ABSTRACT
HYPOTHESIS: Synthetic micro/nanomotors are gaining extensive attention for various biomedical applications (especially in drug delivery) due to their ability to mimic the motion of biological micro/nanoscale swimmers. The feasibility of these applications relies on tight control of propulsion speed, direction, and type of motion (translation, circular, etc.) along with the exerted self-propulsive force. We propose to exploit the variation of both self-propulsion speed and force of active colloids with different patch coverages (with and without supporting layer) for engineering diffusiophoretic micro/nanomotors. EXPERIMENTS: The microswimmers were designed at various patch coverages (10°, 30°, and 90°) with (Ti/Pt) and without (Pt) an adhesion layer for the catalytic patch through glancing angle metal deposition (GLAD) technique. Mean-square displacement (MSD) analysis was performed to obtain the self-propulsion parameters like speed and angular speed. Using optical tweezers (OT), the self-propulsive force was measured from the force power spectral density. FINDINGS: The findings of our experiments suggest the non-requirement of any adhesion layer preceding the catalyst deposition since the Pt 10° colloidal batch had the maximal self-propulsion speed (4.61±0.3µm/s) and force (345±57fN) for 5% w/v H2O2 fuel concentration. Moreover, the self-propulsion speed and force decreased with increasing patch size, contrary to theoretical estimates. Also, the self-propulsive force obtained from MSD is 2 to 4 times lower in magnitude than the OT based force values. We believe that the self-propelling motion of the micromotors is possibly hindered due to interactions with the surface of the quartz cuvette during the optical microscopic analysis. Further, the MSD is limited to the self-propulsive motion in two dimensions. On the other hand, OT based force measurement involve trapping the particles in the bulk of the solution entirely avoiding the particle-substrate interactions. Hence, OT based force measurements are better than the propulsion velocity based stokes drag force estimates. We believe that this study can lay the foundation in designing efficient micro/nanomotors for translational biomedical applications.
ABSTRACT
Optical trapping is a potent tool for achieving precise and noninvasive manipulation of small objects in a vacuum and liquids. However, due to the substantial disparity between optical forces and interfacial adhesion, target objects should be suspended in fluid environments, rendering solid contact surfaces a restricted area for conventional optical tweezers. In this work, by relying on a single continuous wave (CW) laser, we demonstrate an optical manipulation system applicable for both fluid and solid domains, namely, amphibious hybrid laser tweezers. The key to our system lies in modulating the intensity of the CW laser with duration shorter than the dynamic thermal equilibrium time within objects, wherein strong thermal gradient forces with â¼6 orders of magnitude higher than the forces in optical tweezers are produced, enabling moving and trapping micro/nano-objects on solid interfaces. Thereby, CW laser-based optical tweezers and pulsed laser-based photothermal shock tweezers are seamlessly fused with the advantages of cost-effectiveness and simplicity. Our concept breaks the stereotype that CW lasers are limited to generating tiny forces and instead achieve ultrawide force generation spanning from femto-newtons (10-15 N) to (10-6 N). Our work expands the horizon of optical manipulation by seamlessly bridging its applications in fluid and solid environments and holds promise for inspiring optical manipulation techniques to perform more challenging tasks, which may unearth application scenarios in diverse fields such as fundamental physical research, nanofabrication, micro/nanorobotics, biomedicine, and cytology.
ABSTRACT
BACKGROUND: Mouse nodal immotile cilia mechanically sense the bending direction for left-right (L-R) determination and activate the left-side-specific signaling cascade, leading to increased Nodal activity. Asymmetric distribution of Pkd2, a crucial channel for L-R determination, on immotile cilia has been reported recently. However, the causal relationship between the asymmetric Pkd2 distribution and direction-dependent flow sensing is not well understood. Furthermore, the underlying molecular mechanism directing this asymmetric Pkd2 distribution remains unclear. RESULTS: The effects of several recombinant proteins and inhibitors on the Pkd2 distribution were analyzed using super-resolution microscopy. Notably, bone morphogenetic protein 4 (BMP4) affected the Pkd2 distribution. Additionally, three-dimensional manipulation of nodal immotile cilia using optical tweezers revealed that excess BMP4 caused defects in the mechanosensing ability of the cilia. CONCLUSIONS: Experimental data together with model calculations suggest that BMP4 regulates the asymmetric distribution of Pkd2 in nodal immotile cilia, thereby affecting the ability of these cilia to sense the bending direction for L-R determination. This study, for the first time, provides insight into the relationship between the asymmetric protein distribution in cilia and their function.
