Chromatin condensation fluctuations rather than steady-state predict chromatin accessibility.

Chromatin accessibility to protein factors is critical for genome activities. However, the dynamic properties of chromatin higher-order structures that regulate its accessibility are poorly understood. Here, we took advantage of the microenvironment sensitivity of the fluorescence lifetime of EGFP-H4 histone incorporated in chromatin to map in the nucleus of live cells the dynamics of chromatin condensation and its direct interaction with a tail acetylation recognition domain (the double bromodomain module of human TAFII250, dBD). We reveal chromatin condensation fluctuations supported by mechanisms fundamentally distinct from that of condensation. Fluctuations are spontaneous, yet their amplitudes are affected by their sub-nuclear localization and by distinct and competing mechanisms dependent on histone acetylation, ATP and both. Moreover, we show that accessibility of acetylated histone H4 to dBD is not restricted by chromatin condensation nor predicted by acetylation, rather, it is predicted by chromatin condensation fluctuations.

Biomechanical Control of Lysosomal Secretion Via the VAMP7 Hub: A Tug-of-War between VARP and LRRK1.

The rigidity of the cell environment can vary tremendously between tissues and in pathological conditions. How this property may affect intracellular membrane dynamics is still largely unknown. Here, using atomic force microscopy, we show that cells deficient in the secretory lysosome v-SNARE VAMP7 are impaired in adaptation to substrate rigidity. Conversely, VAMP7-mediated secretion is stimulated by more rigid substrate and this regulation depends on the Longin domain of VAMP7. We further find that the Longin domain binds the kinase and retrograde trafficking adaptor LRRK1 and that LRRK1 negatively regulates VAMP7-mediated exocytosis. Conversely, VARP, a VAMP7- and kinesin 1-interacting protein, further controls the availability for secretion of peripheral VAMP7 vesicles and response of cells to mechanical constraints. LRRK1 and VARP interact with VAMP7 in a competitive manner. We propose a mechanism whereby biomechanical constraints regulate VAMP7-dependent lysosomal secretion via LRRK1 and VARP tug-of-war control of the peripheral pool of secretory lysosomes.

Vinculin head-tail interaction defines multiple early mechanisms for cell substrate rigidity sensing.

Rigidity sensing is a critical determinant of cell fate and behavior but its molecular mechanisms are poorly understood. Focal adhesions (FAs) are complexes that anchor cells to the matrix. Among their components, vinculin undergoes an auto-inhibitory head-tail interaction that regulates the recruitment of, and interactions with its partners in a force-dependent manner. It is unknown, however, whether this mechanism is involved in substrate rigidity sensing. Here, we use a range of quantitative fluorescence microscopies on live human Mesenchymal Stem Cells to address this question. We identify two distinct rigidity-sensing molecular modules in FAs, one of which involves vinculin and talin, is regulated by vinculin head-tail interaction, and targets cell morphology. Vinculin and talin are recruited independently in a rigidity-dependent manner to FAs where they directly interact in a rigidity-independent stoichiometry at a site proximal to talin head. Vinculin head-tail interaction is required on soft substrates to destabilize vinculin and talin in FAs, and to allow hMSCs branching. Another module involves paxillin and FAK, which soft substrates also destabilize, but independently of vinculin head-tail interaction. This multi-modularity may be key to allow a versatile response to complex biomechanical cues.

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements.

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis.

Time-domain fluorescence lifetime imaging microscopy: a quantitative method to follow transient protein-protein interactions in living cells.

Quantitative analysis in Förster resonance energy transfer (FRET) imaging studies of protein-protein interactions within live cells is still a challenging issue. Many cellular biology applications aim at the determination of the space and time variations of the relative amount of interacting fluorescently tagged proteins occurring in cells. This relevant quantitative parameter can be, at least partially, obtained at a pixel-level resolution by using fluorescence lifetime imaging microscopy (FLIM). Indeed, fluorescence decay analysis of a two-component system (FRET and no FRET donor species), leads to the intrinsic FRET efficiency value (E) and the fraction of the donor-tagged protein that undergoes FRET (fD). To simultaneously obtain fD and E values from a two-exponential fit, data must be acquired with a high number of photons, so that the statistics are robust enough to reduce fitting ambiguities. This is a time-consuming procedure. However, when fast-FLIM acquisitions are used to monitor dynamic changes in protein-protein interactions at high spatial and temporal resolutions in living cells, photon statistics and time resolution are limited. In this case, fitting procedures are unreliable, even for single lifetime donors. We introduce the concept of a minimal fraction of donor molecules involved in FRET (mfD), obtained from the mathematical minimization of fD. Here, we discuss different FLIM techniques and the compromises that must be made between precision and time invested in acquiring FLIM measurements. We show that mfD constitutes an interesting quantitative parameter for fast FLIM because it gives quantitative information about transient interactions in live cells.

