Cellular Output and Physicochemical Properties of the Membrane-Derived Vesicles Depend on Chemical Stimulants.

Synthetic liposomes are widely used as drug delivery vehicles in biomedical treatments, such as for mRNA-based antiviral vaccines like those recently developed against SARS-CoV-2. Extracellular vesicles (EVs), which are naturally produced by cells, have emerged as a next-generation delivery system. However, key questions regarding their origin within cells remain unresolved. In this regard, plasma membrane vesicles (PMVs), which are essentially produced from the cellular plasma membrane (PM), present a promising alternative. Unfortunately, their properties relevant to biomedical applications have not be extensively studied. Therefore, we conducted a thorough investigation of the methods used in the production of PMVs. By leveraging advanced fluorescence techniques in microscopy and flow cytometry, we demonstrated a strong dependence of the physicochemical attributes of PMVs on the chemicals used during their production. Following established protocols employing chemicals such as paraformaldehyde (PFA), N-ethylmaleimide (NEM) or dl-dithiothreitol (DTT) and by developing a modified NEM-based method that involved a hypotonic shock step, we generated PMVs from THP-1 CD1d cells. We systematically compared key parameters such as vesicle output, their size distribution, vesicular content analysis, vesicular membrane lipid organization and the mobility of a transmembrane protein. Our results revealed distinct trends: PMVs isolated using NEM-based protocols closely resembled natural vesicles, whereas PFA induced significant molecular cross-linking, leading to notable changes in the biophysical properties of the vesicles. Furthermore, our novel NEM protocol enhanced the efficiency of PMV production. In conclusion, our study highlights the unique characteristics of chemically produced PMVs and offers insights into their potentially diverse yet valuable biological functions.

Segmented fluorescence correlation spectroscopy (FCS) on a commercial laser scanning microscope.

Performing accurate Fluorescence Correlation Spectroscopy (FCS) measurements in cells can be challenging due to cellular motion or other intracellular processes. In this respect, it has recently been shown that analysis of FCS data in short temporal segments (segmented FCS) can be very useful to increase the accuracy of FCS measurements inside cells. Here, we demonstrate that segmented FCS can be performed on a commercial laser scanning microscope (LSM), even in the absence of the dedicated FCS module. We show how data can be acquired on a Leica SP8 confocal microscope and then exported and processed with a custom software in MATLAB. The software performs segmentation of the data to extract an average ACF and measure the diffusion coefficient in specific subcellular regions. First of all, we measure the diffusion of fluorophores of different size in solution, to show that good-quality ACFs can be obtained in a commercial LSM. Next, we validate the method by measuring the diffusion coefficient of GFP in the nucleus of HeLa cells, exploiting variations of the intensity to distinguish between nucleoplasm and nucleolus. As expected, the measured diffusion coefficient of GFP is slower in the nucleolus relative to nucleoplasm. Finally, we apply the method to HeLa cells expressing a PARP1 chromobody to measure the diffusion coefficient of PARP1 in different subcellular regions. We find that PARP1 diffusion is slower in the nucleolus compared to the nucleoplasm.

Understanding and manipulating extracellular behaviors of Wnt ligands.

Wnt, a family of secreted signaling proteins, serves diverse functions in embryogenesis, organogenesis, cancer, and stem cell functions. In the context of development, Wnt has been considered a representative morphogen, forming concentration gradients to give positional information to cells or tissues. However, although gradients are often illustrated in schemata, the reality of concentration gradients, or in other words, actual spatial distribution of Wnt ligands, and their behaviors in the extracellular space still remain poorly known. To understand extracellular behavior of Wnt ligands, quantitative analyses such as fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) are highly informative because Wnt dispersal involves physical and biochemical processes, such as diffusion and binding to or dissociation from cell surface molecules, including heparan sulfate proteoglycans (HSPGs). Here, I briefly discuss representative methods to quantify morphogen dynamics. In addition, I discuss molecular manipulations of morphogens, mainly focusing on use of protein binders, and synthetic biology of morphogens as indicators of current and future directions in this field.

Fluorogenic Hyaluronan Nanogels Track Individual Early Protein Aggregates Originated under Oxidative Stress.

