Determinants for Intrinsically Disordered Protein Recruitment into Phase-Separated Protein Condensates

Multivalent interactions between amino acid residues of intrinsically disordered proteins (IDPs) drive phase separation of these proteins into liquid condensates, forming various membrane-less organelles in cells. These interactions between often...

The loader complex Scc2/4 forms co-condensates with DNA as loading sites for cohesin

The genome is organized by diverse packaging mechanisms like nucleosome formation, loop extrusion and phase separation, which all compact DNA in a dynamic manner. Phase separation additionally drives protein recruitment to condensed DNA sites and thus regulates gene transcription. The cohesin complex is a key player in chromosomal organization that extrudes loops to connect distant regions of the genome and ensures sister chromatid cohesion after S-phase. For stable loading onto the DNA and for activation, cohesin requires the loading complex Scc2/4. As the precise loading mechanism remains unclear, we investigated whether phase separation might be the initializer of the cohesin recruitment process. We found that, in absence of cohesin, budding yeast Scc2/4 forms phase separated co-condensates with DNA, which comprise liquid-like properties shown by droplet shape, fusion ability and reversibility. We reveal in DNA curtain and optical tweezer experiments that these condensates are built by DNA bridging and bending through Scc2/4. Importantly, Scc2/4-mediated condensates recruit cohesin efficiently and increase the stability of the cohesin complex. We conclude that phase separation properties of Scc2/4 enhance cohesin loading by molecular crowding, which might then provide a starting point for the recruitment of additional factors and proteins.

Unidirectional recruitment relations between proteins can be determined by a CRISPR/dCas9 recruitment assay

Directional recruitment of protein complexes is critical for proper function of many nuclear processes. Here we present CRISPR-PITA (Protein Interaction and Telomere Recruitment Assay), an assay that determines the ability of a given protein to recruit any other nuclear factor. The protein of interest is directed via CRISPR/dCas9, a dead Cas9 that does not cut DNA, to a repeat sequence, such as telomeres, to obtain dots that are easily detectable by microscopy. The recruitment of endogenous nuclear proteins to these dots can then be visualized using specific antibodies. We determined recruitment abilities in CRISPR-PITA to methyl-CpG binding protein MeCP2, histone deacetylase 1 (HDAC1), heterochromatin protein 1 (HP1α), and the latency-associated nuclear antigen (LANA) encoded by Kaposi’s sarcoma associated herpesvirus (KSHV, HHV-8). LANA was able to recruit its known interactors ORC2 and SIN3A to LANA-telomere dots. In contrast, LANA was unable to recruit MeCP2 whereas MeCP2 was able to recruit LANA. Similarly, HDAC1 that interacts with MeCP2 through the transcriptional-repression domain (TRD) same as LANA, was unable to recruit MeCP2, but MeCP2 recruited HDAC1. One important function of LANA is to tether the viral episomal genomes to the cellular chromosomes during cell division. The unidirectional recruitment of LANA by MeCP2, makes MeCP2 a candidate anchor for KSHV genome tethering by LANA. We found that cells derived from Rett syndrome and express a mutant MeCP2 (T158M), impaired in DNA binding, cannot support KSHV genome maintenance. In summary, we describe a broadly applicable protein recruitment assay based on CRISPR/dCas9.Significance StatementCRISPR/Cas9 is a revolutionary system that has profoundly impacted biology research. Here we present another application for CRISPR/Cas9, in evaluating recruitment relations between nuclear proteins. A protein of interest is directed to a repeat sequence via the catalytically inactive Cas9 (dCas9) to generate easily detectable dots. Then, the recruitment of other nuclear proteins to these dots can be evaluated. Using this assay, we show that some interacting proteins have a unidirectional recruitment property, where only one of the proteins can recruit its partner. We propose that available interacting domains can force this unidirectional recruitment. Using this recruitment assay, we found unidirectional recruitment of the KSHV encoded LANA and HDAC1 by MeCP2. Furthermore, this unidirectional recruitment is critical for viral latency, since LANA fails to maintain the viral genomes in MeCP2 mutant cells.

