The DNA Damage Response: Making It Safe to Play with Knives

In addition to membrane-bound organelles, eukaryotic cells feature various membraneless compartments, including the centrosome, the nucleolus and various granules. Many of these compartments form through liquid–liquid phase separation, and the principles, mechanisms and regulation of their assembly as well as their cellular functions are now beginning to emerge. Biomolecular condensates are micron-scale compartments in eukaryotic cells that lack surrounding membranes but function to concentrate proteins and nucleic acids. These condensates are involved in diverse processes, including RNA metabolism, ribosome biogenesis, the DNA damage response and signal transduction. Recent studies have shown that liquid–liquid phase separation driven by multivalent macromolecular interactions is an important organizing principle for biomolecular condensates. With this physical framework, it is now possible to explain how the assembly, composition, physical properties and biochemical and cellular functions of these important structures are regulated.

Biomolecular condensates: organizers of cellular biochemistry

I. the origin of species by means of natural selection

基礎講座 電気泳動(Electrophoresis)

BACKGROUND Living cells contain distinct subcompartments to facilitate spatiotemporal regulation of biological reactions. In addition to canonical membrane-bound organelles such as secretory vesicles and endoplasmic reticulum, there are many organelles that do not have an enclosing membrane yet remain coherent structures that can compartmentalize and concentrate specific sets of molecules. Examples include assemblies in the nucleus such as the nucleolus, Cajal bodies, and nuclear speckles and also cytoplasmic structures such as stress granules, P-bodies, and germ granules. These structures play diverse roles in various biological processes and are also increasingly implicated in protein aggregation diseases. ADVANCES A number of studies have shown that membrane-less assemblies exhibit remarkable liquid-like features. As with conventional liquids, they typically adopt round morphologies and coalesce into a single droplet upon contact with one another and also wet intracellular surfaces such as the nuclear envelope. Moreover, component molecules exhibit dynamic exchange with the surrounding nucleoplasm and cytoplasm. These findings together suggest that these structures represent liquid-phase condensates, which form via a biologically regulated (liquid-liquid) phase separation process. Liquid phase condensation increasingly appears to be a fundamental mechanism for organizing intracellular space. Consistent with this concept, several membrane-less organelles have been shown to exhibit a concentration threshold for assembly, a hallmark of phase separation. At the molecular level, weak, transient interactions between molecules with multivalent domains or intrinsically disordered regions (IDRs) are a driving force for phase separation. In cells, condensation of liquid-phase assemblies can be regulated by active processes, including transcription and various posttranslational modifications. The simplest physical picture of a homogeneous liquid phase is often not enough to capture the full complexity of intracellular condensates, which frequently exhibit heterogeneous multilayered structures with partially solid-like characters. However, recent studies have shown that multiple distinct liquid phases can coexist and give rise to richly structured droplet architectures determined by the relative liquid surface tensions. Moreover, solid-like phases can emerge from metastable liquid condensates via multiple routes of potentially both kinetic and thermodynamic origins, which has important implications for the role of intracellular liquids in protein aggregation pathologies. OUTLOOK The list of intracellular assemblies driven by liquid phase condensation is growing rapidly, but our understanding of their sequence-encoded biological function and dysfunction lags behind. Moreover, unlike equilibrium phases of nonliving matter, living cells are far from equilibrium, with intracellular condensates subject to various posttranslational regulation and other adenosine triphosphate–dependent biological activity. Efforts using in vitro reconstitution, combined with traditional cell biology approaches and quantitative biophysical tools, are required to elucidate how such nonequilibrium features of living cells control intracellular phase behavior. The functional consequences of forming liquid condensates are likely multifaceted and may include facilitated reaction, sequestration of specific factors, and organization of associated intracellular structures. Liquid phase condensation is particularly interesting in the nucleus, given the growing interest in the impact of nuclear phase behavior on the flow of genetic information; nuclear condensates range from micrometer-sized bodies such as the nucleolus to submicrometer structures such as transcriptional assemblies, all of which directly interact with and regulate the genome. Deepening our understanding of these intracellular states of matter not only will shed light on the basic biology of cellular organization but also may enable therapeutic intervention in protein aggregation disease by targeting intracellular phase behavior.

