Evaluation of functional transbilayer coupling in live cells by controlled lipid exchange and imaging fluorescence correlation spectroscopy.

Biophysical coupling between the inner and outer leaflets, known as inter-leaflet or transbilayer coupling, is a fundamental organizational principle in the plasma membranes of live mammalian cells. Lipid-based interactions between the two leaflets are proposed to be a primary mechanism underlying transbilayer coupling. However, there are only a few experimental evidence supporting the existence of such interactions in live cells.

Overcoming Barriers Associated with Oral Delivery of Differently Sized Fluorescent Core-Shell Silica Nanoparticles.

Oral delivery, while a highly desirable form of nanoparticle-drug administration, is limited by challenges associated with overcoming several biological barriers. Here, the authors study how fluorescent and poly(ethylene glycol)-coated (PEGylated) core-shell silica nanoparticles sized 5 to 50 nm interact with major barriers including intestinal mucus, intestinal epithelium, and stomach acid. From imaging fluorescence correlation spectroscopy studies using quasi-total internal reflection fluorescence microscopy, diffusion of nanoparticles through highly scattering mucus is progressively hindered above a critical hydrodynamic size around 20 nm.

Lipid-driven interleaflet coupling of plasma membrane order regulates FcεRI signaling in mast cells.

Interleaflet coupling-the influence of one leaflet on the properties of the opposing leaflet-is a fundamental plasma membrane organizational principle. This coupling is proposed to participate in maintaining steady-state biophysical properties of the plasma membrane, which in turn regulates some transmembrane signaling processes. A prominent example is antigen (Ag) stimulation of signaling by clustering transmembrane receptors for immunoglobulin E (IgE), FcεRI.

Transbilayer Coupling of Lipids in Cells Investigated by Imaging Fluorescence Correlation Spectroscopy.

Plasma membranes host numerous receptors, sensors, and ion channels involved in cellular signaling. Phase separation within the plasma membrane has emerged as a key biophysical regulator of signaling reactions in multiple physiological and pathological contexts. There is much evidence that plasma membrane composition supports the coexistence of liquid-ordered (Lo) and liquid-disordered (Ld) phases or domains at physiological conditions.

A palmitoylation code controls PI4KIIIα complex formation and PI(4,5)P2 homeostasis at the plasma membrane.

Phosphatidylinositol 4-kinase IIIα (PI4KIIIα) is the major enzyme responsible for generating phosphatidylinositol (4)-phosphate [PI(4)P] at the plasma membrane. This lipid kinase forms two multicomponent complexes, both including a palmitoylated anchor, EFR3. Whereas both PI4KIIIα complexes support production of PI(4)P, the distinct functions of each complex and mechanisms underlying the interplay between them remain unknown.

Lipid-based and protein-based interactions synergize transmembrane signaling stimulated by antigen clustering of IgE receptors.

Antigen (Ag) crosslinking of immunoglobulin E-receptor (IgE-FcεRI) complexes in mast cells stimulates transmembrane (TM) signaling, requiring phosphorylation of the clustered FcεRI by lipid-anchored Lyn tyrosine kinase. Previous studies showed that this stimulated coupling between Lyn and FcεRI occurs in liquid ordered (Lo)-like nanodomains of the plasma membrane and that Lyn binds directly to cytosolic segments of FcεRI that it initially phosphorylates for amplified activity. Net phosphorylation above a nonfunctional threshold is achieved in the stimulated state but not in the resting state, and current evidence supports the hypothesis that this relies on Ag crosslinking to disrupt a balance between Lyn and tyrosine phosphatase activities.

Ultrasmall, Bright, and Photostable Fluorescent Core-Shell Aluminosilicate Nanoparticles for Live-Cell Optical Super-Resolution Microscopy.

Stochastic optical reconstruction microscopy (STORM) is an optical super-resolution microscopy (SRM) technique that traditionally requires toxic and non-physiological imaging buffers and setups that are not conducive to live-cell studies. It is observed that ultrasmall (<10 nm) fluorescent core-shell aluminosilicate nanoparticles (aC' dots) covalently encapsulating organic fluorophores enable STORM with a single excitation source and in a regular (non-toxic) imaging buffer. It is shown that fourfold coordinated aluminum is responsible for dye blinking, likely via photoinduced redox processes.