Mechanical checkpoint for persistent cell polarization in adhesion-naive fibroblasts.

Cell polarization is a fundamental biological process implicated in nearly every aspect of multicellular development. The role of cell-extracellular matrix contacts in the establishment and the orientation of cell polarity have been extensively studied. However, the respective contributions of substrate mechanics and biochemistry remain unclear. Here we propose a believed novel single-cell approach to assess the minimal polarization trigger. Using nonadhered round fibroblast cells, we show that stiffness sensing through single localized integrin-mediated cues are necessary and sufficient to trigger and direct a shape polarization. In addition, the traction force developed by cells has to reach a minimal threshold of 56 ± 1.6 pN for persistent polarization. The polarization kinetics increases with the stiffness of the cue. The polarized state is characterized by cortical actomyosin redistribution together with cell shape change. We develop a physical model supporting the idea that a local and persistent inhibition of actin polymerization and/or myosin activity is sufficient to trigger and sustain the polarized state. Finally, the cortical polarity propagates to an intracellular polarity, evidenced by the reorientation of the centrosome. Our results define the minimal adhesive requirements and quantify the mechanical checkpoint for persistent cell shape and organelle polarization, which are critical regulators of tissue and cell development.

Golgi sorting regulates organization and activity of GPI proteins at apical membranes.

Here we combined classical biochemistry with new biophysical approaches to study the organization of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) with high spatial and temporal resolution at the plasma membrane of polarized epithelial cells. We show that in polarized MDCK cells, after sorting in the Golgi, each GPI-AP reaches the apical surface in homoclusters. Golgi-derived homoclusters are required for their subsequent plasma membrane organization into cholesterol-dependent heteroclusters. By contrast, in nonpolarized MDCK cells, GPI-APs are delivered to the surface as monomers in an unpolarized manner and are not able to form heteroclusters. We further demonstrate that this GPI-AP organization is regulated by the content of cholesterol in the Golgi apparatus and is required to maintain the functional state of the protein at the apical membrane. Thus, in contrast to fibroblasts, in polarized epithelial cells, a selective cholesterol-dependent sorting mechanism in the Golgi regulates both the organization and function of GPI-APs at the apical surface.

Quantitative study of protein-protein interactions in live cell by dual-color fluorescence correlation spectroscopy.

Dual-color FCS is a powerful method to monitor protein-protein interactions in living cells. The main idea is based on the cross-correlation analysis of temporal fluorescence intensity fluctuations of two fluorescent proteins to obtain their co-diffusion and relative concentration. But, when performing these experiments, the spectral overlap in the emission of the two colors produces an artifact that corrupts the cross-correlation data: spectral bleed-through. We have shown that problems with cross talk are overcome with Fluorescence Lifetime Correlation Spectroscopy (FLCS). FLCS applied to dual-color cross-correlation, utilizing for example eGFP and mCherry fluorescent proteins, allows the determination of protein-protein interactions in living cells without the need of spectral bleed-through calibration. Here, we present in detail how this methodology can be implemented using a commercial setup (Microtime from PicoQuant, SP8 SMD from Leica or any conventional confocal with PicoQuant TCSPC module, and also with a Becker and Hickl TCSPC module). The dual-color FLCS experimental procedure where the different laser intensities do not have to be controlled during the experiment constitutes a very powerful technique to quantitatively study protein interactions in live samples.

A molecular network for the transport of the TI-VAMP/VAMP7 vesicles from cell center to periphery.

