scholarly journals Characterization of Single Protein Dynamics in Cell Plasma Membrane Derived Polymer Cushioned Lipid Bilayers

2019 ◽  
Author(s):  
Wai Cheng (Christine) Wong ◽  
Jz-Yuan Juo ◽  
Yi-Hung Liao ◽  
Ching-Ya Cheng ◽  
Chih-Hsiang Lin ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Protein Dynamics ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Diffusion Coefficients ◽  
Transmembrane Protein ◽  
Transmembrane Proteins ◽  
Cell Plasma

AbstractNative cell membrane derived supported lipid bilayers (SLBs) are emerging platforms that have broad applications ranging from fundamental research to next-generation biosensors. Central to the success of the platform is proper accommodation of membrane proteins so that their dynamics and functions are preserved. Polymer cushions have been commonly employed to avoid direct contact of the bilayer membrane to the supporting substrate, and thus the mobility of transmembrane proteins is maintained. However, little is known about how the polymer cushion affects the absolute mobility of membrane molecules. Here, we characterized the dynamics of single membrane proteins in polymer-cushioned lipid bilayers derived from cell plasma membranes and investigated the effects of polymer length. Three membrane proteins of distinct structures, i.e., GPI-anchored protein, single-pass transmembrane protein CD98 heavy chain, and seven-pass transmembrane protein SSTR3, were fused with green fluorescence proteins (GFPs) and their dynamics were measured by fluorescence single-molecule tracking. An automated data acquisition was implemented to study the effects of PEG polymer length to protein dynamics with large statistics. Our data showed that increasing the PEG polymer length (molecular weight from 1,000 to 5,000) enhanced the mobile fraction of the membrane proteins. Moreover, the diffusion coefficients of transmembrane proteins were raised by increasing the polymer length, whereas the diffusion coefficient of GPI-anchored protein remained almost identical with different polymer lengths. Importantly, the diffusion coefficients of the three membrane proteins became identical (2.5 μm2/s approximately) in the cushioned membrane with the longest polymer length (molecular weight of 5,000), indicating that the SLBs were fully suspended from the substrate by the polymer cushion at the microscopic length scale. Transient confinements were observed from all three proteins, and increasing the polymer length reduced the tendency of transient confinements. The measured dynamics of membrane proteins were found to be nearly unchanged after depletion of cholesterol, suggesting that the observed immobilization and transient confinement were not due to cholesterol-enriched membrane nanodomains (lipid rafts). Our single-molecule dynamics elucidate the biophysical properties of polymer cushioned plasma membrane bilayers that are potentially useful for future developments of membrane-based biosensors and analytical assays.

scholarly journals Lateral diffusion of wild-type and mutant Ld antigens in L cells.

1984 ◽  
Vol 99 (6) ◽  
pp. 2333-2335 ◽  
Author(s):  
M Edidin ◽  
M Zuniga
Keyword(s):  
Plasma Membrane ◽  
Diffusion Coefficients ◽  
Transmembrane Protein ◽  
Transmembrane Proteins ◽  
Lateral Diffusion ◽  
Cytoplasmic Domain ◽  
Histocompatibility Antigen ◽  
Wild Type ◽  
Major Histocompatibility Antigen ◽  
Cytoplasmic Side

We have compared the lateral diffusion of intact transmembrane proteins, wild-type H-2Ld antigens, with that of mutants truncated in the cytoplasmic domain. Diffusion coefficients and mobile fractions were similar for all molecules examined, from wild-type Ld antigens with 31 residues on the cytoplasmic side of the plasma membrane to mutants with only four residues in the cytoplasmic domain. This result limits ways in which the lateral diffusion of a major histocompatibility antigen, a transmembrane protein, can be constrained by interactions with other molecules.

scholarly journals Single-molecule imaging of HIV-1 envelope glycoprotein dynamics and Gag lattice association exposes determinants responsible for virus incorporation

2019 ◽  
Vol 116 (50) ◽  
pp. 25269-25277 ◽  
Author(s):  
Nairi Pezeshkian ◽  
Nicholas S. Groves ◽  
Schuyler B. van Engelenburg
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Envelope Glycoprotein ◽  
Virus Assembly ◽  
Single Molecule Tracking ◽  
Virus Particles ◽  
Cell Plasma ◽  
The Matrix ◽  
Resolution Single ◽  
Hiv 1

