Chromatin compartmentalization regulates the response to DNA damage.

The DNA damage response is essential to safeguard genome integrity. Although the contribution of chromatin in DNA repair has been investigated1,2, the contribution of chromosome folding to these processes remains unclear3. Here we report that, after the production of double-stranded breaks (DSBs) in mammalian cells, ATM drives the formation of a new chromatin compartment (D compartment) through the clustering of damaged topologically associating domains, decorated with γH2AX and 53BP1. This compartment forms by a mechanism that is consistent with polymer-polymer phase separation rather than liquid-liquid phase separation. The D compartment arises mostly in G1 phase, is independent of cohesin and is enhanced after pharmacological inhibition of DNA-dependent protein kinase (DNA-PK) or R-loop accumulation. Importantly, R-loop-enriched DNA-damage-responsive genes physically localize to the D compartment, and this contributes to their optimal activation, providing a function for DSB clustering in the DNA damage response. However, DSB-induced chromosome reorganization comes at the expense of an increased rate of translocations, also observed in cancer genomes. Overall, we characterize how DSB-induced compartmentalization orchestrates the DNA damage response and highlight the critical impact of chromosome architecture in genomic instability.

LEAFY homeostasis is regulated via ubiquitin-dependent degradation and sequestration in cytoplasmic condensates.

The transcription factor LEAFY (LFY) plays crucial roles in flower development by activating floral homeotic genes. Activation of LFY targets requires the combined action of LFY and the E3 ubiquitin ligase UFO, although the precise underlying mechanism remains unclear. Here, we show that LFY accumulates in biomolecular condensates within the cytoplasm, while recombinant LFY forms condensates with similar properties in vitro. UFO interacts with LFY within these condensates and marks it for degradation. LFY levels in the nucleus are buffered against changes in total LFY levels induced by proteasome inhibition, UFO overexpression, or mutation of lysine residues in a disordered region of LFY. Perturbation of cytoplasmic LFY condensates by 1,6-hexanediol treatment induces the relocalization of LFY to the nucleus and the subsequent activation of the LFY target AP3 in flowers. Our data suggest that nucleocytoplasmic partitioning, condensation, and ubiquitin-dependent degradation regulate LFY levels in the nucleus to control its activity.

Detecting and quantifying liquid-liquid phase separation in living cells by model-free calibrated half-bleaching.

Cells contain numerous substructures that have been proposed to form via liquid-liquid phase separation (LLPS). It is currently debated how to reliably distinguish LLPS from other mechanisms. Here, we benchmark different methods using well-controlled model systems in vitro and in living cells. We find that 1,6-hexanediol treatment and classical FRAP fail to distinguish LLPS from the alternative scenario of molecules binding to spatially clustered binding sites without phase-separating. In contrast, the preferential internal mixing seen in half-bleach experiments robustly distinguishes both mechanisms. We introduce a workflow termed model-free calibrated half-FRAP (MOCHA-FRAP) to probe the barrier at the condensate interface that is responsible for preferential internal mixing. We use it to study components of heterochromatin foci, nucleoli, stress granules and nuage granules, and show that the strength of the interfacial barrier increases in this order. We anticipate that MOCHA-FRAP will help uncover the mechanistic basis of biomolecular condensates in living cells.

Oligomerization processes limit photoactivation and recovery of the orange carotenoid protein.

The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection by quenching of the excess of light-harvested energy. The photoactivation mechanism remains elusive, in part due to absence of data pertaining to the timescales over which protein structural changes take place. It also remains unclear whether or not oligomerization of the dark-adapted and light-adapted OCP could play a role in the regulation of its energy-quenching activity. Here, we probed photoinduced structural changes in OCP by a combination of static and time-resolved X-ray scattering and steady-state and transient optical spectroscopy in the visible range. Our results suggest that oligomerization partakes in regulation of the OCP photocycle, with different oligomers slowing down the overall thermal recovery of the dark-adapted state of OCP. They furthermore reveal that upon non-photoproductive excitation a numbed state forms, which remains in a non-photoexcitable structural state for at least ≈0.5 µs after absorption of a first photon.

A kaleidoscope of photosynthetic antenna proteins and their emerging roles.

