Single-molecule fluorescence techniques are key for a number of applications, including DNA sequencing, molecular and cell biology and early diagnosis. Unfortunately, observation of single molecules ...by diffraction-limited optics is restricted to detection volumes in the femtolitre range and requires pico- or nanomolar concentrations, far below the micromolar range where most biological reactions occur. This limitation can be overcome using plasmonic nanostructures, which enable the confinement of light down to nanoscale volumes. Although these nanoantennas enhance fluorescence brightness, large background signals and/or unspecific binding to the metallic surface have hampered the detection of individual fluorescent molecules in solution at high concentrations. Here we introduce a novel 'antenna-in-box' platform that is based on a gap-antenna inside a nanoaperture. This design combines fluorescent signal enhancement and background screening, offering high single-molecule sensitivity (fluorescence enhancement up to 1,100-fold and microsecond transit times) at micromolar sample concentrations and zeptolitre-range detection volumes. The antenna-in-box device can be optimized for single-molecule fluorescence studies at physiologically relevant concentrations, as we demonstrate using various biomolecules.
Tissue morphogenesis arises from controlled cell deformations in response to cellular contractility. During Drosophila gastrulation, apical activation of the actomyosin networks drives apical ...constriction in the invaginating mesoderm and cell-cell intercalation in the extending ectoderm. Myosin II (MyoII) is activated by cell-surface G protein-coupled receptors (GPCRs), such as Smog and Mist, that activate G proteins, the small GTPase Rho1, and the kinase Rok. Quantitative control over GPCR and Rho1 activation underlies differences in deformation of mesoderm and ectoderm cells. We show that GPCR Smog activity is concentrated on two different apical plasma membrane compartments, i.e., the surface and plasma membrane invaginations. Using fluorescence correlation spectroscopy, we probe the surface of the plasma membrane, and we show that Smog homo-clusters in response to its activating ligand Fog. Endocytosis of Smog is regulated by the kinase Gprk2 and β-arrestin-2 that clears active Smog from the plasma membrane. When Fog concentration is high or endocytosis is low, Smog rearranges in homo-clusters and accumulates in plasma membrane invaginations that are hubs for Rho1 activation. Lastly, we find higher Smog homo-cluster concentration and numerous apical plasma membrane invaginations in the mesoderm compared to the ectoderm, indicative of reduced endocytosis. We identify that dynamic partitioning of active Smog at the surface of the plasma membrane or plasma membrane invaginations has a direct impact on Rho1 signaling. Plasma membrane invaginations accumulate high Rho1-guanosine triphosphate (GTP) suggesting they form signaling centers. Thus, Fog concentration and Smog endocytosis form coupled regulatory processes that regulate differential Rho1 and MyoII activation in the Drosophila embryo.
•Medial apical Rho1 and Myosin II activation are reduced by Gprk2 and β-arrestin•FCS reveals GPCR Smog homo-clustering induced by the ligand Fog•Gprk2 and β-arrestin clear Smog homo-clusters from the surface via Smog endocytosis•Differential GPCR regulation may involve GPCR endocytosis in addition to Fog levels
Jha et al. study activation of contractility by the motor Myosin II during epithelial tissue morphogenesis. This requires signaling by GPCRs and Rho1. They show that ligand concentration and receptor endocytosis are coupled processes that tune the density of homo-clustered receptors and set the amplitude of apical Rho1 and Myosin II activation.
Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic ...strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We report on a novel design for the fabrication of ultrabright bowtie nanoaperture antenna (BNA) probes to breach the intrinsic trade-off between power transmission and field confinement of circular ...nanoapertures as in near-field scanning optical microscopy (NSOM) or planar zero mode waveguides. The approach relies on the nanofabrication of BNAs at the apex of tapered optical fibers tuned to diameters close to their cutoff region, resulting in 103× total improvement in throughput over conventional NSOM probes of similar confinement area. By using individual fluorescence molecules as optical nanosensors, we show for the first time nanoimaging of single molecules using BNA probes with an optical confinement of 80 nm, measured the 3D near-field emanating from these nanostructures and determined a ∼6-fold enhancement on the single molecule signal. The broadband field enhancement, nanoscale confinement, and background free illumination provided by these nanostructures offer excellent perspectives as ultrabright optical nanosources for a full range of applications, including cellular nanoimaging, spectroscopy, and biosensing.
Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a major class of lipid-anchored plasma membrane proteins. GPI-APs form nanoclusters generated by cortical acto-myosin activity. While our ...understanding of the physical principles governing this process is emerging, the molecular machinery and functional relevance of GPI-AP nanoclustering are unknown. Here, we first show that a membrane receptor signaling pathway directs nanocluster formation. Arg-Gly-Asp motif-containing ligands bound to the β1-integrin receptor activate src and focal adhesion kinases, resulting in RhoA signaling. This cascade triggers actin-nucleation via specific formins, which, along with myosin activity, drive the nanoclustering of membrane proteins with actin-binding domains. Concurrently, talin-mediated activation of the mechano-transducer vinculin is required for the coupling of the acto-myosin machinery to inner-leaflet lipids, thereby generating GPI-AP nanoclusters. Second, we show that these nanoclusters are functional; disruption of their formation either in GPI-anchor remodeling mutants or in vinculin mutants impairs cell spreading and migration, hallmarks of integrin function.
