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1.
Biophys J ; 121(16): 3146-3161, 2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-35841144

RESUMO

Cholesterol plays a unique role in the regulation of membrane organization and dynamics by modulating the membrane phase transition at the nanoscale. Unfortunately, due to their small sizes and dynamic nature, the effects of cholesterol-mediated membrane nanodomains on membrane dynamics remain elusive. Here, using ultrahigh-speed single-molecule tracking with advanced optical microscope techniques, we investigate the diffusive motion of single phospholipids in the live cell plasma membrane at the nanoscale and its dependency on the cholesterol concentration. We find that both saturated and unsaturated phospholipids undergo anomalous subdiffusion on the length scale of 10-100 nm. The diffusion characteristics exhibit considerable variations in space and in time, indicating that the nanoscopic lipid diffusion is highly heterogeneous. Importantly, through the statistical analysis, apparent dual-mobility subdiffusion is observed from the mixed diffusion behaviors. The measured subdiffusion agrees well with the hop diffusion model that represents a diffuser moving in a compartmentalized membrane created by the cytoskeleton meshwork. Cholesterol depletion diminishes the lipid mobility with an apparently smaller compartment size and a stronger confinement strength. Similar results are measured with temperature reduction, suggesting that the more heterogeneous and restricted diffusion is connected to the nanoscopic membrane phase transition. Our conclusion supports the model that cholesterol depletion induces the formation of gel-phase, solid-like membrane nanodomains. These nanodomains undergo restricted diffusion and act as diffusion obstacles to the membrane molecules that are excluded from the nanodomains. This work provides the experimental evidence that the nanoscopic lipid diffusion in the cell plasma membrane is heterogeneous and sensitive to the cholesterol concentration and temperature, shedding new light on the regulation mechanisms of nanoscopic membrane dynamics.


Assuntos
Colesterol , Bicamadas Lipídicas , Membrana Celular/metabolismo , Colesterol/metabolismo , Difusão , Bicamadas Lipídicas/metabolismo , Fosfolipídeos/metabolismo
2.
Nanoscale ; 11(2): 568-577, 2019 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-30548049

RESUMO

Nanoparticles have been used extensively in biology-related research and many applications require direct visualization of individual nanoparticles by optical microscopy. For long-term and high-speed measurements, scattering-based microscopy is a unique technique because of the stable and indefinite scattering signals. In scattering-based single-particle measurements, large nanoparticles are usually needed in order to generate sufficient signals for detection. However, larger nanoparticles introduce greater mass loading, experience stronger steric hindrance, and are more prone to crosslinking. In this work, we demonstrate coherent brightfield (COBRI) microscopy with enhanced contrast and show its capability of direct visualization of very small nanoparticles in scattering at a high speed. COBRI microscopy allows us to visualize and track single metallic and dielectric nanoparticles, as small as 10 nm, at 1000 frames per second. A quantitative relationship between the linear scattering cross section of the nanoparticle and its COBRI contrast is reported. Using COBRI microscopy, we further demonstrate the tracking of 10 nm gold nanoparticles labeled to lipid molecules in supported bilayer membranes, showing that the small nanoparticles may facilitate single-molecule measurements with reduced perturbation. Furthermore, the identical imaging sensitivities of COBRI and interferometric scattering (iSCAT) microscopy, the reflection counterpart of COBRI, is demonstrated at an equal illumination intensity. Finally, future improvements in the speed and sensitivity of scattering-based interference microscopy are discussed.