Proteins are broadly versatile biochemical materials, whose functionality is tightly related to their folding state. Native folding can be lost to yield misfolded conformations, often leading to formation of protein oligomers, aggregates, and biomolecular phase condensates. The fluorogenic hyaluronan HA-RB, a nonsulfonated glycosaminoglycan with a combination of polyanionic character and of hydrophobic spots due to rhodamine B dyes, binds to early aggregates of the model protein cytoplasmic glyceraldehyde-3-phosphate dehydrogenase 1 from Arabidopsis thaliana (AtGAPC1) since the very onset of the oligomeric phase, making them brightly fluorescent. This initial step of aggregation has, until now, remained elusive with other fluorescence- or scattering-based techniques. The information gathered from nanotracking (via light-sheet fluorescence microscopy) and from FCS in a confocal microscope converges to highlight the ability of HA-RB to bind protein aggregates from the very early steps of aggregation and with high affinity. Altogether, this fluorescence-based approach allows one to monitor and track individual early AtGAPC1 aggregates in the size range from 10 to 100 nm with high time (∼10-2 s) and space (∼250 nm) resolution.

CD28 and 41BB Costimulatory Domains Alone or in Combination Differentially Influence Cell Surface Dynamics and Organization of Chimeric Antigen Receptors and Early Activation of CAR T Cells.

Chimeric antigen receptor (CAR)-modified T cells brought a paradigm shift in the treatment of chemotherapy-resistant lymphomas. Conversely, clinical experience with CAR T cells targeting solid tumors has been disheartening, indicating the necessity of their molecular-level optimization. While incorporating CD28 or 41BB costimulatory domains into CARs in addition to the CD3z signaling domain improved the long-term efficacy of T cell products, their influence on early tumor engagement has yet to be elucidated. We studied the antigen-independent self-association and membrane diffusion kinetics of first- (.z), second- (CD28.z, 41BB.z), and third- (CD28.41BB.z) generation HER2-specific CARs in the resting T cell membrane using super-resolution AiryScan microscopy and fluorescence correlation spectroscopy, in correlation with RoseTTAFold-based structure prediction and assessment of oligomerization in native Western blot. While .z and CD28.z CARs formed large, high-density submicron clusters of dimers, 41BB-containing CARs formed higher oligomers that assembled into smaller but more numerous membrane clusters. The first-, second-, and third-generation CARs showed progressively increasing lateral diffusion as the distance of their CD3z domain from the membrane plane increased. Confocal microscopy analysis of immunological synapses showed that both small clusters of highly mobile CD28.41BB.z and large clusters of less mobile .z CAR induced more efficient CD3ζ and pLck phosphorylation than CD28.z or 41BB.z CARs of intermediate mobility. However, electric cell-substrate impedance sensing revealed that the CD28.41BB.z CAR performs worst in sequential short-term elimination of adherent tumor cells, while the .z CAR is superior to all others. We conclude that the molecular structure, membrane organization, and mobility of CARs are critical design parameters that can predict the development of an effective immune synapse. Therefore, they need to be taken into account alongside the long-term biological effects of costimulatory domains to achieve an optimal therapeutic effect.

A cell free biomembrane platform for multimodal study of influenza virus hemagglutinin and for evaluation of entry-inhibitors against hemagglutinin.

Seasonal periodic pandemics and epidemics caused by Influenza A viruses (IAVs) are associated with high morbidity and mortality worldwide. They are frequent and unpredictable in severity so there is a need for biophysical platforms that can be used to provide both mechanistic insights into influenza virulence and its potential treatment by anti-IAV agents. Host membrane viral association through the glycoprotein hemagglutinin (HA) of IAVs is one of the primary steps in infection. HA is thus a potential target for drug discovery and development against influenza. Deconvolution of the multivalent interactions of HA at the interfaces of the host cell membrane can help unravel therapeutic targets. In this contribution, we reported the effect of a multivalent HA glycoprotein association on various glycosphingolipid receptors (GD1a, GM3, GM1) doped asymmetrically into an artificial host membrane spanned across an aqueous filled microcavity array. The extent of HA association and its impact on membrane resistance, capacitance, and diffusivity was measured using highly sensitive electrochemical impedance spectroscopy (EIS) and fluorescence lifetime correlation spectroscopy (FLCS). Furthermore, we investigated the inhibition of the influenza HA glycoprotein association with the host mimetic surface by natural and synthetic sialic acid-based inhibitors (sialic acid, Siaα2,3-GalOMe, FB127, 3-sialyl lactose) using electrochemical impedance spectroscopy and observe that while all inhibit, they do not prevent host binding. Overall, the work demonstrates the platform provides a label-free screening platform for the biophysical evaluation of new inhibitors in the development of potential therapeutics for IAV infection prevention and treatment.

