Live imaging and single-cell analysis reveal differential dynamics of autophagy and apoptosis

Autophagy is induced by many cytotoxic stimuli but it is often unclear whether, under specific conditions, autophagy plays a prosurvival or a prodeath role. To answer this critical question we developed a novel methodology that employs automated live microscopy and image analysis to measure autophagy and apoptosis simultaneously in single cells. We used this approach to perform a systems-level analysis of pathway dynamics for both autophagy and apoptosis. We found that induction of autophagy in response to different stimuli is uniformly unimodal; in contrast, cells induce apoptosis in an all-or-none bimodal fashion. By tracking the fate of single cells we found that autophagy precedes apoptosis, and that within the same population apoptosis is delayed in cells that mount a stronger autophagy response. Inhibition of autophagy by knocking down ATG5 promoted apoptosis, thus confirming that autophagy plays a protective role. We anticipate that our single-cell approach will be a powerful tool for gaining a quantitative understanding of the complex regulation of autophagy, its influence on cell fate decisions and its relationship with other cellular pathways.     

Cellular and molecular basis of decision‐making

People think they are in control of their own decisions: what to eat or drink, whom to marry or pick a fight with, where to live, what to buy. Behavioural economists and neurophysiologists have long studied decision‐making behaviours. However, these behaviours have only recently been studied through the light of molecular genetics. Here, we review recent research in mice, Drosophila melanogaster and Caenorhabditis elegans, that analyses the molecular and cellular mechanisms underlying decision‐making. These studies interrogate decision‐making about food, sexual behaviour, aggression or foraging strategies, and add molecular and cell biology understanding onto the consilience of brain and decision.     

Predation by the carabid beetles Pterostichus madidus and Nebria brevicollis is affected by size and condition of the prey slug Deroceras reticulatum

1 Slugs are important pests in many agricultural crops but molluscicides commonly used to control slugs affect non‐target organisms. Encouraging biological control may help to reduce molluscicide use, but the efficiency of potential natural enemies needs to be investigated. 2 Serological tests have shown that certain carabid species consume slugs. These techniques, however, do not distinguish between scavenging and true predation, nor do they provide information on the size or other characteristics of the prey consumed. The study reported here was undertaken to establish whether scavenging of dead slugs might be an important factor contributing to positive serological test results. 3 Both Pterostichus madidus (Fabricius) and Nebria brevicollis (Fabricius) consumed Deroceras reticulatum (Müller) under laboratory conditions. Dead slugs were scavenged in preference to injured or healthy slugs. 4 Only small, live slugs (< 0.11 g) were killed by both beetle species, which may, therefore, be incapable of killing larger slugs. 5 These generalist beetle species appeared unable to overcome the defence mucus produced by slugs. The data suggest that positive serological results from field collected beetles may reflect scavenging rather than predation on live or injured slugs.     

Veränderungen des intracytoplasmatischen Membransystems (Thylakoide) von Rhodospirillum rubrum unter Stickstoff-Limitierung

Zusammenfassung Ruhende Zellen von Rhodospirillum rubrum formten in Gegenwart von Substrat nach 1–4 Tagen ihre intracytoplasmatischen Vesikel zu Schläuchen und unregelmäßigen Membran-Aggregaten um. Die Veränderungen traten anaerob und aerob im Licht sowie in aerober Dunkelkultur auf und waren von der Art des Substrats abhängig. Ohne Substratzugabe waren keine Veränderungen im intracytoplasmatischen Membransystem zu beobachten. Auch bei einer völligen Umgestaltung der Thylakoidvesikel blieb der Bacteriochlorophyllgehalt der Zellen nahezu konstant. Da in denselben Zellen sich Speicherstoffe anhäuften, kann angenommen werden, daß die ATP-Bildung nicht limitierend war. Die Rate der anaeroben Photophosphorylierung und die O₂-Aufnahme im Dunkeln verringerten sich jedoch. Auch die Zahl der vermehrungsfähigen Bakterien nahm ab.Es wird diskutiert, ob die Veränderungen der intracytoplasmatischen Membranvesikel nur eine Desintegration der Membranen beim Absterben der Bakterien sind oder ob sie als eine vom Stoffwechsel der Zelle kontrollierte Morphogenese ablaufen.

