2500 Ma to approximately 10 types at 1500 Ma and 50 cell types at approximately 1000 Ma. CONCLUSIONS: The results suggest that oxygen levels in the environment, and the ability of eukaryotes to extract energy from oxygen, as well as produce oxygen, were key factors in the rise of complex multicellular life. Mitochondria and organisms with more than 2-3 cell types appeared soon after the initial increase in oxygen levels at 2300 Ma. The addition of plastids at 1500 Ma, allowing eukaryotes to produce oxygen, preceded the major rise in complexity.

Animals↗
PubMed2003

ForC, a novel type of formin family protein lacking an FH1 domain, is involved in multicellular development in Dictyostelium discoideum.

Formins are highly conserved regulators of cytoskeletal organization and share three regions of homology: the FH1, FH2 and FH3 domains. Of the nine known formin genes or pseudogenes carried by Dictyostelium, forC is novel in that it lacks an FH1 domain. Mutant Dictyostelium lacking forC (DeltaforC) grew normally during the vegetative phase and, when starved, migrated normally and formed tight aggregates. Subsequently, however, DeltaforC cells made aberrant fruiting bodies with short stalks and sori that remained unlifted. DeltaforC aggregates were also unable to migrate as slugs, suggesting forC is involved in mediating cell movement during multicellular stages of Dictyostelium development. Consistent with this idea, expression of forC was increased significantly in aggregates of wild-type cells. GFP-ForC expressed in DeltaforC cells was localized at the crowns, which are macropinocytotic structures rich in F-actin, suggesting that, like other formin isoforms, ForC functions in close relation with the actin cytoskeleton. Truncation analysis of GFP-ForC revealed that the FH3 domain is required for ForC localization; moreover, localization of a truncated GFP-ForC mutant at the site of contacts between cells on substrates and along the cortex of cells within a multicellular culminant suggests that ForC is involved in the local actin cytoskeletal reorganization mediating cell-cell adhesion.

Actin Cytoskeleton↗
PubMed2000

Do growth and cell division rates determine cell size in multicellular organisms?

Studies in yeast have provided some clues to how cell size might be determined in unicellular eukaryotes; yet little attention has been paid to this issue in multicellular organisms. Reproducible cell sizes might be achieved in the dividing cells of multicellular organisms by the coordination of growth with cell division. Recently, mutations in genes encoding homologues of components of the mammalian insulin/phosphoinositide 3-kinase signalling pathway have been shown to affect organ growth and cell size during Drosophila melanogaster imaginal disc development. The data suggest that signalling through this pathway alters cell size because it primarily affects the growth of these organs (i.e. their increase in mass) and does not have a proportional impact on cell division. These observations are in keeping with the hypothesis that growth and cell division are regulated independently, and that cell size is just a consequence of the rate at which tissues grow and the cells within them divide. However, signalling through this pathway can affect cell cycle phasing and at least influence cell division. These interactions may provide a means of coordinating growth and cell division, such that cells divide only when they are above a minimum size.

Animals↗
PubMed1986

Radiation response of multicellular spheroids initiated from five human melanoma xenograft lines. Relationship to the radioresponsiveness in vivo.

Multicellular spheroids, initiated from five human melanoma xenograft lines (E.E., E.F., G.E., M.F., V.N.) and grown in liquid-overlay culture, were characterised with regard to radiation response. The principal aim of the work was to search for possible correlations with the radioresponsiveness in vivo of the parent xenografts. The spheroids were 100 +/- 5 microns in diameter at irradiation and did not contain radiobiologically hypoxic cells. Single-cell survival measured in soft agar, specific growth delay and spheroid cure were used as end-points. The cellular radiosensitivity was the same whether a melanoma was grown as spheroids or as xenografts. An intercellular contact effect was found for spheroids of the G.E. melanoma but not for spheroids of the E.E., E.F., M.F. and V.N. melanomas, in agreement with previous observations from studies of the corresponding xenografts in vivo. A positive correlation was found between the radiation response of the spheroids, measured as cell survival after 6 Gy or as specific growth delay after 6 Gy, and the radiation response of the parent tumours, measured as specific growth delay after 15 Gy. There was no correlation between the SCD50 (the dose required to cure 50% of the spheroids) and the radioresponsiveness in vivo. It is concluded that differences in radioresponsiveness in vivo among tumours may be identified from studies of the corresponding multicellular spheroids grown in liquid-overlay culture.

Animals↗
PubMed2006

Bacterial programmed cell death and multicellular behavior in bacteria.

Traditionally, programmed cell death (PCD) is associated with eukaryotic multicellular organisms. However, recently, PCD systems have also been observed in bacteria. Here we review recent research on two kinds of genetic programs that promote bacterial cell death. The first is mediated by mazEF, a toxin-antitoxin module found in the chromosomes of many kinds of bacteria, and mainly studied in Escherichia coli. The second program is found in Bacillus subtilis, in which the skf and sdp operons mediate the death of a subpopulation of sporulating bacterial cells. We relate these two bacterial PCD systems to the ways in which bacterial populations resemble multicellular organisms.

Antitoxins↗
PubMed2006

Confrontation cultures of embryonic stem cells with multicellular tumor spheroids to study tumor-induced angiogenesis.

Human embryonic stem cells efficiently differentiate blood vessels, which allows using this in vitro model to study the interaction of blood vessels with adjacent tissues. Herein, we introduce confrontation cultures of human embryonic stem cells with multicellular tumor spheroids to investigate molecular mechanisms of tumor-induced angiogenesis. Vascularization of tumor tissue by the host is a prerequisite for tumor growth, which has led to the development of antiangiogenic therapy. This promising anti-cancer therapy intends to reduce, halt, or even regress tumor growth by deprivation from blood, oxygen, and nutrient supply. Confrontation cultures of human embryonic stem cells with multicellular tumor spheroids allow the investigation of the time course of endothelial cell invasion into the tumor tissue, the concomitant analysis of changes in angiogenesis-related gene expression, and analysis of the cellular microenvironment (i.e., pericellular oxygen pressure, tissue pH, and levels of tissue reactive oxygen species). The in vitro model of confrontation cultures is suitable for routine screening of antiangiogenic agents in pre-clinical trials and may be used to replace animal experiments applied in antiangiogenesis research.

Animal Use Alternatives↗
PubMed2006

Visualizing signaling and cell movement during the multicellular stages of dictyostelium development.

Time-lapse microscopy provides a powerful tool with which to study cell behavior during Dictyostelium development. On a macroscopic level, the overall cell movement patterns that give rise to the complex multicellular structures such as slugs and fruiting bodies can be studied together with the signal waves that coordinate cell movement. Using green fluorescent protein fusion proteins, it is also possible to visualize the cytoskeleton or signal transduction processes at high resolution in single cells in their multicellular environment.

Animals↗