ABSTRACT
The study aims to determine how chitosan impacts pectin hydrogel's ability to attach peritoneal leukocytes, activate complement, induce hemolysis, and adsorb blood proteins. The hydrogels PEC-Chi0, PEC-Chi25, PEC-Chi50, and PEC-Chi75 were prepared by placing a mixture solution of 4% pectin and 4% chitosan in a ratio of 4:0, 3:1, 2:2, and 1:3 in a solution of 1.0 M CaCl2. Chitosan was found to modify the mechanical properties of pectin-calcium hydrogels, such as hardness and cohesiveness-to-adhesiveness ratio. Chitosan in the pectin-calcium hydrogel caused pH-sensitive swelling in Hanks' solution. The PEC-Chi75 hydrogel was shown to adsorb serum proteins at pH 7.4 to a greater extent than other hydrogels. PEC-Chi75's strong adsorption capacity was related to lower peritoneal leukocyte adherence to its surface when compared to other hydrogels, showing improved biocompatibility. Using the optical tweezers approach, it was shown that the force of interaction between pectin-chitosan hydrogels and plasma proteins increased from 10 to 24 pN with increasing chitosan content from 0 to 75%. Thus, the properties of pectin-calcium hydrogel, which determine interactions with body tissues after implantation, are improved by the addition of chitosan, making pectin-chitosan hydrogel a promising candidate for smart biomaterial development.
ABSTRACT
Microrheology, the study of material flow at micron scales, has advanced significantly since Robert Brown's discovery of Brownian motion in 1827. Mason and Weitz's seminal work in 1995 established the foundation for microrheology techniques, enabling the measurement of viscoelastic properties of complex fluids using light-scattering particles. However, existing techniques face limitations in exploring very slow dynamics, crucial for understanding biological systems. Here, we present a proof of concept for a novel microrheology technique called "Optical Halo", which utilises a ring-shaped Bessel beam created by optical tweezers to overcome existing limitations. Through numerical simulations and theoretical analysis, we demonstrate the efficacy of the Optical Halo in probing viscoelastic properties across a wide frequency range, including low-frequency regimes inaccessible to conventional methods. This innovative approach holds promise for elucidating the mechanical behaviour of complex biological fluids.
ABSTRACT
Mutation in oncogene KRas plays a crucial role in the occurrence and progression of numerous malignant tumors. Malignancy involves changes in cell mechanics for extensive cellular deformation during metastatic dissemination. We hypothesize that oncogene KRas mutations are intrinsic to alterations in cellular mechanics that promote malignant tumor generation and progression. Here, we demonstrate the use of optical tweezers coupled with a confocal fluorescence imaging system and gene interference technique to reveal that the mutant KRas protein can be transported between homogeneous and heterogeneous tumor cells by tunneling nanotubes (TNTs), resulting in a significant reduction of membrane tension and acceleration of membrane phospholipid flow in the recipient cells. Simultaneously, the changes in membrane mechanical properties of the tumor cells also enhance the metastatic and invasive ability of the tumors, which further contribute to the deterioration of the tumors. This finding helps to clarify the association between oncogene mutations and changes in the mechanical properties of tumor cells, which provides a theoretical basis for the development of cancer treatment strategies. STATEMENT OF SIGNIFICANCE: Here, we present a laser confocal fluorescence system integrated with optical tweezers to observe the transfer of mutant KRasG12D protein from mutant cells to wild-type cells through TNTs. Malignancy involves changes in cell mechanics for extensive cellular deformation during metastatic dissemination. Our results demonstrate a significant decrease in membrane tension and an increase in membrane phospholipid flow in recipient cells. These alterations in mechanical properties augment the migration and invasive capabilities of tumor cells, contributing to tumor malignancy. Our findings propose that cellular mechanical properties could serve as new markers for tumor development, and targeting membrane tension may hold potential as a therapeutic strategy.
