Sterol flow between the plasma membrane and the endosome is regulated by the LAM family protein Ltc1

Marek, Magdalena; Vincenzetti, Vincent

doi:10.1101/720383

Cited by 3 publications

(3 citation statements)

References 82 publications

(104 reference statements)

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“…2D). To corroborate this finding, we next employed two versions of the domain 4 of perfringolysin fused to mCherry probe (D4-mCherry), YDA and YQDA, which have different sensitivities in the detection of cholesterol [30][31][32] . We expressed the probes constitutively in the C. elegans intestine and analyzed their cellular distribution.…”

Section: Cholesterol Accumulates Predominantly In the Apical Membranementioning

confidence: 95%

Patched regulates lipid homeostasis by controlling cellular cholesterol levels

Castillo

Hannich

Kaech

et al. 2019

Preprint

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words)Hedgehog (Hh) signaling is essential during development and in organ physiology. In the canonical pathway, Hh binding to Patched (PTCH) relieves the inhibition of Smoothened (SMO). Yet, PTCH may also perform SMO-independent functions. While the PTCH homolog PTC-3 is essential in C. elegans, worms lack SMO, providing an excellent model to probe non-canonical PTCH function. Here, we show that PTC-3 is a cholesterol transporter. ptc-3(RNAi) leads to accumulation of intracellular cholesterol and defects in ER structure and lipid droplet formation. These phenotypes were accompanied by a reduction in acyl chain (FA) length and desaturation. ptc-3(RNAi)induced lethality, fat storage and ER morphology defects were rescued by reducing dietary cholesterol. We provide evidence that cholesterol accumulation modulates the function of nuclear hormone receptors such as of the PPARa homolog NHR-49 and NHR-181, and affects FA composition. Our data uncover a novel role for PTCH in organelle structure maintenance and fat metabolism.No clear SMO homolog is encoded in the genome. In addition, some of the other downstream targets of the canonical Hh signaling pathway are also missing. In fact, it was proposed that SMO and those components were specifically lost during evolution in nematodes 15,21-23 . For example, SUFU is not conserved and the homolog of the transcription factor Gli, TRA-1, is involved in sex determination and gonad development in males and hermaphrodites 24 . Therefore, C. elegans provides an excellent model to study non-canonical, SMO-independent Hh signaling pathways, in particular in somatic tissues. To dissect SMO-independent PTCH functions, we concentrated on PTC-3, which is expressed in somatic tissues, in particular in the hypodermis, glia and gut 20 . We found that reduction of PTC-3 levels causes the accumulation of intracellular cholesterol and reduction in poly unsaturated fatty acids (PUFAs). Moreover, the endoplasmic reticulum lost most of its reticulate tubular form and developed elaborate sheet structures in the intestine. This effect in turn strongly impaired lipid droplet biogenesis, resulting in the inability of the animal to store fat.Reduction of dietary cholesterol rescued fat storage defects, the ER morphology defects, and improved development and survival in ptc-3(RNAi) animals. Cholesterol levels influence nuclear hormone receptor activity such as of the PPARa homolog NHR-49, which is involved in the regulation of FA synthesis. Thus, our data demonstrate that PTCH also controls intracellular cholesterol levels in C. elegans.Moreover, we show that PTCH thereby impinges on FA metabolism, organellar structure and fat storage capacity.

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Section: Cholesterol Accumulates Predominantly In the Apical Membranementioning

confidence: 95%

Patched regulates lipid homeostasis by controlling cellular cholesterol levels

Castillo

Hannich

Kaech

et al. 2019

Preprint

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“…We further used the act1, pak1, rga3 and pom1 promoters, all of which lead to detectable levels of cytosolic GFP, to express Ltc1, a protein localized at ER-PM contact sites ( Fig. 3F; Marek et al, 2019). The localization of Ltc1, which was difficult to detect from its native promoter, showed a more prominent localization pattern when mildly overexpressed from pak1, pom1 or rga3 promoters.…”

Section: Expanding the Usage Of Stable Integration Vectorsmentioning

confidence: 99%

“…However, we refrain from using different constructs to compare between conditions as genomic position effects may become evident only in certain backgrounds (Allshire and Ekwall, 2015). While we present the full vector series for the first time here, some plasmids have been used in published studies (Billault-Chaumartin and Martin, 2019;Gerganova et al, 2019;Lamas et al, 2019;Marek et al, 2019;Vještica et al, 2018) which illustrates their application in fission yeast research.…”

Section: Verify Correct Integration By Diagnostic Pcrs On Both Sides mentioning

confidence: 99%

A toolbox of Stable Integration Vectors (SIV) in the fission yeastSchizosaccharomyces pombe

Vještica

Marek

Nkosi

et al. 2019

Preprint

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Schizosaccharomyces pombe is a widely used model organism that resembles higher eukaryotes in many aspects of cell physiology. Its popularity as an experimental system partially stems from the ease of genetic manipulations, where the innate homology-targeted repair is exploited to precisely edit the genome. While vectors to incorporate exogenous sequences into the chromosomes are available, most are poorly characterized. Here we show that commonly used fission yeast vectors, which upon integration produce repetitive genomic regions, yield unstable genomic loci. We overcome this problem by designing a new series of Stable Integration Vectors (SIV) that target four different prototrophy genes. SIV produce non-repetitive, stable genomic loci and integrate predominantly as single copy. Additionally, we develop a set of complementary auxotrophic alleles that preclude false-positive integration events. We expand the vector series to include antibiotic resistance markers, promoters, fluorescent tags and terminators, and build a highly modular toolbox to introduce heterologous sequences. Finally, as proof of concept, we generate a large set of ready-to-use, fluorescent probes to mark organelles and cellular processes with a wide range of applications in fission yeast research.

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A toolbox of stable integration vectors in the fission yeast Schizosaccharomyces pombe

Vještica

Marek

Nkosi

et al. 2020

Journal of Cell Science

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Schizosaccharomyces pombe is a widely used model organism to study many aspects of eukaryotic cell physiology. Its popularity as an experimental system partially stems from the ease of genetic manipulations, where the innate homology-targeted repair is exploited to precisely edit the genome. While vectors to incorporate exogenous sequences into the chromosomes are available, most are poorly characterized. Here, we show that commonly used fission yeast vectors, which upon integration produce repetitive genomic regions, give rise to unstable genomic loci. We overcome this problem by designing a new series of stable integration vectors (SIVs) that target four different prototrophy genes. SIVs produce non-repetitive, stable genomic loci and integrate predominantly as single copy. Additionally, we develop a set of complementary auxotrophic alleles that preclude false-positive integration events. We expand the vector series to include antibiotic resistance markers, promoters, fluorescent tags and terminators, and build a highly modular toolbox to introduce heterologous sequences. Finally, as proof of concept, we generate a large set of ready-to-use, fluorescent probes to mark organelles and cellular processes with a wide range of applications in fission yeast research.

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Sterol flow between the plasma membrane and the endosome is regulated by the LAM family protein Ltc1

Cited by 3 publications

References 82 publications

Patched regulates lipid homeostasis by controlling cellular cholesterol levels

Patched regulates lipid homeostasis by controlling cellular cholesterol levels

A toolbox of Stable Integration Vectors (SIV) in the fission yeastSchizosaccharomyces pombe

A toolbox of stable integration vectors in the fission yeast Schizosaccharomyces pombe

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