Mechanism of Bloom syndrome complex assembly required for double Holliday junction dissolution and genome stability

The RecQ-like helicase BLM cooperates with topoisomerase IIIα, RMI1, and RMI2 in a heterotetrameric complex (the “Bloom syndrome complex”) for dissolution of double Holliday junctions, key intermediates in homologous recombination. Mutations in any component of the Bloom syndrome complex can cause genome instability and a highly cancer-prone disorder called Bloom syndrome. Some heterozygous carriers are also predisposed to breast cancer. To understand how the activities of BLM helicase and topoisomerase IIIα are coupled, we purified the active four-subunit complex. Chemical cross-linking and mass spectrometry revealed a unique architecture that links the helicase and topoisomerase domains. Using biochemical experiments, we demonstrated dimerization mediated by the N terminus of BLM with a 2:2:2:2 stoichiometry within the Bloom syndrome complex. We identified mutations that independently abrogate dimerization or association of BLM with RMI1, and we show that both are dysfunctional for dissolution using in vitro assays and cause genome instability and synthetic lethal interactions with GEN1/MUS81 in cells. Truncated BLM can also inhibit the activity of full-length BLM in mixed dimers, suggesting a putative mechanism of dominant-negative action in carriers of BLM truncation alleles. Our results identify critical molecular determinants of Bloom syndrome complex assembly required for double Holliday junction dissolution and maintenance of genome stability.

Homologous recombination (HR) is an essential and highly regulated cellular process required for maintenance of genome stability during mitosis and sexual reproduction. A key intermediate in HR and recombinational DNA repair is a four-way DNA junction known as a Holliday junction (HJ). During meiosis, double Holliday junctions (dHJs) are cut by endonucleases (resolution) in order to create the cross-overs necessary for proper chromosome segregation (1, 2). In somatic cells, however, cross-overs are potentially deleterious, and their formation is suppressed by the BLM protein (3). Failure to suppress cross-over formation leads to an increased frequency of sister chromatid exchanges and has the potential to result in translocations and/or somatic cell loss of heterozygosity (4), key drivers of genome instability and cancer. As a likely consequence, individuals with homozygous BLM mutations exhibit Bloom syndrome and are highly cancer prone (5). Heterozygous BLM mutation carriers are also significantly overrepresented in breast cancer families (6, 7).The 1,417-amino acid BLM protein suppresses DNA cross-overs by promoting dHJ “dissolution,” a process that unlinks the recombination intermediates back to their prerecombination state (8). Critical to dHJ dissolution by BLM is a RecQ-type superfamily II helicase domain. The ATPase function of this domain is required for DNA unwinding, translocation, and strand exchange within the HJ (9, 10). Dissolution also requires the concerted action of three additional factors, topoisomerase IIIα (TopoIIIα), RMI1, and RMI2. Together, these four proteins form the Bloom syndrome complex (BS complex; also known as the “dissolvasome” or “BTRR [BLM-TopoIIIα-RMI1-RMI2]”) (11). TopoIIIα belongs to the type IA class of topoisomerases, padlock-shaped enzymes that effect changes in DNA topology through a “strand-passage” mechanism (12). When coupled with TopoIIIα, BLM promotes DNA decatenation under physiological conditions by catalyzing DNA unlinking in an ATPase-dependent manner.The ancillary factors RMI1 (625 amino acids) and RMI2 (147 amino acids) are Replication Protein A–like, OB (oligonucleotide binding) fold–containing proteins that stimulate the dissolution reaction and have been implicated in either nucleic acid engagement (13–15) or protein–protein interactions (16–19). In particular, in the yeast dissolvasome complex, RMI1 stabilizes the covalently bound open form of the TopoIIIα DNA gate in a configuration that favors DNA strand passage (10). The significance of these components to BLM function is underscored by recent discoveries of homozygous mutant TOP3Α, RMI1, or RMI2 individuals who have Bloom syndrome–like disorders (20, 21).X-ray crystal structures of the RecQ helicase domain of BLM [encompassing residues 636 to 1298, Protein Data Bank (PDB) ID codes 4CGZ, 4CDG, and 403M (22, 23)] revealed a unique position of the DNA duplex relative to the helicase core and visualization of both pre- and posttranslocation states that flip out at least three bases of single-strand DNA (ssDNA). An additional structure of TopoIIIα (residues 21 to 639) in complex with RMI1 [residues 2 to 216; PDB ID code 4CHT (12)] also revealed the characteristic toroidal structure of type I topoisomerases, with a gate mechanism that is regulated by an insertion loop within the first OB fold of RMI1. While these structures informed on key residues and the biophysical action of each protein, they did not explain how the BS complex assembles or point to an overall mechanism of dHJ dissolution. Key to understanding this mechanism is a molecular characterization of the number and relative position of helicases and topoisomerases within the complex. However, there are contrasting reports in the literature on the oligomeric state of BLM. Size exclusion chromatography and atomic force microscopy support monomeric, dimeric, and larger oligomeric states, while EM data reveal five- or six-lobed structures reminiscent of ring-shaped multimeric ATPases (24–27). The oligomeric state of BLM within the BS complex has never been investigated, although RMI1:RMI2 purified in isolation forms a 1:1 heterodimer in published reports (16, 18, 19).The primary goal of this study was to establish how the BS complex assembles and how this assembly promotes dHJ dissolution and maintenance of genome stability. To achieve this, we purified an active recombinant BS complex. Using a combination of biochemical, biophysical, and cross-linking mass spectrometry (XL-MS) experiments, we determined that recombinant BS complex exists predominantly in a 2:2:2:2 stoichiometry. We found that dimerization of the BS complex and interaction with TopoIIIα/RMI1 are mediated by the N terminus of BLM, and we define critical residues required for these interactions and dHJ dissolution in vitro. To validate our in vitro experiments, we used cell complementation experiments with mutant BLM proteins and showed that these interactions are also required for genome stability in cells.     