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Changes in the intracytoplasmic membrane system (thylakoids) of Rhodospirillum rubrum under nitrogen limitation

Summary In resting cells of Rhodospirillum rubrum under N-limitation in presence of a substrate the intracytoplasmic vesicles were transformed within 1 to 4 days into tubes and irregular membrane aggregates. The changes were observed either in the light under aerobic and anaerobic conditions or in the dark under aerobic conditions. They were dependent on the substrate supplied. Without substrate no changes in the intracytoplasmic membrane system occurred. Even after a complete morphological change of the thylakoid vesicles the bacteriochlorophyll content of the cells remained nearly constant. It can be assumed that the ATP production was not limiting, because in the same cells storage material accumulated. On the other hand, however, the rate of the anaerobic photophosphorylation, the O₂-consumption in the dark, and the number of viable cells in the culture decreased.It is discussed, whether the variations of the intracytoplasmic membrane vesicles are only a disintegration of the membranes during death of the bacteria or whether they occur as a morphogenesis which is controlled by the metabolism of the cell.

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Abkürzungen BChl Bacteriochlorophyll - CM Cytoplasmamembran - ICM intracytoplasmatische Membranen     

Autosis and autophagic cell death: the dark side of autophagy

It is controversial whether cells truly die via autophagy or whether — in dying cells — autophagy is merely an innocent bystander or a well-intentioned ‘Good Samaritan'' trying to prevent inevitable cellular demise. However, there is increasing evidence that the genetic machinery of autophagy may be essential for cell death in certain settings. We recently identified a novel form of autophagy gene-dependent cell death, termed autosis, which is mediated by the Na⁺,K⁺-ATPase pump and has unique morphological features. High levels of cellular autophagy, as occurs with treatment with autophagy-inducing peptides, starvation, or in vivo during certain types of ischemia, can trigger autosis. These findings provide insights into the mechanisms and strategies for prevention of cell death during extreme stress conditions.     

Comparison of terpenoid indole alkaloid production and degradation in two cell lines of Tabernaemontana divaricata

Two cell lines of Tabernaemontana divaricata derived from the same suspension culture accumulate different amounts of the terpenoid indole alkaloids O-acetylvallesamine and voaphylline. [¹⁵N]O-acetylvallesamine and [¹⁵N]voaphylline were added to the suspension cultures to investigate whether the lack of accumulating capacity of one of the cell lines was due to a low biosynthetic ability or to high turnover rates. The difference was shown to be due to the inability of the cell culture to biosynthesize both alkaloids. Both cell lines were able to metabolize O-acetylvallesamine. This metabolisation occurred mainly during the stationary phase. The alkaloids added were chemically unstable under culture conditions. Under normal batch cell culture conditions chemical breakdown is thought to play a minor role in the total amount of compound transformed.     

Immunogenicity of Ehrlichia ruminantium Grown in Tick Cell Lines

Ehrlichia (previously Cowdria) ruminantium, the pathogen which causes heartwater in domestic and wild ruminants, can now be propagated in cell lines from one vector (Amblyomma variegatum) and five non-vector (Ixodes scapularis, I. ricinus, Boophilus decoloratus, B. microplus and Rhipicephalus appendiculatus) tick species. E. ruminantium isolates from West and South Africa and the Caribbean vary in their cell line preference, growth patterns and immunogenic capability. In laboratory trials, certain combinations of tick cell line and E. ruminantium isolate were highly immunogenic in sheep. These trial vaccines were grown under specific in vitro conditions and administered as a single intravenous dose of freshly harvested whole, live culture. Following immunisation and subsequent exposure to virulent E. ruminantium, protected sheep showed no clinical response and a range of serological responses. This revised version was published online in July 2006 with corrections to the Cover Date.     

