Project description:We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue. Changes in polysomes are easily quantified between treatments, such as the cellular response to amino acid starvation. Ribo Mega-SEC is shown to provide an efficient, convenient and highly reproducible method for studying functional translation complexes. We show that Ribo Mega-SEC is readily combined with high-throughput MS-based proteomics to characterize proteins associated with polysomes and ribosomal subunits. It also facilitates isolation of complexes for electron microscopy and structural studies.
Project description:Starvation is among the most ancient of selection pressures, driving evolution of a robust arsenal of starvation survival defenses. In order to survive starvation stress, organisms must be able to curtail anabolic processes during starvation and judiciously activate catabolic pathways. Although the activation of metabolic defenses in response to nutrient deprivation is an obvious component of starvation survival, less appreciated is the importance of the ability to recover from starvation upon re-exposure to nutrients. In order for organisms to successfully recover from starvation, cells must be kept in a state of ready so that upon the return of nutrients, activities such as growth and reproduction can be resumed. Critical to this state of ready is the lysosome, an organelle that provides essential signals of nutrient sufficiency to cell growth-activating pathways in the fed state. In this issue, Murphy and colleagues provide evidence that exposure of Caenorhabditis elegans roundworms to 2 simple nutrients, glucose and the polyunsaturated fatty acid linoleate, is able to render lysosomal function competent to activate key downstream starvation recovery pathways, bypassing the need for a master transcriptional regulator of lysosomes. These findings provide a quantum leap forward in our understanding of the cellular determinants that permit organisms to survive cycles of feast and famine.
Project description:A universal feature of the response to stress and nutrient limitation is transcriptional upregulation of genes encoding proteins important for survival. Interestingly, under many of these conditions overall protein synthesis levels are reduced, thereby dampening the stress response at the level of protein expression. For example, during glucose starvation in yeast, translation is rapidly and reversibly repressed, yet transcription of many stress- and glucose-repressed genes is increased. Using ribosome profiling and microscopy, we found that this transcriptionally upregulated gene set consists of two classes: (1) one producing mRNAs that are preferentially translated during glucose limitation and are diffusely localized in the cytoplasm – this class includes many heat shock protein mRNAs; and (2) another producing mRNAs that are poorly translated during glucose limitation, have high rates of translation initiation, and are concentrated in foci that co-localize with P bodies and stress granules – this class is enriched for glucose metabolism mRNAs. Remarkably, the information specifying differential localization and translation of these two classes of mRNAs is encoded in the promoter sequence – promoter responsiveness to heat shock factor (Hsf1) specifies diffuse cytoplasmic localization and preferential translation upon glucose starvation, whereas different promoter elements upstream of genes encoding poorly translated glucose metabolism mRNAs direct these mRNAs to RNA granules under glucose starvation. Thus, promoter sequences and transcription factor binding can influence not only mRNA levels, but also subcellular localization of mRNAs and the efficiency with which they are translated, enabling cells to tailor protein production to environmental conditions. Examination of mRNA translation in S. cerevisiae upon glucose starvation.
Project description:How energy deprivation induces macroautophagy/autophagy is not fully understood. Here, we show that Atg11, a receptor protein for cargo recognition in selective autophagy, is required for the initiation of glucose starvation-induced autophagy. Upon glucose starvation, Atg11 facilitates the interaction between Snf1 and Atg1, thus is required for Snf1-dependent Atg1 activation. Phagophore assembly site (PAS) formation requires Atg11 via its control of the association of Atg17 with Atg29-Atg31. The binding of Atg11 with Atg9 is crucial for recruiting Atg9 vesicles to the PAS and, thus, glucose starvation-induced autophagy. We propose Atg11 as a key initiation factor controlling multiple key steps in energy deprivation-induced autophagy. <b>Abbreviations:</b> AMPK: AMP-activated protein kinase; Ams1: α-mannosidase; Ape1: aminopeptidase I; Cvt: cytoplasm-to-vacuole targeting; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent protein; MBP: myelin basic protein; MMS: methanesulfonate; PAS: phagophore assembly site; PNBM: p-nitrobenzyl mesylate; SD-G: glucose starvation medium; SD-N: nitrogen starvation medium; ULK1, unc-51 like autophagy activating kinase 1; WT: wild type.
