The plasma membrane represents an outstanding example of self-organization in biology. It plays a vital role in protecting the integrity of the cell interior and regulates meticulously the import and ...export of diverse substances. Its major building blocks are proteins and lipids, which self-assemble to a fluid lipid bilayer driven mainly by hydrophobic forces. Even if the plasma membrane appears—globally speaking—homogeneous at physiological temperatures, the existence of specialized nano- to micrometre-sized domains of raft-type character within cellular and synthetic membrane systems has been reported. It is hypothesized that these domains are the origin of a plethora of cellular processes, such as signalling or vesicular trafficking. This review intends to highlight the driving forces of lipid self-assembly into a bilayer membrane and the formation of small, transient domains within the plasma membrane. The mechanisms of self-assembly depend on several factors, such as the lipid composition of the membrane and the geometry of lipids. Moreover, the dynamics and organization of glycosphingolipids into nanometre-sized clusters will be discussed, also in the context of multivalent lectins, which cluster several glycosphingolipid receptor molecules and thus create an asymmetric stress between the two membrane leaflets, leading to tubular plasma membrane invaginations.
This article is part of the theme issue ‘Self-organization in cell biology’.
Spectrally‐resolved single molecule localization microscopy (srSMLM) is a recent technique enriching single molecule localization microscopy with the simultaneous recording of spectra of the single ...emitters. srSMLM resolution is limited by the number of photons collected per emitters. Sharing a photon budget to record the localization and the spectroscopic information results in a loss of spatial and spectral resolution—or forces the sacrifice of one at the expense of the other. Here, srUnet—a deep‐learning Unet‐based image processing routine trained to increase the spectral and spatial signals to compensate for the resolution loss inherent in additionally recording the spectral component is reported. Both localization and spectral precision are improved by srUnet—particularly for the low‐emitting species. srUnet increases the fraction of localization whose signal can be both spatially and spectrally characterized. It preserves spectral shifts and the linearity of the dispersion of light. It strongly facilitates wavelength assignment in multicolor experiments. srUnet is a simple post‐processing add‐on boosting srSMLM performance close to conventional SMLM with the potential to turn srSMLM into the new standard for multicolor single molecule imaging.
srUnet is a postprocessing routine based on a deep‐learning network trained to enhanced spectrally resolved single molecule localization microscopy images. srUnet improves raw images and results in better spatial and spectral precision images. Also, srUnet offsets the cost of spectral data acquisition to bring srSMLM performance close to that of classical SMLM.
Fluorescent nucleoside analogues (FNAs) are structurally diverse mimics of the natural essentially non-fluorescent nucleosides which have found numerous applications in probing the structure and ...dynamics of nucleic acids as well as their interactions with various biomolecules. In order to minimize disturbance in the labelled nucleic acid sequences, the FNA chromophoric groups should resemble the natural nucleobases in size and hydrogen-bonding patterns. Isomorphic and expanded FNAs are the two groups that best meet the criteria of non-perturbing fluorescent labels for DNA and RNA. Significant progress has been made over the past decades in understanding the fundamental photophysics that governs the spectroscopic and environmentally sensitive properties of these FNAs. Herein, we review recent advances in the spectroscopic and computational studies of selected isomorphic and expanded FNAs. We also show how this information can be used as a rational basis to design new FNAs, select appropriate sequences for optimal spectroscopic response and interpret fluorescence data in FNA applications.
Understanding and optimization of the photophysics of fluorescent nucleoside analogues are critical for their applications in probing the structure and dynamics of nucleic acids, and studying their interactions with ligands and biomolecules.
Spectrally-resolved single-molecule localization microscopy (srSMLM) has emerged as a powerful tool for exploring the spectral properties of single emitters in localization microscopy. By ...simultaneously capturing the spatial positions and spectroscopic signatures of individual fluorescent molecules, srSMLM opens up the possibility of investigating an additional dimension in super-resolution imaging. However, appropriate and dedicated tools are required to fully capitalize on the spectral dimension. Here, we propose the application of the spectral phasor analysis as an effective method for summarizing and analyzing the spectral information obtained from srSMLM experiments. The spectral phasor condenses the complete spectrum of a single emitter into a two-dimensional space, preserving key spectral characteristics for single-molecule spectral exploration. We demonstrate the effectiveness of spectral phasor in efficiently classifying single Nile Red fluorescence emissions from largely overlapping cyanine fluorescence signals in dual-color PAINT experiments. Additionally, we employed spectral phasor with srSMLM to reveal subtle alterations occurring in the membrane of Gram-positive Enterococcus hirae in response to gramicidin exposure, a membrane-perturbing antibiotic treatment. Spectral phasor provides a robust, model-free analytic tool for the detailed analysis of the spectral component of srSMLM, enhancing the capabilities of multi-color spectrally-resolved single-molecule imaging.
The HIV-1 nucleocapsid protein (NC) is a desirable target in antiretroviral therapy due to its high conservation among HIV-1 strains, and to its multiple and crucial roles in the HIV-1 replication ...cycle. Natural products represent a valuable source of NC inhibitors, with the catechol group being a privileged scaffold in NC inhibition. By coupling molecular modeling with NMR spectroscopy and fluorescence-based assays, we disclosed lithospermic acid, a catechol derivative extracted from
, as a potent and chemically stable non-covalent inhibitor of the NC. Being different from other catechol derivative reported so far, lithospermic acid does not undergo spontaneous oxidation in physiological conditions, thus becoming a profitable starting point for the development of efficient NC inhibitors.