In situ effective diffusion coefficient profiles in live biofilms using pulsed‐field gradient nuclear magnetic resonance

Diffusive mass transfer in biofilms is characterized by the effective diffusion coefficient. It is well documented that the effective diffusion coefficient can vary by location in a biofilm. The current literature is dominated by effective diffusion coefficient measurements for distinct cell clusters and stratified biofilms showing this spatial variation. Regardless of whether distinct cell clusters or surface‐averaging methods are used, position‐dependent measurements of the effective diffusion coefficient are currently: (1) invasive to the biofilm, (2) performed under unnatural conditions, (3) lethal to cells, and/or (4) spatially restricted to only certain regions of the biofilm. Invasive measurements can lead to inaccurate results and prohibit further (time‐dependent) measurements which are important for the mathematical modeling of biofilms. In this study our goals were to: (1) measure the effective diffusion coefficient for water in live biofilms, (2) monitor how the effective diffusion coefficient changes over time under growth conditions, and (3) correlate the effective diffusion coefficient with depth in the biofilm. We measured in situ two‐dimensional effective diffusion coefficient maps within Shewanella oneidensis MR‐1 biofilms using pulsed‐field gradient nuclear magnetic resonance methods, and used them to calculate surface‐averaged relative effective diffusion coefficient (D_rs) profiles. We found that (1) D_rs decreased from the top of the biofilm to the bottom, (2) D_rs profiles differed for biofilms of different ages, (3) D_rs profiles changed over time and generally decreased with time, (4) all the biofilms showed very similar D_rs profiles near the top of the biofilm, and (5) the D_rs profile near the bottom of the biofilm was different for each biofilm. Practically, our results demonstrate that advanced biofilm models should use a variable effective diffusivity which changes with time and location in the biofilm. Biotechnol. Bioeng. 2010;106: 928–937. © 2010 Wiley Periodicals, Inc.     

An analysis of the Impact of <Emphasis Type="Italic">NRG1</Emphasis> Overexpression on the <Emphasis Type="Italic">Candida albicans</Emphasis> Response to Specific Environmental Stimuli

The ability of the opportunistic fungal pathogen Candida albicans to form filaments has been strongly linked to its capacity to cause disease in humans. We previously described the construction of a strain in which filamentation can be modulated both in vitro and in vivo by placing one copy of the NRG1 gene under the control of a tetracycline-regulatable promoter. To further characterize the role of NRG1 in controlling filamentous growth, and in an attempt to determine whether NRG1 downregulation is a requirement for filamentation per se, or is only necessary under certain environmental conditions, we have conducted an analysis of the growth of the tet-NRG1 strain under a variety of in vitro conditions. Through overexpression of NRG1, we were able to block filamentation of C. albicans in both liquid media and on solid media. Filamentation in response to the low-oxygen environment of embedded growth was also inhibited. In all of these conditions, normal filamentation could be restored by down regulating expression from the tet-NRG1 allele. Interestingly, although elevated NRG1 levels were able to inhibit the formation of true hyphae in response to a wide range of environmental stimuli, elevated NRG1 expression did not affect the formation of pseudohyphae on nitrogen-limiting synthetic low ammonia dextrose (SLAD) medium. This work further illustrates the key role played by NRG1 in the control of filamentation and suggests that, although NRG1 repression plays a key role in regulating true hyphal growth, it apparently does not regulate pseudohyphal growth in the same fashion.     