Robust designation of meiotic crossover sites by CDK-2 through phosphorylation of the MutSγ complex

Crossover formation is essential for proper segregation of homologous chromosomes during meiosis. Here, we show that Caenorhabditis elegans cyclin-dependent kinase 2 (CDK-2) partners with cyclin-like protein COSA-1 to promote crossover formation by promoting conversion of meiotic double-strand breaks into crossover–specific recombination intermediates. Further, we identify MutSγ component MSH-5 as a CDK-2 phosphorylation target. MSH-5 has a disordered C-terminal tail that contains 13 potential CDK phosphosites and is required to concentrate crossover–promoting proteins at recombination sites. Phosphorylation of the MSH-5 tail appears dispensable in a wild-type background, but when MutSγ activity is partially compromised, crossover formation and retention of COSA-1 at recombination sites are exquisitely sensitive to phosphosite loss. Our data support a model in which robustness of crossover designation reflects a positive feedback mechanism involving CDK-2–mediated phosphorylation and scaffold-like properties of the MSH5 C-terminal tail, features that combine to promote full recruitment and activity of crossover–promoting complexes.

Sexually reproducing organisms rely on proper chromosome segregation during meiosis to produce gametes with a complete genome. During meiotic prophase I, chromosomes pair and undergo crossover recombination with their homologous partners. This process, together with sister chromatid cohesion, leads to the formation of physical linkages between the homologs and enables their separation during meiosis I. Defects in crossover formation are disastrous, leading to miscarriages and congenital disorders, such as Down syndrome (1).Meiotic recombination initiates with the generation of programmed DNA double-strand breaks (DSBs) by the topoisomerase-like enzyme Spo11 (2). DSBs are resected to yield two 3′-end single-stranded DNA (ssDNA) overhangs, which are rapidly coated by RecA recombinases Dmc1 and Rad51. This nucleoprotein filament then seeks out homology and invades a homologous template, forming a metastable single-end invasion intermediate (D-loop) (3). The invading strand primes DNA synthesis and extends the D-loop. If the extended D-loop is captured by ssDNA on the other side of DSBs in a process known as second-end capture, a double Holliday junction (dHJ) forms (4). While dHJs can be resolved biochemically as either crossovers or non–crossovers (5), during meiosis, the majority of dHJs are specifically resolved as crossovers through the activity of MutLγ (MLH1-MLH3) (6–8) or other structure-selective endonucleases. Although a multitude of DSBs are generated during meiotic prophase, strikingly few are ultimately selected to become crossovers. Early recombination intermediates pare down in pachytene until each homolog pair receives at least one crossover, while the majority of DSBs are repaired as non–crossovers via synthesis-dependent strand annealing (9). However, how meiotic DSBs are chosen to become crossovers remains poorly understood.Throughout eukaryotes, crossover recombination is primarily controlled by a group of proteins collectively termed “ZMM” (10). Notably, homologs of the yeast RING (Really interesting new gene) domain protein Zip3 [ZHP-1, ZHP-2, ZHP-3, and ZHP-4 in Caenorhabditis elegans (11–14), Drosophila Vilya and Narya/Nenya (15, 16), Hei10 in Arabidopsis (17), and Hei10 and RNF212 in mammals (18, 19)] initially localize as abundant foci or long stretches along the synaptonemal complex (SC) but eventually concentrate at crossover sites in late pachytene (20). These SUMO or ubiquitin ligases appear to promote crossover designation by stabilizing the ZMM proteins at crossover sites while removing them from other recombination intermediates (13, 14, 