Inter-molecular interaction kinetics: tale of photon anti-bunching and bunching in fluorescence correlation spectroscopy (FCS).

Molecular interactions are fundamental to any chemical or biological processes, and their rates define the operational sequence and control for any desirable product. Here, we deliberate on a recently developed novel fluorescence spectroscopic method, which combines fluorescence photon anti-bunching, photon bunching, time-correlated single-photon counting (TCSPC), and steady-state fluorescence spectroscopy, to study composite chemical reactions with single molecule sensitivity. The proposed method captures the full picture of the multifaceted quenching kinetics, which involves static quenching by ground state complexation and collisional quenching in the excited state under dynamic exchange of fluorophore in a heterogeneous media, and which cannot be seen by steady-state or lifetime measurements alone. Photon correlation in fluorescence correlation spectroscopy (FCS) provides access to interrogate interaction dynamics from picosecond to seconds, stitching all possible stages of dye-quencher interaction in a micellar media. This is not possible with the limited time window available to conventional ensemble techniques like TCSPC, flash photolysis, transient absorption, stop-flow, etc. The basic premises of such unified global analysis and sanctity of extracted parameters critically depends on the minimum but precise description of reaction scheme, for which careful inspection of ensemble spectroscopy data for photo-physical behaviour is very important. Though in this contribution we discussed and demonstrated the merits of photon antibunching and bunching spectroscopy for dye-quencher interaction in cationic cetyltrimethylammonium bromide (CTAB) micellar solution by photo-induced electron transfer mechanism and the influence of micellar charge and microenvironment on the interaction kinetics, but in principal similar arguments are equally applicable to any other interaction mechanisms which alter fluorescence photon correlations, like Förster resonance energy transfer (FRET), proton transfer, isomerisation, etc.

Yeast-produced fructosamine-3-kinase retains mobility after ex vivo intravitreal injection in human and bovine eyes as determined by Fluorescence Correlation Spectroscopy.

Globally, over 2 billion people suffer from vision impairment. Despite complex multifactorial etiology, advanced glycation end products are involved in the pathogenesis of many causative age- and diabetes-related eye diseases. Deglycating enzyme fructosamine-3-kinase (FN3K) was recently proposed as a potential therapeutic, but for further biopharmaceutical development, knowledge on its manufacturability and stability and mobility in the vitreous fluid of the eye is indispensable. We evaluated recombinant production of FN3K in two host systems, and its diffusion behavior in both bovine and human vitreous. Compared to Escherichia coli, intracellular production in Pichia pastoris yielded more and higher purity FN3K. The yeast-produced enzyme was used in a first attempt to use fluorescence correlation spectroscopy to study protein mobility in non-sonicated bovine vitreous, human vitreous, and intact bovine eyes. It was demonstrated that FN3K retained mobility upon intravitreal injection, although a certain delay in diffusion was observed. Alkylation of free cysteines was tolerated both in terms of enzymatic activity and vitreous diffusion. Ex vivo diffusion data gathered and the availability of yeast-produced high purity enzyme now clear the path for in vivo pharmacokinetics studies of FN3K.

Lipophilic probe behavior in microemulsions evaluated by fluorescence correlation spectroscopy.

