Separating Actin-Dependent Chemokine Receptor Nanoclustering from Dimerization Indicates a Role for Clustering in CXCR4 Signaling and Function.

A current challenge in cell motility studies is to understand the molecular and physical mechanisms that govern chemokine receptor nanoscale organization at the cell membrane, and their influence on cell response. Using single-particle tracking and super-resolution microscopy, we found that the chemokine receptor CXCR4 forms basal nanoclusters in resting T cells, whose extent, dynamics, and signaling strength are modulated by the orchestrated action of the actin cytoskeleton, the co-receptor CD4, and its ligand CXCL12. We identified three CXCR4 structural residues that are crucial for nanoclustering and generated an oligomerization-defective mutant that dimerized but did not form nanoclusters in response to CXCL12, which severely impaired signaling. Overall, our data provide new insights to the field of chemokine biology by showing that receptor dimerization in the absence of nanoclustering is unable to fully support CXCL12-mediated responses, including signaling and cell function in vivo.

A progesterone derivative linked to a stable phospholipid activates breast cancer cell response without leaving the cell membrane.

In hormone-responsive breast cancer cells, progesterone (P4) has been shown to act via its nuclear receptor (nPR), a ligand-activated transcription factor. A small fraction of progesterone receptor is palmitoylated and anchored to the cell membrane (mbPR) forming a complex with estrogen receptor alpha (ERα). Upon hormone exposure, either directly or via interaction with ERα, mbPR activates the SRC/RAS/ERK kinase pathway leading to phosphorylation of nPR by ERK. Kinase activation is essential for P4 gene regulation, as the ERK and MSK1 kinases are recruited by the nPR to its genomic binding sites and trigger chromatin remodeling. An interesting open question is whether activation of mbPR can result in gene regulation in the absence of ligand binding to intracellular progesterone receptor (iPR). This matter has been investigated in the past using P4 attached to serum albumin, but the attachment is leaky and albumin can be endocytosed and degraded, liberating P4. Here, we propose a more stringent approach to address this issue by ensuring attachment of P4 to the cell membrane via covalent binding to a stable phospholipid. This strategy identifies the actions of P4 independent from hormone binding to iPR. We found that a membrane-attached progestin can activate mbPR, the ERK signaling pathway leading to iPR phosphorylation, initial gene regulation and entry into the cell cycle, in the absence of detectable intracellular progestin.

Stochastic particle unbinding modulates growth dynamics and size of transcription factor condensates in living cells.

Liquid-liquid phase separation (LLPS) is emerging as a key physical principle for biological organization inside living cells, forming condensates that play important regulatory roles. Inside living nuclei, transcription factor (TF) condensates regulate transcriptional initiation and amplify the transcriptional output of expressed genes. However, the biophysical parameters controlling TF condensation are still poorly understood. Here we applied a battery of single-molecule imaging, theory, and simulations to investigate the physical properties of TF condensates of the progesterone receptor (PR) in living cells. Analysis of individual PR trajectories at different ligand concentrations showed marked signatures of a ligand-tunable LLPS process. Using a machine learning architecture, we found that receptor diffusion within condensates follows fractional Brownian motion resulting from viscoelastic interactions with chromatin. Interestingly, condensate growth dynamics at shorter times is dominated by Brownian motion coalescence (BMC), followed by a growth plateau at longer timescales that result in nanoscale condensate sizes. To rationalize these observations, we extended on the BMC model by including the stochastic unbinding of particles within condensates. Our model reproduced the BMC behavior together with finite condensate sizes at the steady state, fully recapitulating our experimental data. Overall, our results are consistent with condensate growth dynamics being regulated by the escaping probability of PR molecules from condensates. The interplay between condensation assembly and molecular escaping maintains an optimum physical condensate size. Such phenomena must have implications for the biophysical regulation of other nuclear condensates and could also operate in multiple biological scenarios.

Altered CXCR4 dynamics at the cell membrane impairs directed cell migration in WHIM syndrome patients.

