State Letter

(Summary by Simone Alessio Del Grosso)

Fernando Muzzopappa,Maud Hertzog, and Fabian Erdel

TL;DR: Generally, shorter DNA molecules (≤1kb) form dynamic, liquid-like round shaped condensates exhibiting faster exchange with their dilute surrounding than longer DNA molecules (≥10 kb) which form immobile, solid-like irregularly shaped condensates. The tested conditions suggest an intrinsic tendency for DNA to undergo liquid-liquid phase separation, even in the absence of intrinsically disordered proteins.

DNA compaction and regulated storage is a necessity in living organisms, achieved by condensing and packaging the genomic DNA. Among a plethora of cellular components and processes, liquid-liquid phase separation (LLPS) has been suggested to be a driving force for genome organization. However, it has remained largely unclear how DNA length influences the condensate’s material properties such as fluidity and morphology. In this matter, Muzzopappa et al. set out to systematically investigate DNA condensates using labeled lambda-DNA of different lengths using an integration of confocal fluorescence microscopy, fluorescence recovery after photobleaching (FRAP) and single molecule total internal reflections fluorescence microscopy (TIRF). DNA phase separation was triggered in three different ways, either by polyethylene-glycol and Mg2+ ions (PSI), or by addition of the intrinsically disordered protein linker histone H1 or through nucleosome reconstitution coupled to H1 addition.

They first show that morphology of condensates depends both on DNA length and DNA concentration. Longer DNA fragments (≥10 kb) formed bigger, irregularly shaped condensates at higher concentration (10-100 ug/ml) and smaller, either round or irregular condensates at lower concentration (10-100 ng/ml). Shorter DNA molecules (≤1kb) only formed visible condensates at higher concentrations (1-100ug/ml). This was seen for all three kinds of condensation conditions. Additionally, they showed that upon digestion of condensates consisting of longer DNA molecules, the shape shifts towards a more roundish one. In a second step, they characterized the dynamics of said condensates and saw, again irrespective of condensation condition, that irregularly shaped condensates consisting of longer DNA molecules showed little fluorescence recovery after bleaching, pointing towards very limited exchange between the condensate and its dilute surrounding. Recovery after partial internal photobleaching was also limited, indicating a solid-like material state for such condensations. For round shaped condensates two behaviors were observed: longer DNA molecules exhibit slow dynamics (slow exchange), whereas short ones show fast dynamics. However, these dynamics of short DNA molecules were shown to slow down after addition of longer (unlabeled) DNA molecules. In a last characterization, the authors demonstrated increased stability of longer DNA molecules towards shear stresses than shorter ones in condensates prepared via PSI. Overall, the transition between liquid- and solid-like seems to be gradual, as condensates consisting of DNA molecules of roughly 1-10 kb show intermediate properties.

The authors draw parallels between their found results and congruent previous literature and discuss their findings in potential scenarios in the context of morphology and dynamics of chromatin domains.

Link to paper

Fabian Erdel is a group leader at the Center for Integrative Biology at Toulouse (France).

(Summary by Dr. Wei Chen)

Anas Malki, Jean-Marie Teulon, Aldo R. Camacho-Zarco, Shu-wen W. Chen, Wiktor Adamski, Damien Maurin, Nicola Salvi, Jean-Luc Pellequer, and Martin Blackledge

TL;DR: The conformational and physical properties of the disordered protein CAHS-8, which is important for tardigrade survival in extreme conditions, is characterized by NMR, SAXS, AFM, and DLS. A model of how protein gelation protects tardigrade from desiccation is proposed.

Tardigrades have remarkable abilities to survive extreme conditions including high temperature, high pressure, radiation, and absence of oxygen or water. Although the molecular mechanism is still unknown, two families of proteins have been identified to be important for the protection against extreme stress—cytosolic abundant heat-soluble (CAHS) proteins and secreted abundant heat soluble (SAHS) proteins, both intrinsically disordered and only found in tardigrades. To understand how CAHS proteins respond to stress, the authors used NMR to characterize the conformation and small-angle x-ray scattering (SAXS), atomic force microscope (AFM), and dynamic light scattering (DLS) to follow the formation of oligomer, fibril, and gel.