Insect muscle as a model for programmed cell death

Programmed cell death (PCD) is a fundamental component of development in virtually all animals. Despite the ubiquity of this phenomenon, little is known about what tells a cell to die, and less still about the physiological and molecular mechanisms that bring about death. One system that has proven to be very amenable for the study of PCD is the intersegmental muscle (ISM) of the tobacco hawkmoth Manduca sexta. These giant muscle cells are used during the eclosion (emergence) behavior of the adult moth, and then die during the subsequent 30 h. This review uses the ISMs as a model system to address questions that are basic to any cell death system, including the following: (1) how do cells know when to die; (2) what physiological changes accompany death; (3) what are the molecular mechanisms that mediate death; and (4) do all cells die by the same process? For the ISMs, the trigger for PCD is a decline in the circulating titer of the insect molting hormone, 20-hydroxyecdysone (20-HE). During cell death there are rapid decreases in both the myofibrillar sensitivity to intracellular calcium and the resulting force of fiber contraction. The ability of the ISMs to under go PCD requires the repression and activation of specific genes. Two of the repressed genes encode actin and myosin. One of the upregulated presumptive cell-death genes encodes polyubiquitin, which appears to play a critical role in the rapid proteolysis that accompanies ISM death. One curious aspect of ISM death is that these cells display none of the features that are characteristic of apoptosis, suggesting that they may die by a fundamentally different mechanism. © 1992 John Wiley & Sons, Inc.     

Comparative evaluation of Pseudomonas fluorescens and their lipopolysaccharides as implicated in induction of resistance against pearl millet downy mildew

Two plant growth promoting Pseudomonas fluorescens isolates namely UOM SAR 14 and UOM SAR 80 most effectively induced resistance against downy mildew disease of pearl millet both under greenhouse and field conditions. Relative assessment of live cultures of P. fluorescens UOM SAR 14 and UOM SAR 80 and their lipopolysaccharides (LPS) extracted from their cell walls were evaluated for their ability to induce resistance against pearl millet downy mildew. Treatment with P. fluorescens and their LPS enhanced the seed germination and seedling vigour considerably. Although both live cultures and their LPS treatment induced resistance in pearl millet against downy mildew disease both under greenhouse and field conditions as evidenced by the significant reduction of the disease, live cultures were more effective than the LPS in level of resistance induced. Live cultures of UOM SAR 14 and UOM SAR 80 induced 66% and 57% protection while their respective LPS extracts offered 59 and 53% protection against downy mildew disease under greenhouse conditions. Similarly, under field conditions with very heavy inoculum pressure live cultures offered 75% and 70%, and their LPS offered 71% and 67% protection, respectively. In either case, the time gap required for the building up of resistance was found to be 3 days and nature of the resistance induced was systemic and durable with both live cultures and their lipopolysaccharides. It was also noticed that the live bacteria significantly varied in the degree of protection offered and so also their respective LPS.     

Farnesylation is involved in meristem organization in Arabidopsis

 Although studies in plant and animal cell culture systems indicate farnesylation is required for normal cell cycle progression, how this lipid modification of select proteins translates into whole-organism developmental decisions involving cell proliferation or differentiation is largely unknown. The era1 mutant of the higher plant Arabidopsis thaliana (L.) Heynh. offers a unique opportunity to understand the role farnesylation may play in regulating various processes during the development of a multicellular organism. Loss of farnesylation affects many aspects of Arabidopsis growth and development. In particular, apical and axillary meristem development is altered and these phenotypes are contingent on the growth conditions. Received: 25 October 1999 / Accepted: 22 December 1999     

Einfluß von Mikrofloren verschiedener Böden und von Bakterienreinkulturen auf das Pflanzenwachstum