The HIV-1 envelope glycoprotein (Env) is sparsely incorporated onto assembling virus particles on the host cell plasma membrane in order for the virus to balance infectivity and evade the immune response. Env becomes trapped in a nascent particle on encounter with the polymeric viral protein Gag, which forms a dense protein lattice on the inner leaflet of the plasma membrane. While Env incorporation efficiency is readily measured biochemically from released particles, very little is known about the spatiotemporal dynamics of Env trapping events. Herein, we demonstrate, via high-resolution single-molecule tracking, that retention of Env trimers within single virus assembly sites requires the Env cytoplasmic tail (CT) and the L12 residue in the matrix (MA) domain of Gag but does not require curvature of the viral lattice. We further demonstrate that Env trimers are confined to subviral regions of a budding Gag lattice, supporting a model where direct interactions and/or steric corralling between the Env-CT and a lattice of MA trimers promote Env trapping and infectious HIV-1 assembly.

scholarly journals Invasion by Toxoplasma gondii Establishes a Moving Junction That Selectively Excludes Host Cell Plasma Membrane Proteins on the Basis of Their Membrane Anchoring

1999 ◽  
Vol 190 (12) ◽  
pp. 1783-1792 ◽  
Author(s):  
Dana G. Mordue ◽  
Naishadh Desai ◽  
Michael Dustin ◽  
L. David Sibley
Keyword(s):  
Plasma Membrane ◽  
Toxoplasma Gondii ◽  
Host Cell ◽  
Transmembrane Domain ◽  
Membrane Lipids ◽  
Transmembrane Proteins ◽  
Host Cells ◽  
Cell Plasma Membrane ◽  
Cell Plasma ◽  
Membrane Anchoring

The protozoan parasite Toxoplasma gondii actively penetrates its host cell by squeezing through a moving junction that forms between the host cell plasma membrane and the parasite. During invasion, this junction selectively controls internalization of host cell plasma membrane components into the parasite-containing vacuole. Membrane lipids flowed past the junction, as shown by the presence of the glycosphingolipid GM1 and the cationic lipid label 1.1′-dihexadecyl-3-3′-3-3′-tetramethylindocarbocyanine (DiIC16). Glycosylphosphatidylinositol (GPI)-anchored surface proteins, such as Sca-1 and CD55, were also readily incorporated into the parasitophorous vacuole (PV). In contrast, host cell transmembrane proteins, including CD44, Na+/K+ ATPase, and β1-integrin, were excluded from the vacuole. To eliminate potential differences in sorting due to the extracellular domains, parasite invasion was examined in host cells transfected with recombinant forms of intercellular adhesion molecule 1 (ICAM-1, CD54) that differed in their mechanism of membrane anchoring. Wild-type ICAM-1, which contains a transmembrane domain, was excluded from the PV, whereas both GPI-anchored ICAM-1 and a mutant of ICAM-1 missing the cytoplasmic tail (ICAM-1–Cyt−) were readily incorporated into the PV membrane. Our results demonstrate that during host cell invasion, Toxoplasma selectively excludes host cell transmembrane proteins at the moving junction by a mechanism that depends on their anchoring in the membrane, thereby creating a nonfusigenic compartment.

scholarly journals Single Molecule Analysis Reveals Coexistence of Stable Serotonin Transporter Monomers and Oligomers in the Live Cell Plasma Membrane

2015 ◽  
Vol 108 (2) ◽  
pp. 322a
Author(s):  
Andreas Anderluh ◽  
Enrico Klotzsch ◽  
Vivek Kumar ◽  
Amy H. Newman ◽  
Harald H. Sitte ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Serotonin Transporter ◽  
Single Molecule ◽  
Live Cell ◽  
Cell Plasma Membrane ◽  
Cell Plasma ◽  
Single Molecule Analysis

scholarly journals High-speed single-molecule imaging reveals signal transduction by induced transbilayer raft phases