Project description:The transition from the fed to the fasted state necessitates a shift from carbohydrate to fat metabolism that is thought to be mostly orchestrated by reductions in plasma insulin concentrations. Here, we show in awake rats that insulinopenia per se does not cause this transition but that both hypoleptinemia and insulinopenia are necessary. Furthermore, we show that hypoleptinemia mediates a glucose-fatty acid cycle through activation of the hypothalamic-pituitary-adrenal axis, resulting in increased white adipose tissue (WAT) lipolysis rates and increased hepatic acetyl-coenzyme A (CoA) content, which are essential to maintain gluconeogenesis during starvation. We also show that in prolonged starvation, substrate limitation due to reduced rates of glucose-alanine cycling lowers rates of hepatic mitochondrial anaplerosis, oxidation, and gluconeogenesis. Taken together, these data identify a leptin-mediated glucose-fatty acid cycle that integrates responses of the muscle, WAT, and liver to promote a shift from carbohydrate to fat oxidation and maintain glucose homeostasis during starvation.
Project description:Mitochondria undergo morphological and dynamic changes in response to environmental stresses. Few studies have focused on addressing mitochondrial remodeling under stress. Using the fission yeast Schizosaccharomyces pombe as a model organism, here we investigated mitochondrial remodeling under glucose starvation. We employed live-cell microscopy to monitor mitochondrial morphology and dynamics of cells in profusion chambers under glucose starvation. Our results revealed that mitochondria fragment within minutes after glucose starvation and that the dynamin GTPase Dnm1 is required for promoting mitochondrial fragmentation. Moreover, we found that glucose starvation enhances Dnm1 localization to mitochondria and increases the frequency of mitochondrial fission but decreases PKA activity. We further demonstrate that low PKA activity enhances glucose starvation-induced mitochondrial fragmentation, whereas high PKA activity confers resistance to glucose starvation-induced mitochondrial fragmentation. Moreover, we observed that AMP-activated protein kinase is not involved in regulating mitochondrial fragmentation under glucose starvation. Of note, glucose starvation-induced mitochondrial fragmentation was associated with enhanced reactive oxygen species production. Our work provides detailed mechanistic insights into mitochondrial remodeling in response to glucose starvation.
Project description:<h4>Motivation</h4>Ribosome Profiling (Ribo-seq) has revolutionized the study of RNA translation by providing information on ribosome positions across all translated RNAs with nucleotide-resolution. Yet several technical limitations restrict the sequencing depth of such experiments, the most common of which is the overabundance of rRNA fragments. Various strategies can be employed to tackle this issue, including the use of commercial rRNA depletion kits. However, as they are designed for more standardized RNAseq experiments, they may perform suboptimally in Ribo-seq. In order to overcome this, it is possible to use custom biotinylated oligos complementary to the most abundant rRNA fragments, however currently no computational framework exists to aid the design of optimal oligos.<h4>Results</h4>Here, we first show that a major confounding issue is that the rRNA fragments generated via Ribo-seq vary significantly with differing experimental conditions, suggesting that a "one-size-fits-all" approach may be inefficient. Therefore we developed Ribo-ODDR, an oligo design pipeline integrated with a user-friendly interface that assists in oligo selection for efficient experiment-specific rRNA depletion. Ribo-ODDR uses preliminary data to identify the most abundant rRNA fragments, and calculates the rRNA depletion efficiency of potential oligos. We experimentally show that Ribo-ODDR designed oligos outperform commercially available kits and lead to a significant increase in rRNA depletion in Ribo-seq.<h4>Availability</h4>Ribo-ODDR is freely accessible at https://github.com/fallerlab/Ribo-ODDR.<h4>Supplementary information</h4>Supplementary data are available at Bioinformatics online.
Project description:We investigated the extent to which increases in glucose utilization indices (GUIs) in individual skeletal muscles during chow re-feeding after 6 h, 24 h or 48 h starvation are related to the antecedent duration of starvation. Chow re-feeding after either acute or prolonged starvation led to an increase in glucose disposal by the muscle mass. Glucose intolerance after prolonged starvation was not associated with lower values of GUI in skeletal muscle. In both working and non-working muscles, the increment in GUI during the first 2 h of re-feeding was less after acute than after prolonged starvation. In non-working muscles the differential responses to re-feeding were due to higher GUI values after re-feeding rather than lower pre-prandial GUI values. Therefore the contribution of non-working muscles to glucose clearance is higher as the antecedent period of starvation is extended. Rates of glycogen deposition in non-working muscles after refeeding were similar to absolute values of GUI, and a strong relationship existed between measured GUI values and rates of glycogen deposition.