Subject(s)
Cell Membrane , Nanotubes , Proto-Oncogene Proteins p21(ras) , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Humans , Nanotubes/chemistry , Cell Membrane/metabolism , Animals , Cell Line, Tumor , Mutation/genetics , Optical Tweezers , Mice , Protein Transport , Neoplasms/pathology , Neoplasms/metabolismABSTRACT
The HIV-1 nucleocapsid protein (NC) is a multifunctional viral protein necessary for HIV-1 replication. Recent studies have demonstrated that reverse transcription (RT) completes in the intact viral capsid, and the timing of RT and uncoating are correlated. How the small viral core stably contains the ~10 kbp double stranded (ds) DNA product of RT, and the role of NC in this process, are not well understood. We showed previously that NC binds and saturates dsDNA in a non-specific electrostatic binding mode that triggers uniform DNA self-attraction, condensing dsDNA into a tight globule against extending forces up to 10 pN. In this study, we use optical tweezers and atomic force microscopy to characterize the role of NC's basic residues in dsDNA condensation. Basic residue mutations of NC lead to defective interaction with the dsDNA substrate, with the constant force plateau condensation observed with wild-type (WT) NC missing or diminished. These results suggest that NC's high positive charge is essential to its dsDNA condensing activity, and electrostatic interactions involving NC's basic residues are responsible in large part for the conformation, size, and stability of the dsDNA-protein complex inside the viral core. We observe DNA re-solubilization and charge reversal in the presence of excess NC, consistent with the electrostatic nature of NC-induced DNA condensation. Previous studies of HIV-1 replication in the presence of the same cationic residue mutations in NC showed significant defects in both single- and multiple-round viral infectivity. Although NC participates in many stages of viral replication, our results are consistent with the hypothesis that cationic residue mutations inhibit genomic DNA condensation, resulting in increased premature capsid uncoating and contributing to viral replication defects.
Subject(s)
DNA, Viral , HIV-1 , Reverse Transcription , HIV-1/genetics , HIV-1/physiology , HIV-1/chemistry , HIV-1/metabolism , DNA, Viral/genetics , DNA, Viral/metabolism , gag Gene Products, Human Immunodeficiency Virus/metabolism , gag Gene Products, Human Immunodeficiency Virus/genetics , gag Gene Products, Human Immunodeficiency Virus/chemistry , Humans , Cations/metabolism , Virus Replication , Microscopy, Atomic Force , Virion/metabolism , Virion/genetics , Virion/chemistry , MutationABSTRACT
Robotic nanomanipulation emerges as a cutting-edge technique pivotal for in situ nanofabrication, advanced sensing, and comprehensive material characterization. In this study, we develop an optical robotic platform (ORP) for the dynamic manipulation of colloidal nanoparticles (NPs). The ORP incorporates a human-in-the-loop control mechanism enhanced by real-time visual feedback. This feature enables the generation of custom optical landscapes with adjustable intensity and phase configurations. Based on the ORP, we achieve the parallel and reconfigurable manipulation of multiple NPs. Through the application of spatiotemporal phase gradient-reversals, our platform demonstrates capabilities in trapping, binding, rotating, and transporting NPs across custom trajectories. This presents a previously unidentified paradigm in the realm of in situ nanomanipulation. Additionally, the ORP facilities a "capture-and-print" assembly process, utilizing a strategic interplay of phase and intensity gradients. This process operates under a constant laser power setting, streamlining the assembly of NPs into any targeted configuration. With its precise positioning and manipulation capabilities, underpinned by the spatiotemporal modulation of optical gradients, the ORP will facilitate the development of colloid-based sensors and on-demand fabrication of nanodevices.
ABSTRACT
Molecular motors are machines essential for life since they convert chemical energy into mechanical work. However, the precise mechanism by which nucleotide binding, catalysis, or release of products is coupled to the work performed by the molecular motor is still not entirely clear. This is due, in part, to a lack of understanding of the role of force in the mechanical-structural processes involved in enzyme catalysis. From a mechanical perspective, one promising hypothesis is the Haldane-Pauling hypothesis which considers the idea that part of the enzymatic catalysis is strain-induced. It suggests that enzymes cannot be efficient catalysts if they are fully complementary to the substrates. Instead, they must exert strain on the substrate upon binding, using enzyme-substrate energy interaction (binding energy) to accelerate the reaction rate. A novel idea suggests that during catalysis, significant strain energy is built up, which is then released by a local unfolding/refolding event known as 'cracking'. Recent evidence has also shown that in catalytic reactions involving conformational changes, part of the heat released results in a center-of-mass acceleration of the enzyme, raising the possibility that the heat released by the reaction itself could affect the enzyme's integrity. Thus, it has been suggested that this released heat could promote or be linked to the cracking seen in proteins such as adenylate kinase (AK). We propose that the energy released as a consequence of ligand binding/catalysis is associated with the local unfolding/refolding events (cracking), and that this energy is capable of driving the mechanical work.