We evaluated the dispersion and diffusion of fluorescent-labeled lipophilic vitamin E (VE) in microemulsions (MEs) including water-in-oil (W/O) type ME, oil-in-water (O/W) type ME, and bicontinuous ME (BME), using fluorescence correlation spectroscopy (FCS). We prepared a fluorescent ATTO 488 or BODIPY group labeled VE (VE-ATTO or VE-BODIPY). VE-ATTO possesses lipophilic and hydrophilic parts, while VE-BODIPY consists solely of the lipophilic part. The VE-ATTO dissolved in heptane solution as an oil phase appeared hot pink in color due to the solvatochromism effect under room light and almost no fluorescent signal, which was unlike the VE-ATTO dissolved in ME solutions and all the VE-BODIPY solutions (typical fluorescent green color). The FCS measurement proved that VE-BODIPY diffuses faster than VE-ATTO. This is presumably because the "surfactant-like" VE-ATTO is localized and trapped at the micro-water/micro-oil interface of the MEs, while the VE-BODIPY exists in the ME phase and macro-oil phase with good dispersion. These results demonstrate that FCS is a powerful tool for the rapid evaluation of the lipophilic probe behavior in heterogeneous ME solutions.

Quantifying Intercellular Movement and Protein Stoichiometry for Computational Modeling.

Analyzing protein movement dynamics and their regulation has shown to be important in the study of cell fate decisions. Such analyses can be performed with scanning fluorescence correlation spectroscopy (scanning FCS), a versatile imaging methodology that has been applied in the animal kingdom and recently adapted to the plant kingdom. Specifically, scanning FCS allows for qualitatively capturing protein movement across barriers, such as the active transport through plasmodesmata, the analysis of protein movement rates, and the quantification of the stoichiometry of protein complexes, composed of one or more different proteins. Importantly, the quantifiable data generated with scanning FCS can be used to inform computational models, enhancing model simulations of in vivo events, such as cell fate decisions, during plant development.

Advances in Confocal Microscopy and Selected Applications.

Over the last 30 years, confocal microscopy has emerged as a primary tool for biological investigation across many disciplines. The simplicity of use and widespread accessibility of confocal microscopy ensure that it will have a prominent place in biological imaging for many years to come, even with the recent advances in light sheet and field synthesis microscopy. Since these more advanced technologies still require significant expertise to effectively implement and carry through to analysis, confocal microscopy-based approaches still remain the easiest way for biologists with minimal imaging experience to address fundamental questions about how their systems are arranged through space and time. In this review, we discuss a number of advanced applications of confocal microscopy for probing the spatiotemporal dynamics of biological systems.

Structure, interdomain dynamics, and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes.

Rhodesain is the lysosomal cathepsin L-like cysteine protease of Trypanosoma brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood-brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating prodomain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in Escherichia coli and determined its crystal structure. The trypanosomal prodomain differs from nonparasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose side-chain interactions resemble those of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations, and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH sensitivity of proenzyme cleavage in (trypanosomal) CathL-like proteases.

Transactivation of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs): Recent insights using luminescence and fluorescence technologies.

Alterations in signalling due to bidirectional transactivation of G protein-coupled receptor (GPCRs) and receptor tyrosine kinases (RTKs) are well established. Transactivation significantly diversifies signalling networks within a cell and has been implicated in promoting both advantageous and disadvantageous physiological and pathophysiological outcomes, making the GPCR/RTK interactions attractive new targets for drug discovery programmes. Transactivation has been observed for a plethora of receptor pairings in multiple cell types; however, the precise molecular mechanisms and signalling effectors involved can vary with receptor pairings and cell type. This short review will discuss the recent applications of proximity-based assays, such as resonance energy transfer and fluorescence-based imaging in investigating the dynamics of GPCR/RTK complex formation, subsequent effector protein recruitment and the cellular locations of complexes in living cells.

Clusterin inhibits Aß42 aggregation through a "strawberry model" as detected by FRET-FCS.