Liquid phase condensation in cell physiology and disease.

https://www.zora.uzh.ch/id/eprint/206236/1/1-s2.0-S156878642100135X-main.pdf

Biomolecular condensates at sites of DNA damage: More than just a phase

Formation of poly(ADP-ribose) (PAR) marks intracellular stress signaling and is notably induced upon DNA damage. PAR polymerases (PARPs) catalyze PAR synthesis upon genotoxic stress and thereby recruit multiple proteins to damaged chromatin. PAR induction is transient and antagonized by the action of PAR glycohydrolase (PARG). Given that poly(ADP-ribosyl)ation (PARylation) is involved in genome integrity maintenance and other vital cellular functions, but also in light of the recent approval of PARP inhibitors for cancer treatments, reliable measurements of intracellular PAR formation have gained importance. Here we provide a detailed protocol for PAR measurements by quantitative image-based cytometry. This technique combines the high spatial resolution of single-cell microscopy with the advantages of cell population measurements through automated high-content imaging. Such upscaling of immunofluorescence-based PAR detection not only increases the robustness of the measurements through averaging across large cell populations but also allows for the discrimination of subpopulations and thus enables multivariate measurements of PAR levels and DNA damage signaling. We illustrate how this technique can be used to assess the dynamics of the cellular response to oxidative damage as well as to PARP inhibitor-induced genotoxicity in a cell cycle resolved manner. Due to the possibility to use any automated microscope for quantitative image-based cytometry, the presented method has widespread applicability in the area of PARP biology and beyond.

Cell Cycle Resolved Measurements of Poly(ADP-Ribose) Formation and DNA Damage Signaling by Quantitative Image-Based Cytometry

We measured for the first time the U-turn trajectory of individual magneto-tactic bacteria (MTB) under reversal of the magnetic field, as a function of the field strength. The measurement was performed using shallow 5 micrometer deep microfluidic channels and a setup which allowed for magnetic fields with rapid alternating directions at varying magnitudes up to 60 mT.

Magnetic manipulation of bacteria in microfluidics

System and method for for superheating and/or supercooling of liquids and use of the system and/or method, wherein the liquid is within a capillary tube, wherein there is at least one heating and/or cooling means for heating the liquid above boiling point of the liquid at ambient pressure or cooling the liquid below freezing point of the liquid at ambient pressure, wherein the at least one heating and/or cooling means is in thermal contact with the capillary tube in an area, in which there is liquid inside the capillary tube, and wherein the capillary tube is scratch-free at its inner surface.

System and method for superheating and/or supercooling of liquids and use of the system and/or method

A posttranslational protein modification with increasing relevance for cancer biology is poly(ADP-ribosyl)ation (PARylation) (1). Inhibitors of PARylation show promising effects either as monotherapeutic agents or as chemo- or radiosensitizers to support classical DNA damaging cancer therapies. The targets of these inhibitors are enzymes of the family of poly(ADP-ribose) polymerases (PARPs, also known as ARTDs) (2). PARP inhibitors are analogs of NAD+, which PARP enzymes use as substrate to synthesize poly(ADP-ribose) (PAR). PAR can be covalently attached to or interact non-covalently with target proteins (3,4). In humans, 17 genes encode for PARP/ARTD enzymes, of which 5 can generate PAR (PARP1/2/4 and TNKS1/2), while all others either catalyze mono-ADP-ribosylation (MARylation) or appear to be inactive. PARPs localize to various cellular compartments and participate in a multitude of cancer-relevant cellular functions, such as DNA damage responses, transcription, chromatin organization and regulation of cell death (1).

https://tcr.amegroups.com/article/viewFile/10422/pdf

Matthias Altmeyer

Papers

Biomolecular condensates at sites of DNA damage: More than just a phase

Cell Cycle Resolved Measurements of Poly(ADP-Ribose) Formation and DNA Damage Signaling by Quantitative Image-Based Cytometry

Magnetic manipulation of bacteria in microfluidics

System and method for superheating and/or supercooling of liquids and use of the system and/or method

Identifying ADP-ribosylation targets by chemical genetics