Subject(s)
Molecular Motor Proteins , Animals , Humans , Molecular Motor Proteins/metabolism , Molecular Motor Proteins/chemistry , Protein Unfolding , Enzymes/metabolism , Energy MetabolismABSTRACT
We report the second harmonic generation (SHG) response from a single 34 nm diameter lithium niobate nanoparticle. The experimental setup involves a first beam devoted to the optical trapping of single nanoparticles, whereas a second arm involves a femtosecond laser source leading to the SHG emission from the trapped nanoparticles. SHG operation where one to three nanoparticles are present in the optical trap is first demonstrated, highlighting the transition between coherent and incoherent SHG, the latter known as hyper-Rayleigh scattering (HRS). With a spatial light modulator moving the optical trap in and out of the focus of the femtosecond beam, the SHG intensity is switched back and forth between a low and a high level. This controlled operation opens new avenues for nanoparticle characterization and applications in sensing or communication and information technologies and constitutes the first step in the design of active substrateless metasurfaces.
ABSTRACT
The Sec61 translocon allows the translocation of secretory preproteins from the cytosol to the endoplasmic reticulum lumen during polypeptide biosynthesis. These proteins possess an N-terminal signal peptide (SP) which docks at the translocon. SP mutations can abolish translocation and cause diseases, suggesting an essential role for this SP/Sec61 interaction. However, a detailed biophysical characterization of this binding is still missing. Here, optical tweezers force spectroscopy was used to characterize the kinetic parameters of the dissociation process between Sec61 and the SP of prepro-alpha-factor. The unbinding parameters including off-rate constant and distance to the transition state were obtained by fitting rupture force data to Dudko-Hummer-Szabo models. Interestingly, the translocation inhibitor mycolactone increases the off-rate and accelerates the SP/Sec61 dissociation, while also weakening the interaction. Whereas the translocation deficient mutant containing a single point mutation in the SP abolished the specificity of the SP/Sec61 binding, resulting in an unstable interaction. In conclusion, we characterize quantitatively the dissociation process between the signal peptide and the translocon, and how the unbinding parameters are modified by a translocation inhibitor.
Subject(s)
Optical Tweezers , SEC Translocation Channels , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Kinetics , Protein Binding , Protein Sorting Signals , Protein Transport , SEC Translocation Channels/chemistry , SEC Translocation Channels/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/metabolismABSTRACT
Optical tweezers have elucidated numerous biological processes, particularly by enabling the precise manipulation and measurement of tension. One question concerns the biological relevance of these experiments and the generalizability of these experiments to wider biological systems. Here, we categorize the applicability of the information garnered from optical tweezers in two distinct categories: the direct relevance of tension in biological systems, and what experiments under tension can tell us about biological systems, while these systems do not reach the same tension as the experiment, still, these artificial experimental systems reveal insights into the operations of biological machines and life processes.
ABSTRACT
Pathological processes often change the mechanical properties of cells. Increased rigidity could be a marker of cellular malfunction. Erythrocytes are a type of cell that deforms to squeeze through tiny capillaries; changes in their rigidity can dramatically affect their functionality. Furthermore, differences in the homeostatic elasticity of the cell can be used as a tool for diagnosis and even for choosing the adequate treatment for some illnesses. More accurate types of equipment needed to study biomechanical phenomena at the single-cell level are very costly and thus out of reach for many laboratories around the world. This study presents a simple and low-cost technique to study the rigidity of red blood cells (RBCs) through the application of electric fields in a hand-made microfluidic chamber that uses a capacitor principle. As RBCs are deformed with the application of voltage, cells are observed under a light microscope. From mechanical force vs. deformation data, the elastic constant of the cells is determined. The results obtained with the capacitor-based method were compared with those obtained using optical tweezers, finding good agreement. In addition, P. falciparum-infected erythrocytes were tested with the electric field applicator. Our technique provides a simple means of testing the mechanical properties of individual cells.
