Telomere Loop Dynamics in Chromosome End Protection.

Telomeres regulate DNA damage response (DDR) and DNA repair activity at chromosome ends. How telomere macromolecular structure contributes to ATM regulation and its potential dissociation from control over non-homologous end joining (NHEJ)-dependent telomere fusion is of central importance to telomere-dependent cell aging and tumor suppression. Using super-resolution microscopy, we identify that ATM activation at mammalian telomeres with reduced TRF2 or at human telomeres during mitotic arrest occurs specifically with a structural change from telomere loops (t-loops) to linearized telomeres. Additionally, we find the TRFH domain of TRF2 regulates t-loop formation while suppressing ATM activity. Notably, we demonstrate that ATM activation and telomere linearity occur separately from telomere fusion via NHEJ and that linear DDR-positive telomeres can remain resistant to fusion, even during an extended G1 arrest, when NHEJ is most active. Collectively, these results suggest t-loops act as conformational switches that specifically regulate ATM activation independent of telomere mechanisms to inhibit NHEJ.

Conformational states of the kinase Lck regulate clustering in early T cell signaling.

Phosphorylation of the T cell antigen receptor (TCR) by the tyrosine kinase Lck is an essential step in the activation of T cells. Because Lck is constitutively active, spatial organization may regulate TCR signaling. Here we found that Lck distributions on the molecular level were controlled by the conformational states of Lck, with the open, active conformation inducing clustering and the closed, inactive conformation preventing clustering. In contrast, association with lipid domains and protein networks were not sufficient or necessary for Lck clustering. Conformation-driven Lck clustering was highly dynamic, so that TCR triggering resulted in Lck clusters that contained phosphorylated TCRs but excluded the phosphatase CD45. Our data suggest that Lck conformational states represent an intrinsic mechanism for the intermolecular organization of early T cell signaling.

VAMP7 controls T cell activation by regulating the recruitment and phosphorylation of vesicular Lat at TCR-activation sites.

The mechanisms by which Lat (a key adaptor in the T cell antigen receptor (TCR) signaling pathway) and the TCR come together after TCR triggering are not well understood. We investigate here the role of SNARE proteins, which are part of protein complexes involved in the docking, priming and fusion of vesicles with opposing membranes, in this process. Here we found, by silencing approaches and genetically modified mice, that the vesicular SNARE VAMP7 was required for the recruitment of Lat-containing vesicles to TCR-activation sites. Our results indicated that this did not involve fusion of Lat-containing vesicles with the plasma membrane. VAMP7, which localized together with Lat on the subsynaptic vesicles, controlled the phosphorylation of Lat, formation of the TCR-Lat-signaling complex and, ultimately, activation of T cells. Our findings suggest that the transport and docking of Lat-containing vesicles with target membranes containing TCRs regulates TCR-induced signaling.

Actin dynamics drive membrane reorganization and scission in clathrin-independent endocytosis.

Nascent transport intermediates detach from donor membranes by scission. This process can take place in the absence of dynamin, notably in clathrin-independent endocytosis, by mechanisms that are yet poorly defined. We show here that in cells scission of Shiga toxin-induced tubular endocytic membrane invaginations is preceded by cholesterol-dependent membrane reorganization and correlates with the formation of membrane domains on model membranes, suggesting that domain boundary forces are driving tubule membrane constriction. Actin triggers scission by inducing such membrane reorganization process. Tubule occurrence is indeed increased upon cellular depletion of the actin nucleator component Arp2, and the formation of a cortical actin shell in liposomes is sufficient to trigger the scission of Shiga toxin-induced tubules in a cholesterol-dependent but dynamin-independent manner. Our study suggests that membranes in tubular Shiga toxin-induced invaginations are poised to undergo actin-triggered reorganization leading to scission by a physical mechanism that may function independently from or in synergy with pinchase activity.

Dephosphorylation accelerates the dissociation of ZAP70 from the T cell receptor.