2020 ◽  
Vol 219 (12) ◽  
Author(s):  
Ikuko Koyama-Honda ◽  
Takahiro K. Fujiwara ◽  
Rinshi S. Kasai ◽  
Kenichi G.N. Suzuki ◽  
Eriko Kajikawa ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Single Molecule ◽  
High Speed ◽  
Transmembrane Protein ◽  
Single Molecule Imaging ◽  
Lipid Interactions ◽  
Alkyl Chains ◽  
Outer Leaflet ◽  
Protein Moiety

Using single-molecule imaging with enhanced time resolutions down to 5 ms, we found that CD59 cluster rafts and GM1 cluster rafts were stably induced in the outer leaflet of the plasma membrane (PM), which triggered the activation of Lyn, H-Ras, and ERK and continually recruited Lyn and H-Ras right beneath them in the inner leaflet with dwell lifetimes <0.1 s. The detection was possible due to the enhanced time resolutions employed here. The recruitment depended on the PM cholesterol and saturated alkyl chains of Lyn and H-Ras, whereas it was blocked by the nonraftophilic transmembrane protein moiety and unsaturated alkyl chains linked to the inner-leaflet molecules. Because GM1 cluster rafts recruited Lyn and H-Ras as efficiently as CD59 cluster rafts, and because the protein moieties of Lyn and H-Ras were not required for the recruitment, we conclude that the transbilayer raft phases induced by the outer-leaflet stabilized rafts recruit lipid-anchored signaling molecules by lateral raft–lipid interactions and thus serve as a key signal transduction platform.

scholarly journals Single-molecule fluorescence vistas of how lipids regulate membrane proteins

2021 ◽  
Author(s):  
Alyssa E. Ward ◽  
Yujie Ye ◽  
Jennifer A. Schuster ◽  
Shushu Wei ◽  
Francisco N. Barrera
Keyword(s):  
Membrane Proteins ◽  
Physical Properties ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Short Review ◽  
Single Molecule Fluorescence ◽  
Determining Factors ◽  
Fluorescence Methods ◽  
Basic Understanding ◽  
Sample Heterogeneity

The study of membrane proteins is undergoing a golden era, and we are gaining unprecedented knowledge on how this key group of proteins works. However, we still have only a basic understanding of how the chemical composition and the physical properties of lipid bilayers control the activity of membrane proteins. Single-molecule (SM) fluorescence methods can resolve sample heterogeneity, allowing to discriminate between the different molecular populations that biological systems often adopt. This short review highlights relevant examples of how SM fluorescence methodologies can illuminate the different ways in which lipids regulate the activity of membrane proteins. These studies are not limited to lipid molecules acting as ligands, but also consider how the physical properties of the bilayer can be determining factors on how membrane proteins function.

scholarly journals Full assembly of HIV-1 particles requires assistance of the membrane curvature factor IRSp53

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Kaushik Inamdar ◽  
Feng-Ching Tsai ◽  
Rayane Dibsy ◽  
Aurore de Poret ◽  
John Manzi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Particle Production ◽  
Virus Assembly ◽  
Membrane Curvature ◽  
Particle Assembly ◽  
Gag Protein ◽  
Cell Plasma ◽  
Curved Membranes ◽  
Hiv 1

During HIV-1 particle formation, the requisite plasma membrane curvature is thought to be solely driven by the retroviral Gag protein. Here, we reveal that the cellular I-BAR protein IRSp53 is required for the progression of HIV-1 membrane curvature to complete particle assembly. SiRNA-mediated knockdown of IRSp53 gene expression induces a decrease in viral particle production and a viral bud arrest at half completion. Single molecule localization microscopy at the cell plasma membrane shows a preferential localization of IRSp53 around HIV-1 Gag assembly sites. In addition, we observe the presence of IRSp53 in purified HIV-1 particles. Finally, HIV-1 Gag protein preferentially localizes to curved membranes induced by IRSp53 I-BAR domain on giant unilamellar vesicles. Overall, our data reveal a strong interplay between IRSp53 I-BAR and Gag at membranes during virus assembly. This highlights IRSp53 as a crucial host factor in HIV-1 membrane curvature and its requirement for full HIV-1 particle assembly.

