Unveiling mRNP composition by fluorescence correlation and cross-correlation spectroscopy using cell lysates.

Understanding the mRNA life cycle requires information about the dynamics and macromolecular composition and stoichiometry of mRNPs. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are appealing technologies to study these macromolecular structures because they have single molecule sensitivity and readily provide information about their molecular composition and dynamics. Here, we demonstrate how FCS can be exploited to study cytoplasmic mRNPs with high accuracy and reproducibility in cell lysates. Cellular lysates not only recapitulate data from live cells but provide improved readings and allow investigation of single mRNP analysis under particular conditions or following enzymatic treatments. Moreover, FCCS employing minute amounts of cells closely corroborated previously reported RNA dependent interactions and provided estimates of the relative overlap between factors in the mRNPs, thus depicting their heterogeneity. The described lysate-based FCS and FCCS analysis may not only complement current biochemical approaches but also provide novel opportunities for the quantitative analysis of the molecular composition and dynamics of single mRNPs.

Phosphorylated filamin A regulates actin-linked caveolae dynamics.

Caveolae are relatively stable membrane invaginations that compartmentalize signaling, regulate lipid metabolism and mediate viral entry. Caveolae are closely associated with actin fibers and internalize in response to diverse stimuli. Loss of cell adhesion is known to induce rapid and robust caveolae internalization and trafficking toward a Rab11-positive recycling endosome; however, pathways governing this process are poorly understood. Here, we report that filamin A is required to maintain the F-actin-dependent linear distribution of caveolin-1. High spatiotemporal resolution particle tracking of caveolin-1-GFP vesicles by total internal reflection fluorescence (TIRF) microscopy revealed that FLNa is required for the F-actin-dependent arrest of caveolin-1 vesicles in a confined area and their stable anchorage to the plasma membrane. The linear distribution and anchorage of caveolin-1 vesicles are both required for proper caveolin-1 inwards trafficking. De-adhesion-triggered caveolae inward trafficking towards a recycling endosome is impaired in FLNa-depleted HeLa and FLNa-deficient M2-melanoma cells. Inwards trafficking of caveolin-1 requires both the ability of FLNa to bind actin and cycling PKCα-dependent phosphorylation of FLNa on Ser2152 after cell detachment.

Number and brightness image analysis reveals ATF-induced dimerization kinetics of uPAR in the cell membrane.

We studied the molecular forms of the GPI-anchored urokinase plasminogen activator receptor (uPAR-mEGFP) in the human embryo kidney (HEK293) cell membrane and demonstrated that the binding of the amino-terminal fragment (ATF) of urokinase plasminogen activator is sufficient to induce the dimerization of the receptor. We followed the association kinetics and determined precisely the dimeric stoichiometry of uPAR-mEGFP complexes by applying number and brightness (N&B) image analysis. N&B is a novel fluctuation-based approach for measuring the molecular brightness of fluorophores in an image time sequence in live cells. Because N&B is very sensitive to long-term temporal fluctuations and photobleaching, we have introduced a filtering protocol that corrects for these important sources of error. Critical experimental parameters in N&B analysis are illustrated and analyzed by simulation studies. Control experiments are based on mEGFP-GPI, mEGFP-mEGFP-GPI, and mCherry-GPI, expressed in HEK293. This work provides a first direct demonstration of the dimerization of uPAR in live cells. We also provide the first methodological guide on N&B to discern minor changes in molecular composition such as those due to dimerization events, which are involved in fundamental cell signaling mechanisms.

Real-time multi-parameter spectroscopy and localization in three-dimensional single-particle tracking.

Tracking of single particles in optical microscopy has been employed in studies ranging from material sciences to biophysics down to the level of single molecules. The technique intrinsically circumvents ensemble averaging and may therefore reveal directly mechanistic details of the involved dynamic processes. Such processes range from translational and rotational motion to spectral dynamics. We distinguish between conventional a posteriori tracking of objects (e.g. from the sequences of images) and the experimentally more refined 'on-the-fly' tracking technique. In this technique, the observation volume of the microscope is kept centred with respect to the moving object via a feedback algorithm. This approach brings a series of advantages in comparison with the tracking from images, ranging from a superior spatio-temporal resolution (2-50nm and 1-32ms) to the capability of inferring additional data (e.g. fluorescence lifetime, emission spectrum, polarization, intensity dynamics) from an object as it moves over several microns in three dimensions. In this contribution, we describe the principle of the tracking technique as implemented on a two-photon laser scanning microscope and illustrate its capabilities with experimental data, from particles labelled with different dyes moving in a liquid to the characterization of small fluorescently labelled protein assemblies in living cells.