ABSTRACT
Semiflexible fiber gels such as collagen and fibrin have unique nonlinear mechanical properties that play an important role in tissue morphogenesis, wound healing, and cancer metastasis. Optical tweezers microrheology has greatly contributed to the understanding of the mechanics of fibrous gels at the microscale, including its heterogeneity and anisotropy. However, the explicit relationship between micromechanical properties and gel deformation has been largely overlooked. We introduce a unique gel-stretching apparatus and employ it to study the relationship between microscale strain and stiffening in fibrin and collagen gels, focusing on the development of anisotropy in the gel. We find that gels stretched by as much as 15 % stiffen significantly both in parallel and perpendicular to the stretching axis, and that the parallel axis is 2-3 times stiffer than the transverse axis. We also measure the stiffening and anisotropy along bands of aligned fibers created by aggregates of cancer cells, and find similar effects as in gels stretched with the tensile apparatus. Our results illustrate that the extracellular microenvironment is highly sensitive to deformation, with implications for tissue homeostasis and pathology. STATEMENT OF SIGNIFICANCE: The inherent fibrous architecture of the extracellular matrix (ECM) gives rise to unique strain-stiffening mechanics. The micromechanics of fibrous networks has been studied extensively, but the deformations involved in its stiffening at the microscale were not quantified. Here we introduce an apparatus that enables measuring the deformations in the gel as it is being stretched while simultaneously using optical tweezers to measure its microscale anisotropic stiffness. We reveal that fibrin and collagen both stiffen dramatically already at â¼10 % deformation, accompanied by the emergence of significant, yet moderate anisotropy. We measure similar stiffening and anisotropy in the matrix remodeled by the tensile apparatus to those found between cancer cell aggregates. Our results emphasize that small strains are enough to introduce substantial stiffening and anisotropy. These have been shown to result in directional cell migration and enhanced force propagation, and possibly control processes like morphogenesis and cancer metastasis.
Subject(s)
Fibrin , Gels , Rheology , Anisotropy , Gels/chemistry , Fibrin/chemistry , Humans , Tensile Strength , Stress, Mechanical , Collagen/chemistry , AnimalsABSTRACT
Fundamentally, the heart needs to generate sufficient force and power output to dynamically meet the needs of the body. Cardiomyocytes contain specialized structures referred to as sarcomeres that power and regulate contraction. Disruption of sarcomeric function or regulation impairs contractility and leads to cardiomyopathies and heart failure. Basic, translational, and clinical studies have adapted numerous methods to assess cardiac contraction in a variety of pathophysiological contexts. These tools measure aspects of cardiac contraction at different scales ranging from single molecules to whole organisms. Moreover, these studies have revealed new pathogenic mechanisms of heart disease leading to the development of novel therapies targeting contractility. In this review, the authors explore the breadth of tools available for studying cardiac contractile function across scales, discuss their strengths and limitations, highlight new insights into cardiac physiology and pathophysiology, and describe how these insights can be harnessed for therapeutic candidate development and translational.
ABSTRACT
Organelles and vesicular cargoes are transported by teams of kinesin and dynein motors along microtubules. We isolated endocytic organelles from cells at different stages of maturation and reconstituted their motility along microtubules in vitro. We asked how the sets of motors transporting a cargo determine its motility and response to the microtubule-associated protein tau. Here, we find that phagosomes move in both directions along microtubules, but the directional bias changes during maturation. Early phagosomes exhibit retrograde-biased transport while late phagosomes are directionally unbiased. Correspondingly, early and late phagosomes are bound by different numbers and combinations of kinesins-1, -2, -3, and dynein. Tau stabilizes microtubules and directs transport within neurons. While single-molecule studies show that tau differentially regulates the motility of kinesins and dynein in vitro, less is known about its role in modulating the trafficking of endogenous cargoes transported by their native teams of motors. Previous studies showed that tau preferentially inhibits kinesin motors, which biases late phagosome transport towards the microtubule minus-end. Here, we show that tau strongly inhibits long-range, dynein-mediated motility of early phagosomes. Tau reduces forces generated by teams of dynein motors on early phagosomes and accelerates dynein unbinding under load. Thus, cargoes differentially respond to tau, where dynein complexes on early phagosomes are more sensitive to tau inhibition than those on late phagosomes. Mathematical modeling further explains how small changes in the number of kinesins and dynein on cargoes impact the net directionality but also that cargoes with different sets of motors respond differently to tau.
Subject(s)
Dyneins , Kinesins , Microtubules , tau Proteins , Kinesins/metabolism , Kinesins/genetics , tau Proteins/metabolism , tau Proteins/genetics , Dyneins/metabolism , Dyneins/genetics , Animals , Microtubules/metabolism , Phagosomes/metabolism , Biological Transport , Mice , Humans , Endocytosis/physiologyABSTRACT
Tetraspanin 4, a protein with four transmembrane helices and three connecting loops, senses membrane curvature and localizes to membrane tubes. This enrichment in tubular membranes enhances its diverse interactions. While the transmembrane part of the protein likely contributes to curvature sensitivity, the possible roles of the ectodomains in curvature sensitivity of tetraspanin 4 are still unknown. Here, using micropipette aspiration combined with confocal microscopy and optical tweezers, we show that the extracellular loop 2 contributes to the curvature sensitivity and curvature-induced interactions of tetraspanin 4. To this end, we created truncated tetraspanin 4 mutants by deleting each of the connecting loops. Subsequently, we pulled membrane tubes from giant plasma membrane vesicles containing tetraspanin 4-GFP or its mutants while maintaining controllable membrane tension and curvature. Among the mutations tested, the removal of the extracellular loop 2 had the most significant impact on both the curvature sensitivity and interactions of tetraspanin 4. Based on the results, we suggest that the extracellular loop 2 regulates the affinity of tetraspanin 4 towards curved membranes and affects its lateral interactions.
