Inhibition of myogenesis by transforming growth factor β is density-dependent and related to the translocation of transcription factor MEF2 to the cytoplasm

Transforming growth factor β (TGF-β) was found to inhibit differentiation of myogenic cells only when they were grown to high density. Inhibition also occurred when myogenic cells were cocultured with other types of mesenchymal cells but not when they were cocultured with epithelial cells. It is therefore possible that some density-dependent signaling mediates the intracellular response to TGF-β. Within 30 min of treatment, TGF-β induced translocation of MEF2, but not MyoD, myogenin, or p21, to the cytoplasm of myogenic cells grown to high density. Translocation was reversible on withdrawal of TGF-β. By using immune electron microscopy and Western blot analysis on subcellular fractions, MEF2 was shown to be tightly associated with cytoskeleton membrane components. To test whether MEF2 export from the nucleus was causally related to the inhibitory action of TGF-β, we transfected C2C12 myoblasts with MEF2C containing the nuclear localization signal of simian virus 40 large T antigen (nlsSV40). Myogenic cells expressing the chimerical MEF2C/nlsSV40, but not wild-type MEF2C, retained this transcription factor in the nucleus and were resistant to the inhibitory action of TGF-β. We propose a mechanism in which the inhibition of myogenesis by TGF-β is mediated through MEF2 localization to the cytoplasm, thus preventing it from participating in an active transcriptional complex.

Evolution of foraging behavior in Drosophila by density-dependent selection

One of the rare examples of a single major gene underlying a naturally occurring behavioral polymorphism is the foraging locus of Drosophila melanogaster. Larvae with the rover allele, forR, have significantly longer foraging path lengths on a yeast paste than do those homozygous for the sitter allele, fors. These variants do not differ in general activity in the absence of food. The evolutionary significance of this polymorphism is not as yet understood. Here we examine the effect of high and low animal rearing densities on the larval foraging path-length phenotype and show that density-dependent natural selection produces changes in this trait. In three unrelated base populations the long path (rover) phenotype was selected for under high-density rearing conditions, whereas the short path (sitter) phenotype was selected for under low-density conditions. Genetic crosses suggested that these changes resulted from alterations in the frequency of the fors allele in the low-density-selected lines. Further experiments showed that density-dependent selection during the larval stage rather than the adult stage of development was sufficient to explain these results. Density-dependent mechanisms may be sufficient to maintain variation in rover and sitter behavior in laboratory populations.

Density-dependent decline of host abundance resulting from a new infectious disease

Although many new diseases have emerged within the past 2 decades [Cohen, M. L. (1998) Brit. Med. Bull. 54, 523–532], attributing low numbers of animal hosts to the existence of even a new pathogen is problematic. This is because very rarely does one have data on host abundance before and after the epizootic as well as detailed descriptions of pathogen prevalence [Dobson, A. P. & Hudson, P. J. (1985) in Ecology of Infectious Diseases in Natural Populations, eds. Grenfell, B. T. & Dobson, A. P. (Cambridge Univ. Press, Cambridge, U.K.), pp. 52–89]. Month by month we tracked the spread of the epizootic of an apparently novel strain of a widespread poultry pathogen, Mycoplasma gallisepticum, through a previously unknown host, the house finch, whose abundance has been monitored over past decades. Here we are able to demonstrate a causal relationship between high disease prevalence and declining house finch abundance throughout the eastern half of North America because the epizootic reached different parts of the house finch range at different times. Three years after the epizootic arrived, house finch abundance stabilized at similar levels, although house finch abundance had been high and stable in some areas but low and rapidly increasing in others. This result, not previously documented in wild populations, is as expected from theory if transmission of the disease was density dependent.

Promoter regions involved in density-dependent regulation of basic fibroblast growth factor gene expression in human astrocytic cells.

Expression of mitogenic basic fibroblast growth factor (bFGF) in the central nervous system is inhibited by direct cell contact and is implicated in reactive and neoplastic transformation of astrocytes. The molecular mechanisms controlling expression of bFGF were examined in cultures of human astrocytes. Cell-density-dependent depletion of bFGF mRNA levels parallels changes in bFGF gene protein. Regulation of transcription of a bFGF luciferase reporter gene containing an upstream region (bp -1800 to +314) of the bFGF gene promoter mimicks the density-dependent regulation of the endogenous bFGF gene in transfected astrocytes. Deletion analysis has identified a fragment (bp -650 to -513) and sequences further downstream (bp -274 to +314) as the regions required for the regulation of bFGF gene activity by cell density. Unlike in astrocytes, changing the cell density of glioma cell cultures does not affect the levels of bFGF protein and mRNA. bFGF luciferase constructs were expressed at the same level in high- or low-density cultures of glioma cells, indicating altered regulation of the bFGF gene promoter. Electrophoretic mobility shift assays showed binding of nuclear proteins to a fragment of bFGF gene promoter from bp -650 to -453. This binding was abolished by a deletion of the upstream cell-density-responsive region (bp -650 to -512). Binding was observed with nuclear extracts from subconfluent astrocytes but was reduced in extracts from confluent astrocytes. Our results indicate that induction of bFGF in astrocytes upon reduction of cell density is mediated transcriptionally by positive trans-acting factors interacting with bFGF promoter. In contrast, nuclear proteins from glioma cells bind to the promoter region from bp -650 to -453 independent of cell density. Thus, the constitutive binding of trans-acting factor(s) to the region of the bFGF promoter from bp -650 to -453 may be responsible for the continuous expression of bFGF that leads to the uncontrolled growth of glioma cells.