Extracellular plaque deposits of ß-amyloid peptide (Aß) are one of the main pathological features of Alzheimer's disease (AD). The aggregation of Aß42 species, especially Aß42 oligomers, is still an active research field in AD pathogenesis. Secretory clusterin protein (sCLU), an extracellular chaperone, plays an important role in AD pathogenesis. Although sCLU interacts directly with Aß42 in vitro and in vivo, the mechanism is not clear. In this paper, His-tagged sCLU (sCLU-His) was cloned, expressed and purified, and we applied florescence resonance energy transfer-fluorescence correlation spectroscopy (FRET-FCS) to investigate the direct interaction of sCLU-His and Aß42 at the single-molecule fluorescence level in vitro. Here, we chose four different fluorescently labeled Aß42 oligomers to form two different groups of aggregation models, easy or difficult to aggregate. The results showed that sCLU-His could form complexes with both aggregation models, and sCLU-His inhibited the aggregation of Aß42/RB  ~ Aß42/Atto647 (easy to aggregate model). The complexes were produced as the Aß42/Label adhered to the sCLU-His, which is similar to a "strawberry model," as strawberry seeds are dotted on the outer surface of strawberries. This work provided additional insight into the interaction mechanism of sCLU and Aß42 .

Fluorescence Correlation Spectroscopy Reveals Interaction of Some Microdomain-Associated Lipids with Cellular Focal Adhesion Sites.

Cells adhere to the extracellular matrix at distinct anchoring points, mostly focal adhesions. These are rich in immobile transmembrane- and cytoskeletal-associated proteins, some of which are known to interact with lipids of the plasma membrane. To investigate their effect on lipid mobility and molecular interactions, fluorescently labeled lipids were incorporated into the plasma membranes of primary myofibroblasts using fusogenic liposomes. With fluorescence correlation spectroscopy, we tested mobilities of labeled microdomain-associated lipids such as sphingomyelin (SM), ganglioside (GM1), and cholesterol as well as of a microdomain-excluded phospholipid (PC) and a lipid-like molecule (DiIC18(7)) in focal adhesions (FAs) and in neighboring non-adherent membrane areas. We found significantly slower diffusion of SM and GM1 inside FAs but no effect on cholesterol, PC, and DiIC18(7). These data were compared to the molecular behavior in Lo/Ld-phase separated giant unilamellar vesicles, which served as a model system for microdomain containing lipid membranes. In contrast to the model system, lipid mobility changes in FAs were molecularly selective, and no particle enrichment occurred. Our findings suggest that lipid behavior in FAs cannot be described by Lo/Ld-phase separation. The observed slow-down of some molecules in FAs is potentially due to transient binding between lipids and some molecular constituent(s).

Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments.

Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis, and self-powered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. To date, experimental observations of catalysis-enhanced enzyme diffusion have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an anti-Brownian electrokinetic (ABEL) trap and in-solution single-particle tracking, we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the single-molecule level, in sharp contrast to the ∼20% enhancement seen in parallel FCS experiments using p-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte Carlo simulations, we establish that pNPP-induced dye blinking at the ∼10-ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models--including those of our own--in the field, and indicate that in-solution single-particle tracking and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale.

In Vivo Single-Molecule Detection of Nanoparticles for Multiphoton Fluorescence Correlation Spectroscopy to Quantify Cerebral Blood Flow.

We present the application of multiphoton in vivo fluorescence correlation spectroscopy (FCS) of fluorescent nanoparticles for the measurement of cerebral blood flow with excellent spatial and temporal resolution. Through the detection of single nanoparticles within the complex vessel architecture of a live mouse, this new approach enables the quantification of nanoparticle dynamics occurring within the vasculature along with simultaneous measurements of blood flow properties in the brain. In addition to providing high resolution blood flow measurements, this approach enables real-time quantification of nanoparticle concentration, degradation, and transport. This method is capable of quantifying flow rates at each pixel with submicron resolution to enable monitoring of dynamic changes in flow rates in response to changes in the animal's physiological condition. Scanning the excitation beam using FCS provides pixel by pixel mapping of flow rates with subvessel resolution across capillaries 300 µm deep in the brains of mice.

Quantification of native mRNA dynamics in living neurons using fluorescence correlation spectroscopy and reduction-triggered fluorescent probes.