3.
ACS Nano ; 13(10): 10918-10928, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31259529

RESUMO

Single-molecule tracking is a powerful method to study molecular dynamics in living systems including biological membranes. High-resolution single-molecule tracking requires a bright and stable signal, which has typically been facilitated by nanoparticles due to their superb optical properties. However, there are concerns about using a nanoparticle to label a single molecule because of its relatively large size and the possibility of cross-linking multiple target molecules, both of which could affect the original molecular dynamics. In this work, using various labeling schemes, we investigate the effects using nanoparticles to measure the diffusion of single-membrane molecules. By conjugating a low density of streptavidin (sAv) to gold nanoparticles (AuNPs) of different sizes (10, 15, 20, 30, and 40 nm), we isolate and quantify the effect of the particle size on the diffusion of biotinylated lipids in supported lipid bilayers (SLBs). We find that single sAv tends to cross-link two biotinylated lipids, leading to a much slower diffusion in SLBs. We further demonstrate a simple and robust strategy for the monovalent and oriented labeling of a single lipid molecule with a AuNP by using naturally dimeric rhizavidin (rAv) as a bridge, thus connecting the biotinylated nanoparticle surface and biotinylated target molecule. The rAv-AuNP conjugate demonstrates fast and free diffusion in SLBs (2-3 µm2/s for rAv-AuNP sizes of 10-40 nm), which is comparable to the diffusion of dye-labeled lipids, indicating that the adverse size and cross-linking effects are successfully avoided. We also note that the diffusion of dye-labeled lipids critically depends on the choice of dye, which could report different diffusion coefficients by about 20% (2.2 µm2/s of ATTO647N and 2.6 µm2/s of ATTO532). By comparing the diffusion of the uniformly and randomly oriented labeling of a single lipid molecule with a AuNP, we conclude that oriented labeling is favorable for measuring the diffusion of single-membrane molecules. Our work shows that the measured diffusion of the membrane molecule is highly sensitive to the molecular design of the cross-linker for labeling. The demonstrated approach of monovalent and oriented AuNP labeling provides the opportunity to study single-molecule membrane dynamics at much higher spatiotemporal resolutions and, most importantly, without labeling artifacts.


Assuntos
Membrana Celular/química , Bicamadas Lipídicas/química , Nanotecnologia , Imagem Individual de Molécula/métodos , Ouro/química , Nanopartículas Metálicas/química , Simulação de Dinâmica Molecular , Tamanho da Partícula , Estreptavidina/química
4.
J Phys Chem B ; 123(30): 6492-6504, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31290322

RESUMO

Native cell-membrane-derived supported lipid bilayers (SLBs) are an emerging platform with broad applications ranging from fundamental research to next-generation biosensors. Central to the success of the platform is the proper accommodation of membrane proteins so that their dynamics and functions are preserved. Polymer cushions have been commonly employed to avoid direct contact between the bilayer membrane and the supporting substrate, and thus, the mobility of the 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 with distinct structures, i.e., a GPI-anchored protein, single-pass transmembrane protein CD98 heavy chain, and seven-pass transmembrane protein SSTR3, were fused with green fluorescent protein (GFP), and their dynamics were measured by fluorescent single-molecule tracking. An automated data acquisition was implemented to study the effects of PEG polymer length on protein dynamics with large statistics. Our data showed that increasing the PEG polymer length (molecular weight from 1000 to 5000) enhanced the mobile fraction of the membrane proteins. Moreover, the diffusion coefficients of transmembrane proteins were augmented with the polymer length, whereas the diffusion coefficient of the GPI-anchored protein remained almost identical for different polymer lengths. Importantly, the diffusion coefficients of the three membrane proteins became identical (2.5 µm2/s approximately) for the cushioned membrane with the longest polymer length (molecular weight of 5000), indicating that at the microscopic length scale, the SLBs were fully suspended from the substrate by the polymer cushion. Transient confinements were observed for all three proteins, and increasing the polymer length reduced the tendency of transient confinement. The measured dynamics of membrane proteins were found to be nearly unchanged after the 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 the future developments of membrane-based biosensors and analytical assays.


Assuntos
Membrana Celular/química , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Polímeros/química , Colesterol , Células HeLa , Humanos , Lipossomos , Modelos Moleculares , Conformação Proteica
5.
ACS Appl Mater Interfaces ; 11(22): 19774-19781, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31081612

RESUMO

Real-time tracking of membrane proteins is essential to gain an in-depth understanding of their dynamics on the cell surface. However, conventional fluorescence imaging with molecular probes like organic dyes and fluorescent proteins often suffers from photobleaching of the fluorophores, thus hindering their use for continuous long-term observations. With the availability of fluorescent nanodiamonds (FNDs), which have superb biocompatibility and excellent photostability, it is now possible to conduct the imaging in both short and long terms with high temporal and spatial resolution. To realize the concept, we have developed a facile method (e.g., one-pot preparation) to produce alkyne-functionalized hyperbranched-polyglycerol-coated FNDs for bioorthogonal labeling of azide-modified membrane proteins and azide-modified antibodies of membrane proteins. The high specificity of this labeling method has allowed us to continuously monitor the movements of the proteins of interest (such as integrin α5) on/in living cells over 2 h. The results open a new horizon for live cell imaging with functional nanoparticles and fluorescence microscopy.


Assuntos
Química Click/métodos , Glicoproteínas/química , Proteínas de Membrana/química , Nanodiamantes/química , Imagem Óptica/métodos , Linhagem Celular , Citometria de Fluxo , Células HeLa , Humanos , Microscopia Confocal , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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