NMR revealed that CAHS-8, a protein with 227 residues, is comprised of a central helical domain in residues 95 to 195 flanked with disordered regions at both ends. Chemical shifts, ¹⁵N relaxation, and residual dipolar coupling showed that the helical domain is divided into two regions, α₁ and α₂, connected by a more flexible linker. Absence of paramagnetic relaxation enhancement with labels either in the helical region or in the C-terminal disordered region indicated that there are no long-range interactions to support a tertiary structure.

SAXS showed that CAHS-8 exhibited transitions from monomers to oligomers to fibril as the concentration increased. The authors used DLS to map the phase diagram of CAHS-8 by measuring the decay of autocorrelation of scattering (contains information of diffusion coefficient, and therefore information of the particle size) as a function of temperature and concentration. In addition, a hydrogel phase was observed at low temperature and high concentration.

The phase transition of CAHS-8 into fibril and further into gel-like mesh was observed by AFM. NMR analyses on the CAHS-8 hydrogel showed the disordered regions remain flexible in gel as their resonances remained in the ¹H-¹⁵N HSQC spectrum, while the resonances for helix α₁ disappeared, suggesting the involvement of α₁ in the formation of fibrous gel. Comparison of ¹⁵N relaxation between CAHS-8 monomer and gel showed higher relaxation rates for some disordered regions and helix α₂ in the gel state, suggesting these regions are tethered to larger immobile objects. NMR analysis of other proteins incorporated into the CAHS hydrogel showed no conformational differences from their solution form.

The authors proposed a model in which CAHS-8 utilized the highly conserved helix α₁ to form hydrogel in desiccated conditions (low temperature, high protein concentrations) to minimize water volume required in such state, and that other proteins can remain their native conformations to function in the gel environment, supporting tardigrade survival.

Link to paper

Martin Blackledge is a group leader at the Institute of Structural Biology, Grenoble (France).

(Summary by Dr. Sujit Basak)

Pu-Yun Shih, Yu-Lun Fang, Sahana Shankar, Sue-Ping Lee, Hsiao-Tang Hu, Hsin Chen, Ting-Fang Wang, Kuo-Chiang Hsia, and Yi-Ping Hsueh

TL;DR: The study demonstrates the relevance of condensate formation and zinc-induced phase transition to the synaptic distribution and function of ASD-linked proteins.

Autism spectrum disorders (ASD) associated genes participate in synapse remodeling and formation, in balancing excitatory and inhibitory neurotransmitter, protein synthesis in the neuronal cells, and in their activation and adherence. In recent times, synaptopathies have been explained through the formation of liquid-liquid phase separation (LLPS) at active zones and as reconstituted post-synaptic density in vitro. Interestingly, the conformation and dynamics of these LLPS candidate proteins, including epsin1, dynamin, synaptojanin, complexin, rabphilin-3A from presynapse and PSD-95, SynGAP, SHANK, HOMER, cytoplasmic GluN2B, Stargazin, and CAMKII from post-synapse reveal compelling information about protein-protein interaction in the multi-component condensates. The role of several genes that increase the susceptibility to such neurodevelopmental disorders, is still elusive, although more efforts are being undertaken in this direction. Additionally, these protein-protein interactions influenced by the presence of mono- and di-valent cations at synapse is another venue which are remained unexplored.