The compartmental organization of eukaryotic cells is maintained dynamically by vesicular trafficking. SNARE proteins play a crucial role in intracellular membrane fusion and need to be targeted to their proper donor or acceptor membrane. The molecular mechanisms that allow for the secretory vesicles carrying the v-SNARE TI-VAMP/VAMP7 to leave the cell center, load onto microtubules, and reach the periphery to mediate exocytosis are largely unknown. Here, we show that the TI-VAMP/VAMP7 partner Varp, a Rab21 guanine nucleotide exchange factor, interacts with GolginA4 and the kinesin 1 Kif5A. Activated Rab21-GTP in turn binds to MACF1, an actin and microtubule regulator, which is itself a partner of GolginA4. These components are required for directed movement of TI-VAMP/VAMP7 vesicles from the cell center to the cell periphery. The molecular mechanisms uncovered here suggest an integrated view of the transport of vesicles carrying a specific v-SNARE toward the cell surface.

Live-cell imaging reveals multiple interactions between Epstein-Barr virus nuclear antigen 1 and cellular chromatin during interphase and mitosis.

Epstein-Barr virus (EBV) establishes a life-long latent infection in humans. In proliferating latently infected cells, EBV genomes persist as multiple episomes that undergo one DNA replication event per cell cycle and remain attached to the mitotic chromosomes. EBV nuclear antigen 1 (EBNA-1) binding to the episome and cellular genome is essential to ensure proper episome replication and segregation. However, the nature and regulation of EBNA-1 interaction with chromatin has not been clearly elucidated. This activity has been suggested to involve EBNA-1 binding to DNA, duplex RNA, and/or proteins. EBNA-1 binding protein 2 (EBP2), a nucleolar protein, has been proposed to act as a docking protein for EBNA-1 on mitotic chromosomes. However, there is no direct evidence thus far for EBP2 being associated with EBNA-1 during mitosis. By combining video microscopy and Förster resonance energy transfer (FRET) microscopy, we demonstrate here for the first time that EBNA-1 and EBP2 interact in the nucleoplasm, as well as in the nucleoli during interphase. However, in strong contrast to the current proposed model, we were unable to observe any interaction between EBNA-1 and EBP2 on mitotic chromosomes. We also performed a yeast double-hybrid screening, followed by a FRET analysis, that led us to identify HMGB2 (high-mobility group box 2), a well-known chromatin component, as a new partner for EBNA-1 on chromatin during interphase and mitosis. Although the depletion of HMGB2 partly altered EBNA-1 association with chromatin in HeLa cells during interphase and mitosis, it did not significantly impact the maintenance of EBV episomes in Raji cells.

Dual-color fluorescence lifetime correlation spectroscopy to quantify protein-protein interactions in live cell.

Dual-color fluorescence correlation spectroscopy is an interesting method to quantify protein interaction in living cells. But, when performing these experiments, one must compensate for a known spectral bleed through artifact that corrupts cross-correlation data. In this article, problems with crosstalk were overcome with an approach based on fluorescence lifetime correlation spectroscopy (FLCS). We show that FLCS applied to dual-color EGFP and mCherry cross-correlation allows the determination of protein-protein interactions in living cells without the need of spectral bleed through calibration. The methodology was validated by using EGFP-mCherry tandem in comparison with coexpressed EGFP and mCherry in live cell. The dual-color FLCS experimental procedure where the different laser intensities do not have to be controlled during experiment is really very helpful to study quantitatively protein interactions in live sample.

Non fitting based FRET-FLIM analysis approaches applied to quantify protein-protein interactions in live cells.

New imaging methodologies in quantitative fluorescence microscopy and nanoscopy have been developed in the last few years and are beginning to be extensively applied to biological problems, such as the localization and quantification of protein interactions. Fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM) is currently employed not only in biophysics or chemistry but also in bio-medicine, thanks to new advancements in technology and also new developments in data treatment. FRET-FLIM can be a very useful tool to ascertain protein interactions occurring in single living cells. In this review, we stress the importance of increasing the acquisition speed when working in vivo employing Time-Domain FLIM. The development of the new mathematical-based non-fitting methods allows the determining of the fraction of interacting donor without the requirement of high count statistics, and thus allows the performing of high speed acquisitions in FRET-FLIM to still be quantitative.

Parallel heterodyne detection of dynamic light-scattering spectra from gold nanoparticles diffusing in viscous fluids.

We developed a microscope intended to probe, using a parallel heterodyne receiver, the fluctuation spectrum of light quasi-elastically scattered by gold nanoparticles diffusing in viscous fluids. The cutoff frequencies of the recorded spectra scale up linearly with those expected from single-scattering formalism in a wide range of dynamic viscosities (1 to 15 times water viscosity at room temperature). Our scheme enables ensemble-averaged optical fluctuations measurements over multispeckle recordings in low light, at temporal frequencies up to 10 kHz, with a 12 Hz framerate array detector.

