Starved cells use mitochondria for autophagosome biogenesis

Comment on: Hailey DW, et al. Cell 2010; 141:656-67.     

Methanol Improves Methane Uptake in Starved Methanotrophic Microorganisms

Methanotrophs in enrichment cultures grew and sustained atmospheric methane oxidation when supplied with methanol. If they were not supplied with methanol or formate, their atmospheric methane oxidation came to a halt, but it was restored within hours in response to methanol or formate. Indigenous forest soil methanotrophs were also dependent on a supply of methanol upon reduced methane access but only when exposed to a methane-free atmosphere. Their immediate response to each methanol addition, however, was to shut down the oxidation of atmospheric methane and to reactivate atmospheric methane oxidation as the methanol was depleted.     

The Induction of Endocytosis in Starved Tetrahymena pyriformis

SYNOPSIS. The formation of digestive vacuoles by starved Tetrahymena pyriformis could be induced by mixtures of latex particles and a variety of potentially digestible solutes. Latex particles themselves had little effect in inducing vacuole formation. Protein, polypeptide, and RNA were highly effective inducers, while glutamate, amino acid mixtures, polysacharides, and glucose were moderately effective. Sodium-β-glycerophosphate had a slight effect and sodium acetate was ineffective. The possible stimulus to endocytosis is discussed. The endocytic response to inducers does not appear to be an all-or-none phenomenon and varies with the concentration of inducer. The stimulatory effect for protein-related inducers seems to be produced by a large number of stimulatory molecules acting upon a single cell and the magnitude of the response appears to be related to molecular size.     

Messenger Ribonucleic Acid Stability in Relaxed and Stringent Escherichia coli Starved for Methionine

During starvation for each of four amino acids, relaxed and stringent strains of Escherichia coli showed exponential decay of pulse-labeled unstable messenger ribonucleic acid (mRNA), with RNA degraded more slowly in the relaxed strain. An additional unique difference was observed during starvation for methionine: the relaxed strain showed non-exponential decay of mRNA, with a survival curve similar to that of an aging process.     

The Hemolymph Proteome of Fed and Starved Drosophila Larvae

The co-operation of specialized organ systems in complex multicellular organisms depends on effective chemical communication. Thus, body fluids (like blood, lymph or intraspinal fluid) contain myriads of signaling mediators apart from metabolites. Moreover, these fluids are also of crucial importance for immune and wound responses. Compositional analyses of human body fluids are therefore of paramount diagnostic importance. Further improving their comprehensiveness should increase our understanding of inter-organ communication. In arthropods, which have trachea for gas exchange and an open circulatory system, the single dominating interstitial fluid is the hemolymph. Accordingly, a detailed analysis of hemolymph composition should provide an especially comprehensive picture of chemical communication and defense in animals. Therefore we used an extensive protein fractionation workflow in combination with a discovery-driven proteomic approach to map out the detectable protein composition of hemolymph isolated from Drosophila larvae. Combined mass spectrometric analysis revealed more than 700 proteins extending far beyond the previously known Drosophila hemolymph proteome. Moreover, by comparing hemolymph isolated from either fed or starved larvae, we provide initial provisional insights concerning compositional changes in response to nutritional state. Storage proteins in particular were observed to be strongly reduced by starvation. Our hemolymph proteome catalog provides a rich basis for data mining, as exemplified by our identification of potential novel cytokines, as well as for future quantitative analyses by targeted proteomics.     

Effect of Interfaces on Small, Starved Marine Bacteria

The copiotrophic marine Vibrio sp. strain DW1, shown previously in batch culture to increase in numbers at the onset of starvation and then to form viable small cells with low endogenous respiration, appears to have a survival advantage at interfaces. Vibrio sp. strain DW1 behaved differently at interfaces compared with the aqueous phase under starvation conditions: (i) small cells were observed at an air-water interface without nutrients, (ii) nutrients added to the air-water interface quickly produced larger cells at the surface, (iii) motility persisted many hours longer at the solid-water interface of a dialysis membrane in a microchamber at the onset of starvation, and (iv) regrowth and division at the solid-liquid interface occurred quickly and at nutrient concentrations too low to permit growth in the aqueous phase. It was concluded that, if small starved cells from copiotrophic bacteria can reach an interface, additional survival mechanisms become available to them: (i) interfaces constitute areas of favorable nutrient conditions, and (ii) interfaces lacking a sufficient amount of nutrient, nevertheless, trigger cells to become smaller, thus increasing their surface/volume ratio and the packing density.     