From patterns to processes: Phase and density dependencies in the Canadian lynx cycle

Across the boreal forest of North America, lynx populations undergo 10-year cycles. Analysis of 21 time series from 1821 to the present demonstrates that these fluctuations are generated by nonlinear processes with regulatory delays. Trophic interactions between lynx and hares cause delayed density-dependent regulation of lynx population growth. The nonlinearity, in contrast, appears to arise from phase dependencies in hunting success by lynx through the cycle. Using a combined approach of empirical, statistical, and mathematical modeling, we highlight how shifts in trophic interactions between the lynx and the hare generate the nonlinear process primarily by shifting functional response curves during the increase and the decrease phases.

Control of time-dependent biological processes by temporally patterned input

Temporal patterning of biological variables, in the form of oscillations and rhythms on many time scales, is ubiquitous. Altering the temporal pattern of an input variable greatly affects the output of many biological processes. We develop here a conceptual framework for a quantitative understanding of such pattern dependence, focusing particularly on nonlinear, saturable, time-dependent processes that abound in biophysics, biochemistry, and physiology. We show theoretically that pattern dependence is governed by the nonlinearity of the input–output transformation as well as its time constant. As a result, only patterns on certain time scales permit the expression of pattern dependence, and processes with different time constants can respond preferentially to different patterns. This has implications for temporal coding and decoding, and allows differential control of processes through pattern. We show how pattern dependence can be quantitatively predicted using only information from steady, unpatterned input. To apply our ideas, we analyze, in an experimental example, how muscle contraction depends on the pattern of motorneuron firing.

Directionality principles in thermodynamics and evolution

Directionality in populations of replicating organisms can be parametrized in terms of a statistical concept: evolutionary entropy. This parameter, a measure of the variability in the age of reproducing individuals in a population, is isometric with the macroscopic variable body size. Evolutionary trends in entropy due to mutation and natural selection fall into patterns modulated by ecological and demographic constraints, which are delineated as follows: (i) density-dependent conditions (a unidirectional increase in evolutionary entropy), and (ii) density-independent conditions, (a) slow exponential growth (an increase in entropy); (b) rapid exponential growth, low degree of iteroparity (a decrease in entropy); and (c) rapid exponential growth, high degree of iteroparity (random, nondirectional change in entropy). Directionality in aggregates of inanimate matter can be parametrized in terms of the statistical concept, thermodynamic entropy, a measure of disorder. Directional trends in entropy in aggregates of matter fall into patterns determined by the nature of the adiabatic constraints, which are characterized as follows: (i) irreversible processes (an increase in thermodynamic entropy) and (ii) reversible processes (a constant value for entropy). This article analyzes the relation between the concepts that underlie the directionality principles in evolutionary biology and physical systems. For models of cellular populations, an analytic relation is derived between generation time, the average length of the cell cycle, and temperature. This correspondence between generation time, an evolutionary parameter, and temperature, a thermodynamic variable, is exploited to show that the increase in evolutionary entropy that characterizes population processes under density-dependent conditions represents a nonequilibrium analogue of the second law of thermodynamics.

Protein misfolding and temperature up-shift cause G1 arrest via a common mechanism dependent on heat shock factor in Saccharomyces cerevisiae

