Fast fluorescence microscopy for imaging the dynamics of embryonic development

Live imaging has gained a pivotal role in developmental biology since it increasingly allows real-time observation of cell behavior in intact organisms. Microscopes that can capture the dynamics of ever-faster biological events, fluorescent markers optimal for in vivo imaging, and, finally, adapted reconstruction and analysis programs to complete data flow all contribute to this success. Focusing on temporal resolution, we discuss how fast imaging can be achieved with minimal prejudice to spatial resolution, photon count, or to reliably and automatically analyze images. In particular, we show how integrated approaches to imaging that combine bright fluorescent probes, fast microscopes, and custom post-processing techniques can address the kinetics of biological systems at multiple scales. Finally, we discuss remaining challenges and opportunities for further advances in this field

RNA polymerase II clusters form in line with surface condensation on regulatory chromatin

It is essential for cells to control which genes are transcribed into RNA. In eukaryotes, two major control points are recruitment of RNA polymerase II (Pol II) into a paused state, and subsequent pause release toward transcription. Pol II recruitment and pause release occur in association with macromolecular clusters, which were proposed to be formed by a liquid–liquid phase separation mechanism. How such a phase separation mechanism relates to the interaction of Pol II with DNA during recruitment and transcription, however, remains poorly understood. Here, we use live and super-resolution microscopy in zebrafish embryos to reveal Pol II clusters with a large variety of shapes, which can be explained by a theoretical model in which regulatory chromatin regions provide surfaces for liquid-phase condensation at concentrations that are too low for canonical liquid–liquid phase separation. Model simulations and chemical perturbation experiments indicate that recruited Pol II contributes to the formation of these surface-associated condensates, whereas elongating Pol II is excluded from these condensates and thereby drives their unfolding

Tropical – software for quantitative analysis of FRAP experiments : identifying the dynamics of linker histone H1°

Fluorescence recovery after photobleaching (FRAP) experiments using laser scanning microscopes to follow the in vivo dynamics of proteins tagged to fluorescent markers like the green fluorescent protein (GFP) has become a standard method in cell biology. FRAP perturbs the fluorescence distribution by photobleaching GFPtagged proteins in a specific area of a cell and monitors the fluorescence redistribution. Adequate methods to quantify the results of FRAP experiments have recently became available. Those methods allow the extraction of diffusion coefficients and dissociation constants of proteins diffusing inside distinct cellular compartments and undergoing dynamic binding and dissociation with immobile or mobile binding sites. However, software incorporating such methods was not available until now. Therefore I developed Tropical, a software for simulation and parameter estimation of reaction–diffusion models. Based on spatio-temporal microscopy images, Tropical estimates reaction and diffusion coefficients for user-defined models. Tropical allows the investigation of systems with an inhomogeneous distribution of molecules, making it well suited for quantitative analyses of microscopy experiments such as FRAP. Tropical was used in this thesis to analyze the dynamic behavior of linker histone H1°, which plays a crucial role in the dynamic organization of chromatin by stabilizing the nucleosome, a structure involved in DNA packing in eucaryotic cells. FRAP experiments were performed using three forms of linker histone H1°, the wild type, and two forms with mutated sites, that are likely to play a major role in DNA binding. Diffusion coefficients on the three forms were estimated with Tropical by fitting a pure diffusion model, assuming binding to happen instantaneously. The model showed a very good fit to the experimental data. It could be shown that lysine 52 significantly influences the DNA binding properties of H1° and its mutation resulted in a 3-fold enhanced diffusion coefficient. The H1° form containing six point mutations however showed an even higher diffusion coefficient, about 15 times higher than the one of the wild type histone, revealing a much larger contribution to DNA binding of these six mutated sites. Using Tropical to estimate the diffusion coefficients of linker histone H1° was another proof for the power and functionality of Tropical, besides the recently published one (Ulrich et al. 2006). Tropicals’ main advantages are (1) that it directly operates on microscopy images, (2) an inhomogeneous distribution of binding partners can be considered and (3) the obtained result can directly be verified. This thesis will first line out the current knowledge of eucaryotic chromatin organization, to clarify the role of linker histone H1. I will then give an overview of microscopy techniques available to reveal protein dynamics and their quantitative analysis using mathematical models. The next part will explain Tropical and its implemented methods in detail. Finally I will present the results obtained on the dynamics of H1° and critically discuss the applicability of Tropical to analyze FRAP data and FRAP as a method to reveal protein dynamics

The interaction of promoter chromatin architecture with the cell cycle regulates transcription activation kinetics