18, 19, 21, 22). Although many meiotic proteins are shown to be SUMO modified (23), key targets of the Zip3 family proteins remain largely unknown.The meiosis-specific MutS homologs MSH4 and MSH5 form a heterodimeric MutSγ complex and play essential roles in crossover formation in diverse eukaryotes (24–31). MutSγ localizes to recombination intermediates as numerous foci but ultimately accumulates at sites that are destined to become crossovers (32, 33). Biochemical analyses using recombinant MSH4 and MSH5 have shown that MutSγ recognizes single-end invasion intermediates and HJs in vitro (34, 35). HJs activate the ATP hydrolysis of MutSγ and promote the exchange of bound ADP for ATP, inducing the formation of a sliding clamp that dissociates from HJs (35, 36). By iterative loading and embracing DNA duplexes within a dHJ, MutSγ is thought to stabilize crossover–specific recombination intermediates (33, 35). In addition, MutSγ recruits and activates the resolvase activity of MutLγ, enabling biased processing of dHJs into crossovers during meiosis (37, 38).A genetic screen in C. elegans identified a cyclin-like protein COSA-1 as a component essential for processing meiotic DSBs into crossovers (39). The mammalian ortholog CNTD1 was subsequently identified (40), and both COSA-1 and CNTD1 have been shown to localize to crossover sites (39, 41, 42). In the absence of COSA-1/CNTD1, MutSγ components persist as numerous foci in pachytene, and crossover formation is eliminated or severely compromised (33, 40), demonstrating a crucial role of COSA-1/CNTD1 in converting early recombination intermediates into crossovers. Because both COSA-1 and CNTD1 are members of the cyclin family, it is plausible that they form a complex with a cyclin-dependent kinase (CDK) and regulate the recombination process through phosphorylation.Several lines of evidence have suggested that CDK2 might be a relevant kinase partner for CNTD1. CDK2 interacts with CNTD1 in yeast-two hybrid assays (41, 42) and localizes to interstitial chromosome sites (43, 44) in a CNTD1-dependent manner (40). Reduced CDK2 activity leads to a failure in crossover formation, while a hyperactive form of CDK2 causes an increased number of MLH1 foci (45). However, due to its requirement at telomeres in tethering chromosomes to the nuclear envelope, deletion of CDK2 leads to severe defects in SC assembly between paired homologs (synapsis) and pachytene arrest (46–49). Further, while a full-length CNTD1-specific protein of the excepted size was detected (using CNTD1 antibodies) in one study (42), a short CNTD1 isoform that cannot interact with CDK2 was the predominant isoform detected in another study (using hemagglutinin antibodies in Cntd1^FH/FH mice with an epitope tag sequence inserted into the endogenous Cntd1 locus) (41), raising questions regarding the extent to which CDK2 and CNTD1 might act as functional partners. Thus, it has been difficult to determine the role of CDK2 in crossover recombination. Moreover, key meiotic targets of CDK2 have not yet been identified.We reasoned that CDK-2, the C. elegans homolog of CDK2, might also localize and function at crossover sites. However, global knockdown of C. elegans CDK-2 by RNA interference leads to cell cycle arrest of mitotically proliferating germ cells (50), thereby precluding the analysis of its requirement during meiotic prophase. To overcome this limitation and establish the meiotic function of CDK-2, we use the auxin-inducible degradation system to deplete CDK-2 from the adult germline, demonstrating that CDK-2 partners with COSA-1 to promote crossover formation during C. elegans meiosis. Moreover, we identify MSH-5 as a key substrate for CDK-2 and provide evidence that CDK-2 and COSA-1 partner to promote crossover designation through phosphorylation and activation of the MutSγ complex.     