Display omitted
•Integrin signaling triggers RhoA-Formin to drive acto-myosin based nanoclustering•Force-mediated vinculin activation promotes GPI-anchored protein nanoclustering•GPI-anchored protein nanoclusters regulate Fibronectin-mediated integrin function•Integrins represent an active element of the actin-membrane composite model
Nanodomains of GPI-anchored proteins at the plasma membrane are constructed downstream of a cytoplasmic machinery triggered by membrane receptor signaling cascade. They are also sensitive to the lipid composition of the membrane. These domains in turn regulate receptor-mediated physiological responses, modulating the functions of the signaling receptor.
Recently, the implementation of plasmonic nanoantennas has opened new possibilities to investigate the nanoscale dynamics of individual biomolecules in living cells. However, studies so far have been ...restricted to single molecular species as the narrow wavelength resonance of gold‐based nanostructures precludes the simultaneous interrogation of different fluorescently labeled molecules. Here, broadband aluminum‐based nanoantennas carved at the apex of near‐field probes are exploited to resolve nanoscale‐dynamic molecular interactions on living cell membranes. Through multicolor excitation, the authors simultaneously recorded fluorescence fluctuations of dual‐color labeled transmembrane receptors known to form nanoclusters. Fluorescence cross‐correlation studies revealed transient interactions between individual receptors in regions of ≈60 nm. Moreover, the high signal‐to‐background ratio provided by the antenna illumination allowed the authors to directly detect fluorescent bursts arising from the passage of individual receptors underneath the antenna. Remarkably, by reducing the illumination volume below the characteristic receptor nanocluster sizes, the molecular diffusion within nanoclusters is resolved and distinguished from nanocluster diffusion. Spatiotemporal characterization of transient interactions between molecules is crucial to understand how they communicate with each other to regulate cell function. This work demonstrates the potential of broadband photonic antennas to study multi‐molecular events and interactions in living cell membranes with unprecedented spatiotemporal resolution.
A plasmonic antenna is engineered at the apex of a near‐field probe to illuminate a living cell at the nanoscale. This nanosource of light allows monitoring the fluorescence fluctuations of diffusing molecules inside this tiny illumination volume, which allows resolving molecular diffusion within nanoclusters and distinguishes it from nanocluster diffusion.
Broadband Plasmonic Nanoantennas
A plasmonic antenna is engineered at the apex of a near‐field probe to illuminate a living cell at the nanoscale. This nanosource of light allows monitoring the ...fluorescence fluctuations of diffusing molecules inside this tiny illumination volume, which allows resolving molecular diffusion within nanoclusters and distinguishes it from nanocluster diffusion. More details can be found in article number 2207977 by Mathieu Mivelle, Maria F. Garcia‐Parajo, and co‐workers.
Recruitment of receptor proteins to lipid rafts has been proposed as an important mechanism to regulate their cellular function. In particular, rafts have been implicated in regulation of ...integrin-mediated cell adhesion, although the underlying mechanism remains elusive. We used single-molecule near-field optical microscopy (NSOM) with localization accuracy of approximately 3 nm, to capture the spatio-functional relationship between the integrin LFA-1 and raft components (GPI-APs) on immune cells. Dual color nanoscale imaging revealed the existence of a nanodomain GPI-AP subpopulation that further concentrated in regions smaller than 250 nm, suggesting a hierarchical prearrangement of GPI-APs on resting monocytes. We previously demonstrated that in quiescent monocytes, LFA-1 preorganizes in nanoclusters. We now show that integrin nanoclusters are spatially different but reside proximal to GPI-AP nanodomains, forming hotspots on the cell surface. Ligand-mediated integrin activation resulted in an interconversion from monomers to nanodomains of GPI-APs and the generation of nascent adhesion sites where integrin and GPI-APs colocalized at the nanoscale. Cholesterol depletion significantly affected the reciprocal distribution pattern of LFA-1 and GPI-APs in the resting state, and LFA-1 adhesion to its ligand. As such, our data demonstrate the existence of nanoplatforms as essential intermediates in nascent cell adhesion. Since raft association with a variety of membrane proteins other than LFA-1 has been documented, we propose that hotspots regions enriched with raft components and functional receptors may constitute a prototype of nanoscale inter-receptor assembly and correspond to a generic mechanism to offer cells with privileged areas for rapid cellular function and responses to the outside world.
Quantitative fluorescence emission anisotropy microscopy reveals the organization of fluorescently labeled cellular components and allows their characterization in terms of changes in either ...rotational diffusion or homo-Förster's energy transfer characteristics in living cells. These properties provide insights into molecular organization, such as orientation, confinement, and oligomerization in situ. Here we elucidate how quantitative measurements of anisotropy using multiple microscope systems may be made by bringing out the main parameters that influence the quantification of fluorescence emission anisotropy. We focus on a variety of parameters that contribute to errors associated with the measurement of emission anisotropy in a microscope. These include the requirement for adequate photon counts for the necessary discrimination of anisotropy values, the influence of extinction ratios of the illumination source, the detector system, the role of numerical aperture, and excitation wavelength. All these parameters also affect the ability to capture the dynamic range of emission anisotropy necessary for quantifying its reduction due to homo-FRET and other processes. Finally, we provide easily implementable tests to assess whether homo-FRET is a cause for the observed emission depolarization.
PDF
