The nucleus of eukaryotes is organized into functional compartments, the two most prominent being heterochromatin and nucleoli. These structures are highly enriched in DNA, proteins or RNA, and thus thought to be crowded. In vitro, molecular crowding induces volume exclusion, hinders diffusion and enhances association, but whether these effects are relevant in vivo remains unclear. Here, we establish that volume exclusion and diffusive hindrance occur in dense nuclear compartments by probing the diffusive behaviour of inert fluorescent tracers in living cells. We also demonstrate that chromatin-interacting proteins remain transiently trapped in heterochromatin due to crowding induced enhanced affinity. The kinetic signatures of these crowding consequences allow us to derive a fractal model of chromatin organization, which explains why the dynamics of soluble nuclear proteins are affected independently of their size. This model further shows that the fractal architecture differs between heterochromatin and euchromatin, and predicts that chromatin proteins use different target-search strategies in the two compartments. We propose that fractal crowding is a fundamental principle of nuclear organization, particularly of heterochromatin maintenance.

Mammalian genomes contain several billion base pairs of DNA that are packaged in chromatin fibres. At selected gene loci, cohesin complexes have been proposed to arrange these fibres into higher-order structures, but how important this function is for determining overall chromosome architecture and how the process is regulated are not well understood. Using conditional mutagenesis in the mouse, here we show that depletion of the cohesin-associated protein Wapl stably locks cohesin on DNA, leads to clustering of cohesin in axial structures, and causes chromatin condensation in interphase chromosomes. These findings reveal that the stability of cohesin-DNA interactions is an important determinant of chromatin structure, and indicate that cohesin has an architectural role in interphase chromosome territories. Furthermore, we show that regulation of cohesin-DNA interactions by Wapl is important for embryonic development, expression of genes such as c-myc (also known as Myc), and cell cycle progression. In mitosis, Wapl-mediated release of cohesin from DNA is essential for proper chromosome segregation and protects cohesin from cleavage by the protease separase, thus enabling mitotic exit in the presence of functional cohesin complexes.

The organization and biophysical properties of the cytosol implicitly govern molecular interactions within cells. However, little is known about mechanisms by which cells regulate cytosolic properties and intracellular diffusion rates. Here, we demonstrate that the intracellular environment of budding yeast undertakes a startling transition upon glucose starvation in which macromolecular mobility is dramatically restricted, reducing the movement of both chromatin in the nucleus and mRNPs in the cytoplasm. This confinement cannot be explained by an ATP decrease or the physiological drop in intracellular pH. Rather, our results suggest that the regulation of diffusional mobility is induced by a reduction in cell volume and subsequent increase in molecular crowding which severely alters the biophysical properties of the intracellular environment. A similar response can be observed in fission yeast and bacteria. This reveals a novel mechanism by which cells globally alter their properties to establish a unique homeostasis during starvation.DOI: http://dx.doi.org/10.7554/eLife.09376.001

The GTPase Rab27A interacts with myosin-VIIa and myosin-Va via MyRIP or melanophilin and mediates melanosome binding to actin. Here we show that Rab27A and MyRIP are associated with secretory granules (SGs) in adrenal chromaffin cells and PC12 cells. Overexpression of Rab27A, GTPase-deficient Rab27A-Q78L, or MyRIP reduced secretory responses of PC12 cells. Amperometric recordings of single adrenal chromaffin cells revealed that Rab27A-Q78L and MyRIP reduced the sustained component of release. Moreover, these effects on secretion were partly suppressed by the actin-depolymerizing drug latrunculin but strengthened by jasplakinolide, which stabilizes the actin cortex. Finally, MyRIP and Rab27A-Q78L restricted the motion of SGs in the subplasmalemmal region of PC12 cells, as measured by evanescent-wave fluorescence microscopy. In contrast, the Rab27A-binding domain of MyRIP and a MyRIP construct that interacts with myosin-Va but not with actin increased the mobility of SGs. We propose that Rab27A and MyRIP link SGs to F-actin and control their motion toward release sites through the actin cortex.

Analysis of trajectories of dynamical biological objects, such as breeding ants or cell organelles, is essential to reveal the interactions they develop with their environments. Many previous works used a global characterization based on parameters calculated for entire trajectories. In cases where transient behavior was detected, this usually concerned only a particular type, such as confinement or directed motion. However, these approaches are not appropriate in situations in which the tracked objects may display many different types of transient motion. We have developed a method to exhaustively analyze different kinds of transient behavior that the tracked objects may exhibit. The method discriminates stalled periods, constrained and directed motions from random dynamics by evaluating the diffusion coefficient, the mean-square displacement curvature, and the trajectory asymmetry along individual trajectories. To detect transient motions of various durations, these parameters are calculated along trajectories using a rolling analysis window whose width is variable. The method was applied to the study of secretory vesicle dynamics in the subplasmalemmal region of human carcinoid BON cells. Analysis of transitions between transient motion periods, combined with plausible assumptions about the origin of each motion type, leads to a model of dynamical subplasmalemmal organization.

PARP1 and its effector, the ATP-dependent chromatin remodeler Alc1/Chd1L, are identified as key players during the rapid chromatin relaxation at DNA damage sites.

Ensconsin cooperates with its binding partner, Kinesin-1, during interphase to trigger centrosome separation, but it promotes microtubule polymerization independently of Kinesin-1 to control spindle length during mitosis.

Chromatin is a multiscale structure on which transcription, replication, recombination and repair of the genome occur. To fully understand any of these processes at the molecular level under physiological conditions, a clear picture of the polymorphic and dynamic organization of chromatin in the eukaryotic nucleus is required. Recent studies indicate that a fractal model of chromatin architecture is consistent with both the reaction-diffusion properties of chromatin interacting proteins and with structural data on chromatin interminglement. In this study, we provide a critical overview of the experimental evidence that support a fractal organization of chromatin. On this basis, we discuss the functional implications of a fractal chromatin model for biological processes and propose future experiments to probe chromatin organization further that should allow to strongly support or invalidate the fractal hypothesis.