Specific Engineered G Protein Coupling to Histamine Receptors Revealed from Cellular Assay Experiments and Accelerated Molecular Dynamics Simulations

G protein-coupled receptors (GPCRs) are targets of extracellular stimuli and hence occupy a key position in drug discovery. By specific and not yet fully elucidated coupling profiles with α subunits of distinct G protein families, they regulate cellular responses. The histamine H2 and H4 receptors (H2R and H4R) are prominent members of Gs- and Gi-coupled GPCRs. Nevertheless, promiscuous G protein and selective Gi signaling have been reported for the H2R and H4R, respectively, the molecular mechanism of which remained unclear. Using a combination of cellular experimental assays and Gaussian accelerated molecular dynamics (GaMD) simulations, we investigated the coupling profiles of the H2R and H4R to engineered mini-G proteins (mG). We obtained coupling profiles of the mGs, mGsi, or mGsq proteins to the H2R and H4R from the mini-G protein recruitment assays using HEK293T cells. Compared to H2R–mGs expressing cells, histamine responses were weaker (pEC50, Emax) for H2R–mGsi and –mGsq. By contrast, the H4R selectively bound to mGsi. Similarly, in all-atom GaMD simulations, we observed a preferential binding of H2R to mGs and H4R to mGsi revealed by the structural flexibility and free energy landscapes of the complexes. Although the mG α5 helices were consistently located within the HR binding cavity, alternative binding orientations were detected in the complexes. Due to the specific residue interactions, all mG α5 helices of the H2R complexes adopted the Gs-like orientation toward the receptor transmembrane (TM) 6 domain, whereas in H4R complexes, only mGsi was in the Gi-like orientation toward TM2, which was in agreement with Gs- and Gi-coupled GPCRs structures resolved by X-ray/cryo-EM. These cellular and molecular insights support (patho)physiological profiles of the histamine receptors, especially the hitherto little studied H2R function in the brain, as well as of the pharmacological potential of H4R selective drugs.

New Methodologies to Study DNA Repair Processes in Space and Time Within Living Cells

DNA repair requires a coordinated effort from an array of factors that play different roles in the DNA damage response from recognizing and signaling the presence of a break, creating a repair competent environment, and physically repairing the lesion. Due to the rapid nature of many of these events, live-cell microscopy has become an invaluable method to study this process. In this review we outline commonly used tools to induce DNA damage under the microscope and discuss spatio-temporal analysis tools that can bring added information regarding protein dynamics at sites of damage. In particular, we show how to go beyond the classical analysis of protein recruitment curves to be able to assess the dynamic association of the repair factors with the DNA lesions as well as the target-search strategies used to efficiently find these lesions. Finally, we discuss how the use of mathematical models, combined with experimental evidence, can be used to better interpret the complex dynamics of repair proteins at DNA lesions.

Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy v1

Lysophagy-the selective elimination of damaged lysosomes by the autophagy pathway-is a critical housekeeping mechanism in cells. This pathway surveils lysosomes and selectively demarcates terminally damaged lysosomes for elimination. Among the most upstream signaling proteins in this pathway are the glycan binding proteins-Galectins-which recognize N and O linked glycan chains on the luminal side of transmembrane lysosomal proteins. These glycosyl modifications are only accessible to galectin proteins upon extensive lysosomal membrane rupture and serve as a sensitive measure of lysosomal damage and eventual clearance by selective autophagy. Indeed, prior work has shown that immunofluorescence of Galectin-3 serves as a convenient proxy for lysophagic flux in tissue culture cells (Aits et al., 2015; Maejima et al., 2013). Here we describe a method for monitoring protein recruitment to damaged lysosomes via immunofluorescence and confocal imaging.

SGnn: A Web Server for the Prediction of Prion-Like Domains Recruitment to Stress Granules Upon Heat Stress

Proteins bearing prion-like domains (PrLDs) are essential players in stress granules (SG) assembly. Analysis of data on heat stress-induced recruitment of yeast PrLDs to SG suggests that this propensity might be connected with three defined protein biophysical features: aggregation propensity, net charge, and the presence of free cysteines. These three properties can be read directly in the PrLDs sequences, and their combination allows to predict protein recruitment to SG under heat stress. On this basis, we implemented SGnn, an online predictor of SG recruitment that exploits a feed-forward neural network for high accuracy classification of the assembly behavior of PrLDs. The simplicity and precision of our strategy should allow its implementation to identify heat stress-induced SG-forming proteins in complete proteomes.

ARF GTPases and Their Ubiquitous Role in Intracellular Trafficking Beyond the Golgi

Molecular switches of the ADP-ribosylation factor (ARF) GTPase family coordinate intracellular trafficking at all sorting stations along the secretory pathway, from the ER-Golgi-intermediate compartment (ERGIC) to the plasma membrane (PM). Their GDP-GTP switch is essential to trigger numerous processes, including membrane deformation, cargo sorting and recruitment of downstream coat proteins and effectors, such as lipid modifying enzymes. While ARFs (in particular ARF1) had mainly been studied in the context of coat protein recruitment at the Golgi, COPI/clathrin-independent roles have emerged in the last decade. Here we review the roles of human ARF1-5 GTPases in cellular trafficking with a particular emphasis on their roles in post-Golgi secretory trafficking and in sorting in the endo-lysosomal system.