RNA localization in subcellular compartments is essential for spatial and temporal regulation of protein expression in neurons. Several techniques have been developed to visualize mRNAs inside cells, but the study of the behavior of endogenous and nonengineered mRNAs in living neurons has just started. In this study, we combined reduction-triggered fluorescent (RETF) probes and fluorescence correlation spectroscopy (FCS) to investigate the diffusion properties of activity-regulated cytoskeleton-associated protein (Arc) and inositol 1,4,5-trisphosphate receptor type 1 (Ip3r1) mRNAs. This approach enabled us to discriminate between RNA-bound and unbound fluorescent probes and to quantify mRNA diffusion parameters and concentrations in living rat primary hippocampal neurons. Specifically, we detected the induction of Arc mRNA production after neuronal activation in real time. Results from computer simulations with mRNA diffusion coefficients obtained in these analyses supported the idea that free diffusion is incapable of transporting mRNA of sizes close to those of Arc or Ip3r1 to distal dendrites. In conclusion, the combined RETF-FCS approach reported here enables analyses of the dynamics of endogenous, unmodified mRNAs in living neurons, affording a glimpse into the intracellular dynamics of RNA in live cells.

Conformational Switching in Bcl-xL: Enabling Non-Canonic Inhibition of Apoptosis Involves Multiple Intermediates and Lipid Interactions.

The inhibition of mitochondrial permeabilization by the anti-apoptotic protein Bcl-xL is crucial for cell survival and homeostasis. Its inhibitory role requires the partitioning of Bcl-xL to the mitochondrial outer membrane from an inactive state in the cytosol, leading to its extensive refolding. The molecular mechanisms behind these events and the resulting conformations in the bilayer are unclear, and different models have been proposed to explain them. In the most recently proposed non-canonical model, the active form of Bcl-xL employs its N-terminal BH4 helix to bind and block its pro-apoptotic target. Here, we used a combination of various spectroscopic techniques to study the release of the BH4 helix (α1) during the membrane insertion of Bcl-xL. This refolding was characterized by a gradual increase in helicity due to the lipid-dependent partitioning-coupled folding and formation of new helix αX (presumably in the originally disordered loop between helices α1 and α2). Notably, a comparison of various fluorescence and circular dichroism measurements suggested the presence of multiple Bcl-xL conformations in the bilayer. This conclusion was explicitly confirmed by single-molecule measurements of FÓ§rster Resonance Energy Transfer from Alexa-Fluor-488-labeled Bcl-xL D189C to a mCherry fluorescent protein attached at the N-terminus. These measurements clearly indicated that the refolding of Bcl-xL in the bilayer is not a two-state transition and involves multiple membranous intermediates of variable compactness.

The cortical actin network regulates avidity-dependent binding of hyaluronan by the lymphatic vessel endothelial receptor LYVE-1.

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) mediates the docking and entry of dendritic cells to lymphatic vessels through selective adhesion to its ligand hyaluronan in the leukocyte surface glycocalyx. To bind hyaluronan efficiently, LYVE-1 must undergo surface clustering, a process that is induced efficiently by the large cross-linked assemblages of glycosaminoglycan present within leukocyte pericellular matrices but is induced poorly by the shorter polymer alone. These properties suggested that LYVE-1 may have limited mobility in the endothelial plasma membrane, but no biophysical investigation of these parameters has been carried out to date. Here, using super-resolution fluorescence microscopy and spectroscopy combined with biochemical analyses of the receptor in primary lymphatic endothelial cells, we provide the first evidence that LYVE-1 dynamics are indeed restricted by the submembranous actin network. We show that actin disruption not only increases LYVE-1 lateral diffusion but also enhances hyaluronan-binding activity. However, unlike the related leukocyte HA receptor CD44, which uses ERM and ankyrin motifs within its cytoplasmic tail to bind actin, LYVE-1 displays little if any direct interaction with actin, as determined by co-immunoprecipitation. Instead, as shown by super-resolution stimulated emission depletion microscopy in combination with fluorescence correlation spectroscopy, LYVE-1 diffusion is restricted by transient entrapment within submembranous actin corrals. These results point to an actin-mediated constraint on LYVE-1 clustering in lymphatic endothelium that tunes the receptor for selective engagement with hyaluronan assemblages in the glycocalyx that are large enough to cross-bridge the corral-bound LYVE-1 molecules and thereby facilitate leukocyte adhesion and transmigration.