Photosynthetic light-harvesting antennae are pigment-binding proteins that perform one of the most fundamental tasks on Earth, capturing light and transferring energy that enables life in our biosphere. Adaptation to different light environments led to the evolution of an astonishing diversity of light-harvesting systems. At the same time, several strategies have been developed to optimize the light energy input into photosynthetic membranes in response to fluctuating conditions. The basic feature of these prompt responses is the dynamic nature of antenna complexes, whose function readily adapts to the light available. High-resolution microscopy and spectroscopic studies on membrane dynamics demonstrate the crosstalk between antennae and other thylakoid membrane components. With the increased understanding of light-harvesting mechanisms and their regulation, efforts are focusing on the development of sustainable processes for effective conversion of sunlight into functional bio-products. The major challenge in this approach lies in the application of fundamental discoveries in light-harvesting systems for the improvement of plant or algal photosynthesis. Here, we underline some of the latest fundamental discoveries on the molecular mechanisms and regulation of light harvesting that can potentially be exploited for the optimization of photosynthesis.

Elucidation of the essential amino acids involved in the binding of the cyanobacterial Orange Carotenoid Protein to the phycobilisome.

The Orange Carotenoid Protein (OCP) is a soluble photoactive protein involved in cyanobacterial photoprotection. It is formed by the N-terminal domain (NTD) and C-terminal (CTD) domain, which establish interactions in the orange inactive form and share a ketocarotenoid molecule. Upon exposure to intense blue light, the carotenoid molecule migrates into the NTD and the domains undergo separation. The free NTD can then interact with the phycobilisome (PBS), the extramembrane cyanobacterial antenna, and induces thermal dissipation of excess absorbed excitation energy. The OCP and PBS amino acids involved in their interactions remain undetermined. To identify the OCP amino acids essential for this interaction, we constructed several OCP mutants (23) with modified amino acids located on different NTD surfaces. We demonstrated that only the NTD surface that establishes interactions with the CTD in orange OCP is involved in the binding of OCP to PBS. All amino acids surrounding the carotenoid ß1 ring in the OCPR-NTD (L51, P56, G57, N104, I151, R155, N156) are important for binding OCP to PBS. Additionally, modification of the amino acids influences OCP photoactivation and/or recovery rates, indicating that they are also involved in the translocation of the carotenoid.

DNA length tunes the fluidity of DNA-based condensates.

Living organisms typically store their genomic DNA in a condensed form. Mechanistically, DNA condensation can be driven by macromolecular crowding, multivalent cations, or positively charged proteins. At low DNA concentration, condensation triggers the conformational change of individual DNA molecules into a compacted state, with distinct morphologies. Above a critical DNA concentration, condensation goes along with phase separation into a DNA-dilute and a DNA-dense phase. The latter DNA-dense phase can have different material properties and has been reported to be rather liquid-like or solid-like depending on the characteristics of the DNA and the solvent composition. Here, we systematically assess the influence of DNA length on the properties of the resulting condensates. We show that short DNA molecules with sizes below 1 kb can form dynamic liquid-like assemblies when condensation is triggered by polyethylene glycol and magnesium ions, binding of linker histone H1, or nucleosome reconstitution in combination with linker histone H1. With increasing DNA length, molecules preferentially condense into less dynamic more solid-like assemblies, with phage λ-DNA with 48.5 kb forming mostly solid-like assemblies under the conditions assessed here. The transition from liquid-like to solid-like condensates appears to be gradual, with DNA molecules of roughly 1-10 kb forming condensates with intermediate properties. Titration experiments with linker histone H1 suggest that the fluidity of condensates depends on the net number of attractive interactions established by each DNA molecule. We conclude that DNA molecules that are much shorter than a typical human gene are able to undergo liquid-liquid phase separation, whereas longer DNA molecules phase separate by default into rather solid-like condensates. We speculate that the local distribution of condensing factors can modulate the effective length of chromosomal domains in the cell. We anticipate that the link between DNA length and fluidity established here will improve our understanding of biomolecular condensates involving DNA.

Structural dynamics in the C terminal domain homolog of orange carotenoid Protein reveals residues critical for carotenoid uptake.

The structural features enabling carotenoid translocation between molecular entities in nature is poorly understood. Here, we present the three-dimensional X-ray structure of an expanded oligomeric state of the C-terminal domain homolog (CTDH) of the orange carotenoid protein, a key water-soluble protein in cyanobacterial photosynthetic photo-protection, at 2.9 Å resolution. This protein binds a canthaxanthin carotenoid ligand and undergoes structural reorganization at the dimeric level, which facilitates cargo uptake and delivery. The structure displays heterogeneity revealing the dynamic nature of its C-terminal tail (CTT). Molecular dynamics (MD) simulations based on the CTDH structures identified specific residues that govern the dimeric transition mechanism. Mutagenesis based on the crystal structure and these MD simulations then confirmed that these specific residues within the CTT are critical for carotenoid uptake, encapsulation and delivery processes. We present a mechanism that can be applied to other systems that require cargo uptake.