Protein-protein binding domains are critical in signaling networks. Src homology 2 (SH2) domains are binding domains that interact with sequences containing phosphorylated tyrosines. A subset of SH2 domain-containing proteins has tandem domains, which are thought to enhance binding affinity and specificity. However, a trade-off exists between long-lived binding and the ability to rapidly reverse signaling, which is a critical requirement of noise-filtering mechanisms such as kinetic proofreading. Here, we use modeling to show that the unbinding rate of tandem, but not single, SH2 domains can be accelerated by phosphatases. Using surface plasmon resonance, we show that the phosphatase CD45 can accelerate the unbinding rate of zeta chain-associated protein kinase 70 (ZAP70), a tandem SH2 domain-containing kinase, from biphosphorylated peptides from the T cell receptor (TCR). An important functional prediction of accelerated unbinding is that the intracellular ZAP70-TCR half-life in T cells will not be fixed but rather, dependent on the extracellular TCR-antigen half-life, and we show that this is the case in both cell lines and primary T cells. The work highlights that tandem SH2 domains can break the trade-off between signal fidelity (requiring long half-life) and signal reversibility (requiring short half-life), which is a key requirement for T cell antigen discrimination mediated by kinetic proofreading.

Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events.

Engaged T cell antigen receptors (TCRs) initiate signaling through the adaptor protein Lat. In quiescent T cells, Lat is segregated into clusters on the cell surface, which raises the question of how TCR triggering initiates signaling. Using super-resolution fluorescence microscopy, we found that pre-existing Lat domains were neither phosphorylated nor laterally transported to TCR activation sites, which suggested that these clusters do not participate in TCR signaling. Instead, TCR activation resulted in the recruitment and phosphorylation of Lat from subsynaptic vesicles. Studies of Lat mutants confirmed that recruitment preceded and was essential for phosphorylation and that both processes were independent of surface clustering of Lat. Our data suggest that TCR ligation preconditions the membrane for vesicle recruitment and bulk activation of the Lat signaling network.

Biomolecular Binding under Confinement: Statistical Predictions of Steric Influence in Absence of Long-Distance Interactions.

We propose a theoretical model for the influence of confinement on biomolecular binding at the single-molecule scale at equilibrium, based on the change of the number of microstates (localization and orientation) upon reaction. Three cases are discussed: DNA sequences shorter and longer than the single strain DNA Kuhn length and spherical proteins, confined into a spherical container (liposome, droplet, etc.). The influence of confinement is found to be highly dependent on the molecular structure and significant for large molecules (relative to container size).

Statistical predictions on the encapsulation of single molecule binding pairs into sized-dispersed nanocontainers.

Single molecule experiments have recently attracted enormous interest. Many of these studies involve the encapsulation of a single molecule into nanoscale containers (such as vesicles, droplets and nanowells). In such cases, the single molecule encapsulation efficiency is a key parameter to consider in order to get a statistically significant quantitative information. It has been shown that such encapsulation typically follows a Poisson distribution and such theory of encapsulation has only been applied to the encapsulation of single molecules into perfectly sized monodispersed containers. However, experimentally nanocontainers are usually characterized by a size distribution, and often just a single binding pair (rather than a single molecule) is required to be encapsulated. Here the use of Poisson distribution is extended to predict the encapsulation efficiency of two different molecules in an association equilibrium. The Poisson distribution is coupled with a log-normal distribution in order to consider the effect of the container size distribution, and the effect of adsorption to the container is also considered. This theory will allow experimentalists to determine what single molecule encapsulation efficiency can be expected as a function of the experimental conditions. Two case studies, based on experimental data, are given to support the theoretical predictions.

Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response.