Accumulation of misfolded proteins in the cell at high temperature may cause entry into a nonproliferating, heat-shocked state. The imino acid analog azetidine 2-carboxylic acid (AZC) is incorporated into cellular protein competitively with proline and can misfold proteins into which it is incorporated. AZC addition to budding yeast cells at concentrations sufficient to inhibit proliferation selectively activates heat shock factor (HSF). We find that AZC treatment fails to cause accumulation of glycogen and trehalose (Msn2/4-dependent processes) or to induce thermotolerance (a protein kinase C-dependent process). However, AZC-arrested cells can accumulate glycogen and trehalose and can acquire thermotolerance in response to a subsequent heat shock. We find that AZC treatment arrests cells in a viable state and that this arrest is reversible. We find that cells at high temperature or cells deficient in the ubiquitin-conjugating enzymes Ubc4 and Ubc5 are hypersensitive to AZC-induced proliferation arrest. We find that AZC treatment mimics temperature up-shift in arresting cells in G1 and represses expression of CLN1 and CLN2. Mutants with reduced G1 cyclin-Cdc28 activity are hypersensitive to AZC-induced proliferation arrest. Expression of the hyperstable Cln3–2 protein prevents G1 arrest upon AZC treatment and temperature up-shift. Finally, we find that the EXA3–1 mutation, encoding a defective HSF, prevents efficient G1 arrest in response to both temperature up-shift and AZC treatment. We conclude that nontoxic levels of misfolded proteins (induced by AZC treatment or by high temperature) selectively activate HSF, which is required for subsequent G1 arrest.

Myofibroblasts differentiate from fibroblasts when plated at low density.

Myofibroblasts, defined by their expression of smooth muscle alpha-actin, appear at corneal and dermal incisions and promote wound contraction. We report here that cultured fibroblasts differentiate into myofibroblasts by a cell density-dependent mechanism. Fibroblasts seeded at low density (5 cells per mm2) produced a cell culture population consisting of 70-80% myofibroblasts, 5-7 days after seeding. In contrast, fibroblasts seeded at high density (500 cells per mm2) produced cultures with only 5-10% myofibroblasts. When the myofibroblast-enriched cultures were subsequently passaged at high density, the smooth muscle alpha-actin phenotype was lost within 3 days. Furthermore, initially 60% of the low density-cultured cells incorporated BrdUrd compared to 30% of cells passaged at high density. Media from myofibroblast-enriched cultures had more latent and active transforming growth factor beta (TGF-beta) than did media from fibroblast-enriched cultures. Although there was a trend towards increased numbers of myofibroblasts after addition of exogenous TGF-beta, the results did not reach statistical significance. We conclude that myofibroblast differentiation can be induced in fibroblasts by plating at low density. We propose a cell density-dependent model of myofibroblast differentiation during wounding and healing in which at least two factors interact: loss of cell contact and the presence of TGF-beta.

Nuclear/cytoplasmic localization of the von Hippel-Lindau tumor suppressor gene product is determined by cell density.

The product of the von Hippel-Lindau (VHL) tumor suppressor gene, the gene inactivated in VHL disease and in sporadic clear-cell renal carcinomas, has recently been shown to have as a functional target the transcription elongation complex, elongin (also called SIII). Here it is shown that there is a tightly regulated, cell-density-dependent transport of VHL into and/or out of the nucleus. In densely grown cells, the VHL protein is predominantly in the cytoplasm, whereas in sparse cultures, most of the protein can be detected in the nucleus. We have identified a putative nuclear localization signal in the first 60 and first 28 amino acids of the human and rat VHL protein, respectively. Sequences in the C-terminal region of the VHL protein may also be required for localization to the cytosol. These findings provide the initial indication of a novel cell density-dependent pathway that is responsible for the regulation of VHL cellular localization.

A negative regulator mediates quorum-sensing control of exopolysaccharide production in Pantoea stewartii subsp. stewartii

Classical quorum-sensing (autoinduction) regulation, as exemplified by the lux system of Vibrio fischeri, requires N-acyl homoserine lactone (AHL) signals to stimulate cognate transcriptional activators for the cell density-dependent expression of specific target gene systems. For Pantoea stewartii subsp. stewartii, a bacterial pathogen of sweet corn and maize, the extracellular polysaccharide (EPS) stewartan is a major virulence factor, and its production is controlled by quorum sensing in a population density-dependent manner. Two genes, esaI and esaR, encode essential regulatory proteins for quorum sensing. EsaI is the AHL signal synthase, and EsaR is the cognate gene regulator. esaI, ΔesaR, and ΔesaI-esaR mutations were constructed to establish the regulatory role of EsaR. We report here that strains containing an esaR mutation produce high levels of EPS independently of cell density and in the absence of the AHL signal. Our data indicate that quorum-sensing regulation in P. s. subsp. stewartii, in contrast to most other described systems, uses EsaR to repress EPS synthesis at low cell density, and that derepression requires micromolar amounts of AHL. In addition, derepressed esaR strains, which synthesize EPS constitutively at low cell densities, were significantly less virulent than the wild-type parent. This finding suggests that quorum sensing in P. s. subsp. stewartii may be a mechanism to delay the expression of EPS during the early stages of infection so that it does not interfere with other mechanisms of pathogenesis.

