No abstract

The association of nuclear DNA with histones to form chromatin is essential to the temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of chromatin in vitro and in vivo. Our in vitro studies demonstrate that MgCl2-dependent self-association of native chromatin fragments or reconstituted nucleosomal arrays produced supramolecular condensates whose constituents are physically constrained and solid-like. Liquid chromatin condensates could be generated in vitro, but only using non-physiological conditions. By measuring DNA mobility within heterochromatin and euchromatin in living cells, we show that chromatin also exhibits solid-like behavior in vivo. Representative heterochromatin proteins, however, displayed liquid-like behavior and coalesced around a solid chromatin scaffold. Remarkably, both euchromatin and heterochromatin showed solid-like behavior even when transmission electron microscopy revealed limited interactions between chromatin fibers. Our results therefore argue that chromatin is not liquid but exists in a solid-like material state whose properties are tuned by fiber-fiber interactions. INTRODUCTION:

The dynamic structure of chromatin is linked to gene regulation and many other biological functions. Consequently, it is of importance to understand the factors that regulate chromatin dynamics. While the in vivo analysis of chromatin has verified that histone post-translational modifications play a role in modulating DNA accessibility, the complex nuclear environment and multiplicity of modifications prevents clear conclusions as to how individual modifications influence chromatin dynamics in the cell. For this reason, in vitro analyses of model reconstituted nucleosomal arrays has been pivotal in understanding the dynamic nature of chromatin compaction and the affects that specific post-translational modifications can have on the higher order chromatin structure. In this mini-review, we briefly describe the dynamic chromatin structures that have been observed in vitro and the environmental conditions that give rise to these various conformational states. Our focus then turns to a discussion of the specific histone post-translational modifications that have been shown to alter formation of these higher order chromatin structures in vitro and how this may relate to the biological state and accessibility of chromatin in vivo.

During clathrin-mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott–Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G-actin) and a central-acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (Las17) is the strongest of five factors promoting Arp2/3-dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G-actin-binding motif in Las17. Here, we describe the in vitro and in vivo characterization of such Las17 G-actin-binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two-hybrid system, GST-pulldown, fluorescence polarization and pyrene-actin polymerization assays, we show that LGM binds G-actin and is necessary for normal Arp2/3-mediated actin polymerization in vitro. Live-cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known Las17 G-actin-binding motif, WH2. These results establish a second G-actin-binding motif in Las17 and advance our knowledge on the mechanism of actin assembly during CME.

Clathrin-mediated endocytosis is a fundamental transport pathway that depends on numerous protein-protein interactions. Testing the importance of the adaptor protein-clathrin interaction for coat formation and progression of endocytosis in vivo has been difficult due to experimental constrains. Here, we addressed this question using the yeast clathrin adaptor Sla1, which is unique in showing a cargo endocytosis defect upon substitution of 3 amino acids in its clathrin-binding motif (sla1 ) that disrupt clathrin binding. Live-cell imaging showed an impaired Sla1-clathrin interaction causes reduced clathrin levels but increased Sla1 levels at endocytic sites. Moreover, the rate of Sla1 recruitment was reduced indicating proper dynamics of both clathrin and Sla1 depend on their interaction. sla1 cells showed a delay in progression through the various stages of endocytosis. The Arp2/3-dependent actin polymerization machinery was present for significantly longer time before actin polymerization ensued, revealing a link between coat formation and activation of actin polymerization. Ultimately, in sla1 cells a larger than normal actin network was formed, dramatically higher levels of various machinery proteins other than clathrin were recruited, and the membrane profile of endocytic invaginations was longer. Thus, the Sla1-clathrin interaction is important for coat formation, regulation of endocytic progression and membrane bending.