Princz, Andrea et al. (2017) International Worm Meeting "SUMOylation regulates lifespan and mitochondrial homeostasis in C. elegans."

Princz, Andrea, Tavernarakis, Nektarios

[International Worm Meeting, 2017]

Posttranslational modifications have pivotal roles in numerous cellular processes. SUMOylation, the attachment of SUMO (small ubiquitin-related modifier) to a protein, is implicated in the regulation of DNA damage response, cell division, sub-cellular protein localization and protein-protein interactions, among others. Protein SUMOylation levels increase progressively during ageing. Nevertheless, whether elevated SUMOylation is only an unrelated consequence of the ageing process or it serves a causative, regulatory role in senescent decline is not understood. The C. elegans genome contains a single gene encoding SUMO (smo-1), rendering the nematode a convenient model in which to genetically dissect the role of SUMOylation in organismal physiology and ageing. The insulin/IGF-1 pathway is a well-characterized and conserved signal transduction cascade, which has a major role in determining the lifespan of animals, mainly through the DAF-16/FOXO transcription factor and the stress response-related transcription factor SKN-1/NRF2. Interestingly, these two key transcription factors contain putative SUMOylation sites. Deletion of smo-1 causes embryonic lethality. However, we find that RNAi knockdown of smo-1 initiated at the L4 stage shortens the lifespan of both wild type and long-lived animals. Notably, knockdown of a SUMO protease gene (ulp-1), extends the lifespan of long-lived mutants (clk-1, daf-2, ife-2), but does not modify the longevity of wild type animals. The lifespan-altering effect of SUMOylation is tissue-specific. In addition, we observed that manipulation of SUMOylation levels by either knockdown of smo-1 or ulp-1 influences the activity of DAF-16 and SKN-1, as well as, stress resistance, energy metabolism and mitochondrial homeostasis, in a genetic background- and age-dependent manner. These findings directly implicate SUMOylation in the regulation of key stress responses and longevity.

Princz, Andrea et al. (2015) International Worm Meeting "Modulation of longevity and stress response pathways by SUMOylation in C. elegans."

Princz, Andrea, Tavernarakis, Nektarios

[International Worm Meeting, 2015]

SUMOylation, the attachment of SUMO (small ubiquitin-related modifier) to a protein, is a posttranslational modification implicated in the regulation of diverse cellular processes, including the DNA damage response, sub-cellular protein localization and protein-protein interactions, among others. Protein SUMOylation levels increase progressively during ageing. However, whether elevated SUMOylation is merely a corollary of the ageing process or it serves a causative, regulatory role in senescent decline is not understood. Interestingly, DAF-16/FOXO and SKN-1/NRF2, two key transcription factors regulating various stress responses and longevity in the nematode Caenorhabditis elegans, contain putative SUMOylation sites. The C. elegans genome contains only one gene encoding SUMO (smo-1), rendering the nematode a convenient model in which to genetically dissect the role of SUMOylation in organismal physiology and ageing. Elimination of smo-1 causes embryonic lethality. Nevertheless, we find that RNAi knockdown of smo-1 initiated at the L4 stage shortens the lifespan of both wild type and long-lived animals. This effect is not dependent on neuron-specific knockdown of smo-1. Notably, knockdown of the SUMO protease encoding gene (ulp-1), extents lifespan in long-lived mutants (clk-1, daf-2, ife-2), but not in wild type animals. In addition, we observed that manipulation of SUMOylation levels by either knockdown of smo-1 or ulp-1 influences the activity of DAF-16 and SKN-1, as well as stress resistance and energy metabolism, in a genetic background- and age-dependent manner. We are currently investigating the mechanisms by which SUMOylation modulates the activity of key, stress response transcription regulators, and how these mechanisms interface with main signalling pathways that impinge on longevity.

Rieckher, Matthias et al. (2015) International Worm Meeting "mRNA decay interfaces with protein synthesis to modulate stress resistance and longevity in C. elegans."

Tavernarakis, Nektarios, Princz, Andrea, Markaki, Maria, Rieckher, Matthias

[International Worm Meeting, 2015]

Attenuation of protein synthesis has been demonstrated to increase lifespan across taxa. Recent findings show that activators of mRNA degradation suppress mRNA translation by targeting mRNAs for storage and decapping in cytoplasmic Processing (P) bodies. We hypothesized that storage and degradation of mRNAs in P bodies may regulate physiological processes such as stress responses and ageing via the control of mRNA translation. We observed that somatic P bodies increase in size and number during ageing in C. elegans. IFE-2, an isoform of the translation initiation factor eIF4E, progressively localizes to P bodies upon stress and during ageing. Loss of the P body factor "enhancer of mRNA decapping" EDC-3 promotes longevity and resistance to stress. EDC-3 mutants display reduced protein synthesis in somatic cells. Furthermore, lack of EDC-3 leads to increased P body formation. Notably, longevity and stress resistance in EDC-3-depleted animals is rescued by neuron-specific EDC-3 expression. We find that the transcription factor SKN-1, which also mediates lifespan extension by eIF4E deficiency in somatic C. elegans tissues, is required for the effects of EDC-3 depletion on stress resistance and lifespan. Further, loss of SKN-1 robustly enhances P body formation in EDC-3 mutants. We conclude that upon mRNA decapping impairment, eIF4E becomes sequestered in P bodies, which in turn downregulates general protein synthesis in the soma, thereby increasing organismal lifespan. Our findings indicate a causative role for P bodies in the regulation of stress resistance and ageing.

