Dynamic organization of transcription compartments is dependent on functional nuclear architecture.

Transcription in higher organisms requires spatiotemporal coordination of transcription machinery and the transcription factors at promoter sites. Toward this, recent evidence suggests that both static compartmentalization and dynamic self-organization of transcriptional apparatus are in effect at sites of transcription. Although the dynamics of transcription machinery is essential to genome regulation, the principles underlying this organization and its functional coupling to nuclear architecture is unclear. In a recent study we revealed that Uridine-5'-triphosphate (UTP) uptake in living cells labeled transcription-related compartments. In this article, we quantitatively establish multicolor labeling strategies for UTP-enriched transcription compartments (TCs) and probe their dynamic organization. UTP-enriched TCs were found to be in two distinct fractions: one colocalized with phosphorylated RNA pol II and the other as nascent aggregates. The fraction colocalized with the phosphorylated RNA pol II decreased with the inhibition of transcription initiation or elongation. Fluorescence anisotropy imaging and photobleaching experiments suggest that TCs are functional aggregates of nascent transcripts that are assembled in a transcription-dependent manner. Fluorescence correlation spectroscopy analysis revealed the relative fraction and sizes of fluorescent UTP-labeled transcripts in the nucleoplasm. Time-lapse imaging experiments of TCs exhibited pause and a mobile nature of these compartments within interchromosome territories. Perturbation of either nucleoskeletal protein or the cytoskeleton resulted in reduced active mobility of TCs, whereas inhibitors of transcription enhanced the mobile fraction of TCs. Further, high temporal resolution imaging showed evidence of stepping dynamics of TCs regulated by nucleoskeleton and chromatin modifications. Taken together, our experiments suggest the transient compartmentalization of UTP-enriched aggregates and their dynamic reorganization in a transcription-dependent manner. These results may have important implications for understanding spatiotemporal control of eukaryotic transcription.

ImFCS: a software for imaging FCS data analysis and visualization.

The multiplexing of fluorescence correlation spectroscopy (FCS), especially in imaging FCS using fast, sensitive array detectors, requires the handling of large amounts of data. One can easily collect in excess of 100,000 FCS curves a day, too many to be treated manually. Therefore, ImFCS, an open-source software which relies on standard image files was developed and provides a wide range of options for the calculation of spatial and temporal auto- and cross-correlations, as well as differences in Cross-Correlation Functions (ΔCCF). ImFCS permits fitting of standard models to correlation functions and provides optimized histograms of fitted parameters. Applications include the measurement of diffusion and flow with Imaging Total Internal Reflection FCS (ITIR-FCS) and Single Plane Illumination Microscopy FCS (SPIM-FCS) in biologically relevant samples. As a compromise between ITIR-FCS and SPIM-FCS, we extend the applications to Imaging Variable Angle-FCS (IVA-FCS) where sub-critical oblique illumination provides sample sectioning close to the cover slide.

Single plane illumination fluorescence correlation spectroscopy (SPIM-FCS) probes inhomogeneous three-dimensional environments.

The life sciences require new highly sensitive imaging tools, which allow the quantitative measurement of molecular parameters within a physiological three-dimensional (3D) environment. Therefore, we combined single plane illumination microscopy (SPIM) with camera based fluorescence correlation spectroscopy (FCS). SPIM-FCS provides contiguous particle number and diffusion coefficient images with a high spatial resolution in homo- and heterogeneous 3D specimens and live zebrafish embryos. Our SPIM-FCS recorded up to 4096 spectra within 56 seconds at a laser power of 60 microW without damaging the embryo. This new FCS modality provides more measurements per time and more, less photo-toxic measurements per sample than confocal based methods. In essence, SPIM-FCS offers new opportunities to observe biomolecular interactions quantitatively and functions in a highly multiplexed manner within a physiologically relevant 3D environment.

Probing events with single molecule sensitivity in zebrafish and Drosophila embryos by fluorescence correlation spectroscopy.