Chemokine receptor nanoscale organization at the cell membrane is orchestrated by the actin cytoskeleton and influences cell responses. Using single-particle tracking analysis we show that CXCR4R334X, a truncated mutant chemokine receptor linked to WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis), fails to nanoclusterize after CXCL12 stimulation, and alters the lateral mobility and spatial organization of CXCR4 when coexpressed. These findings correlate with multiple phalloidin-positive protrusions in cells expressing CXCR4R334X, and their inability to correctly sense chemokine gradients. The underlying mechanisms involve inappropriate actin cytoskeleton remodeling due to the inadequate ß-arrestin1 activation by CXCR4R334X, which disrupts the equilibrium between activated and deactivated cofilin. Overall, we provide insights into the molecular mechanisms governing CXCR4 nanoclustering, signaling and cell function, and highlight the essential scaffold role of ß-arrestin1 to support CXCL12-mediated actin reorganization and receptor clustering. These defects associated with CXCR4R334X expression might contribute to the severe immunological symptoms associated with WHIM syndrome.

Tensing Flipper: Photosensitized Manipulation of Membrane Tension, Lipid Phase Separation, and Raft Protein Sorting in Biological Membranes.

The lateral organization of proteins and lipids in the plasma membrane is fundamental to regulating a wide range of cellular processes. Compartmentalized ordered membrane domains enriched with specific lipids, often termed lipid rafts, have been shown to modulate the physicochemical and mechanical properties of membranes and to drive protein sorting. Novel methods and tools enabling the visualization, characterization, and/or manipulation of membrane compartmentalization are crucial to link the properties of the membrane with cell functions. Flipper, a commercially available fluorescent membrane tension probe, has become a reference tool for quantitative membrane tension studies in living cells. Here, we report on a so far unidentified property of Flipper, namely, its ability to photosensitize singlet oxygen (1O2) under blue light when embedded into lipid membranes. This in turn results in the production of lipid hydroperoxides that increase membrane tension and trigger phase separation. In biological membranes, the photoinduced segregated domains retain the sorting ability of intact phase-separated membranes, directing raft and nonraft proteins into ordered and disordered regions, respectively, in contrast to radical-based photo-oxidation reactions that disrupt raft protein partitioning. The dual tension reporting and photosensitizing abilities of Flipper enable simultaneous visualization and manipulation of the mechanical properties and lateral organization of membranes, providing a powerful tool to optically control lipid raft formation and to explore the interplay between membrane biophysics and cell function.

Broadband Plasmonic Nanoantennas for Multi-Color Nanoscale Dynamics in Living Cells.

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.

Shear forces induce ICAM-1 nanoclustering on endothelial cells that impact on T-cell migration.

The leukocyte-specific ß2-integrin LFA-1 and its ligand ICAM-1, expressed on endothelial cells (ECs), are involved in the arrest, adhesion, and transendothelial migration of leukocytes. Although the role of mechanical forces on LFA-1 activation is well established, the impact of forces on its major ligand ICAM-1 has received less attention. Using a parallel-plate flow chamber combined with confocal and super-resolution microscopy, we show that prolonged shear flow induces global translocation of ICAM-1 on ECs upstream of flow direction. Interestingly, shear forces caused actin rearrangements and promoted actin-dependent ICAM-1 nanoclustering before LFA-1 engagement. T cells adhered to mechanically prestimulated ECs or nanoclustered ICAM-1 substrates developed a promigratory phenotype, migrated faster, and exhibited shorter-lived interactions with ECs than when adhered to non mechanically stimulated ECs or to monomeric ICAM-1 substrates. Together, our results indicate that shear forces increase ICAM-1/LFA-1 bonds because of ICAM-1 nanoclustering, strengthening adhesion and allowing cells to exert higher traction forces required for faster migration. Our data also underscore the importance of mechanical forces regulating the nanoscale organization of membrane receptors and their contribution to cell adhesion regulation.