Fatty Tissue in Starved Lambs a Histoquantitative and Statistical Study

The kidney fat was histologically examined of 27 spontaneously dead lambs of which 14 had starved. The lambs were born with mature fat, but in young animals a starvation period of more than 24 hrs. reduced the fat tissues and changed its cells towards the embryonal type built up of preadipocytes. These cells were smaller than the mature fat cells. Nucleus was large, round and situated in the centre of the cell. The slight eosinophilic, strongly diminished cytoplasm was some granulated and had some small fat droplets. The starvation changes of fat cells did not depend on the weight of animals or on the age of lambs less than two weeks.     

Cycloheximide Insensitive Amoebo-Flagellate Transformation in Starved Amoebae of Physarum polycephalum

The requirement of protein synthesis for amoebo-flagellate transformation of Physarum polycephalum was re-examined. When amoebae were grown on nutrient agar in association with live food bacteria and harvested in mid-exponential phase of growth, it took ca. 2 hours for half the cells to form flagella after suspension in phosphate buffer. The transformation was completely inhibited by 5 μg/ml cycloheximide. To the contrary, when the amoebae in mid-exponential phase were starved for 3 hr on non-nutrient agar and then suspended in phosphate buffer, the duration required for this process was shortened to ca. 8 min and it was not inhibited by up to 100 μg/ml cycloheximide. A similar result was obtained using bactobolin, another inhibitor of protein synthesis. When amoebae were starved on non-nutrient agar containing 5 μg/ml cycloheximide, however, the starvation effect described above was not observed. The results indicate that protein(s) necessary for the transformation might be synthesized during the starvation period, and that the amoebo-flagellate transformation may or may not require concomitant protein synthesis depending upon preculture conditions.     

Kinetic Differences Between Fed and Starved Chinese Hamster Ovary Cells

When Chinese hamster (CHO-K1) cells are grown as monolayer cultures, they eventually reach a population-density plateau after which no net increase in cell numbers occurs. the kinetics of aged cells in nutritionally deprived (starved) or density-inhibited (fed) late plateau-phase cultures were studied by four methods: (i) Reproductive integrity and cell viability were monitored daily by clonogenic-cell assay and erythrosin-b dye-exclusion techniques. (ii) Mitotic frequencies of cells from 18 day old cultures were determined during regrowth by analysing time-lapse video microscope records of dividing cells. (iii) Tritiated-thymidine ([³H]TdR) auto-radiography was used to determine the fractions of DNA-synthesizing cells in cultures entering plateau phase and during regrowth after harvest. (iv) the rate of labelled nucleoside uptake and incorporation into DNA was measured using liquid scintillation or sodium iodide crystal counters after labelling with [³H]TdR or [¹²⁵]UdR. Non-cycling cells in starved cultures accumulate primarily as G₁, phase cells. Most cells not in G₁ phase had stopped in G₂, phase. Very few cells (< 2%) were found in S phase. In contrast, about half of the cells in periodically fed cultures were found to be in DNA-synthetic phase, and the percentage of these S phase cells fluctuated in a manner reflecting the frequency of medium replacement. Populations of both types of plateau-phase cultures demonstrate extremely coherent cyclic patterns of DNA synthesis upon harvest and reculturing. They retain this high degree of synchrony for more than three generations after the resumption of growth. From these data it is concluded that nutritionally deprived (starved) late plateau-phase cells generally stop in either G₁, or G₂, phase, whereas periodically fed late plateau-phase cultures contain a very large fraction of cycling cells. Populations of cells from these two types of non-expanding cultures are kinetically dissimilar, and should not be expected to respond to extracellular stimuli in the same manner.     

Ultrastructural Localization of Acid Phosphatase in Starved Tokophrya infusionum*

SYNOPSIS. Young organisms of Tokophrya infusionum starved for several hr, are best suited for a study of the fine structure of this organism including the distribution of its organelles. Acid phosphatase was localized by a combined electron microscopy and cytochemical approach using modified Gomori methods. The enzyme was found in small dense bodies, spheroid vesicles, missile-like bodies, rough-surfaced endoplasmic reticulum, residue and autophagic vacuoles. The small dense bodies are thought to be primary lysosomes since electron micrographs show a) a continuity between the membrane of the rough-surfaced endoplasmic reticulum and that of the dense bodies and b) a connection between the contents of both structures when the dense bodies form from the endoplasmic reticulum.     