Changing Color for Photoprotection: The Orange Carotenoid Protein.

Under high irradiance, light becomes dangerous for photosynthetic organisms and they must protect themselves. Cyanobacteria have developed a simple mechanism, involving a photoactive soluble carotenoid protein, the orange carotenoid protein (OCP), which increases thermal dissipation of excess energy by interacting with the cyanobacterial antenna, the phycobilisome. Here, we summarize our knowledge of the OCP-related photoprotective mechanism, including the remarkable progress that has been achieved in recent years on OCP photoactivation and interaction with phycobilisomes, as well as with the fluorescence recovery protein, which is necessary to end photoprotection. A recently discovered unique mechanism of carotenoid transfer between soluble proteins related to OCP is also described.

Interdomain interactions reveal the molecular evolution of the orange carotenoid protein.

The photoactive orange carotenoid protein (OCP) is a blue-light intensity sensor involved in cyanobacterial photoprotection. Three OCP families co-exist (OCPX, OCP1 and OCP2), having originated from the fusion of ancestral domain genes. Here, we report the characterization of an OCPX and the evolutionary characterization of OCP paralogues focusing on the role of the linker connecting the domains. The addition of the linker with specific amino acids enabled the photocycle of the OCP ancestor. OCPX is the paralogue closest to this ancestor. A second diversification gave rise to OCP1 and OCP2. OCPX and OCP2 present fast deactivation and weak antenna interaction. In OCP1, OCP deactivation became slower and interaction with the antenna became stronger, requiring a further protein to detach OCP from the antenna and accelerate its deactivation. OCP2 lost the tendency to dimerize, unlike OCPX and OCP1, and the role of its linker is slightly different, giving less controlled photoactivation.

Different roles for ApcD and ApcF in Synechococcus elongatus and Synechocystis sp. PCC 6803 phycobilisomes.

The phycobilisome, the cyanobacterial light harvesting complex, is a huge phycobiliprotein containing extramembrane complex, formed by a core from which rods radiate. The phycobilisome has evolved to efficiently absorb sun energy and transfer it to the photosystems via the last energy acceptors of the phycobilisome, ApcD and ApcE. ApcF also affects energy transfer by interacting with ApcE. In this work we studied the role of ApcD and ApcF in energy transfer and state transitions in Synechococcus elongatus and Synechocystis PCC6803. Our results demonstrate that these proteins have different roles in both processes in the two strains. The lack of ApcD and ApcF inhibits state transitions in Synechocystis but not in S. elongatus. In addition, lack of ApcF decreases energy transfer to both photosystems only in Synechocystis, while the lack of ApcD alters energy transfer to photosystem I only in S. elongatus. Thus, conclusions based on results obtained in one cyanobacterial strain cannot be systematically transferred to other strains and the putative role(s) of phycobilisomes in state transitions need to be reconsidered.

Light-controlled carotenoid transfer between water-soluble proteins related to cyanobacterial photoprotection.

Carotenoids are lipophilic pigments with multiple biological functions from coloration to vision and photoprotection. Still, the number of water-soluble carotenoid-binding proteins described to date is limited, and carotenoid transport and carotenoprotein maturation processes are largely underexplored. Recent studies revealed that CTDHs, which are natural homologs of the C-terminal domain (CTD) of the orange carotenoid protein (OCP), a photoswitch involved in cyanobacterial photoprotection, are able to bind carotenoids, with absorption shifted far into the red region of the spectrum. Despite the recent discovery of their participation in carotenoid transfer processes, the functional roles of the diverse family of CTDHs are not well understood. Here, we characterized CTDH carotenoproteins from Anabaena variabilis (AnaCTDH) and Thermosynechococcus elongatus and examined their ability to participate in carotenoid transfer processes with a set of OCP-derived proteins. This revealed that carotenoid transfer occurs in several directions guided by different affinities for carotenoid and specific protein-protein interactions. We show that CTDHs have higher carotenoid affinity compared to the CTD of OCP from Synechocystis, which results in carotenoid translocation from the CTD into CTDH via a metastable heterodimer intermediate. Activation of OCP by light, or mutagenesis compromising the OCP structure, provides AnaCTDH with an opportunity to extract carotenoid from the full-length OCP, either from Synechocystis or Anabaena. These previously unknown reactions between water-soluble carotenoproteins demonstrate multidirectionality of carotenoid transfer, allowing for efficient and reversible control over the carotenoid-mediated protein oligomerization by light, which gives insights into the physiological regulation of OCP activity by CTDH and suggests multiple applications.