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•HIV-1 Gag protein adopts a compact (C-Gag) form in cells.•C-Gag formation requires both MA and NC RNA-binding domains.•C-Gag is stabilized by an intramolecular interaction between MA ...and CA domains.•Mutation altering C-Gag formation reduce infectious pseudoparticles production and infectivity.
HIV-1 Gag polyprotein plays a pivotal role in assembly and budding of new particles, by specifically packaging two copies of viral gRNA in the host cell cytoplasm and selecting the cell plasma membrane for budding. Both gRNA and membrane selections are thought to be mediated by the compact form of Gag. This compact form binds to gRNA through both its matrix (MA) and nucleocapsid (NC) domains in the cytoplasm. At the plasma membrane, the membrane competes with gRNA for Gag binding, resulting in a transition to the extended form of Gag found in immature particles with MA bound to membrane lipids and NC to gRNA. The Gag compact form was previously evidenced in vitro. Here, we demonstrated the compact form of Gag in cells by confocal microscopy, using a bimolecular fluorescence complementation approach with a split-GFP bipartite system. Using wild-type Gag and Gag mutants, we showed that the compact form is highly dependent on the binding of MA and NC domains to RNA, as well as on interactions between MA and CA domains. In contrast, Gag multimerization appears to be less critical for the accumulation of the compact form. Finally, mutations altering the formation of Gag compact form led to a strong reduction in viral particle production and infectivity, revealing its key role in the production of infectious viral particles.
Delivery systems able to coencapsulate both hydrophilic and hydrophobic species are of great interest in both fundamental research and industrial applications. Water-in-oil-in-water (w1/O/W2) ...emulsions are interesting systems for this purpose, but they suffer from limited stability. In this study, we propose an innovative approach to stabilize double emulsions by the synthesis of a silica membrane at the water/oil interface of the primary emulsion (i.e., inner w1/O emulsion). This approach allows the formulation of stable double emulsions through a two-step process, enabling high encapsulation efficiencies of model hydrophilic dyes encapsulated in the internal droplets. This approach also decreases the scale of the double droplets up to the nanoscale, which is not possible without silica stabilization. Different formulation and processing parameters were explored in order to optimize the methodology. Physicochemical characterization was performed by dynamic light scattering, encapsulation efficiency measurements, release profiles, and optical and transmission electron microscopies.
Ostreolysin A6 (OlyA6) is an oyster mushroom-derived membrane-binding protein that, upon recruitment of its partner protein, pleurotolysin B, forms a cytolytic membrane pore complex. OlyA6 itself is ...not cytolytic but has been reported to exhibit pro-apoptotic activities in cell culture. Here we report the formation dynamics and the structure of OlyA6 assembly on a lipid membrane containing an OlyA6 high-affinity receptor, ceramide phosphoethanolamine, and cholesterol. High-speed atomic force microscopy revealed the reorganization of OlyA6 dimers from initial random surface coverage to 2D protein crystals composed of hexameric OlyA6 repeat units. Crystal growth took place predominantly in the longitudinal direction by the association of OlyA6 dimers, forming a hexameric unit cell. Molecular-level examination of the OlyA6 crystal elucidated the arrangement of dimers within the unit cell and the structure of the dimer that recruits pleurotolysin B for pore formation.
Many molecular processes within a cell are carried out by molecular machines built from a large number of proteins organized by their protein-protein interactions (PPIs). Exploring PPIs in their ...cellular context is critical to better understand the proteins functions. Förster resonance energy transfer measured by fluorescence lifetime imaging (FLIM-FRET) enables to monitor PPIs and to map their spatial organization in a living cell with high spatial and temporal specificity. But both the accurate measurement and the interpretation of multi-exponential FLIM-FRET data associated to mixtures of interacting and non-interacting proteins are difficult. Here we show that a simple diagram plot can find interesting visualization properties by clustering pixels with similar decay signatures. FLIM diagram plot can be used to provide valuable information about stoichiometry and binding mode in PPIs, even in the presence of large differences in protein expression levels of the different interacting partners. The proposed FLIM diagram plot is a useful visual approach for a more straightforward interpretation of complex lifetime data. This approach was applied for revealing critical features of PPIs in live Pseudomonas aeruginosa.
Ensemble and single particle studies of the excitation power density (
P
)-dependent upconversion luminescence (UCL) of core and core-shell β-NaYF
4
:Yb,Er upconversion nanoparticles (UCNPs) doped ...with 20% Yb
3+
and 1% or 3% Er
3+
performed over a
P
regime of 6 orders of magnitude reveal an increasing contribution of the emission from high energy Er
3+
levels at
P
> 1 kW/cm
2
. This changes the overall emission color from initially green over yellow to white. While initially the green and with increasing
P
the red emission dominate in ensemble measurements at
P
< 1 kW/cm
2
, the increasing population of higher Er
3+
energy levels by multiphotonic processes at higher
P
in single particle studies results in a multitude of emission bands in the ultraviolet/visible/near infrared (UV/vis/NIR) accompanied by a decreased contribution of the red luminescence. Based upon a thorough analysis of the P-dependence of UCL, the emission bands activated at high
P
were grouped and assigned to 2–3, 3–4, and 4 photonic processes involving energy transfer (ET), excited-state absorption (ESA), cross-relaxation (CR), back energy transfer (BET), and non-radiative relaxation processes (nRP). This underlines the
P
-tunability of UCNP brightness and color and highlights the potential of
P
-dependent measurements for mechanistic studies required to manifest the population pathways of the different Er
3+
levels.
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