In vitro selection of a 7-methyl-guanosine binding RNA that inhibits translation of capped mRNA molecules

Using systematic evolution of ligands by exponential enrichment (SELEX), an RNA molecule was isolated that displays a 1,000-fold higher affinity for guanosine residues that carry an N-7 methyl group than for nonmethylated guanosine residues. The methylated guanosine residue closely resembles the 5′ terminal cap structure present on all eukaryotic mRNA molecules. The cap-binding RNA specifically inhibited the translation of capped but not uncapped mRNA molecules in cell-free lysates prepared from either human HeLa cells or from Saccharomyces cerevisiae. These findings indicate that the cap-binding RNA will also be useful in studies of other cap-dependent processes such as pre-mRNA splicing and nucleocytoplasmic mRNA transport.

Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa

Bacteria communicate with each other to coordinate expression of specific genes in a cell density-dependent fashion, a phenomenon called quorum sensing and response. Although we know that quorum sensing via acyl-homoserine lactone (HSL) signals controls expression of several virulence genes in the human pathogen Pseudomonas aeruginosa, the number and types of genes controlled by quorum sensing have not been studied systematically. We have constructed a library of random insertions in the chromosome of a P. aeruginosa acyl-HSL synthesis mutant by using a transposon containing a promoterless lacZ. This library was screened for acyl-HSL induction of lacZ. Thirty-nine quorum sensing-regulated genes were identified. The genes were organized into classes depending on the pattern of regulation. About half of the genes appear to be in seven operons, some seem organized in large patches on the genome. Many of the quorum sensing-regulated genes code for putative virulence factors or production of secondary metabolites. Many of the genes identified showed a high level of induction by acyl-HSL signaling.

RacF1, a Novel Member of the Rho Protein Family in Dictyostelium discoideum, Associates Transiently with Cell Contact Areas, Macropinosomes, and Phagosomes

Using a PCR approach we have isolated racF1, a novel member of the Rho family in Dictyostelium. The racF1 gene encodes a protein of 193 amino acids and is constitutively expressed throughout the Dictyostelium life cycle. Highest identity (94%) was found to a RacF2 isoform, to Dictyostelium Rac1A, Rac1B, and Rac1C (70%), and to Rac proteins of animal species (64–69%). To investigate the role of RacF1 in cytoskeleton-dependent processes, we have fused it at its amino-terminus with green fluorescent protein (GFP) and studied the dynamics of subcellular redistribution using a confocal laser scanning microscope and a double-view microscope system. GFP–RacF1 was homogeneously distributed in the cytosol and accumulated at the plasma membrane, especially at regions of transient intercellular contacts. GFP–RacF1 also localized transiently to macropinosomes and phagocytic cups and was gradually released within <1 min after formation of the endocytic vesicle or the phagosome, respectively. On stimulation with cAMP, no enrichment of GFP–RacF1 was observed in leading fronts, from which it was found to be initially excluded. Cell lines were obtained using homologous recombination that expressed a truncated racF1 gene lacking sequences encoding the carboxyl-terminal region responsible for membrane targeting. These cells displayed normal phagocytosis, endocytosis, and exocytosis rates. Our results suggest that RacF1 associates with dynamic structures that are formed during pinocytosis and phagocytosis. Although RacF1 appears not to be essential, it might act in concert and/or share functions with other members of the Rho family in the regulation of a subset of cytoskeletal rearrangements that are required for these processes.

An α-Tubulin Mutant Destabilizes the Heterodimer: Phenotypic Consequences and Interactions with Tubulin-binding Proteins

Many effectors of microtubule assembly in vitro enhance the polymerization of subunits. However, several Saccharomyces cerevisiae genes that affect cellular microtubule-dependent processes appear to act at other steps in assembly and to affect polymerization only indirectly. Here we use a mutant α-tubulin to probe cellular regulation of microtubule assembly. tub1-724 mutant cells arrest at low temperature with no assembled microtubules. The results of several assays reported here demonstrate that the heterodimer formed between Tub1-724p and β-tubulin is less stable than wild-type heterodimer. The unstable heterodimer explains several conditional phenotypes conferred by the mutation. These include the lethality of tub1-724 haploid cells when the β-tubulin–binding protein Rbl2p is either overexpressed or absent. It also explains why the TUB1/tub1-724 heterozygotes are cold sensitive for growth and why overexpression of Rbl2p rescues that conditional lethality. Both haploid and heterozygous tub1-724 cells are inviable when another microtubule effector, PAC2, is overexpressed. These effects are explained by the ability of Pac2p to bind α-tubulin, a complex we demonstrate directly. The results suggest that tubulin-binding proteins can participate in equilibria between the heterodimer and its components.