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      Membrane order in the plasma membrane and endocytic recycling compartment

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      PLoS ONE
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          Abstract

          The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles.

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          Most cited references12

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          Switchable nile red-based probe for cholesterol and lipid order at the outer leaflet of biomembranes.

          Cholesterol and sphingomyelin form together a highly ordered membrane phase, which is believed to play important biological functions in plasma membranes of mammalian cells. Since sphingomyelin is present mainly at the outer leaflet of cell membranes, monitoring its lipid order requires molecular probes capable to bind specifically at this leaflet and exhibit negligibly slow flip-flop. In the present work, such a probe was developed by modifying the solvatochromic fluorescent dye Nile Red with an amphiphilic anchor group. To evaluate the flip-flop of the obtained probe (NR12S), we developed a methodology of reversible redox switching of its fluorescence at one leaflet using sodium dithionite. This method shows that NR12S, in contrast to parent Nile Red, binds exclusively the outer membrane leaflet of model lipid vesicles and living cells with negligible flip-flop in the time scale of hours. Moreover, the emission maximum of NR12S in model vesicles exhibits a significant blue shift in liquid ordered phase (sphingomyelin-cholesterol) as compared to liquid disordered phase (unsaturated phospholipids). As a consequence, these two phases could be clearly distinguished in NR12S-stained giant vesicles by fluorescence microscopy imaging of intensity ratio between the blue and red parts of the probe emission spectrum. Being added to living cells, NR12S binds predominantly, if not exclusively, their plasma membranes and shows an emission spectrum intermediate between those in liquid ordered and disordered phases of model membranes. Importantly, the emission color of NR12S correlates well with the cholesterol content in cell membranes, which allows monitoring the cholesterol depletion process with methyl-beta-cyclodextrin by fluorescence spectroscopy and microscopy. The attractive photophysical and switching properties of NR12S, together with its selective outer leaflet staining and sensitivity to cholesterol and lipid order, make it a new powerful tool for studying model and cell membranes.
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            Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process

            A central question in the endocytic process concerns the mechanism for sorting of recycling components (such as transferrin or low density lipoprotein receptors) from lysosomally directed components; membrane- associated molecules including receptors are generally directed towards the recycling pathway while the luminal content of sorting endosomes, consisting of the acid-released ligands, are lysosomally targeted. However, it is not known whether recycling membrane receptors follow bulk membrane flow or if these proteins are actively sorted from lysosomally directed material because of specific protein sequences and/or structural features. Using quantitative fluorescence microscopy we have determined the endocytic route and kinetics of traffic of the bulk carrier, membrane lipids, to address this issue directly. We show that N-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-epsilon-aminohexanoyl]- sphingosylphosphorylcholine (C6-NBD-SM) in endocytosed as bulk membrane, and it transits the endocytic system kinetically and morphologically identically to fluorescently labeled transferrin in a CHO cell line. With indistinguishable kinetics, the two labeled markers sort from lysosomally destined molecules in peripherally located sorting endosomes, accumulate in a peri-centriolar recycling compartment, and finally exit the cell. Other fluorescently labeled lipids, C6-NBD-phosphatidylcholine and galactosylceramide also traverse the same pathway. The constitutive nature of sorting of bulk membrane towards the recycling pathway and the lysosomal direction of fluid phase implies a geometric basis of sorting.
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              To see or not to see: lateral organization of biological membranes and fluorescence microscopy.

              In the last few years several experimental strategies based on epi-, confocal and two photon excitation fluorescence microscopy techniques have been employed to study the lateral structure of membranes using giant vesicles as model systems. This review article discusses the methodological aspects of the aforementioned experimental approaches, particularly stressing the information obtained by the use of UV excited fluorescent probes using two-photon excitation fluorescence microscopy. Additionally, the advantages of utilizing visual information, to correlate the lateral structure of compositionally simple membranes with complex situations, i.e., biological membranes, will be addressed.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: ResourcesRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                10 November 2017
                2017
                : 12
                : 11
                : e0188041
                Affiliations
                [1 ] Department of Biochemistry, Weill Cornell Medical College, New York, NY, United States of America
                [2 ] Weill Cornell Medical College, Rockefeller University, and Memorial Sloan Kettering Cancer Center Tri-Institutional Chemical Biology Program, New York, NY, United States of America
                University of Nebraska Medical Center, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                [¤]

                Current address: Genentech, South San Francisco, CA, United States of America

                Article
                PONE-D-17-31641
                10.1371/journal.pone.0188041
                5681288
                29125865
                4fffdbd0-dced-480e-8572-96f31f345616
                © 2017 Iaea, Maxfield

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 28 August 2017
                : 31 October 2017
                Page count
                Figures: 3, Tables: 0, Pages: 8
                Funding
                Funded by: NIDDK
                Award ID: F31-DK104631
                Award Recipient :
                Funded by: NIDDK
                Award ID: R37-DK27083
                Award Recipient :
                This work was supported by National Institutes of Health grants F31-DK104631 (DBI) and R37-DK27083 (FRM). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Cell Membranes
                Biology and Life Sciences
                Biochemistry
                Lipids
                Cholesterol
                Biology and Life Sciences
                Biochemistry
                Lipids
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Cell Membranes
                Membrane Characteristics
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Physical Sciences
                Physics
                Electromagnetic Radiation
                Luminescence
                Fluorescence
                Biology and Life Sciences
                Cell Biology
                Cell Physiology
                Membrane Trafficking
                Biology and Life Sciences
                Biophysics
                Physical Sciences
                Physics
                Biophysics
                Custom metadata
                All relevant data are within the paper.

                Uncategorized
                Uncategorized

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