To investigate how chromatin architecture is spatiotemporally organized at a double-strand break (DSB) repair locus, we established a biophysical method to quantify chromatin compaction at the nucleosome level during the DNA damage response (DDR). The method is based on phasor image-correlation spectroscopy of histone fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) microscopy data acquired in live cells coexpressing H2B-eGFP and H2B-mCherry. This multiplexed approach generates spatiotemporal maps of nuclear-wide chromatin compaction that, when coupled with laser microirradiation-induced DSBs, quantify the size, stability, and spacing between compact chromatin foci throughout the DDR. Using this technology, we identify that ataxia-telangiectasia mutated (ATM) and RNF8 regulate rapid chromatin decompaction at DSBs and formation of compact chromatin foci surrounding the repair locus. This chromatin architecture serves to demarcate the repair locus from the surrounding nuclear environment and modulate 53BP1 mobility.

How does T cell receptor clustering impact on signal transduction?

The essential function of the T cell receptor (TCR) is to translate the engagement of peptides on the major histocompatibility complex (pMHC) into appropriate intracellular signals through the associated cluster of differentiation 3 (CD3) complex. The spatial organization of the TCR-CD3 complex in the membrane is thought to be a key regulatory element of signal transduction, raising the question of how receptor clustering impacts on TCR triggering. How signal transduction at the TCR-CD3 complex encodes the quality and quantity of pMHC molecules is not fully understood. This question can be approached by reconstituting T cell signaling in model and cell membranes and addressed by single-molecule imaging of endogenous proteins in T cells. We highlight such methods and further discuss how TCR clustering could affect pMHC rebinding rates, the local balance between kinase and phosphatase activity and/or the lipid environment to regulate the signal efficiency of the TCR-CD3 complex. We also examine whether clustering could affect the conformation of cytoplasmic CD3 tails through a biophysical mechanism. Taken together, we highlight how the spatial organization of the TCR-CD3 complex - addressed by reconstitution approaches - has emerged as a key regulatory element in signal transduction of this archetypal immune receptor.

Investigating Spatial Heterogeneity of Nanoparticles Movement in Live Cells with Pair-Correlation Microscopy and Phasor Analysis.

How nanoparticles distribute in living cells and overcome cellular barriers are important criteria in the design of drug carriers. Pair-correlation microscopy is a correlation analysis of fluctuation in the fluorescence intensity obtained by a confocal line scan that can quantify the dynamic properties of nanoparticle diffusion including the number of mobile nanoparticles, diffusion coefficient, and transit time across a spatial distance. Due to the potential heterogeneities in nanoparticle properties and the complexity within the cellular environment, quantification of averaged auto- and pair-correlation profiles may obscure important insights into the ability of nanoparticles to deliver drugs. To overcome this issue, we used phasor analysis to develop a data standardizing method, which can segment the scanned line into several subregions according to diffusion and address the spatial heterogeneity of nanoparticles moving inside cells. The phasor analysis is a fit-free method that represents autocorrelation profiles for each pixel relative to free diffusion on the so-called phasor plots. Phasor plots can then be used to select subpopulations for which the auto- and pair-correlation analysis can be performed separately. We demonstrate the phasor analysis for pair-correlation microscopy for investigating 16 nm, Cy5-labeled silica nanoparticles diffusing across the plasma membrane and green fluorescent proteins (GFP) diffusing across nuclear envelope in MCF-7 cells.

Annexin A6 Is Critical to Maintain Glucose Homeostasis and Survival During Liver Regeneration in Mice.