Correlative nanophotonic approaches to enlighten the nanoscale dynamics of living cell membranes.

Dynamic compartmentalization is a prevailing principle regulating the spatiotemporal organization of the living cell membrane from the nano- up to the mesoscale. This non-arbitrary organization is intricately linked to cell function. On living cell membranes, dynamic domains or 'membrane rafts' enriched with cholesterol, sphingolipids and other certain proteins exist at the nanoscale serving as signaling and sorting platforms. Moreover, it has been postulated that other local organizers of the cell membrane such as intrinsic protein interactions, the extracellular matrix and/or the actin cytoskeleton synergize with rafts to provide spatiotemporal hierarchy to the membrane. Elucidating the intricate coupling of multiple spatial and temporal scales requires the application of correlative techniques, with a particular need for simultaneous nanometer spatial precision and microsecond temporal resolution. Here, we review novel fluorescence-based techniques that readily allow to decode nanoscale membrane dynamics with unprecedented spatiotemporal resolution and single-molecule sensitivity. We particularly focus on correlative approaches from the field of nanophotonics. Notably, we introduce a versatile planar nanoantenna platform combined with fluorescence correlation spectroscopy to study spatiotemporal heterogeneities on living cell membranes at the nano- up to the mesoscale. Finally, we outline remaining future technological challenges and comment on potential directions to advance our understanding of cell membrane dynamics under the influence of the actin cytoskeleton and extracellular matrix in uttermost detail.

Excitation-multiplexed multicolor superresolution imaging with fm-STORM and fm-DNA-PAINT.

Recent advancements in single-molecule-based superresolution microscopy have made it possible to visualize biological structures with unprecedented spatial resolution. Determining the spatial coorganization of these structures within cells under physiological and pathological conditions is an important biological goal. This goal has been stymied by the current limitations of carrying out superresolution microscopy in multiple colors. Here, we develop an approach for simultaneous multicolor superresolution imaging which relies solely on fluorophore excitation, rather than fluorescence emission properties. By modulating the intensity of the excitation lasers at different frequencies, we show that the color channel can be determined based on the fluorophore's response to the modulated excitation. We use this frequency multiplexing to reduce the image acquisition time of multicolor superresolution DNA-PAINT while maintaining all its advantages: minimal color cross-talk, minimal photobleaching, maximal signal throughput, ability to maintain the fluorophore density per imaged color, and ability to use the full camera field of view. We refer to this imaging modality as "frequency multiplexed DNA-PAINT," or fm-DNA-PAINT for short. We also show that frequency multiplexing is fully compatible with STORM superresolution imaging, which we term fm-STORM. Unlike fm-DNA-PAINT, fm-STORM is prone to color cross-talk. To overcome this caveat, we further develop a machine-learning algorithm to correct for color cross-talk with more than 95% accuracy, without the need for prior information about the imaged structure.

A DNA origami platform for quantifying protein copy number in super-resolution.

Single-molecule-based super-resolution microscopy offers researchers a unique opportunity to quantify protein copy number with nanoscale resolution. However, while fluorescent proteins have been characterized for quantitative imaging using calibration standards, similar calibration tools for immunofluorescence with small organic fluorophores are lacking. Here we show that DNA origami, in combination with GFP antibodies, is a versatile platform for calibrating fluorophore and antibody labeling efficiency to quantify protein copy number in cellular contexts using super-resolution microscopy.

Inhomogeneous membrane receptor diffusion explained by a fractional heteroscedastic time series model.

Single particle tracking experiments have recently uncovered that the motion of cell membrane components can undergo changes of diffusivity as a result of the heterogeneous environment, producing subdiffusion and nonergodic behavior. In this paper, we show that an autoregressive fractionally integrated moving average (ARFIMA) with noise given by generalized autoregressive conditional heteroscedasticity (GARCH) can describe inhomogeneous diffusion in the cell membrane, where the ARFIMA process models anomalous diffusion and the GARCH process explains a fluctuating diffusion parameter.

