Methods for Physical Characterization of Phase-Separated Bodies and Membrane-less Organelles

Dynamic regulation of nucleolar architecture.

The transformation of the structural organization of interphase nuclei of a plant cell, depending on the type of tissue, ploidy, and the action of abiotic factors of the medium, has been studied by light and transmission electron microscopy. It is shown that the location and the quantitative relationship between condensed and decondensed chromatin, the presence and localization of nuclear bodies in the plant nucleus, and the presence of invaginations of the nuclear membrane and inclusions depend on the tissue type and cell age as well as the intensity, time, and type of exposure to abiotic factors. Examples of different degrees of chromatin condensation in one plant organism are given. It has been established that the degree of chromatin condensation and decondensation, the state of other domains and nonspecific inclusions of the cell nucleus can be artificially modeled for research purposes or subsequent modification. The issue of application of such technologies for the creation and selection of stable forms of agricultural plants taking into account the controlled modification of their genome is discussed. A possible mechanism for incorporation of nonspecific inclusions in a nuclear compartment is proposed.

Ultrastructural organization of the domains in the cell nucleus of dicotyledonous and monocotyledonous plants under abiotic stress

Liquid–liquid phase separation (LLPS) causes the formation of membraneless condensates, which play important roles in diverse cellular processes. Currently, optical microscopy is the most commonly used method to visualize micron-scale phase-separated condensates. Because the optical spatial resolution is restricted by the diffraction limit (~200 nm), dynamic formation processes from individual biomolecules to micron-scale condensates are still mostly unknown. Herein, we provide a detailed protocol applying dual-color fluorescence cross-correlation spectroscopy (dcFCCS) to detect and quantify condensates at the nanoscale, including their size, growth rate, molecular stoichiometry, and the binding affinity of client molecules within condensates. We expect that the quantitative dcFCCS method can be widely applied to investigate many other important phase separation systems.

Quantifying phase separation at the nanoscale by dual-color fluorescence cross-correlation spectroscopy (dcFCCS)

Nucleoli form around RNA polymerase I transcribed ribosomal RNA (rRNA) genes. The direct electron microscopy observation of rRNA genes after nucleolar chromatin spreading (Miller's spreads) constitutes to date the only system to quantitatively assess transcription at a single molecule level. However, the spreading procedure is likely generating artifact and despite being informative, these spread rRNA genes are far from their in vivo situation. The integration of the structural characterization of spread rRNA genes in the three-dimensional (3D) organization of the nucleolus would represent an important scientific achievement. Here, we describe a correlative light and electron microscopy (CLEM) protocol allowing detection of tagged-Pol I by fluorescent microscopy and high-resolution imaging of the nucleolar ultrastructural context. This protocol can be implemented in laboratories equipped with conventional fluorescence and electron microscopes and does not require sophisticated "pipeline" for imaging.

Maxime Berthaud

Papers

Correlative Light and Electron Microscopy of Nucleolar Transcription in Saccharomyces cerevisiae