BACKGROUND AND AIMS: Liver regeneration requires the organized and sequential activation of events that lead to restoration of hepatic mass. During this process, other vital liver functions need to be preserved, such as maintenance of blood glucose homeostasis, balancing the degradation of hepatic glycogen stores, and gluconeogenesis (GNG). Under metabolic stress, alanine is the main hepatic gluconeogenic substrate, and its availability is the rate-limiting step in this pathway. Na+ -coupled neutral amino acid transporters (SNATs) 2 and 4 are believed to facilitate hepatic alanine uptake. In previous studies, we demonstrated that a member of the Ca2+ -dependent phospholipid binding annexins, Annexin A6 (AnxA6), regulates membrane trafficking along endo- and exocytic pathways. Yet, although AnxA6 is abundantly expressed in the liver, its function in hepatic physiology remains unknown. In this study, we investigated the potential contribution of AnxA6 in liver regeneration. APPROACH AND RESULTS: Utilizing AnxA6 knockout mice (AnxA6-/- ), we challenged liver function after partial hepatectomy (PHx), inducing acute proliferative and metabolic stress. Biochemical and immunofluorescent approaches were used to dissect AnxA6-/- mice liver proliferation and energetic metabolism. Most strikingly, AnxA6-/- mice exhibited low survival after PHx. This was associated with an irreversible and progressive drop of blood glucose levels. Whereas exogenous glucose administration or restoration of hepatic AnxA6 expression rescued AnxA6-/- mice survival after PHx, the sustained hypoglycemia in partially hepatectomized AnxA6-/- mice was the consequence of an impaired alanine-dependent GNG in AnxA6-/- hepatocytes. Mechanistically, cytoplasmic SNAT4 failed to recycle to the sinusoidal plasma membrane of AnxA6-/- hepatocytes 48 hours after PHx, impairing alanine uptake and, consequently, glucose production. CONCLUSIONS: We conclude that the lack of AnxA6 compromises alanine-dependent GNG and liver regeneration in mice.

FRET theoretical predictions concerning freely diffusive dyes inside spherical container: how to choose the best pair?

FRET has been massively used to see if biomolecules were bounded or not by labelling both biomolecules by one dye of a FRET pair. This should give a digital answer to the question (fluorescence of the acceptor: high FRET efficency: molecules associated, fluorescence of the donor: low FRET efficency: molecules dissociated). This has been used, inter alia, at the single-molecule scale in containers, such as liposomes. One perspective of the field is to reduce the container's size to study the effect of confinement on binding. The problem is that if the two dyes are encapsulated inside a small liposome, they could have a significant probability to be close one from the other one (and thus to undergo a high FRET efficiency event without binding). This is why we suggest here a theoretical model which gives mean FRET efficiency as a function of liposome radius (the model applies to any spherical container) and Förster radius to help the experimentalist to choose their experimental set-up. Besides, the influence of side effect on mean FRET efficiency has been studied as well. We show here that if this "background FRET" is most of the time non-quantitative, it can remain significant and which makes data analysis trickier. We could show as well that if this background FRET obviously increases when liposome radius decreases, this variation was lower than the one which could be expected because of side effect. We show as well the FRET efficiency function distribution which let the experimentalist know the probability to get one FRET efficiency value.

Mechanobiology of antigen-induced T cell arrest.

To mount an immune response, T cells must first find rare antigens present at the surface of antigen-presenting cells (APCs). They achieve this by migrating rapidly through the crowded space of tissues and constantly sampling the surface of APCs. Upon antigen recognition, T cells decelerate and polarise towards the APC, ultimately forming a specialised interface known as the immunological synapse. These conjugates form as the result of the interaction between pairs of receptors/ligands that are under mechanical stress due to the continuously reorganising cell cytoskeleton. In this review, we discuss the involvement of mechanical forces during antigen recognition by migrating T cells. We will explore this question from a conceptual and technical perspective, with the aim of providing new insights into the emerging field of mechanobiology.

Conformational States Control Lck Switching between Free and Confined Diffusion Modes in T Cells.

T cell receptor phosphorylation by Lck is an essential step in T cell activation. It is known that the conformational states of Lck control enzymatic activity; however, the underlying principles of how Lck finds its substrate over the plasma membrane remain elusive. Here, single-particle tracking is paired with photoactivatable localization microscopy to observe the diffusive modes of Lck in the plasma membrane. Individual Lck molecules switched between free and confined diffusion in both resting and stimulated T cells. Lck mutants locked in the open conformation were more confined than Lck mutants in the closed conformation. Further confinement of kinase-dead versions of Lck suggests that Lck confinement was not caused by phosphorylated substrates. Our data support a model in which confined diffusion of open Lck results in high local phosphorylation rates, and inactive, closed Lck diffuses freely to enable long-range distribution over the plasma membrane.