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2013.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student-submitted PDF version of thesis.Includes bibliographical references.The relationship between regulatory trans-factors binding a gene's cis-regulatory sequence elements and the transcriptional output of that gene is fundamental to even the most complex network behaviors such as metabolism and differentiation. In eukaryotes, chromatin dynamics on gene promoter sequences is an integral part of regulation, and nucleosome remodeling is often required for transcription activation. Though the transient response of these regulated genes is often important in biological contexts, the role of promoter chromatin architecture in activation kinetics is still unclear. We sought to investigate this relationship as well as possible links to the cell cycle, over which chromatin state experiences dramatic changes. To study the activation kinetics of individual promoters, we develop a method to infer real-time transcription rates from protein expression in single Saccharomyces cerevisiae cells using time-lapse fluorescence microscopy. Comparison between the instantaneous transcription rate and cell-cycle phase in each cell demonstrates the majority of transcriptional variability is due to cell cycle-dependent effects with noisy expression restricted to S/G2/M. This is in stark contrast to current stochastic models of gene expression, most of which do not account for extrinsic effects, and reveals a permissive activation period beginning each S-phase. We then employ a switchable transactivator system to probe transient response kinetics as a function of promoter chromatin architecture at the PHO5 promoter, a well-established model system for chromatin-regulated expression. While we show transactivator binding site affinity and location relative to nucleosomes influences promoter response kinetics, the effect is primarily through architecture-dependent reliance on a dominant, permissive activation period in S/G2. Together with similar observations at synthetic promoters using a chimerical, switchable transactivator, these results suggest the cell cycle has a general role in transcription activation. Based on the timing of the permissive period, DNA replication may play a direct role in transactivation. Thus, in network topologies involving noisy genes and positive feedback, the cell cycle-dependent transcription would lead to distinct predictions between frequently- and non-dividing cells. This work reveals an unappreciated yet dominant role for the cell cycle as a general regulator of transcription in eukaryotes with direct implications in better modeling and design of biological networks.by Christopher J. Zopf.Ph.D

Quantitative Analysis of the Structural Dynamics of Mitotic Chromosomes in Live Mammalian Cells

Chromatin, organized into individual chromosomes, is the physiological carrier of the genetic and epigenetic information in eukaryotes. In the nucleus of an intact cell, the structure of chromatin is dynamic and essential for genomic activities. The most dramatic changes in chromatin structure occur in mitosis, when compact metaphase chromosomes are formed, organized and partitioned equally to two daughter cells. How this vital reorganization of chromatin is accomplished remains poorly understood in vivo. To address this, in the first part of my thesis I developed quantitative assays to determine the kinetics of mitotic chromosome compaction, using multidimensional confocal microscopy of live cells stably expressing fluorescent histone 2b. Condensation was measured at three different scales: Large-scale (~800 nm), where the chromatin volume was measured by high resolution 4D imaging; medium scale (~200 nm) by statistical analysis of pixel intensities; and molecular scale (~10 nm) by a FRET reporter of histone tail environment. These measurements show that (i) mitotic compaction may start at least 20 min before prometaphase; (ii) it correlates with changes in histone tail conformation; (iii) chromatin density is not highest in metaphase but in late anaphase chromosomes. In the second part, I focused on the novel finding of highest compaction in anaphase. Single chromosome measurements revealed that chromatids compact in anaphase by a mechanism of lengthwise shortening that starts only after segregation of the sister chromatids is complete. This axial shortening was not affected in condensin-depleted cells, and was independent of the poleward pulling motion on kinetochores, but it nevertheless depended on dynamic microtubules. Perturbation of this shortening caused a severe phenotype of multi-lobulated daughter nuclei, strongly suggesting a function in post-mitotic nuclear assembly and architecture. In addition, if anaphase compaction was perturbed in condensin-depleted cells, segregation defects increased 3-fold, suggesting a second role for anaphase compaction as a rescue mechanism for segregation defects. In the third part, the quantitative compaction assays were used to probe the role of PNUTS, a major protein phosphatase 1 nuclear-targeting subunit, in the regulation of chromatin structure. In live cells depleted of PNUTS by RNAi, compaction was slowed at least 3-fold. Our collaborators in the group of Philippe Collas at the University of Oslo had shown that PNUTS accelerates chromatin decompaction in vitro. PNUTS is thus involved in mitotic chromatin structure in vivo and in vitro, and my findings make it an interesting target for future research to understand the molecular mechanism of chromosome compaction