The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells.

Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such "tonic" activation of iNKT cells remain unclear. Here, we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen-presenting cells (APCs) modulates activation of iNKT cells. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton. Dual-color single-particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton, preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the Toll-like receptor 7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a previously unidentified mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen-loaded CD1d nanoclusters.

Enhancing Magnetic Light Emission with All-Dielectric Optical Nanoantennas.

Electric and magnetic optical fields carry the same amount of energy. Nevertheless, the efficiency with which matter interacts with electric optical fields is commonly accepted to be at least 4 orders of magnitude higher than with magnetic optical fields. Here, we experimentally demonstrate that properly designed photonic nanoantennas can selectively manipulate the magnetic versus electric emission of luminescent nanocrystals. In particular, we show selective enhancement of magnetic emission from trivalent europium-doped nanoparticles in the vicinity of a nanoantenna tailored to exhibit a magnetic resonance. Specifically, by controlling the spatial coupling between emitters and an individual nanoresonator located at the edge of a near-field optical scanning tip, we record with nanoscale precision local distributions of both magnetic and electric radiative local densities of states (LDOS). The map of the radiative LDOS reveals the modification of both the magnetic and electric quantum environments induced by the presence of the nanoantenna. This manipulation and enhancement of magnetic light-matter interaction by means of nanoantennas opens up new possibilities for the research fields of optoelectronics, chiral optics, nonlinear and nano-optics, spintronics, and metamaterials, among others.

Frequency-Encoded Multicolor Fluorescence Imaging with Single-Photon-Counting Color-Blind Detection.

Standard fluorescence microscopy relies on filter-based detection of emitted photons after fluorophore excitation at the appropriate wavelength. Although of enormous utility to the biological community, the implementation of approaches for simultaneous multicolor fluorescence imaging is commonly challenged by the large spectral overlap between different fluorophores. Here, we describe an alternative multicolor fluorescence imaging methodology that exclusively relies on the absorption spectra of the fluorophores instead of their fluorescence emissions. The method is based on multiplexing optical excitation signals in the frequency domain and using single color-blind detection. Because the spectral information is fully encoded during excitation, the method requires minimal spectral filtering on detection. This enables the simultaneous identification of multiple color channels in a single measurement with only one color-blind detector. We demonstrate simultaneous three-color confocal imaging of individual molecules and of four-target imaging on cells with excellent discrimination. Moreover, we have implemented a non-negative matrix factorization algorithm for spectral unmixing to extend the number of color targets that can be discriminated in a single measurement. Using this algorithm, we resolve six spectrally and spatially overlapping fluorophores on fixed cells using four excitation wavelengths. The methodology is fully compatible with live imaging of biological samples and can be easily extended to other imaging modalities, including super-resolution microscopy, making simultaneous multicolor imaging more accessible to the biological research community.

PLANT: A Method for Detecting Changes of Slope in Noisy Trajectories.

Time traces obtained from a variety of biophysical experiments contain valuable information on underlying processes occurring at the molecular level. Accurate quantification of these data can help explain the details of the complex dynamics of biological systems. Here, we describe PLANT (Piecewise Linear Approximation of Noisy Trajectories), a segmentation algorithm that allows the reconstruction of time-trace data with constant noise as consecutive straight lines, from which changes of slopes and their respective durations can be extracted. We present a general description of the algorithm and perform extensive simulations to characterize its strengths and limitations, providing a rationale for the performance of the algorithm in the different conditions tested. We further apply the algorithm to experimental data obtained from tracking the centroid position of lymphocytes migrating under the effect of a laminar flow and from single myosin molecules interacting with actin in a dual-trap force-clamp configuration.

Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells.

Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 µs. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.

In-Plane Plasmonic Antenna Arrays with Surface Nanogaps for Giant Fluorescence Enhancement.