Zusammenfassung Der Einfluß verschiedener Bodenmikrofloren und Bakterienreinkulturen wurde in Wasserkultur auf das Wachstum von Rotklee untersucht.Sämtliche geprüften Bodenmikrofloren bewirkten eine Erhöhung des Sproß/Wurzel-Verhältnisses der Pflanzen, allerdings in unterschiedlicher Weise. Unter den Mikrofloren befanden sich solche, die auf das Wurzelwachstum eine gleich starke Hemmung ausübten, das Sproßwachstum hingegen unterschiedlich beeinflußten. Bei geringer oder fehlender Hemmung des Wurzelwachstums war das Sproßwachstum unter nichtsterilen Versuchsbedingungen größer als bei steril kultivierten Pflanzen. Damit konnte gezeigt werden, daß außer den allgemein bekannten Waschstumsfaktoren die Bodenmikroflora als weiterer Wachstumsfaktor eine nicht unwesentliche Rolle zu spielen vermag.Mit Reinkulturen von Bacillus mycoides, Mycobacterium phlei, Pseudomonas fluorescens, Rhizobium spp. und drei weiteren nicht identifizierten Bakterien wurde keine statistisch gesicherte Verschiebung des Sproß/Wurzel-Verhältnisses der Pflanzen beobachtet.Während die beiden erstgenannten Bakterienreinkulturen sich nur schwach in der Rhizosphäre vermehrten, fanden die Rhizobium spp. und die drei nicht identifizierten Bakterien unter gleichen Wachstumsverhältnissen sehr günstige Lebensbedingungen. Pseudomonas fluorescens wies ähnlich wie die Bakterienpopulation der verschiedenen Bodenmikrofloren mittlere Keimzahlen auf.Aus einem Versuch über den Belichtungseffekt auf das Wurzelwachstum wird auf die Beteiligung eines lichtempfindlichen von gewissen Rhizosphärenmikroorganismen gebildeten Wuchsstoffes geschlossen.

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Summary The effect of different soil microflores and bacteria pure cultures on the growth of red clover (Trifolium pratense) has been investigated in aqueous culture.All examined microflores increased the shoot/root ratio of the plants, however in a different way. Among the microflores were those which effected an equally strong inhibition on the root growth while influencing the shoot growth differently. If no or only a slight inhibition of the root growth occured the shoot growth was more extended under non-sterile experimental conditions than under sterile ones. Thus it could be demonstrated that beside the generally known growth factors the soil microflora as a further growth factor is able to play a not unimportant role.With pure cultures of Bacillus mycoides, Mycobacterium phlei, Pseudomonas fluorescens, Rhizobium spp. and three other bacteria not identified no significant variation of the shoot/root ratio of the plants could be observed.While pure cultures of the two forementioned bacteria could only multiply themselves weakly in the rhizosphere the Rhizobia species and the three not-identified bacteria found very favourable life conditions under equal growth situations. Pseudomonas fluorescens showed medium germ numbers similar to the bacteria population of the different soil microflores.As concluded from an experiment concerning the effect of light on the root growth, the participation of light sensitive growth promoting substance formed by certain rhizosphere microorganisms is supposed.

     

Viability determination of Helicobacter pylori using propidium monoazide quantitative PCR

Background: While Helicobacter pylori exists in a bacillary form in both the natural habitat and the human host, detrimental environmental circumstances have been observed to lead to the conversion of H. pylori from the bacillary to the coccoid form. However, the viability or nonviability of coccoid forms remains to be established in H. pylori. The aim of this study was to determine whether the quantitative PCR combined with propidium monoazide could be an alternative and good technique to determine H. pylori viability in environmental samples and, to contribute to understanding of the role of the H. pylori forms. Materials and Methods: Viability, morphological distribution, and the number of live H. pylori cells were determined using a propidium monoazide‐based quantitative PCR method, at various time points. Results: Under adverse environmental conditions was observed the conversion of H. pylori from the bacillary to the coccoid form, and the decrease in amplification signal, in samples that were treated with propidium monoazide, over the time. Conclusions: Incorporation of propidium monoazide indicates that there is an increase in H. pylori cells with the damaged membrane over the study, leading to the manifestation of cellular degeneration and death. Consequently, quantitative PCR combined with propidium monoazide contributes to our understanding of the role of H. pylori cells, under adverse environmental conditions.     

