Nano-scale resolution of native retinal rod disk membranes reveals differences in lipid composition

Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to coimmunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each sample's copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which was enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein–lipid interactions. High-resolution profiling of the ROS disk lipid composition gives new insights into how intricate membrane structure and protein activity are balanced within the ROS, and provides a model for future studies of other complex cellular structures.

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Dissecting lipid contents in the distinct regions of native retinal rod disk membranes

10.1101/2021.01.11.426250 ◽

2021 ◽

Author(s):

Christopher L. Sander ◽

Avery E. Sears ◽

Antonio F. M. Pinto ◽

Elliot H. Choi ◽

Shirin Kahremany ◽

...

Keyword(s):

Fatty Acid ◽

Membrane Proteins ◽

Lipid Composition ◽

Protein Localization ◽

Specific Protein ◽

Fatty Acid Analysis ◽

Protein Activity ◽

Domain Specific ◽

Retinal Rod ◽

Membrane Compartments

ABSTRACTPhotoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to co-immunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each sample’s copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which were enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein-lipid interactions. High-resolution profiling of the ROS disk lipid composition provides a model for future studies of other complex cellular structures, and gives new insights into how intricate membrane structure and protein activity are balanced within the ROS.SUMMARYSander et al. have parsed the lipid composition of native-source photoreceptor disks and find large differences in fatty acid unsaturation and chain length between the center and rim regions. They selectively copurify membrane proteins and lipids from each region in SMALPs using nanobodies and antibodies.

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THE ULTRASTRUCTURE OF LIPID-DEPLETED ROD PHOTORECEPTOR MEMBRANES

The Journal of Cell Biology ◽

10.1083/jcb.63.2.587 ◽

1974 ◽

Vol 63 (2) ◽

pp. 587-598 ◽

Cited By ~ 27

Author(s):

Izhak Nir ◽

Michael O. Hall

Keyword(s):

Lipid Extraction ◽

Fatty Acid Analysis ◽

Major Protein ◽

Rod Photoreceptor ◽

Thin Sections ◽

Retinal Rod ◽

Layer Chromatography ◽

Chloroform Methanol ◽

Photoreceptor Membranes

The structure of lipid-depleted retinal rod photoreceptor membranes was studied by means of electron microscopy. Aldehyde-fixed retinas were exhaustively extracted with acetone, chloroform-methanol, and acidified chloroform-methanol. The effect of prefixation on the extractability of lipids was evaluated by means of thin-layer chromatography and fatty acid analysis. Prefixation with glutaraldehyde rendered 38% of the phospholipids unextractable, while only 7% were unextractable after formaldehyde fixation. Embedding the retina in a lipid-retaining, polymerizable glutaraldehyde-urea mixture allows a comparison of the interaction of OsO4 with lipid-depleted membranes and rod disk membranes which contain all their lipids. A decrease in electron density and a deterioration of membrane fine structure in lipid-depleted tissue are correlated with the extent of lipid extraction. These observations are indicative of the role of the lipid bilayer in the ultrastructural visualization of membrane structure with OsO4. Negatively stained thin sections of extracted tissue reveal substructures in the lipid-depleted rod membranes. These substructures are probably the opsin molecules which are the major protein component of retinal rod photoreceptor membranes.

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Peptide-tags for site-specific protein labelling in vitro and in vivo

Molecular BioSystems ◽

10.1039/c6mb00023a ◽

2016 ◽

Vol 12 (6) ◽

pp. 1731-1745 ◽

Cited By ~ 96

Author(s):

Jonathan Lotze ◽

Ulrike Reinhardt ◽

Oliver Seitz ◽

Annette G. Beck-Sickinger

Keyword(s):

Metal Ions ◽

Small Molecules ◽

Protein Localization ◽

Specific Protein ◽

Site Specific ◽

Protein Labelling ◽

Peptide Interactions ◽

Peptide Tags

Peptide-tag based labelling can be achieved by (i) enzymes (ii) recognition of metal ions or small molecules and (iii) peptide–peptide interactions and enables site-specific protein visualization to investigate protein localization and trafficking.