Aberrant Protein Sorting to the Nucleus during Erythroblast Enucleation: Mechanistic Basis for Membrane Protein Loss in Hereditary Elliptocytosis and Spherocytosis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1559-1559
Author(s):  
Marcela A. Salomao ◽  
Sarah Short ◽  
Gloria Lee ◽  
Xiuli An ◽  
Mohandas Narla ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Plasma Membranes ◽  
Molecular Mechanisms ◽  
Protein Sorting ◽  
Transmembrane Proteins ◽  
Hereditary Elliptocytosis ◽  
Skeletal Protein ◽  
Aberrant Protein ◽  
Protein 4.1R

Abstract During erythroblast enucleation, nuclei surrounded by plasma membrane separate from erythroblast cytoplasm. A key aspect of this process is sorting of membrane components to plasma membranes surrounding expelled nuclei and young reticulocytes. This protein partitioning performs a crucial role in regulating the protein content of reticulocyte plasma membranes. Although it is known that cytoskeletal actin, spectrin and protein 4.1R distribute to reticulocytes, little is known about the sorting patterns of erythroblast transmembrane proteins. In hereditary spherocytosis (HS) and hereditary elliptocytosis (HE), erythrocytes contain well-described deficiencies of various transmembrane proteins, in addition to those encoded by the mutant genes. For example, elliptocytic human and murine erythrocytes resulting from mutations in the 4.1R gene lack not only protein 4.1R but also transmembrane protein glycophorin C (GPC), known to be a 4.1R binding partner with a key role in linking cytoskeleton to bilayer. Similarly, in HS resulting from mutations in the ankyrin gene, deficiencies of band 3, Rh and GPA have been documented. Various molecular mechanisms could explain deficiencies of membrane proteins in HS and HE erythrocytes including: perturbed trafficking to the erythroblast membrane; aberrant protein sorting during erythroblast enucleation; and selective loss during reticulocyte membrane remodeling. We explored whether aberrant protein sorting during enucleation might be responsible for GPC deficiency in HE. First we performed immunochemical analysis of the sorting pattern of GPC using highly purified extruded nuclei and immature reticulocytes derived from terminally differentiated murine erythroblast cultures. Proteins from equivalent numbers of expelled nuclei and reticulocytes were analyzed by Western blotting. Using antibodies specific for GPC we determined that 90% of GPC sorted to reticulocyte plasma membranes. To validate these results we used live cell, three-color immunofluorescent microscopy and analyzed enucleating erythroblasts, reticulocytes and extruded nuclei from freshly harvested murine wild type bone marrow. Independently confirming the Western blot data, we found that GPC sorted almost exclusively to reticulocytes with little or no GPC in association with nuclear plasma membrane. Strikingly, in 4.1R null erythroblasts GPC was distributed exclusively to expelled nuclei. These findings unequivocally establish that skeletal protein 4.1R is critical for normal sorting of GPC to young reticulocytes and provide clear evidence that specific skeletal protein associations can regulate protein sorting during enucleation. Moreover, our data provide a molecular explanation for the underlying basis of GPC deficiency observed in 4.1R-deficient individuals with HE. We speculate that aberrant protein sorting may be a prevalent mechanism for the deficiencies of various membrane proteins in HS and HE and that their differential loss could contribute to the variable phenotypic expression of these hemolytic disorders.

scholarly journals Correction: Live cell single molecule tracking and localization microscopy of bioorthogonally labeled plasma membrane proteins

Nanoscale ◽  
2020 ◽  
Vol 12 (35) ◽  
pp. 18476-18477
Author(s):  
Andres I. König ◽  
Raya Sorkin ◽  
Ariel Alon ◽  
Dikla Nachmias ◽  
Kalyan Dhara ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Single Molecule ◽  
Live Cell ◽  
Plasma Membrane Proteins ◽  
Single Molecule Tracking ◽  
Localization Microscopy

Correction for ‘Live cell single molecule tracking and localization microscopy of bioorthogonally labeled plasma membrane proteins’ by Andres I. König et al., Nanoscale, 2020, 12, 3236–3248, DOI: 10.1039/C9NR08594G.

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