Imaging FCS delineates subtle heterogeneity in plasma membranes of resting mast cells.

A myriad of transient, nanoscopic lipid- and protein-based interactions confer a steady-state organization of the plasma membrane in resting cells that is poised to orchestrate assembly of key signaling components upon reception of an extracellular stimulus. Although difficult to observe directly in live cells, these subtle interactions can be discerned by their impact on the diffusion of membrane constituents. Here, we quantified the diffusion properties of a panel of structurally distinct lipid, lipid-anchored, and transmembrane (TM) probes in RBL mast cells by imaging fluorescence correlation spectroscopy (ImFCS).

A Funneled Conformational Landscape Governs Flavivirus Fusion Peptide Interaction with Lipid Membranes.

During host cell infection by flaviviruses such as dengue and Zika, acidic pH within the endosome triggers a conformational change in the envelope protein on the outer surface of the virion. This results in exposure of the ∼15 residue fusion peptide (FP) region, freeing it to induce fusion between the viral and endosomal membranes. A better understanding of the conformational dynamics of the FP in the presence of membranes, and the basis for its selectivity for anionic lipid species present within the endosome, would facilitate its therapeutic targeting with antiviral drugs and antibodies.

Spatiotemporal mapping of diffusion dynamics and organization in plasma membranes.

Imaging fluorescence correlation spectroscopy (FCS) and the related FCS diffusion law have been applied in recent years to investigate the diffusion modes of lipids and proteins in membranes. These efforts have provided new insights into the membrane structure below the optical diffraction limit, new information on the existence of lipid domains, and on the influence of the cytoskeleton on membrane dynamics. However, there has been no systematic study to evaluate how domain size, domain density, and the probe partition coefficient affect the resulting imaging FCS diffusion law parameters.

Conformational changes in intact dengue virus reveal serotype-specific expansion.

Dengue virus serotype 2 (DENV2) alone undergoes structural expansion at 37 °C (associated with host entry), despite high sequence and structural homology among the four known serotypes. The basis for this differential expansion across strains and serotypes is unknown and necessitates mapping of the dynamics of dengue whole viral particles to describe their coordinated motions and conformational changes when exposed to host-like environments. Here we capture the dynamics of intact viral particles of two serotypes, DENV1 and DENV2, by amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and time resolved Förster Resonance Energy Transfer.

Plasma membrane organization and dynamics is probe and cell line dependent.

The action and interaction of membrane receptor proteins take place within the plasma membrane. The plasma membrane, however, is not a passive matrix. It rather takes an active role and regulates receptor distribution and function by its composition and the interaction of its lipid components with embedded and surrounding proteins.

Imaging fluorescence (cross-) correlation spectroscopy in live cells and organisms.

Single-plane illumination (SPIM) or total internal reflection fluorescence (TIRF) microscopes can be combined with fast and single-molecule-sensitive cameras to allow spatially resolved fluorescence (cross-) correlation spectroscopy (FCS or FCCS, hereafter referred to FCS/FCCS). This creates a powerful quantitative bioimaging tool that can generate spatially resolved mobility and interaction maps with hundreds to thousands of pixels per sample. These massively parallel imaging schemes also cause less photodamage than conventional single-point confocal microscopy-based FCS/FCCS.

Plasma Membrane Organization of Epidermal Growth Factor Receptor in Resting and Ligand-Bound States.

The spatial arrangement of the epidermal growth factor receptor (EGFR) on the cellular plasma membrane is one of the prime factors that control its downstream signaling pathways and related functions. However, the molecular organization, which spans the scale from nanometers to micrometer-size clusters, has not been resolved in detail, mainly due to a lack of techniques with the required spatiotemporal resolution. Therefore, we used imaging total internal reflection-fluorescence correlation spectroscopy to investigate EGFR dynamics on live CHO-K1 plasma membranes in resting and ligand-bound states.

