Bassam Hajj

Single-molecule dynamics of enhanceosome assembly in embryonic stem cells.

Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. Here, we combine in vivo and in vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84-97 events of 3D diffusion (3.3-3.7 s) interspersed with brief nonspecific collisions (0.75-0.9 s) before acquiring and dwelling at specific target DNA (12.0-14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters.

Fast multicolor 3D imaging using aberration-corrected multifocus microscopy.

Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.

Real-Time Dynamics of RNA Polymerase II Clustering in Live Human Cells

Pol II Micro Clusters In higher eukaryotes, messenger RNA (mRNA) synthesis is thought to involve foci of clustered RNA polymerase II (Pol II) called transcription factories. However, clustered Pol II have not been resolved in living cells, raising the debate about their existence in vivo and what role, if any, they play in nuclear organization and regulation of gene expression. Cisse et al. (p. 664, published online 4 July; see the Perspective by Rickman and Bickmore) developed single-molecule in vivo analyses revealing the distribution and dynamics of Pol II clustering in living cells. Pol II clusters were smaller than the diffraction limit (<250 nm). Transient dynamics of the Pol II clusters, and correlation with changes in transcription, pointed to a role in transcription initiation rather than in elongation. A single-cell quantitative method reveals changes in the distribution of proteins with single-molecule sensitivity. [Also see Perspective by Rickman and Bickmore] Transcription is reported to be spatially compartmentalized in nuclear transcription factories with clusters of RNA polymerase II (Pol II). However, little is known about when these foci assemble or their relative stability. We developed a quantitative single-cell approach to characterize protein spatiotemporal organization, with single-molecule sensitivity in live eukaryotic cells. We observed that Pol II clusters form transiently, with an average lifetime of 5.1 (± 0.4) seconds, which refutes the notion that they are statically assembled substructures. Stimuli affecting transcription yielded orders-of-magnitude changes in the dynamics of Pol II clusters, which implies that clustering is regulated and plays a role in the cell’s ability to effect rapid response to external signals. Our results suggest that transient crowding of enzymes may aid in rate-limiting steps of gene regulation.

Imaging the fate of histone Cse4 reveals de novo replacement in S phase and subsequent stable residence at centromeres

The budding yeast centromere contains Cse4, a specialized histone H3 variant. Fluorescence pulse-chase analysis of an internally tagged Cse4 reveals that it is replaced with newly synthesized molecules in S phase, remaining stably associated with centromeres thereafter. In contrast, C-terminally-tagged Cse4 is functionally impaired, showing slow cell growth, cell lethality at elevated temperatures, and extra-centromeric nuclear accumulation. Recent studies using such strains gave conflicting findings regarding the centromeric abundance and cell cycle dynamics of Cse4. Our findings indicate that internally tagged Cse4 is a better reporter of the biology of this histone variant. Furthermore, the size of centromeric Cse4 clusters was precisely mapped with a new 3D-PALM method, revealing substantial compaction during anaphase. Cse4-specific chaperone Scm3 displays steady-state, stoichiometric co-localization with Cse4 at centromeres throughout the cell cycle, while undergoing exchange with a nuclear pool. These findings suggest that a stable Cse4 nucleosome is maintained by dynamic chaperone-in-residence Scm3. DOI: http://dx.doi.org/10.7554/eLife.02203.001

Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy

Significance A major challenge in modern biological studies is in the determination of the 3D molecular architecture of cellular organelles. In recent years, much progress in nanoscale imaging has been made because of the advent of superresolution optical microscopy. However, many superresolution techniques are still limited to 2D acquisition. Here, we show a volumetric approach for superresolution imaging based on the simultaneous imaging of multiple sample planes using multifocal microscopy. The depth over which structures can be reconstructed reaches 4 µm, comparable with the thickness of many cellular organelles or even whole cells. Single molecule-based superresolution imaging has become an essential tool in modern cell biology. Because of the limited depth of field of optical imaging systems, one of the major challenges in superresolution imaging resides in capturing the 3D nanoscale morphology of the whole cell. Despite many previous attempts to extend the application of photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) techniques into three dimensions, effective localization depths do not typically exceed 1.2 µm. Thus, 3D imaging of whole cells (or even large organelles) still demands sequential acquisition at different axial positions and, therefore, suffers from the combined effects of out-of-focus molecule activation (increased background) and bleaching (loss of detections). Here, we present the use of multifocus microscopy for volumetric multicolor superresolution imaging. By simultaneously imaging nine different focal planes, the multifocus microscope instantaneously captures the distribution of single molecules (either fluorescent proteins or synthetic dyes) throughout an ∼4-µm-deep volume, with lateral and axial localization precisions of ∼20 and 50 nm, respectively. The capabilities of multifocus microscopy to rapidly image the 3D organization of intracellular structures are illustrated by superresolution imaging of the mammalian mitochondrial network and yeast microtubules during cell division.

Phase resolved interferometric spectral modulation (PRISM) for ultrafast pulse measurement and compression

We show through experiments and simulations that parallel phase modulation, a technique developed in the field of adaptive optics, can be employed to quickly determine the spectral phase profile of ultrafast laser pulses and to perform phase compensation as well as pulse shaping. Different from many existing ultrafast pulse measurement methods, the technique reported here requires no spectrum measurements of nonlinear signals. Instead, the power of nonlinear signals is used directly to quickly measure the spectral phase, a convenient feature for applications such as two-photon fluorescence microscopy. The method is found to work with both smooth and even completely random distortions. The experimental results are verified with MIIPS measurements.

Highly efficient multicolor multifocus microscopy by optimal design of diffraction binary gratings

Multifocus microscopy (MFM) allows sensitive and fast three-dimensional imaging. It relies on the efficient design of diffraction phase gratings yielding homogeneous intensities in desired diffraction orders. Such performances are however guaranteed only for a specific wavelength. Here, we discuss a novel approach for designing binary phase gratings with dual color properties and improved diffraction efficiency for MFM. We simulate binary diffraction gratings with tunable phase shifts to explore its best diffraction performances. We report the design and fabrication of a binary array generator of 3 × 3 equal-intensity diffraction orders with 74% efficiency, 95% uniformity and dual color capability. The multicolor properties of this new design are highlighted by two-color MFM imaging. Finally, we discuss the basics of extending this approach to a variety of diffraction pattern designs.

Multi-focus microscopy for fast and ultrasensitive three-dimensional imaging in live cells

A major challenge in imaging lies in fast 3D data acquisition. We present here a multifocus microscopy where nine Z planes are imaged in parallel. Its benefits are illustrated using fast single molecules 3D tracking in cells and super-resolution 3D imaging.