Single mRNP Analysis Reveals that Small Cytoplasmic mRNP Granules Represent mRNA Singletons.

Small cytoplasmic mRNP granules are implicated in mRNA transport, translational control, and decay. Using super-resolution microscopy and fluorescence correlation spectroscopy, we analyzed the molecular composition and dynamics of single cytoplasmic YBX1_IMP1 mRNP granules in live cells. Granules appeared elongated and branched, with patches of IMP1 and YBX1 distributed along mRNA, reflecting the attachment of the two RNA-binding proteins in cis. Particles form at the nuclear pore and do not associate with translating ribosomes, so the mRNP is a repository for mRNAs awaiting translation. In agreement with the average number of mRNA-binding sites derived from crosslinked immunoprecipitation (CLIP) analyses, individual mRNPs contain 5-15 molecules of YBX1 and IMP1 and a single poly(A) tail identified by PABPC1. Taken together, we conclude that small cytoplasmic mRNP granules are mRNA singletons, thus depicting the cellular transcriptome. Consequently, expression of functionally related mRNAs in RNA regulons is unlikely to result from coordinated assembly.

Nanometer-scale optical imaging of collagen fibers using gold nanoparticles.

We describe 3D single particle tracking of gold nanoparticles (AuNPs) moving along collagen fibers in aqueous environment with two-photon excitation conditions. The photoacoustic effect at the collagen fiber caused by the irradiation with ultrashort, near-infrared laser pulses propels the particles adsorbed to the surface of the collagen fibers. We report the tracking of individual AuNPs in three dimensions with high spatial and temporal resolution, of few nanometers and milliseconds, respectively. Due to the emission signal caused by the interaction between the AuNPs and the weak chromophores in the collagen fiber, the trajectories of individual AuNPs reveal the fiber topography with nanometric resolution. The intensity along the trajectory shows that we are sensitive to the distribution of the weak chromophores on the fiber.

Simultaneous measurement of orientational and spectral dynamics of single molecules in nanostructured host-guest materials.

Nanostructured host-guest materials are important for various applications in nanoscience, and therefore, a thorough understanding of the dynamics of the guest molecules within the host matrix is needed. To this aim we used single-molecule fluorescence techniques to simultaneously examine the spectral and the orientational behavior of single molecules in nanostructured porous host materials. Two types of host-guest systems have been investigated. First, oxazine-1 dye molecules were fixed rigidly in the channels of microporous AlPO4-5 crystals. Second, it was shown that terrylenediimide (TDI) dye molecules move in the mesoporous network of an uncalcined M41S thin film. In the first sample both spectral fluctuations ( approximately 5 nm) and rare spectral jumps (>10 nm) of the emission maximum were observed. However, the orientation of the emission dipole of the dye molecules remained constant. In contrast, the second system showed orientational dynamics as well as substantially more spectral dynamics. In this system the molecules were found to move between different regions in the host. The typical motion of the TDI molecules in the pores of M41S was not continuous but characterized by jumps between specific sites. Moreover, the spectral and orientational dynamics were correlated and arose directly from the different environments that were being explored by the mobile molecule.

Vibronic excitation of single molecules: a new technique for studying low-temperature dynamics.

Herein, we present vibronic excitation and detection of purely electronic zero-phonon lines (ZPL) of single molecules as a new tool for investigating dynamics at cryogenic temperatures. Applications of this technique to study crystalline and amorphous matrix materials are presented. In the crystalline environment, spectrally stable ZPLs are observed at moderate excitation powers. By contrast, investigations at higher excitation intensities reveal the opening of local degrees of freedom and spectral jumps, which we interpret as the observation of elementary steps in the melting of a crystal. We compare these results to spectral single-molecule trajectories recorded in a polymer. The way in which much more complicated spectral features can be analysed is shown. Surprisingly, pronounced spectral shifts on a previously not accessible large energy scale are observed, which are hard to reconcile with the standard two-level model system used to describe low-temperature dynamics in disordered systems.