Wilhelm Palm

How Shelterin Protects Mammalian Telomeres

The genomes of prokaryotes and eukaryotic organelles are usually circular as are most plasmids and viral genomes. In contrast, the nuclear genomes of eukaryotes are organized on linear chromosomes, which require mechanisms to protect and replicate DNA ends. Eukaryotes navigate these problems with the advent of telomeres, protective nucleoprotein complexes at the ends of linear chromosomes, and telomerase, the enzyme that maintains the DNA in these structures. Mammalian telomeres contain a specific protein complex, shelterin, that functions to protect chromosome ends from all aspects of the DNA damage response and regulates telomere maintenance by telomerase. Recent experiments, discussed here, have revealed how shelterin represses the ATM and ATR kinase signaling pathways and hides chromosome ends from nonhomologous end joining and homology-directed repair.

Telomere protection by mammalian Pot1 requires interaction with Tpp1

The shelterin complex at mammalian telomeres contains the single-stranded DNA–binding protein Pot1, which regulates telomere length and protects chromosome ends. Pot1 binds Tpp1, the shelterin component that connects Pot1 to the duplex telomeric DNA–binding proteins Trf1 and Trf2. Control of telomere length requires that Pot1 binds Tpp1 as well as the single-stranded telomeric DNA, but it is not known whether the protective function of Pot1 depends on Tpp1. Alternatively, Pot1 might function similarly to the Pot1-like proteins of budding and fission yeast, which have no known Tpp1-like connection to the duplex telomeric DNA. Using mutant mouse cells with diminished Tpp1 levels, RNA interference directed to mouse Tpp1 and Pot1, and complementation of mouse Pot1 knockout cells with human and mouse Pot1 variants, we show here that Tpp1 is required for the protective function of mammalian Pot1 proteins.

Effects of diet and development on the Drosophila lipidome

Cells produce tens of thousands of different lipid species, but the importance of this complexity in vivo is unclear. Analysis of individual tissues and cell types has revealed differences in abundance of individual lipid species, but there has been no comprehensive study comparing tissue lipidomes within a single developing organism. Here, we used quantitative shotgun profiling by high‐resolution mass spectrometry to determine the absolute (molar) content of 250 species of 14 major lipid classes in 6 tissues of animals at 27 developmental stages raised on 4 different diets. Comparing these lipidomes revealed unexpected insights into lipid metabolism. Surprisingly, the fatty acids present in dietary lipids directly influence tissue phospholipid composition throughout the animal. Furthermore, Drosophila differentially regulates uptake, mobilization and tissue accumulation of specific sterols, and undergoes unsuspected shifts in fat metabolism during larval and pupal development. Finally, we observed striking differences between tissue lipidomes that are conserved between phyla. This study provides a comprehensive, quantitative and expandable resource for further pharmacological and genetic studies of metabolic disorders and molecular mechanisms underlying dietary response.

Engineered telomere degradation models dyskeratosis congenita

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by cutaneous symptoms, including hyperpigmentation and nail dystrophy. Some forms of DC are caused by mutations in telomerase, the enzyme that counteracts telomere shortening, suggesting a telomere-based disease mechanism. However, mice with extensively shortened telomeres due to telomerase deficiency do not develop the characteristics of DC, raising questions about the etiology of DC and/or mouse models for human telomere dysfunction. Here we describe mice engineered to undergo telomere degradation due to the absence of the shelterin component POT1b. When combined with reduced telomerase activity, POT1b deficiency elicits several characteristics of DC, including hyperpigmentation and fatal bone marrow failure at 4-5 mo of age. These results provide experimental support for the notion that DC is caused by telomere dysfunction, and demonstrate that key aspects of a human telomere-based disease can be modeled in the mouse.

Functional Dissection of Human and Mouse POT1 Proteins

ABSTRACT The single-stranded telomeric DNA binding protein POT1 protects mammalian chromosome ends from the ATR-dependent DNA damage response, regulates telomerase-mediated telomere extension, and limits 5′-end resection at telomere termini. Whereas most mammals have a single POT1 gene, mice have two POT1 proteins that are functionally distinct. POT1a represses the DNA damage response, and POT1b controls 5′-end resection. In contrast, as we report here, POT1a and POT1b do not differ in their ability to repress telomere recombination. By swapping domains, we show that the DNA binding domain of POT1a specifies its ability to repress the DNA damage response. However, no differences were detected in the in vitro DNA binding features of POT1a and POT1b. In contrast to the repression of ATR signaling by POT1a, the ability of POT1b to control 5′-end resection was found to require two regions in the C terminus, one corresponding to the TPP1 binding domain and a second representing a new domain located between amino acids (aa) 300 and 350. Interestingly, the DNA binding domain of human POT1 can replace that of POT1a to repress ATR signaling, and the POT1b region from aa 300 to 350 required for the regulation of the telomere terminus is functionally conserved in human POT1. Thus, human POT1 combines the features of POT1a and POT1b.