Quantitative comparison of different fluorescent protein couples for fast FRET-FLIM acquisition.

The fluorescent-protein based fluorescence resonance energy transfer (FRET) approach is a powerful method for quantifying protein-protein interactions in living cells, especially when combined with fluorescence lifetime imaging microscopy (FLIM). To compare the performance of different FRET couples for FRET-FLIM experiments, we first tested enhanced green fluorescent protein (EGFP) linked to different red acceptors (mRFP1-EGFP, mStrawberry-EGFP, HaloTag (TMR)-EGFP, and mCherry-EGFP). We obtained a fraction of donor engaged in FRET (f(D)) that was far from the ideal case of one, using different mathematical models assuming a double species model (i.e., discrete double exponential fixing the donor lifetime and double exponential stretched for the FRET lifetime). We show that the relatively low f(D) percentages obtained with these models may be due to spectroscopic heterogeneity of the acceptor population, which is partially caused by different maturation rates for the donor and the acceptor. In an attempt to improve the amount of donor protein engaged in FRET, we tested mTFP1 as a donor coupled to mOrange and EYFP, respectively. mTFP1 turned out to be at least as good as EGFP for donor FRET-FLIM experiments because 1), its lifetime remained constant during light-induced fluorescent changes; 2), its fluorescence decay profile was best fitted with a single exponential model; and 3), no photoconversion was detected. The f(D) value when combined with EYFP as an acceptor was the highest of all tandems tested (0.7). Moreover, in the context of fast acquisitions, we obtained a minimal f(D) (mf(D)) for mTFP1-EYFP that was almost two times greater than that for mCherry-EGFP (0.65 vs. 0.35). Finally, we compared EGFP and mTFP1 in a biological situation in which the fusion proteins were highly immobile, and EGFP and mTFP1 were linked to the histone H4 (EGFP-H4 and mTFP1-H4) in fast FLIM acquisitions. In this particular case, the fluorescence intensity was more stable for EGFP-H4 than for mTFP1-H4. Nevertheless, we show that mTFP1/EYFP stands alone as the best FRET-FLIM couple in terms of f(D) analysis.

Dynamic interaction of amphiphysin with N-WASP regulates actin assembly.

Amphiphysin 1, an endocytic adaptor concentrated at synapses that couples clathrin-mediated endocytosis to dynamin-dependent fission, was also shown to have a regulatory role in actin dynamics. Here, we report that amphiphysin 1 interacts with N-WASP and stimulates N-WASP- and Arp2/3-dependent actin polymerization. Both the Src homology 3 and the N-BAR domains are required for this stimulation. Acidic liposome-triggered, N-WASP-dependent actin polymerization is strongly impaired in brain cytosol of amphiphysin 1 knock-out mice. FRET-FLIM analysis of Sertoli cells, where endogenously expressed amphiphysin 1 co-localizes with N-WASP in peripheral ruffles, confirmed the association between the two proteins in vivo. This association undergoes regulation and is enhanced by stimulating phosphatidylserine receptors on the cell surface with phosphatidylserine-containing liposomes that trigger ruffle formation. These results indicate that actin regulation is a key function of amphiphysin 1 and that such function cooperates with the endocytic adaptor role and membrane shaping/curvature sensing properties of the protein during the endocytic reaction.

Spatiotemporal analysis of cell response to a rigidity gradient: a quantitative study using multiple optical tweezers.

We investigate the dynamic response of single cells to weak and local rigidities, applied at controlled adhesion sites. Using multiple latex beads functionalized with fibronectin, and each trapped in its own optical trap, we study the reaction in real time of single 3T3 fibroblast cells to asymmetrical tensions in the tens of pN x microm(-1) range. We show that the cell feels a rigidity gradient even at this low range of tension, and over time develops an adapted change in the force exerted on each adhesion site. The rate at which force increases is proportional to trap stiffness. Actomyosin recruitment is regulated in space and time along the rigidity gradient, resulting in a linear relationship between the amount of recruited actin and the force developed independently in trap stiffness. This time-regulated actomyosin behavior sustains a constant and rigidity-independent velocity of beads inside the traps. Our results show that the strengthening of extracellular matrix-cytoskeleton linkages along a rigidity gradient is regulated by controlling adhesion area and actomyosin recruitment, to maintain a constant deformation of the extracellular matrix.

Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells.

Quantitative analysis in Förster resonance energy transfer (FRET) experiments in live cells for protein interaction studies is still a challenging issue. In a two-component system (FRET and no FRET donor species), fitting of fluorescence lifetime imaging microscopy (FLIM) data gives the fraction of donor molecules involved in FRET (f(D)) and the intrinsic transfer efficiency. But when fast FLIM acquisitions are used to monitor dynamic changes in protein-protein interactions at high spatial and temporal resolutions in living cells, photon statistics and time resolution are limited. In this case, fitting procedures are not reliable, even for single lifetime donors. We introduce the new concept of a minimal fraction of donor molecules involved in FRET (mf(D)), coming from the mathematical minimization of f(D). We find particular advantage in the use of mf(D) because it can be obtained without fitting procedures and it is derived directly from FLIM data. mf(D) constitutes an interesting quantitative parameter for live cell studies because it is related to the minimal relative concentration of interacting proteins. For multi-lifetime donors, the process of fitting complex fluorescence decays to find at least four reliable lifetimes is a near impossible task. Here, mf(D) extension for multi-lifetime donors is the only quantitative determinant. We applied this methodology for imaging the interaction between the bromodomains of TAF(II250) and acetylated histones H4 in living cells at high resolution. We show the existence of discrete acetylated chromatin domains where the minimal fraction of bromodomain interacting with acetylated H4 oscillates from 0.26 to 0.36 and whose size is smaller than half of one micron cube. We demonstrate that mf(D) by itself is a useful tool to investigate quantitatively protein interactions in live cells, especially when using fast FRET-FLIM acquisition times.

Heterodyne holographic microscopy of gold particles.

We report experimental results on heterodyne holographic microscopy of subwavelength-size gold particles. The apparatus uses continuous green-laser illumination of the metal beads in a total internal reflection configuration for dark-field operation. Detection of the scattered light at the illumination wavelength on a charge-coupled-device array detector enables 3D localization of brownian particles in water.

Fluorescence anisotropy imaging microscopy for homo-FRET in living cells.

In this chapter, we present the basic physical principles of the fluorescence anisotropy imaging microscopy (FAIM) and its application to study FP-tagged protein dynamics and interaction in live cells. The Förster mechanism of electronic energy transfer can occur between like chromophores (homo-fluorescence resonance energy transfer, homo-FRET) inducing fluorescence depolarization and can be monitored by fluorescence anisotropy. The energy transfer rate is fast compared to the rotational time of proteins, and therefore its detection as a fast depolarization process in the fluorescence anisotropy can be easily discriminated from rotational motion. Quantitative analysis of fluorescence anisotropy decays provides information on structural parameters: distance between the two interacting chromophores and spatial orientation between the chromophores within dimeric proteins. Fluorescence anisotropy decay is not easy to measure in living cells under the microscope and the instrumentations are necessarily sophisticated. In contrast, any type of microscope can be used to measure the steady-state anisotropy. Interestingly, two-photon excitation steady-state FAIM is a powerful tool for qualitative analysis of macromolecule interactions in living cells and can be used easily for time-lapse homo-FRET.

beta-arrestin 2 oligomerization controls the Mdm2-dependent inhibition of p53.

beta-arrestins (beta-arrs), two ubiquitous proteins involved in serpentine heptahelical receptor regulation and signaling, form constitutive homo- and heterooligomers stabilized by inositol 1,2,3,4,5,6-hexakisphosphate (IP6). Monomeric beta-arrs are believed to interact with receptors after agonist activation, and therefore, beta-arr oligomers have been proposed to represent a resting biologically inactive state. In contrast to this, we report here that the interaction with and subsequent titration out of the nucleus of the protooncogene Mdm2 specifically require beta-arr2 oligomers together with the previously characterized nucleocytoplasmic shuttling of beta-arr2. Mutation of the IP6-binding sites impair oligomerization, reduce interaction with Mdm2, and inhibit p53-dependent antiproliferative effects of beta-arr2, whereas the competence for receptor regulation and signaling is maintained. These observations suggest that the intracellular concentration of beta-arr2 oligomers might control cell survival and proliferation.