Ignacio Izeddin

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.

Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe.

The actin cytoskeleton of dendritic spines plays a key role in morphological aspects of synaptic plasticity. The detailed analysis of the spine structure and dynamics in live neurons, however, has been hampered by the diffraction-limited resolution of conventional fluorescence microscopy. The advent of nanoscopic imaging techniques thus holds great promise for the study of these processes. We implemented a strategy for the visualization of morphological changes of dendritic spines over tens of minutes at a lateral resolution of 25 to 65 nm. We have generated a low-affinity photoconvertible probe, capable of reversibly binding to actin and thus allowing long-term photoactivated localization microscopy of the spine cytoskeleton. Using this approach, we resolve structural parameters of spines and record their long-term dynamics at a temporal resolution below one minute. Furthermore, we have determined changes in the spine morphology in response to pharmacologically induced synaptic activity and quantified the actin redistribution underlying these changes. By combining PALM imaging with quantum dot tracking, we could also simultaneously visualize the cytoskeleton and the spine membrane, allowing us to record complementary information on the morphological changes of the spines at super-resolution.

Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleus

Gene regulation relies on transcription factors (TFs) exploring the nucleus searching their targets. So far, most studies have focused on how fast TFs diffuse, underestimating the role of nuclear architecture. We implemented a single-molecule tracking assay to determine TFs dynamics. We found that c-Myc is a global explorer of the nucleus. In contrast, the positive transcription elongation factor P-TEFb is a local explorer that oversamples its environment. Consequently, each c-Myc molecule is equally available for all nuclear sites while P-TEFb reaches its targets in a position-dependent manner. Our observations are consistent with a model in which the exploration geometry of TFs is restrained by their interactions with nuclear structures and not by exclusion. The geometry-controlled kinetics of TFs target-search illustrates the influence of nuclear architecture on gene regulation, and has strong implications on how proteins react in the nucleus and how their function can be regulated in space and time. DOI: http://dx.doi.org/10.7554/eLife.02230.001

Quantitative nanoscopy of inhibitory synapses: counting gephyrin molecules and receptor binding sites.

The strength of synaptic transmission is controlled by the number and activity of neurotransmitter receptors. However, little is known about absolute numbers and densities of receptor and scaffold proteins and the stoichiometry of molecular interactions at synapses. Here, we conducted three-dimensional and quantitative nanoscopic imaging based on single-molecule detections to characterize the ultrastructure of inhibitory synapses and to count scaffold proteins and receptor binding sites. We observed a close correspondence between the spatial organization of gephyrin scaffolds and glycine receptors at spinal cord synapses. Endogenous gephyrin was clustered at densities of 5,000-10,000 molecules/μm(2). The stoichiometry between gephyrin molecules and receptor binding sites was approximately 1:1, consistent with a two-dimensional scaffold in which all gephyrin molecules can contribute to receptor binding. The competition of glycine and GABAA receptor complexes for synaptic binding sites highlights the potential of single-molecule imaging to quantify synaptic plasticity on the nanoscopic scale.

PSF shaping using adaptive optics for three-dimensional single-molecule super-resolution imaging and tracking

We present a novel approach for three-dimensional localization of single molecules using adaptive optics. A 52-actuator deformable mirror is used to both correct aberrations and induce two-dimensional astigmatism in the point-spread-function. The dependence of the z-localization precision on the degree of astigmatism is discussed. We achieve a z-localization precision of 40 nm for fluorescent proteins and 20 nm for fluorescent dyes, over an axial depth of ~800 nm. We illustrate the capabilities of our approach for three-dimensional high-resolution microscopy with super-resolution images of actin filaments in fixed cells and single-molecule tracking of quantum-dot labeled transmembrane proteins in live HeLa cells.

Wavelet analysis for single molecule localization microscopy

Localization of single molecules in microscopy images is a key step in quantitative single particle data analysis. Among them, single molecule based super-resolution optical microscopy techniques require high localization accuracy as well as computation of large data sets in the order of 10(5) single molecule detections to reconstruct a single image. We hereby present an algorithm based on image wavelet segmentation and single particle centroid determination, and compare its performance with the commonly used gaussian fitting of the point spread function. We performed realistic simulations at different signal-to-noise ratios and particle densities and show that the calculation time using the wavelet approach can be more than one order of magnitude faster than that of gaussian fitting without a significant degradation of the localization accuracy, from 1 nm to 4 nm in our range of study. We propose a simulation-based estimate of the resolution of an experimental single molecule acquisition.