Characterization of Lipid and Cell Membrane Organization by the Fluorescence Correlation Spectroscopy Diffusion Law.

The plasma membrane organization of live cells defines a plethora of cellular processes important for cell functionality. Many membrane structures that define this organization exist at a spatial resolution below the optical diffraction limit and are highly dynamic. Therefore, a method with millisecond time resolution and nanometer spatial resolution is required for the investigation of plasma membrane organization.

Temperature dependence of diffusion in model and live cell membranes characterized by imaging fluorescence correlation spectroscopy.

The organization of the plasma membrane is regulated by the dynamic equilibrium between the liquid ordered(Lo) and liquid disordered (Ld) phases. The abundance of the Lo phase is assumed to be a consequence of the interaction between cholesterol and the other lipids, which are otherwise in either the Ld or gel (So) phase.The characteristic lipid packing in these phases results in significant differences in their respective lateral dynamics.

Bayesian total internal reflection fluorescence correlation spectroscopy reveals hIAPP-induced plasma membrane domain organization in live cells.

Amyloid fibril deposition of human islet amyloid polypeptide (hIAPP) in pancreatic islet cells is implicated in the pathogenesis of type II diabetes. A growing number of studies suggest that small peptide aggregates are cytotoxic via their interaction with the plasma membrane, which leads to membrane permeabilization or disruption. A recent study using imaging total internal reflection-fluorescence correlation spectroscopy (ITIR-FCS) showed that monomeric hIAPP induced the formation of cellular plasma membrane microdomains containing dense lipids, in addition to the modulation of membrane fluidity.

Imaging fluorescence fluctuation spectroscopy: new tools for quantitative bioimaging.

Fluorescence fluctuation spectroscopy (FFS) techniques provide information at the single-molecule level with excellent time resolution. Usually applied at a single spot in a sample, they have been recently extended into imaging formats, referred to as imaging FFS. They provide spatial information at the optical diffraction limit and temporal information in the microsecond to millisecond range.

Membrane destabilization by monomeric hIAPP observed by imaging fluorescence correlation spectroscopy.

Monomeric hIAPP significantly destabilizes both model and live cell membranes by increasing membrane fluidity. This interaction with membranes happens via carpet formation followed by lipid extraction in a concentration dependent manner and thus we propose that hIAPP aggregation prior to membrane interaction may not be necessary for its cytotoxicity.

Accuracy and precision in camera-based fluorescence correlation spectroscopy measurements.

Imaging fluorescence correlation spectroscopy (FCS) performed using array detectors has been successfully used to quantify the number, mobility, and organization of biomolecules in cells and organisms. However, there have not been any systematic studies on the errors in these estimates that are introduced due to instrumental and experimental factors. State-of-the-art array detectors are still restricted in the number of frames that can be recorded per unit time, sensitivity and noise characteristics, and the total number of frames that can be realistically recorded.

Calibration and limits of camera-based fluorescence correlation spectroscopy: a supported lipid bilayer study.

Camera-based fluorescence correlation spectroscopy (FCS) approaches allow the measurement of thousands of contiguous points yielding excellent statistics and details of sample structure. Imaging total internal reflection FCS (ITIR-FCS) provides these measurements on lipid membranes. Herein, we determine the influence of the point spread function (PSF) of the optical system, the laser power used, and the time resolution of the camera on the accuracy of diffusion coefficient and concentration measurements.

Fluorescence correlation methods for imaging cellular behavior of sphingolipid-interacting probes.

For cell biologists interested in the properties of cell membranes, their composition, and dynamics, the realization that sphingolipids and cholesterol have the capacity to self-organize into ordered domains has given rise to a need to visualize these lipids in actual living cell membranes. In order to find out how various classes of lipids distribute in the membrane and what their behaviors are, it is extremely useful to apply fluorescent probes that either interact with these lipids, or that themselves behave like naturally occurring lipids. At the same time, imaging modalities to observe their behaviors require the appropriate spatial and temporal resolution, on the milli- or microsecond timescale.