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Polarized Proteins in Endothelium and Their Contribution to Function

Journal of Vascular Research ◽

10.1159/000512618 ◽

2021 ◽

pp. 1-27

Author(s):

Abigail G. Wolpe ◽

Claire A. Ruddiman ◽

Phillip J. Hall ◽

Brant E. Isakson

Keyword(s):

Endothelial Cells ◽

Endothelial Function ◽

Planar Cell Polarity ◽

Protein Localization ◽

Peripheral Resistance ◽

Specific Protein ◽

Resistance Arteries ◽

Extracellular Stimuli ◽

Small Resistance ◽

Signaling Domains

Protein localization in endothelial cells is tightly regulated to create distinct signaling domains within their tight spatial restrictions including luminal membranes, abluminal membranes, and interendothelial junctions, as well as caveolae and calcium signaling domains. Protein localization in endothelial cells is also determined in part by the vascular bed, with differences between arteries and veins and between large and small arteries. Specific protein polarity and localization is essential for endothelial cells in responding to various extracellular stimuli. In this review, we examine protein localization in the endothelium of resistance arteries, with occasional references to other vessels for contrast, and how that polarization contributes to endothelial function and ultimately whole organism physiology. We highlight the protein localization on the luminal surface, discussing important physiological receptors and the glycocalyx. The protein polarization to the abluminal membrane is especially unique in small resistance arteries with the presence of the myoendothelial junction, a signaling microdomain that regulates vasodilation, feedback to smooth muscle cells, and ultimately total peripheral resistance. We also discuss the interendothelial junction, where tight junctions, adherens junctions, and gap junctions all convene and regulate endothelial function. Finally, we address planar cell polarity, or axial polarity, and how this is regulated by mechanosensory signals like blood flow.

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Developing Practical Therapeutic Strategies that Target Protein SUMOylation

Current Drug Targets ◽

10.2174/1389450119666181026151802 ◽

2019 ◽

Vol 20 (9) ◽

pp. 960-969 ◽

Cited By ~ 3

Author(s):

Olivia F. Cox ◽

Paul W. Huber

Keyword(s):

Birth Defects ◽

Inflammatory Disease ◽

Intracellular Localization ◽

Specific Protein ◽

Therapeutic Strategies ◽

Biological Processes ◽

Post Translational Modification ◽

Protein Activity ◽

Multiple Components ◽

Congenital Birth Defects

Post-translational modification by small ubiquitin-like modifier (SUMO) has emerged as a global mechanism for the control and integration of a wide variety of biological processes through the regulation of protein activity, stability and intracellular localization. As SUMOylation is examined in greater detail, it has become clear that the process is at the root of several pathologies including heart, endocrine, and inflammatory disease, and various types of cancer. Moreover, it is certain that perturbation of this process, either globally or of a specific protein, accounts for many instances of congenital birth defects. In order to be successful, practical strategies to ameliorate conditions due to disruptions in this post-translational modification will need to consider the multiple components of the SUMOylation machinery and the extraordinary number of proteins that undergo this modification.

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Syntaxin 1A inhibits CFTR chloride channels by means of domain-specific protein-protein interactions

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.95.18.10972 ◽

1998 ◽

Vol 95 (18) ◽

pp. 10972-10977 ◽

Cited By ~ 107

Author(s):

A. P. Naren ◽

M. W. Quick ◽

J. F. Collawn ◽

D. J. Nelson ◽

K. L. Kirk

Keyword(s):

Protein Interactions ◽

Chloride Channels ◽

Specific Protein ◽

Protein Protein Interactions ◽

Syntaxin 1A ◽

Domain Specific

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A Molecular Mechanism for Membrane Geometry-Specific Protein Localization

Biophysical Journal ◽

10.1016/j.bpj.2018.11.2036 ◽

2019 ◽

Vol 116 (3) ◽

pp. 374a-375a

Author(s):

Gabriele Kockelkoren

Keyword(s):

Molecular Mechanism ◽

Protein Localization ◽

Specific Protein ◽

Membrane Geometry

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The transbilayer distribution of polyunsaturated phospholipids determines their facilitating effect on membrane deformation

Soft Matter ◽

10.1039/c9sm02107h ◽

2020 ◽

Vol 16 (7) ◽

pp. 1722-1730 ◽

Cited By ~ 4

Author(s):

Marion L. Tiberti ◽

Bruno Antonny ◽

Romain Gautier

Keyword(s):

Cell Membrane ◽

Lipid Composition ◽

Specific Protein ◽

Membrane Deformation

In the cell, membrane deformation and fission (collectively referred to as ‘budding’) is driven by specific protein machineries but is also influenced by lipid composition.

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