Optical nanoantennas have a great potential for enhancing light-matter interactions at the nanometer scale, yet fabrication accuracy and lack of scalability currently limit ultimate antenna performance and applications. In most designs, the region of maximum field localization and enhancement (i.e., hotspot) is not readily accessible to the sample because it is buried into the nanostructure. Moreover, current large-scale fabrication techniques lack reproducible geometrical control below 20 nm. Here, we describe a new nanofabrication technique that applies planarization, etch back, and template stripping to expose the excitation hotspot at the surface, providing a major improvement over conventional electron beam lithography methods. We present large flat surface arrays of in-plane nanoantennas, featuring gaps as small as 10 nm with sharp edges, excellent reproducibility and full surface accessibility of the hotspot confined region. The novel fabrication approach drastically improves the optical performance of plasmonic nanoantennas to yield giant fluorescence enhancement factors up to 104-105 times, together with nanoscale detection volumes in the 20 zL range. The method is fully scalable and adaptable to a wide range of antenna designs. We foresee broad applications by the use of these in-plane antenna geometries ranging from large-scale ultrasensitive sensor chips to microfluidics and live cell membrane investigations.

Lateral Mobility and Nanoscale Spatial Arrangement of Chemokine-activated α4ß1 Integrins on T Cells.

Chemokine stimulation of integrin α4ß1-dependent T lymphocyte adhesion is a key step during lymphocyte trafficking. A central question regarding α4ß1 function is how its lateral mobility and organization influence its affinity and avidity following cell stimulation with chemokines and/or ligands. Using single particle tracking and superresolution imaging approaches, we explored the lateral mobility and spatial arrangement of individual α4ß1integrins on T cells exposed to different activating stimuli. We show that CXCL12 stimulation leads to rapid and transient α4ß1activation, measured by induction of the activation epitope recognized by the HUTS-21 anti-ß1antibody and by increased talin-ß1 association. CXCL12-dependent α4ß1 activation directly correlated with restricted lateral diffusion and integrin immobilization. Moreover, co-stimulation by CXCL12 together with soluble VCAM-1 potentiated integrin immobilization with a 5-fold increase in immobile integrins compared with unstimulated conditions. Our data indicate that docking by talin of the chemokine-activated α4ß1 to the actin cytoskeleton favors integrin immobilization, which likely facilitates ligand interaction and increased adhesiveness. Superresolution imaging showed that the nanoscale organization of high-affinity α4ß1 remains unaffected following chemokine and/or ligand addition. Instead, newly activated α4ß1 integrins organize on the cell membrane as independent units without joining pre-established integrin sites to contribute to cluster formation. Altogether, our results provide a rationale to understand how the spatiotemporal organization of activated α4ß1 integrins regulates T lymphocyte adhesion.

Enhanced receptor-clathrin interactions induced by N-glycan-mediated membrane micropatterning.

Glycan-protein interactions are emerging as important modulators of membrane protein organization and dynamics, regulating multiple cellular functions. In particular, it has been postulated that glycan-mediated interactions regulate surface residence time of glycoproteins and endocytosis. How this precisely occurs is poorly understood. Here we applied single-molecule-based approaches to directly visualize the impact of glycan-based interactions on the spatiotemporal organization and interaction with clathrin of the glycosylated pathogen recognition receptor dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). We find that cell surface glycan-mediated interactions do not influence the nanoscale lateral organization of DC-SIGN but restrict the mobility of the receptor to distinct micrometer-size membrane regions. Remarkably, these regions are enriched in clathrin, thereby increasing the probability of DC-SIGN-clathrin interactions beyond random encountering. N-glycan removal or neutralization leads to larger membrane exploration and reduced interaction with clathrin, compromising clathrin-dependent internalization of virus-like particles by DC-SIGN. Therefore, our data reveal that cell surface glycan-mediated interactions add another organization layer to the cell membrane at the microscale and establish a novel mechanism of extracellular membrane organization based on the compartments of the membrane that a receptor is able to explore. Our work underscores the important and complex role of surface glycans regulating cell membrane organization and interaction with downstream partners.