Two-dimensional electronic–vibrational sum frequency spectroscopy for interactions of electronic and nuclear motions at interfaces

Interactions of electronic and vibrational degrees of freedom are essential for understanding excited-states relaxation pathways of molecular systems at interfaces and surfaces. Here, we present the development of interface-specific two-dimensional electronic–vibrational sum frequency generation (2D-EVSFG) spectroscopy for electronic–vibrational couplings for excited states at interfaces and surfaces. We demonstrate this 2D-EVSFG technique by investigating photoexcited interface-active (E)-4-((4-(dihexylamino) phenyl)diazinyl)-1-methylpyridin-1- lum (AP3) molecules at the air–water interface as an example. Our 2D-EVSFG experiments show strong vibronic couplings of interfacial AP3 molecules upon photoexcitation and subsequent relaxation of a locally excited (LE) state. Time-dependent 2D-EVSFG experiments indicate that the relaxation of the LE state, S₂, is strongly coupled with two high-frequency modes of 1,529.1 and 1,568.1 cm⁻¹. Quantum chemistry calculations further verify that the strong vibronic couplings of the two vibrations promote the transition from the S₂ state to the lower excited state S₁. We believe that this development of 2D-EVSFG opens up an avenue of understanding excited-state dynamics related to interfaces and surfaces.

Electronic and vibrational degrees of freedom are the most important physical quantities in molecular systems at interfaces and surfaces. Knowledge of interactions between electronic and vibrational motions, namely electronic–vibrational couplings, is essential to understanding excited-states relaxation pathways of molecular systems at interfaces and surfaces. Many excited-states relaxation processes occur at interfaces and surfaces, including charge transfer, energy transfer, proton transfer, proton-coupled electron transfer, configurational dynamics, and so on (1–11). These relaxation processes are intimately related to the electronic–vibrational couplings at interfaces and surfaces. Strong electronic–vibrational couplings could promote nonadiabatic evolution of excited potential energy and thus, facilitate chemical reactions or intramolecular structural changes of interfacial molecules (10, 12, 13). Furthermore, these interactions of electronic and vibrational degrees of freedom are subject to solvent environments (e.g., interfaces/surfaces with a restricted environment of unique physical and chemical properties) (9, 14, 15). Despite the importance of interactions of electronic and vibrational motions, little is known about excited-state electronic–vibrational couplings at interfaces and surfaces.Interface-specific electronic and vibrational spectroscopies enable us to characterize the electronic and vibrational structures separately. As interface-specific tools, second-order electronic sum frequency generation (ESFG) and vibrational sum frequency generation (VSFG) spectroscopies have been utilized for investigating molecular structure, orientational configurations, chemical reactions, chirality, static potential, environmental issues, and biological systems at interfaces and surfaces (16–52). Recently, structural dynamics at interfaces and surfaces have been explored using time-resolved ESFG and time-resolved VSFG with a visible pump or an infrared (IR) pump thanks to the development of ultrafast lasers (6–9, 13–15, 49, 53–61). Doubly resonant sum frequency generation (SFG) has been demonstrated to probe both electronic and vibration transitions of interfacial molecular monolayer (15, 62–71). This frequency-domain two-dimensional (2D) interface/surface spectroscopy could provide information regarding electronic–vibrational coupling of interfacial molecules. However, contributions from excited states are too weak to be probed due to large damping rates of vibrational states in excited states (62, 63). As such, the frequency-domain doubly resonant SFG is used only for electronic–vibrational coupling of electronic ground states. Ultrafast interface-specific electronic–vibrational spectroscopy could allow us to gain insights into how specific nuclear motions drive the relaxation of electronic excited states. Therefore, development of interface-specific electronic–vibrational spectroscopy for excited states is needed.In this work, we integrate the specificity of interfaces and surfaces into the capabilities of ultrafast 2D spectroscopy for dynamical electronic–vibrational couplings in excited states of molecules; 2D interface-specific spectroscopies are analogous to those 2D spectra in bulk that spread the information contained in a pump−probe spectrum over two frequency axes. Thus, one can better interpret congested one-dimensional signals. Two-dimensional vibrational sum frequency generation (2D-VSFG) spectroscopy was demonstrated a few year ago (72–74). Furthermore, heterodyne 2D-VSFG spectroscopy using middle infrared (mid-IR) pulse shaping and noncollinear geometry 2D-VSFG experiments have also been developed to study vibrational structures and dynamics at interfaces (31, 75–78). Recently, two-dimensional electronic sum frequency generation (2D-ESFG) spectroscopy has also been demonstrated for surfaces and interfaces (79). On the other hand, bulk two-dimensional electronic–vibrational (2D-EV) spectroscopy has been extensively used to investigate the electronic relaxation and energy transfer dynamics of molecules, biological systems, and nanomaterials (80–90). The 2D-EV technique not only provides electronic and vibrational interactions between excitons or different excited electronic states of systems but also, identifies fast nonradiative transitions through nuclear motions in molecules, aggregations, and nanomaterials. However, an interface-specific technique for two-dimensional electronic–vibrational sum frequency generation (2D-EVSFG) spectroscopy has yet to be developed.Here, we present the development of 2D-EVSFG spectroscopy for the couplings of electronic and nucleic motions at interfaces and surfaces. The purpose of developing 2D-EVSFG spectroscopy is to bridge the gap between the visible and IR regions to reveal how structural dynamics for photoexcited electronic states are coupled with vibrations at interfaces and surfaces. As an example, we applied this 2D-EVSFG experimental method to time evolution of electronic–vibrational couplings at excited states of interface-active molecules at the air–water interface.     

