Condensed Chromatin Behaves like a Solid on the Mesoscale In Vitro and in Living Cells.

The association of nuclear DNA with histones to form chromatin is essential for temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of condensed chromatin in vitro and in vivo. Our in vitro studies demonstrate that self-association of nucleosomal arrays under a wide range of solution conditions produces supramolecular condensates in which the chromatin is physically constrained and solid-like. By measuring DNA mobility in living cells, we show that condensed chromatin also exhibits solid-like behavior in vivo. Representative heterochromatin proteins, however, display liquid-like behavior and coalesce around the solid chromatin scaffold. Importantly, euchromatin and heterochromatin show solid-like behavior even under conditions that produce limited interactions between chromatin fibers. Our results reveal that condensed chromatin exists in a solid-like state whose properties resist external forces and create an elastic gel and provides a scaffold that supports liquid-liquid phase separation of chromatin binding proteins.

Cargo-mediated recruitment of the endocytic adaptor protein Sla1 in S. cerevisiae.

Endocytosis of plasma membrane proteins is mediated by their interaction with adaptor proteins. Conversely, emerging evidence suggests that adaptor protein recruitment to the plasma membrane may depend on binding to endocytic cargo. To test this idea, we analyzed the yeast adaptor protein Sla1, which binds membrane proteins harboring the endocytic signal NPFxD via the Sla1 SHD1 domain. Consistently, SHD1 domain point mutations that disrupted NPFxD binding caused a proportional reduction in Sla1-GFP recruitment to endocytic sites. Furthermore, simultaneous SHD1 domain point mutation and deletion of the C-terminal LxxQxTG repeat (SR) region linking Sla1 to coat proteins Pan1 and End3 resulted in total loss of Sla1-GFP recruitment to the plasma membrane. These data suggest that multiple interactions are needed for recruitment of Sla1 to the membrane. Interestingly, a Sla1 fragment containing just the third SH3 domain, which binds ubiquitin, and the SHD1 domain displayed broad surface localization, suggesting plasma membrane recruitment is mediated by interaction with both NPFxD-containing and ubiquitylated plasma membrane proteins. Our results also imply that a Sla1 NPF motif adjacent to the SR region might regulate the Sla1-cargo interaction, mechanistically linking Sla1 cargo binding to endocytic site recruitment.

Post-translational modifications and chromatin dynamics.

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.

The Sla1 adaptor-clathrin interaction regulates coat formation and progression of endocytosis.

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 (sla1AAA ) 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. sla1AAA 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 sla1AAA 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.

A second Las17 monomeric actin-binding motif functions in Arp2/3-dependent actin polymerization during endocytosis.

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.