Organismal evolution of the 3D genome: The conformation of chromosomes within the nucleus can reflect a cell's type or state. However, studies of the conservation and evolutionary history of the mechanisms regulating genome structure across species are lacking. Hoencamp et al. mapped three-dimensional (3D) genome organization in 24 eukaryote species, including animals, fungi, and plants. At interphase, species' telomeres and centromeres either clustered across chromosomes or oriented in a polarized state maintaining individual chromosomal territories within the cell, a difference attributed to condensin II. An experimental loss of condensin II in human cells promotes the formation of centromere clusters but has no effect on loop or compartment formation. Whether the structure of the 3D genome varies across species may thus depend on whether they carry a functional condensin II gene. Science , abe2218, this issue p. 984

Many genetic processes depend on proteins interacting with specific sequences on DNA. Despite the large excess of nonspecific DNA in the cell, proteins can locate their targets rapidly. After initial nonspecific binding, they are believed to find the target site by 1D diffusion (“sliding”) interspersed by 3D dissociation/reassociation, a process usually referred to as facilitated diffusion. The 3D events combine short intrasegmental “hops” along the DNA contour, intersegmental “jumps” between nearby DNA segments, and longer volume “excursions.” The impact of DNA conformation on the search pathway is, however, still unknown. Here, we show direct evidence that DNA coiling influences the specific association rate of EcoRV restriction enzymes. Using optical tweezers together with a fast buffer exchange system, we obtained association times of EcoRV on single DNA molecules as a function of DNA extension, separating intersegmental jumping from other search pathways. Depending on salt concentration, targeting rates almost double when the DNA conformation is changed from fully extended to a coiled configuration. Quantitative analysis by an extended facilitated diffusion model reveals that only a fraction of enzymes are ready to bind to DNA. Generalizing our results to the crowded environment of the cell we predict a major impact of intersegmental jumps on target localization speed on DNA.

When DNA-binding proteins search for their specific binding site on a DNA molecule they alternate between linear 1-dimensional diffusion along the DNA molecule, mediated by nonspecific binding, and 3-dimensional volume excursion events between successive dissociation from and rebinding to DNA. If the DNA molecule is kept in a straight configuration, for instance, by optical tweezers, these 3-dimensional excursions may be divided into long volume excursions and short hops along the DNA. These short hops correspond to immediate rebindings after dissociation such that a rebinding event to the DNA occurs at a site that is close to the site of the preceding dissociation. When the DNA molecule is allowed to coil up, immediate rebinding may also lead to so-called intersegmental jumps, i.e., immediate rebindings to a DNA segment that is far away from the unbinding site when measured in the chemical distance along the DNA, but close by in the embedding 3-dimensional space. This effect is made possible by DNA looping. The significance of intersegmental jumps was recently demonstrated in a single DNA optical tweezers setup. Here we present a theoretical approach in which we explicitly take the effect of DNA coiling into account. By including the spatial correlations of the short hops we demonstrate how the facilitated diffusion model can be extended to account for intersegmental jumping at varying DNA densities. It is also shown that our approach provides a quantitative interpretation of the experimentally measured enhancement of the target location by DNA-binding proteins.

Clathrin lattices at the plasma membrane coat both invaginated and flat regions forming clathrin-coated pits and clathrin plaques, respectively. The function and regulation of clathrin-coated pits in endocytosis are well understood but clathrin plaques remain enigmatic nanodomains. Here we use super-resolution microscopy, molecular genetics and cell biology to show that clathrin plaques contain the machinery for clathrin-mediated endocytosis and cell adhesion, and associate with both clathrin-coated pits and filamentous actin. We also find that actin polymerization promoted by N-WASP through the Arp2/3 complex is crucial for the regulation of plaques but not pits. Clathrin plaques oppose cell migration and undergo actin- and N-WASP-dependent disassembly upon activation of LPA receptor 1, but not EGF receptor. Most importantly, plaque disassembly correlates with the endocytosis of LPA receptor 1 and down-modulation of AKT activity. Thus, clathrin plaques serve as dynamic actin-controlled hubs for clathrin-mediated endocytosis and signalling that exhibit receptor specificity.

A photodegradable, covalently crosslinked hydrogel system has been constructed from the biocompatible polymers dextran and poly(ethylene glycol) using the acrylate–thiol Michael addition as the crosslinking method. Light sensitivity of the hydrogel was introduced by placing a non-toxic photolabile o-nitrobenzyl moiety in between dextran backbone and acrylate group. Hydrogels were prepared under physiological conditions without the need of any additional reagents by mixing solutions of dextran functionalized with acrylate-modified o-nitrobenzyl moieties (Dex-AN) and dithiolated poly(ethylene glycol) (DSPEG). The degradation of the hydrogels due to UV irradiation was investigated with scanning electron microscopy, infrared and UV-vis spectroscopy. Using green fluorescent protein (GFP) as a model protein, light triggered protein release from the obtained gel matrices was investigated in different forms. Furthermore, photodegradation of the hydrogel via two photon excitation was also examined using focused pulsed near infrared (NIR) laser beam as a light source.