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The Lipid Transporter TMEM24/C2CD2L is a Cat+- Regulated Component of ER-Plasma Membrane Contact Sites in Mammalian Pancreatic Beta Cells and Neurons.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	The Lipid Transporter TMEM24/C2CD2L is a Cat+- Regulated Component of ER-Plasma Membrane Contact Sites in Mammalian Pancreatic Beta Cells and Neurons./
作者:	Sun, Elizabeth Wen.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2019,
面頁冊數:	198 p.
附註:	Source: Dissertations Abstracts International, Volume: 81-01, Section: B.
Contained By:	Dissertations Abstracts International81-01B.
標題:	Biology. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=13918041
ISBN:	9781392328712

The Lipid Transporter TMEM24/C2CD2L is a Cat+- Regulated Component of ER-Plasma Membrane Contact Sites in Mammalian Pancreatic Beta Cells and Neurons.
Sun, Elizabeth Wen.

The Lipid Transporter TMEM24/C2CD2L is a Cat+- Regulated Component of ER-Plasma Membrane Contact Sites in Mammalian Pancreatic Beta Cells and Neurons. - Ann Arbor : ProQuest Dissertations & Theses, 2019 - 198 p.

Source: Dissertations Abstracts International, Volume: 81-01, Section: B.

Thesis (Ph.D.)--Yale University, 2019.

This item is not available from ProQuest Dissertations & Theses.

The Endoplasmic reticulum (ER) is the largest intracellular organelle with a multiplicity of functions, including regulation of Ca 2+ homeostasis and synthesis of membrane lipids. It is functionally interconnected with most subcellular compartments and with the plasma membrane (PM) through vesicular transport. In addition, the ER makes close appositions with other cell membranes via protein tethers. Some of these proteins also contain a lipid transport module and mediate local exchange of lipids between the ER and the apposed membrane. The constituents of ER-PM contacts, their functions and regulations at these sites have been under investigation in recent years but the picture is far from complete. TMEM24/C2CD2L is a member of the SMP (synaptotagmin-like mitochondrial and lipid-binding proteins) domain-containing protein family of lipid transport proteins, reported to be enriched in pancreatic islets and in the nervous system and shown to regulate insulin secretion in pancreatic beta cells. However, the precise mechanism by which TMEM24 impacts insulin secretion and its role in the nervous system were not understood. To begin address these questions, we have performed a functional characterization of this protein using biochemical approaches and studies in living cells. In a first set of studies, which complemented structural and lipid transport studies by our collaborators, we showed that TMEM24 is an ER-anchored PM tether, thus identifying ER-PM contacts as the sites where TMEM24 mediates its lipid transport function. Pharmacological studies showed that binding of TMEM24 to the PM contact is regulated by cytosolic Ca 2+ through a phosphorylation/dephosphorylation switch mediated by of Protein Kinase C (PKC) and calcineurin, respectively. Using TMEM24 knockout (KO) insulinoma cells, we confirmed a defect in glucose-stimulated insulin secretion upon loss of TMEM24 and showed that this defect correlated with an impairment of spontaneous calcium oscillations in the presence of glucose. In a second study, I have characterized TMEM24 in the mammalian central nervous system. I showed that levels of TMEM24 in brain increase with neuronal maturation and that TMEM24 is selectively expressed in neuronal cells. Expression of exogenous TMEM24 with fluorescent tags in primary cultured neurons showed its enrichment at ER-PM contacts, which in these cells are also enriched in Kv2.1 channels. Such localization was confirmed by EGFP-tagging TMEM24 at its endogenous locus in human neuroblastoma cells. Moreover, evoked and spontaneous activity in primary cultured neurons displayed Ca 2+ dependent regulation of TMEM24 localization similar to what we have observed in beta cells. I have identified a cluster of positively charged amino acids close to the C-terminus of the protein as a critical region for PM binding and several serine residues in th is region as responsible for its PKC-dependent regulation. I have also characterized the TMEM24 paralog, C2CD2. We have found that this protein, like TMEM24, is localized at ER-PM contact sites, but differs from TMEM24 in tissue distribution, Ca 2 +-dependent regulation and in vitro lipid transport activity. Finally, I have helped establish colonies of TMEM24 and C2CD2 KO mice and began to explore potential phenotypic defects in such mice. I did not observe any obvious neurological or behavioral impairments but observed perturbations in insulin levels in TMEM24 KO mice. Collectively, my studies, together with studies of my collaborators demonstrating lipid transport function of TMEM24 with a preference for inositol phospholipids, have advanced the understanding of the cross-talk between the ER and the PM. They have suggested a role of TMEM24 in the fine modulation of phospholipid transport between the two membranes in response to acute activity changes in pancreatic beta cells and neurons. They suggest an interplay between lipid transport at ER-PM contacts and PM excitability that will need to be addressed in future studies.