Lipoproteins in Drosophila melanogaster—Assembly, Function, and Influence on Tissue Lipid Composition

Interorgan lipid transport occurs via lipoproteins, and altered lipoprotein levels correlate with metabolic disease. However, precisely how lipoproteins affect tissue lipid composition has not been comprehensively analyzed. Here, we identify the major lipoproteins of Drosophila melanogaster and use genetics and mass spectrometry to study their assembly, interorgan trafficking, and influence on tissue lipids. The apoB-family lipoprotein Lipophorin (Lpp) is the major hemolymph lipid carrier. It is produced as a phospholipid-rich particle by the fat body, and its secretion requires Microsomal Triglyceride Transfer Protein (MTP). Lpp acquires sterols and most diacylglycerol (DAG) at the gut via Lipid Transfer Particle (LTP), another fat body-derived apoB-family lipoprotein. The gut, like the fat body, is a lipogenic organ, incorporating both de novo–synthesized and dietary fatty acids into DAG for export. We identify distinct requirements for LTP and Lpp-dependent lipid mobilization in contributing to the neutral and polar lipid composition of the brain and wing imaginal disc. These studies define major routes of interorgan lipid transport in Drosophila and uncover surprising tissue-specific differences in lipoprotein lipid utilization.

Survival strategies of a sterol auxotroph

The high sterol concentration in eukaryotic cell membranes is thought to influence membrane properties such as permeability, fluidity and microdomain formation. Drosophila cannot synthesize sterols, but do require them for development. Does this simply reflect a requirement for sterols in steroid hormone biosynthesis, or is bulk membrane sterol also essential in Drosophila? If the latter is true, how do they survive fluctuations in sterol availability and maintain membrane homeostasis? Here, we show that Drosophila require both bulk membrane sterol and steroid hormones in order to complete adult development. When sterol availability is restricted, Drosophila larvae modulate their growth to maintain membrane sterol levels within tight limits. When dietary sterol drops below a minimal threshold, larvae arrest growth and development in a reversible manner. Strikingly, membrane sterol levels in arrested larvae are dramatically reduced (dropping sixfold on average) in most tissues except the nervous system. Thus, sterols are dispensable for maintaining the basic membrane biophysical properties required for cell viability; these functions can be performed by non-sterol lipids when sterols are unavailable. However, bulk membrane sterol is likely to have essential functions in specific tissues during development. In tissues in which sterol levels drop, the overall level of sphingolipids increases and the proportion of different sphingolipid variants is altered. These changes allow survival, but not growth, when membrane sterol levels are low. This relationship between sterols and sphingolipids could be an ancient and conserved principle of membrane homeostasis.

Secretion and Signaling Activities of Lipoprotein-Associated Hedgehog and Non-Sterol-Modified Hedgehog in Flies and Mammals

We identify two distinct Hh secretion forms with complementary signaling activities produced in both mammals and flies: one sterol-modified and lipoprotein-associated, and another lacking sterol modification.

Microsomal triacylglycerol transfer protein (MTP) is required to expand tracheal lumen in Drosophila in a cell-autonomous manner.

Summary The Drosophila tracheal system is a useful model for dissecting the molecular mechanisms controlling the assembly and expansion of tubular organs. We have identified microsomal triacylglycerol transfer protein (MTP) as a new player involved in the lumen expansion in unicellular tubes. MTP is an endoplasmic reticulum resident protein that can transfer triglycerides and phospholipids between membranes in vitro. MTP lipid transfer activity is crucial for the assembly and secretion of apoB family lipoproteins, which are carriers of lipids between different tissues. Here we describe an unexpected role of MTP in tracheal development, which we postulate to be independent of its known function in lipoprotein secretion. We propose that, in tracheal cells, MTP is involved in regulation of de novo apical membrane delivery to the existing lumen and thus promotes proper expansion of the larval tracheal system.