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Enzymatic fluorometric assays for quantifying all major phospholipid classes in cells and intracellular organelles.

ABSTRACT: Cell membrane phospholipids regulate various biological functions. We previously reported enzymatic fluorometric methods for quantifying phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, phosphatidylglycerol and cardiolipin. In the present report, a new enzymatic fluorometric assay was developed for quantifying phosphatidylinositol. These simple, sensitive and high-throughput methods enabled us to quantify all major phospholipid classes in cultured cells and intracellular organelles. By conducting comprehensive quantitative analyses of major phospholipid classes, we demonstrated that the contents of phospholipid classes in HEK293 cells changed with cell density and that overexpression of phosphatidylinositol synthase or CDP-diacylglycerol synthase significantly affected the phospholipid compositions of microsomal and mitochondrial membranes. These enzymatic fluorometric assays for measuring all major phospholipid classes may be applicable to tissues, fluids, lipoproteins, extracellular vesicles and intracellular organelles of many organisms and will further our understanding of cellular, physiological and pathological processes.

SUBMITTER: Tsuji T

PROVIDER: S-EPMC6565719 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Enzymatic fluorometric assays for quantifying all major phospholipid classes in cells and intracellular organelles.

Tsuji Tokuji T Morita Shin-Ya SY Ikeda Yoshito Y Terada Tomohiro T

Scientific reports 20190613 1

Cell membrane phospholipids regulate various biological functions. We previously reported enzymatic fluorometric methods for quantifying phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, phosphatidylglycerol and cardiolipin. In the present report, a new enzymatic fluorometric assay was developed for quantifying phosphatidylinositol. These simple, sensitive and high-throughput methods enabled us to quantify all major phospholipid classes in cultu ...[more]

PMID: 31197208

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Project description:UnlabelledOwing to their small size and enhanced stability, nanobodies derived from camelids have previously been used for the construction of intracellular "nanotraps," which enable redirection and manipulation of green fluorescent protein (GFP)-tagged targets within living plant and animal cells. By taking advantage of intracellular compartmentalization in the magnetic bacterium Magnetospirillum gryphiswaldense, we demonstrate that proteins and even entire organelles can be retargeted also within prokaryotic cells by versatile nanotrap technology. Expression of multivalent GFP-binding nanobodies on magnetosomes ectopically recruited the chemotaxis protein CheW1-GFP from polar chemoreceptor clusters to the midcell, resulting in a gradual knockdown of aerotaxis. Conversely, entire magnetosome chains could be redirected from the midcell and tethered to one of the cell poles. Similar approaches could potentially be used for building synthetic cellular structures and targeted protein knockdowns in other bacteria.ImportanceIntrabodies are commonly used in eukaryotic systems for intracellular analysis and manipulation of proteins within distinct subcellular compartments. In particular, so-called nanobodies have great potential for synthetic biology approaches because they can be expressed easily in heterologous hosts and actively interact with intracellular targets, for instance, by the construction of intracellular "nanotraps" in living animal and plant cells. Although prokaryotic cells also exhibit a considerable degree of intracellular organization, there are few tools available equivalent to the well-established methods used in eukaryotes. Here, we demonstrate the ectopic retargeting and depletion of polar membrane proteins and entire organelles to distinct compartments in a magnetotactic bacterium, resulting in a gradual knockdown of magneto-aerotaxis. This intracellular nanotrap approach has the potential to be applied in other bacteria for building synthetic cellular structures, manipulating protein function, and creating gradual targeted knockdowns. Our findings provide a proof of principle for the universal use of fluorescently tagged proteins as targets for nanotraps to fulfill these tasks.

Dataset Information

Enzymatic fluorometric assays for quantifying all major phospholipid classes in cells and intracellular organelles.

Publications

Enzymatic fluorometric assays for quantifying all major phospholipid classes in cells and intracellular organelles.

Similar Datasets

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