Ribosome Degradation and the Degradation Products in Starved Escherichia coli

The properties of the abnormal ribonucleoprotein particles produced by Escherichia coli Q-13 starved for glucose were studied. Smaller species of these partially deproteinized particles separable to six distinct sizes contained partially degraded ribonucleic acids. The mode of ribosome degradation under this condition is discussed in terms of differential appearance of these intermediate particles.     

Plugging of a Model Rock System by Using Starved Bacteria

The effects of starvation on bacterial penetration through artificial rock cores were examined. Klebsiella pneumoniae was starved in a simple salts solution for a duration of up to 4 weeks. These cell suspensions were injected into sintered glass bead cores, and the resulting reductions in core permeabilities were recorded. Vegetative cell cultures of K. pneumoniae grown in a sodium citrate medium were injected into other, similar cores, and the reductions in core permeabilities were recorded. The starved cell suspensions did not completely block the core pores, whereas the vegetative cultures reduced core permeability to less than 1%. Scanning electron microscopy of core sections infiltrated with either vegetative or starved cells showed that the former produced shallow “skin” plugs and copious amounts of glycocalyx at the inlet face, whereas the latter produced very little glycocalyx and the cells were distributed evenly throughout the length of the core. The use of a DNA assay to produce a cell distribution profile showed that, compared with the vegetative cells, starved bacteria were able to penetrate deeper into the cores. This was due to the smaller size of the cells and the reduction in biofilm production. This ability of starved bacteria to penetrate further into cores than the normal-size vegetative cells can be usefully applied to selective plugging for enhanced oil recovery. To further test the suitability of starved cells for use in selective plugging, the activities of starved cells present within cores were monitored before and after nutrient stimulation. Our data indicate that with nutrient stimulation, the starved cells lose their metabolic dormancy and produce reductions in core permeability due to cell growth and polymer production.     

Synthesis of Unmethylated Deoxyribonucleic Acid in a dnaB Temperature-Sensitive Mutant of Escherichia coli Starved for Methionine

Strain CRT 266, a polyauxotrophic dnaB temperature-sensitive mutant of Escherichia coli K, was investigated for residual deoxyribonucleic acid (DNA) synthesis when returned to the permissive temperature in the absence of protein synthesis. In the presence of methionine, a delayed extra-initiation occurs as well as an erratic long-lasting synthesis. In the absence of methionine, there is no evidence for extra-initiation, whereas the long-lasting synthesis is only slightly depressed. A direct role of methionine independent of protein synthesis in the extra-initiation process is postulated. The largest residual syntheses, with or without methionine, are obtained when (i) bacteria are first grown in a rich medium, (ii) bacteria are shifted to the nonpermissive temperature for 2 h in the same medium, and (iii) bacteria are then starved for aminoacid for 20 h at the permissive temperature. Under these conditions, DNA extracted from methionine-starved cells appears to be a mixture of half-methylated and unmethylated products. The possibility of the occurence of a few methyl groups on the so-called unmethylated DNA is discussed.     

Eukaryotic Translation Initiation Factor 4E-Dependent Translation Is Not Essential for Survival of Starved Yeast Cells

The eukaryotic translation initiation factor 4E (eIF4E) interacts with the mRNA 5' cap structure (m(7)GpppX) and is essential for the appropriate translation of the vast majority of eukaryotic mRNAs. Most studies of the yeast Saccharomyces cerevisiae CDC33 gene product, eIF4E, have been carried out with logarithmically growing cells, and little is known about its role in starved, nonproliferating cells that enter the stationary phase (SP). It has previously been found that the rate of translation in SP cells is more than 2 orders of magnitude lower than it is in dividing yeast cells. Here we show that this low rate of translation is essential for maintaining the viability of starved yeast cells that enter SP. Specifically, starved cells whose eIF4A is inactive or treated with cycloheximide rapidly lose viability. Moreover, after heat inactivation of the cdc33 temperature-sensitive product, the synthesis of most proteins is abolished and only a small group of proteins is still produced. Unexpectedly, starved cdc33 mutant cells whose eIF4E is inactive and which therefore fail to synthesize the bulk of their proteins remain viable for long periods of time, indistinguishable from their isogenic wild-type counterparts. Taken together, our results indicate that eIF4E-independent translation is necessary and sufficient for survival of yeast cells during long periods of starvation.