Photoactivation Mechanism, Timing of Protein Secondary Structure Dynamics and Carotenoid Translocation in the Orange Carotenoid Protein.

The orange carotenoid protein (OCP) is a two-domain photoactive protein that noncovalently binds an echinenone (ECN) carotenoid and mediates photoprotection in cyanobacteria. In the dark, OCP assumes an orange, inactive state known as OCPO; blue light illumination results in the red active state, known as OCPR. The OCPR state is characterized by large-scale structural changes that involve dissociation and separation of C-terminal and N-terminal domains accompanied by carotenoid translocation into the N-terminal domain. The mechanistic and dynamic-structural relations between photon absorption and formation of the OCPR state have remained largely unknown. Here, we employ a combination of time-resolved UV-visible and (polarized) mid-infrared spectroscopy to assess the electronic and structural dynamics of the carotenoid and the protein secondary structure, from femtoseconds to 0.5 ms. We identify a hereto unidentified carotenoid excited state in OCP, the so-called S* state, which we propose to play a key role in breaking conserved hydrogen-bond interactions between carotenoid and aromatic amino acids in the binding pocket. We arrive at a comprehensive reaction model where the hydrogen-bond rupture with conserved aromatic side chains at the carotenoid ß1-ring in picoseconds occurs at a low yield of <1%, whereby the ß1-ring retains a trans configuration with respect to the conjugated π-electron chain. This event initiates structural changes at the N-terminal domain in 1 µs, which allow the carotenoid to translocate into the N-terminal domain in 10 µs. We identified infrared signatures of helical elements that dock on the C-terminal domain ß-sheet in the dark and unfold in the light to allow domain separation. These helical elements do not move within the experimental range of 0.5 ms, indicating that domain separation occurs on longer time scales, lagging carotenoid translocation by at least 2 decades of time.

Structural rearrangements in the C-terminal domain homolog of Orange Carotenoid Protein are crucial for carotenoid transfer.

A recently reported family of soluble cyanobacterial carotenoproteins, homologs of the C-terminal domain (CTDH) of the photoprotective Orange Carotenoid Protein, is suggested to mediate carotenoid transfer from the thylakoid membrane to the Helical Carotenoid Proteins, which are paralogs of the N-terminal domain of the OCP. Here we present the three-dimensional structure of a carotenoid-free CTDH variant from Anabaena (Nostoc) PCC 7120. This CTDH contains a cysteine residue at position 103. Two dimer-forming interfaces were identified, one stabilized by a disulfide bond between monomers and the second between each monomer's ß-sheets, both compatible with small-angle X-ray scattering data and likely representing intermediates of carotenoid transfer processes. The crystal structure revealed a major positional change of the C-terminal tail. Further mutational analysis revealed the importance of the C-terminal tail in both carotenoid uptake and delivery. These results have allowed us to suggest a detailed model for carotenoid transfer via these soluble proteins.

Paralogs of the C-Terminal Domain of the Cyanobacterial Orange Carotenoid Protein Are Carotenoid Donors to Helical Carotenoid Proteins.

The photoactive Orange Carotenoid Protein (OCP) photoprotects cyanobacteria cells by quenching singlet oxygen and excess excitation energy. Its N-terminal domain is the active part of the protein, and the C-terminal domain regulates the activity. Recently, the characteristics of a family of soluble carotenoid-binding proteins (Helical Carotenoid Proteins [HCPs]), paralogs of the N-terminal domain of OCP, were described. Bioinformatics studies also revealed the existence of genes coding for homologs of CTD. Here, we show that the latter genes encode carotenoid proteins (CTDHs). This family of proteins contains two subgroups with distinct characteristics. One CTDH of each clade was further characterized, and they proved to be very good singlet oxygen quenchers. When synthesized in Escherichia coli or Synechocystis PCC 6803, CTDHs formed dimers that share a carotenoid molecule and are able to transfer their carotenoid to apo-HCPs and apo-OCP. The CTDHs from clade 2 have a cysteine in position 103. A disulfide bond is easily formed between the monomers of the dimer preventing carotenoid transfer. This suggests that the transfer of the carotenoid could be redox regulated in clade 2 CTDH. We also demonstrate here that apo-OCPs and apo-CTDHs are able to take the carotenoid directly from membranes, while HCPs are unable to do so. HCPs need the presence of CTDH to become holo-proteins. We propose that, in cyanobacteria, the CTDHs are carotenoid donors to HCPs.