-
[
J Biol Chem,
2004]
Studies using the nematode Caenorhabditis elegans as a model system to investigate the aging process have implicated the insulin/insulin-like growth factor-I signaling pathway in the regulation of organismal longevity through its action on a subset of target genes. These targets can be classified into genes that shorten or extend life-span upon their induction. Genes that shorten life-span include a variety of stress response genes, among them genes encoding catalases; however, no evidence directly implicates catalases in the aging process of nematodes or other organisms. Using genetic mutants, we show that lack of peroxisomal catalase CTL-2 causes a progeric phenotype in C. elegans. Lack of peroxisomal catalase also affects the developmental program of C. elegans, since Deltactl-2 mutants exhibit decreased egg laying capacity. In contrast, lack of cytosolic catalase CTL-1 has no effect on either nematode aging or egg laying capacity. The Deltactl-2 mutation also shortens the maximum life-span of the long lived Deltaclk-1 mutant and accelerates the onset of its egg laying period. The more rapid aging of Deltactl-2 worms is apparently not due to increased carbonylation of the major C. elegans proteins, although altered peroxisome morphology in the Deltactl-2 mutant suggests that changes in peroxisomal function, including increased production of reactive oxygen species, underlie the progeric phenotype of the Deltactl-2 mutant. Our findings support an important role for peroxisomal catalase in both the development and aging of C. elegans and suggest the utility of the Deltactl-2 mutant as a convenient model for the study of aging and the human
-
[
J Mol Biol,
2004]
All organisms except the nematode Caenorhabditis elegans have been shown to possess an import system for peroxisomal proteins containing a peroxisome targeting signal type 2 (PTS2). The currently accepted consensus sequence for this amino-terminal nonapeptide is -(R/K)(L/V/I)X-5(H/Q)(L/A)-. Some C. elegans proteins contain putative PTS2 motifs, including the ortholog (CeMeK) of human mevalonate kinase, an enzyme known to be targeted by PTS2 to mammalian peroxisomes. We cloned the gene for CeMeK (open reading frame Y42G9A.4) and examined the subcellular localization of CeMeK and of two other proteins with putative PTS2s at their amino termini encoded by the open reading frames D1053.2 and W10G11.11. All three proteins localized to the cytosol, confirming and extending the finding that C. elegans lacks PTS2-dependent peroxisomal protein import. The putative PTS2s of the proteins encoded by D1053.2 and W10G11.11 did not function in targeting to peroxisomes in yeast or mammalian cells, suggesting that the current PTS2 consensus sequence is too broad. Analysis of available experimental data on both functional and nonfunctional PTS2s led to two re-evaluated PTS2 consensus sequences: -R(L/V/I/Q)XX(L/V/I/H)(L/S/G/A)X(H/Q)(L/A)-, describes the most common variants of PTS2, while -(R/K)(L/V/I/Q)XX(L/V/I/H/ Q)(L/S/G/A/K)X(H/Q)(L/A/F)-, describes essentially all variants of PTS2. These redefined PTS2 consensus sequences will facilitate the identification of proteins of unknown cellular localization as possible peroxisomal proteins.
-
[
Physiol Genomics,
2002]
RNA-mediated interference (RNAi) for the posttranscriptional silencing of genes was used to evaluate the importance of various peroxisomal enzymes and peroxins for the development of Caenorhabditis elegans and to compare the roles of these proteins in the nematode to their roles in yeasts and humans. The nematode counterparts of the human ATP-binding cassette half-transporters, the enzymes alkyldihydroxyacetonephosphate synthase and Delta(3,5-)Delta(2,4) di-enoyl- CoA isomerase, the receptors for peroxisomal membrane and matrix proteins (Pex19p and Pex5p), and components of the docking and translocation machineries for matrix proteins (Pex13p and Pex12p) are essential for the development of C. elegans. Unexpectedly, RNAi silencing of the acyl-CoA synthetase-mediated activation of fatty acids, the alpha- and beta-oxidation of fatty acids, the intraperoxisomal decomposition of hydrogen peroxide, and the peroxins Pex1p, Pex2p, and Pex6p had no apparent effect on C. elegans development. The described analysis of functional gene knockouts through RNAi provides a basis for the use of C. elegans as a valuable model system with which to study the molecular and physiological defects underlying the human
-
[
Exp Cell Res,
2015]
In order to obtain fine details in 3 dimensions (3D) over time, it is critical for motile biological specimens to be appropriately immobilized. Of the many immobilization options available, the mechanical microcompressor offers many benefits. Our device, previously described, achieves gentle flattening of a cell, allowing us to image finely detailed structures of numerous organelles and physiological processes in living cells. We have imaged protozoa and other small metazoans using differential interference contrast (DIC) microscopy, orientation-independent (OI) DIC, and real-time birefringence imaging using a video-enhanced polychromatic polscope. We also describe an enhancement of our previous design by engineering a new device where the coverslip mount is fashioned onto the top of the base; so the entire apparatus is accessible on top of the stage. The new location allows for easier manipulation of the mount when compressing or releasing a specimen on an inverted microscope. Using this improved design, we imaged immobilized bacteria, yeast, paramecia, and nematode worms and obtained an unprecedented view of cell and specimen details. A variety of microscopic techniques were used to obtain high resolution images of static and dynamic cellular and physiological events.