Zebrafish and Drosophila are animal models widely used in developmental biology. High-resolution microscopy and live imaging techniques have allowed the investigation of biological processes down to the cellular level in these models. Here, using fluorescence correlation spectroscopy (FCS), we show that even processes on a molecular level can be studied in these embryos. The two animal models provide different advantages and challenges. We first characterize their autofluorescence pattern and determine usable penetration depth for FCS especially in the case of zebrafish, where tissue thickness is an issue. Next, the applicability of FCS to study molecular processes is shown by the determination of blood flow velocities with high spatial resolution and the determination of diffusion coefficients of cytosolic and membrane-bound enhanced green fluorescent protein-labeled proteins in different cell types. This work provides an approach to study molecular processes in vivo and opens up the possibility to relate these molecular processes to developmental biology questions.

Determination of dissociation constants in living zebrafish embryos with single wavelength fluorescence cross-correlation spectroscopy.

The quantification of biological interactions is very important in life sciences. Here we report for the first time, to our knowledge, the determination of a biomolecular dissociation constant (K(D)) in living zebrafish embryos at physiological protein expression levels. For that purpose, we extend the application of single wavelength fluorescence cross-correlation spectroscopy into small organisms and measure the interaction of Cdc42, a small Rho-GTPase, and IQGAP1, an actin-binding scaffolding protein. Cdc42 and IQGAP1 were labeled with monomeric red fluorescent protein and enhanced green fluorescent protein, respectively. Both fluorophores were excited at a single wavelength of 514 nm, simplifying the fluorescence spectroscopy measurements and allowing quantification. For the determination of the interaction, we used two Cdc42 mutants, the constitutively active Cdc42(G12V) which is in a predominantly GTP-bound form and the dominant-negative GDP-bound Cdc42(T17N). While Cdc42(G12V) binds to IQGAP1 with an apparent K(D) of approximately 100 nM, Cdc42(T17N) has at least a one-order-of-magnitude lower affinity for the same protein. As a comparison, we measure the same protein-protein interactions in Chinese hamster ovary cell cultures but observe significant differences in protein mobility and K(D) from the zebrafish measurements, supporting the notion that bimolecular interactions depend on the biological system under investigation and are best performed under physiologically relevant conditions.

Line scan fluorescence correlation spectroscopy for three-dimensional microfluidic flow velocity measurements.

The flow direction of microfluidics in biological applications is not limited to two dimensions, but often extends to three dimensions. Currently there are optical methods available for the measurement of 3-D microfluidic flow vectors, but with low spatial resolution. Line scan fluorescence correlation spectroscopy (FCS) was proposed to determine flow directions in 2-D within microchannels and small blood vessels in our previous work. Importantly, its spatial resolution was demonstrated to be as good as 0.5 microm. In this work, we extend line scan FCS to the third dimension for the characterization of 3-D flow velocity vectors. The spatial resolution is close to the diffraction limit using a scan length of 0.5 microm in all three dimensions. The feasibility of line scan FCS for 3-D microfluidic flow is verified by measurements in microchannels and small blood vessels of zebrafish embryos.

Characterization of flow direction in microchannels and zebrafish blood vessels by scanning fluorescence correlation spectroscopy.

The investigation of flow profiles in microstructures and tissues by fluorescence correlation spectroscopy (FCS) has been a challenging topic in the past decade. Due to its inherent optical configuration, a circular focused laser beam, FCS is unable to resolve microfluidic flow directions. Earlier schemes reported the use of two laser beams or the use of nonsymmetrical laser foci to break the symmetry of the measurement system. This, however, is difficult to combine with confocal systems since it would require modifications that interfere with the imaging capabilities. We propose a method called line-scan FCS to measure different flow angles in microchannels and tissues. This method is implemented on a combined laser scanning confocal microscopy (LSCM) and FCS system that enables uncompromised imaging and spectroscopy measurements. We demonstrate that by scanning the laser beam with a defined speed and direction we can measure flow direction with the current system at an optimal resolution of at least 3 microm. The combination system is assessed by measuring flow profiles in a microchannel with and without obstruction. To extend the technique to live tissue measurements we demonstrate that line-scan FCS can determine the flow direction in zebrafish small blood vessels in a label-free approach.