Cortactin-binding protein 2 (CTTNBP2) is also an ASD associated gene and is involved in dendritic spine formation and maintenance. Being a neuron-specific cytoskeleton-associated protein, it interacts with cortactin, F-actin and striatins. Shih et al. have shown that CTTNBP2 contains coiled-coil N-terminal and intrinsically disordered (IDR) C-terminal from sequence analysis. The IDR C-terminal undergoes LLPS to form self-assembled condensate, while zinc facilitates liquid-to-gel phase transition through its interaction with coiled-coil N-terminal. The absence of several zinc binding proteins in CTTNBP2 knockout mice, and the reduction in zinc concentration in low expressed COS1 cells indicates the importance of zinc in understanding the function of CTTNBP2. Although the wild-type (WT), ASD linked variants (mainly D570Y, M120I and R533*) exhibit differential behaviors in the formation of condensates through altered LLPS, they still respond to zinc in the same manner. As a postsynaptic protein, CTTBP2 also interacts with other synaptic proteins like SHANK3 to regulate various signaling pathways. Interestingly, this synaptic protein-protein interaction also triggers condensate formation similar to the self-assembled CTTNBP2 and shows liquid-to-gel phase transition in response to zinc through multimerization or clustering. Thus, this study explores the importance of CTTNBP2 in maintaining zinc concentration at post-synapse to retain synaptic responses.

Link to paper

Kuo-Chiang Hsia is an associate research fellow at the Institute of Molecular Biology Taipei (Taiwan, ROC).

Yi-Ping Hsueh is a distinguished research fellow at the Institute of Molecular Biology Taipei (Taiwan, ROC).

(Summary by Dr. Aritra Chowdhury)

Xiangze Zeng, Kiersten M. Ruff, and Rohit V. Pappu

TL;DR: Well-mixed poly-ampholytic IDP sequences samples two distinct classes of conformational ensembles, globule like and self-avoiding walk (SAW) chain like, and the co-existence of these two conformational ensembles can give encode an apparent Gaussian chain (GC) like behaviour.

Sequence ensemble relationships of IDPs are crucial to deciphering their dynamics and functions. In this regard, the concept of scaling exponents, a concept borrowed from polymer physics that describes the dimensions R of an IDP in terms of the scaling exponent ν and the degree of polymerization N by R∝N^ν, is widely used as a descriptor of IDP dimensions; the scaling exponent can assume four limiting values of 0.33, 0.5, 0.59 and 1, corresponding to a globule, a GC, a SAW and an extended rod, respectively. In this work, Zeng et al. shows how well-mixed poly-ampholytic IDP sequences can have an apparent scaling exponent of 0.5, encoded by simultaneous sampling of SAW chain and globule like conformers.

Dimensions of well mixed polyampholytic IDP sequences, that is sequences with a high fraction of charged residues (FCR), low net charge per residue (NCPR) and with minimal linear segregation of charges, as inferred from scaling exponent ν has been shown to be ~0.59 from simulations, while experiments on such sequences yielded ν values of 0.33 and 0.5 depending on whether the positive charged residue was arginine or lysine. In an attempt to reconcile these results, here the authors show using simulations that in well mixed polyampholytic sequences competing interactions, electrostatic attraction between the oppositely charged side chains and favourable hydration of charged side chains, simultaneously encode globular (ν~0.33) and SAW (ν~0.59) ensembles, respectively; and for a given sequence, the two classes of ensemble are equally populated at a given temperature. They further uncover sequence specific nuances; arginine vis-à-vis a lysine shows a preference for globular ensembles, while aspartate and not glutamate, encode preferences for metastable, necklace-like conformations. These sequence specific nuances can be explained from the specifics of side chain physical chemistry. The co-existence of SAW and globular ensembles reconciles the discrepant estimates of ν for such well mixed polyampholytes, from experiments and previous simulations.

These results provide new insights into polyampholytic IDP dimenisons; it is worth noting that most IDPs in the proteome have polyampholytic character. Furthemore; the results highlight the need for going beyond an apparent scaling exponent estimate to fully understand sequence ensemble relationships in IDPs.

Link to paper

Rohit V. Pappu is the Gene K. Beare Distinguished Professor of Engineering at the McKelvey School of Engineering, a Professor of Biomedical Engineering and the Director of Center for Science & Engineering of Living Systems at the Washington University in St. Louis (USA).