Foraging of multimammate mice, Mastomys natalensis, under different predation pressure: cover, patch-dependent decisions and density-dependent GUDs

Patch use under predation risk often results in a change of feeding behaviour in the prey animals. However, such changes only appear if the animals are able to assess under which predation pressure they live. We investigated patch use of Mastomys natalensis under different conditions of avian predation pressure.
In replicated maize field plots in Morogoro, Tanzania, avian predators were allowed under natural conditions (control), attracted with perches and nest boxes or kept out with nets. During four one‐week periods in late 1999, we measured rodent feeding decisions with the giving‐up density (GUD) method. Trays with known amounts of millet seeds in sand were placed in pairs, one of them under a cover, the other one in the open. M. natalensis mice were expected to give up sooner in the open trays than in those with cover. We hypothesised that M. natalensis mice could assess the ambient predation pressure leading to larger difference in GUD between covered and non‐covered trays in the plots where predators were attracted. We also made video recordings of the rodent activity at a pair of trays in each treatment. The GUD‐values were significantly lower for the covered trays but predation pressure did not affect this difference. The video observations showed that in the control and netted plots the animals visited trays equally frequently regardless of the cover, while the visits in the predator‐attracted plots occurred significantly more often in the covered trays. We conclude that M. natalensis can assess the ambient predation pressure and adapt its behaviour at a feeding patch. However, the variation in predation pressure in our experiment was not obvious from the GUD. Moreover, we found a strong relation between rodent density and GUD, which may mask variations in perceived predation pressure. Similar GUD values may be reached in different ways and we present models to investigate whether animals’ decision to forage at a food patch is only affected by the seed density at that patch, not by that at a neighbour patch.     

Expression of the SOS system in Escherichia coli growing under nitrate respiration conditions

Induction of several SOS functions by mitomycin C, bleomycin or thermal treatment of a recA441 mutant growing under nitrate respiration conditions was studied in Escherichia coli. Mitomycin C caused inhibition of cell division, induction of prophages and expression of umuC gene but like in aerobically growing cells, it did not trigger the cessation of cell repiration. On the contrary, both recA⁺ and recA441 cultures either treated with bleomycin or incubated at 42°C failed to induce any of the different SOS functions cited above.Furthermore, after bleomycin addition or thermal treatment both recA⁺ and recA441 cultures did not present any variation in the cellular ATP level, contrary to what happens under aerobic growth. The blocking of the expression of some SOS functions under nitrate respiration conditions is not an irreversible process because cells incubated under these anaerobic conditions were able to induce the SOS system when changed to an aerobic medium 30 min after the SOS-inducing treatment had been applied.     

Target muscles and sensory afferents do not influence steroid-regulated,segment-specific death of identified motoneurons in Manduca sexta

Programmed cell death plays a critical role in sculpting the nervous system during embryonic development. In holometabolous insects, cell death also plays an important role in the reorganization of the nervous system during metamorphosis. In Manduca sexta, cell death and the factors that regulate it can be studied at the level of individually identified neurons. The accessory planta retractor (APR) motoneurons undergo segment-specific death during the larval-pupal transformation. APRs in abdominal segments 1, 5, and 6 die at pupation; those in abdominal segments 2, 3, and 4 survive until adulthood. Juvenile hormone and ecdysteroids regulate the metamorphic restructuring of the nervous system, but the factors that determine which APRs will live and which will die are not known. The present study assessed the possible importance of cell-cell interactions in determining APR survival at pupation by removing APR's target muscle or mechanosensory input early in the final larval instar, prior to the hormonal cues that trigger the larval-pupal transformation. The motoneurons showed their normal, segment-specific pattern of death in nearly all cases. These results suggest that target muscles and sensory input play little or no role in determining the segment-specific pattern of APR survival at pupation. © 1996 John Wiley & Sons, Inc.