Unveiling the Relationship between the Perovskite Precursor Solution and the Resulting Device Performance.

For the fabrication of perovskite solar cells (PSCs) using a solution process, it is essential to understand the characteristics of the perovskite precursor solution to achieve high performance and reproducibility. The colloids (iodoplumbates) in the perovskite precursors under various conditions were investigated by UV-visible absorption, dynamic light scattering, photoluminescence, and total internal reflection fluorescence microscopy techniques. Their local structure was examined by in situ X-ray absorption fine structure studies. Perovskite thin films on a substrate with precursor solutions were characterized by transmission electron microscopy, X-ray diffraction analysis, space-charge-limited current, and Kelvin probe force microscopy. The colloidal properties of the perovskite precursor solutions were found to be directly correlated with the defect concentration and crystallinity of the perovskite film. This work provides guidelines for controlling perovskite films by varying the precursor solution, making it possible to use colloid-engineered lead halide perovskite layers to fabricate efficient PSCs.

Clustering of the ζ-Chain Can Initiate T Cell Receptor Signaling.

T cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of ζ-chain in a light controlled manner. We showed that spatial clustering of the ζ-chain intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of ζ-chain, Zap70, PLCγ, ERK and initiated Ca2+ flux. In reconstituted COS-7 cells, only Lck expression was required to initiate ζ-chain phosphorylation upon ζ-chain clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed ζ-chain clusters at the plasma membrane. Taken together, our data demonstrated the biophysical relevance of receptor clustering in TCR signaling.

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination.

Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR-CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR-CD3 complexes. We found that only TCR-CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR-pMHC affinity determined the density of TCR-CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR-CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.

Observing the Reversible Single Molecule Electrochemistry of Alexa Fluor 647 Dyes by Total Internal Reflection Fluorescence Microscopy.

Alexa Fluor 647 is a widely used fluorescent probe for cell bioimaging and super-resolution microscopy. Herein, the reversible fluorescence switching of Alexa Fluor 647 conjugated to bovine serum albumin (BSA) and adsorbed onto indium tin oxide (ITO) electrodes under electrochemical potential control at the level of single protein molecules is reported. The modulation of the fluorescence as a function of potential was observed using total internal reflectance fluorescence (TIRF) microscopy. The fluorescence intensity of the Alexa Fluor 647 decreased, or reached background levels, at reducing potentials but returned to normal levels at oxidizing potentials. These electrochemically induced changes in fluorescence were sensitive to pH despite that BSA-Alexa Fluor 647 fluorescence without applied potential is insensitive to pH between values of 4-10. The observed pH dependence indicated the involvement of electron and proton transfer in the fluorescence switching mechanism.

Time-Resolved Laurdan Fluorescence Reveals Insights into Membrane Viscosity and Hydration Levels.

Membrane viscosity and hydration levels characterize the biophysical properties of biological membranes and are reflected in the rate and extent of solvent relaxation, respectively, of environmentally sensitive fluorophores such as Laurdan. Here, we first developed a method for a time-resolved general polarization (GP) analysis with fluorescence-lifetime imaging microscopy that captures both the extent and rate of Laurdan solvent relaxation. We then conducted time-resolved GP measurements with Laurdan-stained model membranes and cell membranes. These measurements revealed that cholesterol levels in lipid vesicles altered membrane hydration and viscosity, whereas curvature had little effect on either parameter. We also applied the method to the plasma membrane of live cells using a supercritical angle fluorescence objective, to our knowledge the first time fluorescence-lifetime imaging microscopy images were generated with supercritical angle fluorescence. Here, we found that local variations in membrane cholesterol most likely account for the heterogeneity of Laurdan lifetime in plasma membrane. In conclusion, time-resolved GP measurements provide additional insights into the biophysical properties of membranes.