Sterol effects and sites of sterol accumulation in Caenorhabditis elegans

Abstract

Caenorhabditis elegans requires sterol, usually supplied as cholesterol, but this is enzymatically modified, and different sterols can substitute. Sterol deprivation decreased brood size and adult growth in the first generation, and completely, reversibly, arrested growth as larvae in the second. After one generation of sterol deprivation, 10 ng/ml cholesterol allowed delayed laying of a few eggs, but full growth required 300 ng/ml. C. elegans synthesizes two unusual 4 ␣ -methyl sterols (4MSs), but each 4MS supported only limited growth as the sole sterol. However, addition of only 10 ng of cholesterol to 1,000 ng of 4MS restored full growth and egg-laying, suggesting that both a 4MS and an unmethylated sterol are required for development. Filipin stained sterols in only a few specific cells: the excretory gland cell, two amphid socket cells, two phasmid socket cells and, in males, spicule socket cells. Sterols were also present in the pharynx and in the intestine of feeding animals in a proximal-to-distal gradient.This non-random sterol distribution, the low concentration requirements, and the effects of 4MSs argues that sterols are unlikely to be used for bulk structural modification of cell membranes, but may be required as hormone precursors and/or developmental effectors. Dietary sterol is required by Caenorhabditis elegans (1, 2) because, like insects, C. elegans is incapable of synthesizing the four-ring sterol nucleus, but its functions remain largely unknown. The existence of sterol-based hormones in C. elegans has recently been suggested, but no hormone has yet been identified (3, 4). Cholesterol is known to be extensively metabolized by C. elegans to form several other sterols, including two unusual 4 ␣ -methyl sterols (4MSs), which are present in substantial amounts (5-8, 9). These sterols might thus be functional, instead of or in addition to cholesterol itself.Insects resemble these nematodes in requiring sterols but being unable to synthesize them. Two functions for cholesterol are known in insects: as the metabolic precursor of the molting hormone ecdysone (10), and as the moiety required for activation by covalent attachment to the morphogen protein hedgehog (11). Insect cells, unlike vertebrate cells, grow normally under sterol-free conditions, and thus do not need cholesterol in their plasma membranes (12)(13)(14).We have now characterized the sterol requirements of C. elegans in some detail. Conditions for stringent sterol deprivation were developed, and the consequences are described. The use of these conditions allowed us to investigate minimum cholesterol requirements, and the ability of other sterols to substitute. Partial and synergistic effects were found, suggesting that different sterols have diverse effects mediated by several pathways. The accumulation of sterol in the intestinal tract and in a few specific cells in C. elegans was also demonstrated by filipin staining, which stains all 3 ␤ -hydroxy sterols.These observations provide a basis for a comprehensive study of ster...

RAB-10 Regulates Glutamate Receptor Recycling in a Cholesterol-dependent Endocytosis Pathway

“…Interestingly, a stronger requirement for RAB-10 is observed for the recycling of clathrin-independent cargo versus clathrin-dependent cargo in intestinal epithelia, suggesting that RAB-10 likely recycles receptors that are endocytosed primarily by clathrin-independent endocytosis . Most forms of clathrin-independent endocytosis proceed through lipid rafts rich in cholesterol; cholesterol depletion inhibits lipid raft formation and depresses clathrin-independent endocytosis (Johannes and Lamaze, 2002;Merris et al, 2003;Parton and Richards, 2003;Kirkham and Parton, 2005;Le Roy and Wrana, 2005). We find that growth on cholesterol-depleted media suppresses the accumulation of GLR-1 in rab-10 but not lin-10 mutants.…”

“…Clathrin-independent endocytosis is highly sensitive to cholesterol levels, and it is depressed under conditions of low cholesterol (Johannes and Lamaze, 2002;Parton and Richards, 2003;Kirkham and Parton, 2005;Le Roy and Wrana, 2005). We grew wild-type, lin-10, and rab-10 animals on cholesteroldepleted media for one generation, and then we examined GLR-1 localization in these animals (Merris et al, 2003(Merris et al, , 2004. Because cholesterol depletion did not affect all animals equally, we categorized GLR-1 localization for each animal as either "no phenotype" (most GLR-1 puncta are small, 0.4 -0.8 m in diameter), "medium phenotype" (animals had mostly small puncta but some larger GLR-1 puncta, 0.8 -1.2 m in diameter), or "strong phenotype" (most GLR-1 is in large accretions, usually Ͼ1.5 m in length).…”

“…For cholesterol-depleted growth of C. elegans, both OP50 bacterial media and C. elegans plate media were prepared using ether-extracted peptone as described previously (Merris et al, 2003). Gravid animals were placed on plates containing cholesterol-depleted media, and they were allowed to lay eggs overnight.…”

The Caenorhabditis elegans IMPAS gene, imp-2, is essential for development and is functionally distinct from related presenilins

“…Worms were observed by using a dissecting microscope with a maximum magnification of ϫ40-60 or a fluorescent dissecting stereo-microscope with a Nomarski optic and magnification of up to ϫ100. Filipin staining was performed as described (19). the cDNA template by PCR reaction with corresponding primers.…”

Endocrine targets for pharmacological intervention in aging in Caenorhabditis elegans

“…Under laboratory conditions C. elegans must be supplemented with exogenous cholesterol since nematodes are unable to synthesize their own sterols (Fabian & Johnson, 1994). Cholesterol deprivation in wild-type animals leads to a variety of phenotypes including developmental arrest and reduced fertility (Shim et al ., 2002;Merris et al ., 2003). Additionally, weak alleles of daf-9 that are thought to be compromised in their ability to synthesize the hormone often show an enhanced phenotype under conditions of cholesterol deprivation (Gerisch et al ., 2001;Jia et al ., 2002).…”