The SNARE Sec22b has a non-fusogenic function in plasma membrane expansion.

Development of the nervous system requires extensive axonal and dendritic growth during which neurons massively increase their surface area. Here we report that the endoplasmic reticulum (ER)-resident SNARE Sec22b has a conserved non-fusogenic function in plasma membrane expansion. Sec22b is closely apposed to the plasma membrane SNARE syntaxin1. Sec22b forms a trans-SNARE complex with syntaxin1 that does not include SNAP23/25/29, and does not mediate fusion. Insertion of a long rigid linker between the SNARE and transmembrane domains of Sec22b extends the distance between the ER and plasma membrane, and impairs neurite growth but not the secretion of VSV-G. In yeast, Sec22 interacts with lipid transfer proteins, and inhibition of Sec22 leads to defects in lipid metabolism at contact sites between the ER and plasma membrane. These results suggest that close apposition of the ER and plasma membrane mediated by Sec22 and plasma membrane syntaxins generates a non-fusogenic SNARE bridge contributing to plasma membrane expansion, probably through non-vesicular lipid transfer.

Assessing the localization of centrosomal proteins by PALM/STORM nanoscopy†‡

The structure of the centrosome was resolved by EM many years ago to reveal a pair of centrioles embedded in a dense network of proteins. More recently, the molecular composition of the centrosome was catalogued by mass spectroscopy and many novel components were identified. Determining precisely where a novel component localizes to within the centrosome remains a challenge, and until now it has required the use of immuno‐EM. This technique is both time‐consuming and unreliable, as it often fails due to problems with antigen accessibility. We have investigated the use of two nanoscopic techniques, photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), as alternative techniques for localizing centrosomal proteins. The localization of a known centrosomal component, the distal appendage protein Cep164 was investigated by direct STORM (dSTORM) and resolved with a high spatial resolution. We further validated the use of nanoscopic PALM imaging by showing that the previously uncharacterized centrosomal protein CCDC123 (Cep123) localizes to the distal appendages, forming ring‐like structures with a diameter of 500 nm. Our results demonstrate that both PALM and STORM imaging have great potential as alternatives to immuno‐EM.

Geometry of the nucleus: a perspective on gene expression regulation

Gene expression control results from the combined interactions of the nearly hundred proteins forming the pre-initiation complex, thousands of transcription regulators, and genomic DNA. In the recent years, new technologies have revealed several key aspects of nuclear spatial organization that showed a fine interplay between the function of nuclear proteins, their 3D organization, and their dynamics. Here we review several concepts that link biochemical reactivity in the nucleus to its 3D spatial organization. We present the analogies between the emerging understanding of nuclear organization in the field of cell biology, and the more established disciplines of heterogeneous catalysis and the physics of random walks. We provide several recent examples showing how nuclear geometry affects protein reactivity in the nucleus.

Single cell correlation fractal dimension of chromatin: a framework to interpret 3D single molecule super-resolution.

Chromatin is a major nuclear component, and it is an active matter of debate to understand its different levels of spatial organization, as well as its implication in gene regulation. Measurements of nuclear chromatin compaction were recently used to understand how DNA is folded inside the nucleus and to detect cellular dysfunctions such as cancer. Super-resolution imaging opens new possibilities to measure chromatin organization in situ. Here, we performed a direct measure of chromatin compaction at the single cell level. We used histone H2B, one of the 4 core histone proteins forming the nucleosome, as a chromatin density marker. Using photoactivation localization microscopy (PALM) and adaptive optics, we measured the three-dimensional distribution of H2B with nanometric resolution. We computed the distribution of distances between every two points of the chromatin structure, namely the Ripley K(r) distribution. We found that the K(r) distribution of H2B followed a power law, leading to a precise measurement of the correlation fractal dimension of chromatin of 2.7. Moreover, using photoactivable GFP fused to H2B, we observed dynamic evolution of chromatin sub-regions compaction. As a result, the correlation fractal dimension of chromatin reported here can be interpreted as a dynamically maintained non-equilibrium state.