Pierleoni A et al. (2006) Bioinformatics "BaCelLo: a balanced subcellular localization predictor."

Casadio R, Martelli PL, Fariselli P, Pierleoni A

[Bioinformatics, 2006]

Motivation. The knowledge of the subcellular localization of a protein is fundamental for elucidating its function. It is difficult to determine the subcellular location for eukaryotic cells with experimental high-throughput procedures. Computational procedures are then needed for annotating the subcellular location of proteins in large scale genomic projects. Results. BaCelLo is a predictor for five classes of subcellular localization (secretory pathway, cytoplasm, nucleus, mitochondrion and chloroplast) and it is based on different SVMs organized in a decision tree. The system exploits the information derived from the residue sequence and from the evolutionary information contained in alignment profiles. It analyzes the whole sequence composition and the compositions of both the N- and C-termini. The training set is curated in order to avoid redundancy. For the first time a balancing procedure is introduced in order to mitigate the effect of biased training sets. Three kingdom-specific predictors are implemented: for animals, plants and fungi, respectively. When distributing the proteins from animals and fungi into four classes, accuracy of BaCelLo reach 74% and 76%, respectively; a score of 67% is obtained when proteins from plants are distributed into five classes. BaCelLo outperforms the other presently available methods for the same task and gives more balanced accuracy and coverage values for each class. We also predict the subcellular localization of five whole proteomes, Homo sapiens, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae and Arabidopsis thaliana, comparing the protein content in each different compartment. Availability. BaCelLo can be accessed at http://www.biocomp.unibo.it/bacello/ Contact. casadio@alma.unibo.it, andrea@biocomp.unibo.it, gigi@biocomp.unibo.it, piero@biocomp.unibo.it.

Siddiqui SS et al. (1990) Worm Breeder's Gazette "Scanning Tunneling Microscopy (STM) of C. elegans Microtubules"

McKuskey B, Hameroff S, Siddiqui SS, Ward S, Sarid D

[Worm Breeder's Gazette, 1990]

We have been studying C. elegans microtubules from cellular and genetic aspects. Since scanning tunneling microscopy (STM) can provide atomic resolution of metals and semiconductive surfaces; our objective is to obtain images of C. elegans microtubules resolved at the atomic level. However, application of STM to biomolecules is faced with a variety of problems, including poor electrical conductivity, flexible stability under STM imaging conditions. In spite of limitations, recently, impressive images of DNA biomolecules have been obtained at atomic resolution. Microtubules (MT) were isolated from 30g of freshly grown N2 worms according to the procedure of Aamdot and Culotti 1986, and Siddiqui et al 1989, using cycles of temperature dependent assembly and disassembly (Shelanski, et al. 1973). Assembled MT were taken in MT isolation buffer (containing 0.1mM GTP, 20 M taxol, and 2.0 mM DTE), diluted tenfold and fixed in glutaraldehyde (0.1%). The samples were kept frozen at -20 C, for 4-5 days. The STM imaging samples were thawed, and a drop was smeared onto a chip of freshly cleaved HOPG ( highly oriented pyrolitic graphite, Union Carbide), and allowed to air dry at room temperature. The STM images were obtained using 'Nanascope II Digital Instruments, California (Sam Howell and C. Lee, kindly helped with the STM settings). All STM imaging was conducted at atmospheric pressure and at room temperature (22 C). Two different STM images of C. elegans MT are shown here. [See Figure 1] Most of our STM scans of MT correspond to one or two microtubules, but they appear flattened and bent along the seams of the protofilaments. The dimensions of alpha and -tubulin dimer subunits are about 8 nm, and each protofilament has a width of 4.6 nm. The flattening and buckeling of protofilaments along the seams could arise due to the fixation conditions, or caused by the STM current flow through the specimen. At present, we are unable to attribute this observation to any single factor, and the images of MT do not provide any higher resolution than conventional electron microscopy using negative staining. But it is increasingly possible that one may be able to resolve individual amino acids or even atoms using STM. We plan to continue looking for improved conditions for better STM or AFM images of C. elegans MT. Brief description of STM: Scanning tunneling microscopy (STM) traces three dimensional images of surfaces - even at the level of individual atoms. Gerd Binnig & Heinrich Rohrer of IBM Zurich received the Nobel Prize in 1986 for STM (Refer, e.g.: Scientific American, August,1985). Briefly in STM, the 'aperture' is a small tungston probe, its tip extremely fine (about 0. 2 nm in width, and may consist of a single atom). Piezoelectric controls move the tip within a nanometer or two of the surface of the conducting sample, allowing an overlap between the electron clouds of the atom at the probe tip and the closest atom of the sample placed on the surface. As a small voltage is applied to the tip, electrons 'tunnel' across this gap, creating a tiny tunneling current. The strength of the tunneling current is exponentially sensitive to the width of the gap (about an order of magnitude per angstrom). Depending on the substrate, typical currents and voltages are in the range of nanoamperes and millivolts. A servo system uses a feedback control that keeps the tip to substrate gap constant, by modulating the voltage across a piezoelectric positioning system (Piezoceramic materials expand or shrink extremely small distances (i.e. angstroms ) in response to the applied voltage. As the tip scans the surface, variations in this voltage, when plotted correspond to surface structure. I wish to thank members of Ward lab (Alicia, Andrea, Bill, Bonnie, Bruce, Jacob, John and Paul); Hameroff Lab (Dolly, Larry, Mohammad, and Richard); and Sarid Lab (Sam and Lee).