ABSTRACT
The organization of nucleosomes into chromatin and their accessibility are shaped by local DNA mechanics. Conversely, nucleosome positions shape genetic variations, which may originate from mismatches during replication and chemical modification of DNA. To investigate how DNA mismatches affect the mechanical stability and the exposure of nucleosomal DNA, we used an optical trap combined with single-molecule FRET and a single-molecule FRET cyclization assay. We found that a single base-pair C-C mismatch enhances DNA bendability and nucleosome mechanical stability for the 601-nucleosome positioning sequence. An increase in force required for DNA unwrapping from the histone core is observed for single base-pair C-C mismatches placed at three tested positions: at the inner turn, at the outer turn, or at the junction of the inner and outer turn of the nucleosome. The results support a model where nucleosomal DNA accessibility is reduced by mismatches, potentially explaining the preferred accumulation of single-nucleotide substitutions in the nucleosome core and serving as the source of genetic variation during evolution and cancer progression. Mechanical stability of an intact nucleosome, that is mismatch-free, is also dependent on the species as we find that yeast nucleosomes are mechanically less stable and more symmetrical in the outer turn unwrapping compared to Xenopus nucleosomes.
Subject(s)
Base Pair Mismatch , DNA , Nucleosomes , Nucleosomes/metabolism , Nucleosomes/chemistry , Nucleosomes/genetics , DNA/chemistry , DNA/metabolism , DNA/genetics , Base Pair Mismatch/genetics , Animals , Fluorescence Resonance Energy Transfer , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Xenopus laevisABSTRACT
Transmembrane transporter proteins are essential for maintaining cellular homeostasis and, as such, are key drug targets. Many transmembrane transporter proteins are known to undergo large structural rearrangements during their functional cycles. Despite the wealth of detailed structural and functional data available for these systems, our understanding of their dynamics and, consequently, how they function is generally limited. We introduce an innovative approach that enables us to directly measure the dynamics and stability of interdomain interactions of transmembrane proteins using optical tweezers. Focusing on the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis, we examine the mechanical properties and potential interactions of its substrate-binding domains. Our measurements are performed in lipid nanodiscs, providing a native-mimicking environment for the transmembrane protein. The technique provides high spatial and temporal resolution and allows us to study the functionally relevant motions and interdomain interactions of individual transmembrane transporter proteins in real time in a lipid bilayer.
Subject(s)
ATP-Binding Cassette Transporters , Bacterial Proteins , Lactococcus lactis , Optical Tweezers , ATP-Binding Cassette Transporters/metabolism , ATP-Binding Cassette Transporters/chemistry , Lactococcus lactis/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Protein Binding , Protein Domains , Single Molecule Imaging , Protein Stability , Lipid Bilayers/metabolism , Lipid Bilayers/chemistryABSTRACT
Ebola virus (EBOV) is an enveloped virus that must fuse with the host cell membrane in order to release its genome and initiate infection. This process requires the action of the EBOV envelope glycoprotein (GP), encoded by the virus, which resides in the viral envelope and consists of a receptor binding subunit, GP1, and a membrane fusion subunit, GP2. Despite extensive research, a mechanistic understanding of the viral fusion process is incomplete. To investigate GP-membrane association, a key step in the fusion process, we used two approaches: high-throughput measurements of single-particle diffusion and single-molecule measurements with optical tweezers. Using these methods, we show that the presence of the endosomal Niemann-Pick C1 (NPC1) receptor is not required for primed GP-membrane binding. In addition, we demonstrate this binding is very strong, likely attributed to the interaction between the GP fusion loop and the membrane's hydrophobic core. Our results also align with previously reported findings, emphasizing the significance of acidic pH in the protein-membrane interaction. Beyond Ebola virus research, our approach provides a powerful toolkit for studying other protein-membrane interactions, opening new avenues for a better understanding of protein-mediated membrane fusion events.