ISBN: 9781392328712Subjects--Topical Terms:

522710
Biology.
Subjects--Index Terms:

ER/PM

The Lipid Transporter TMEM24/C2CD2L is a Cat+- Regulated Component of ER-Plasma Membrane Contact Sites in Mammalian Pancreatic Beta Cells and Neurons.
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245 1 4 $a The Lipid Transporter TMEM24/C2CD2L is a Cat+- Regulated Component of ER-Plasma Membrane Contact Sites in Mammalian Pancreatic Beta Cells and Neurons.
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500 $a Source: Dissertations Abstracts International, Volume: 81-01, Section: B.
500 $a Publisher info.: Dissertation/Thesis.
500 $a Advisor: Camilli, Pietro De.
502 $a Thesis (Ph.D.)--Yale University, 2019.
506 $a This item is not available from ProQuest Dissertations & Theses.
506 $a This item must not be added to any third party search indexes.
506 $a This item must not be sold to any third party vendors.
520 $a The Endoplasmic reticulum (ER) is the largest intracellular organelle with a multiplicity of functions, including regulation of Ca 2+ homeostasis and synthesis of membrane lipids. It is functionally interconnected with most subcellular compartments and with the plasma membrane (PM) through vesicular transport. In addition, the ER makes close appositions with other cell membranes via protein tethers. Some of these proteins also contain a lipid transport module and mediate local exchange of lipids between the ER and the apposed membrane. The constituents of ER-PM contacts, their functions and regulations at these sites have been under investigation in recent years but the picture is far from complete. TMEM24/C2CD2L is a member of the SMP (synaptotagmin-like mitochondrial and lipid-binding proteins) domain-containing protein family of lipid transport proteins, reported to be enriched in pancreatic islets and in the nervous system and shown to regulate insulin secretion in pancreatic beta cells. However, the precise mechanism by which TMEM24 impacts insulin secretion and its role in the nervous system were not understood. To begin address these questions, we have performed a functional characterization of this protein using biochemical approaches and studies in living cells. In a first set of studies, which complemented structural and lipid transport studies by our collaborators, we showed that TMEM24 is an ER-anchored PM tether, thus identifying ER-PM contacts as the sites where TMEM24 mediates its lipid transport function. Pharmacological studies showed that binding of TMEM24 to the PM contact is regulated by cytosolic Ca 2+ through a phosphorylation/dephosphorylation switch mediated by of Protein Kinase C (PKC) and calcineurin, respectively. Using TMEM24 knockout (KO) insulinoma cells, we confirmed a defect in glucose-stimulated insulin secretion upon loss of TMEM24 and showed that this defect correlated with an impairment of spontaneous calcium oscillations in the presence of glucose. In a second study, I have characterized TMEM24 in the mammalian central nervous system. I showed that levels of TMEM24 in brain increase with neuronal maturation and that TMEM24 is selectively expressed in neuronal cells. Expression of exogenous TMEM24 with fluorescent tags in primary cultured neurons showed its enrichment at ER-PM contacts, which in these cells are also enriched in Kv2.1 channels. Such localization was confirmed by EGFP-tagging TMEM24 at its endogenous locus in human neuroblastoma cells. Moreover, evoked and spontaneous activity in primary cultured neurons displayed Ca 2+ dependent regulation of TMEM24 localization similar to what we have observed in beta cells. I have identified a cluster of positively charged amino acids close to the C-terminus of the protein as a critical region for PM binding and several serine residues in th is region as responsible for its PKC-dependent regulation. I have also characterized the TMEM24 paralog, C2CD2. We have found that this protein, like TMEM24, is localized at ER-PM contact sites, but differs from TMEM24 in tissue distribution, Ca 2 +-dependent regulation and in vitro lipid transport activity. Finally, I have helped establish colonies of TMEM24 and C2CD2 KO mice and began to explore potential phenotypic defects in such mice. I did not observe any obvious neurological or behavioral impairments but observed perturbations in insulin levels in TMEM24 KO mice. Collectively, my studies, together with studies of my collaborators demonstrating lipid transport function of TMEM24 with a preference for inositol phospholipids, have advanced the understanding of the cross-talk between the ER and the PM. They have suggested a role of TMEM24 in the fine modulation of phospholipid transport between the two membranes in response to acute activity changes in pancreatic beta cells and neurons. They suggest an interplay between lipid transport at ER-PM contacts and PM excitability that will need to be addressed in future studies.
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653 $a Lipid Transfer Protein
653 $a Membrane Metabolism
653 $a TMEM24/C2CD2L
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