Coulomb interaction,phonons, and superconductivity in twisted bilayer graphene

The polarizability of twisted bilayer graphene, due to the combined effect of electron–hole pairs, plasmons, and acoustic phonons, is analyzed. The screened Coulomb interaction allows for the formation of Cooper pairs and superconductivity in a significant range of twist angles and fillings. The tendency toward superconductivity is enhanced by the coupling between longitudinal phonons and electron–hole pairs. Scattering processes involving large momentum transfers, Umklapp processes, play a crucial role in the formation of Cooper pairs. The magnitude of the superconducting gap changes among the different pockets of the Fermi surface.

Twisted bilayer graphene (TBG) shows a complex phase diagram which combines superconducting and insulating phases (1, 2) and resembles strongly correlated materials previously encountered in condensed matter physics (3–6). On the other hand, superconductivity seems more prevalent in TBG (7–11), while in other strongly correlated materials magnetic phases are dominant.The pairing interaction responsible for superconductivity in TBG has been intensively studied. Among other possible pairing mechanisms, the effect of phonons (12–19) (see also ref. 20), the proximity of the chemical potential to a van Hove singularity in the density of states (DOS) (21–25) and excitations of insulating phases (26–28) (see also refs. 29–31), and the role of electronic screening (32–35) have been considered.In the following, we analyze how the screened Coulomb interaction induces pairing in TBG. The calculation is based on the Kohn–Luttinger formalism (36) for the study of anisotropic superconductivity via repulsive interactions. The screening includes electron–hole pairs (37), plasmons (38), and phonons (note that acoustic phonons overlap with the electron–hole continuum in TBG). Our results show that the repulsive Coulomb interaction, screened by plasmons and electron–hole pairs only, leads to anisotropic superconductivity, although with critical temperatures of order T_c ∼ 10⁻³ to 10⁻² K. The inclusion of phonons in the screening function substantially enhances the critical temperature, to T_c ∼ 1 to 10 K.     

Class-specific interactions between Sis1 J-domain protein and Hsp70 chaperone potentiate disaggregation of misfolded proteins

Protein homeostasis is constantly being challenged with protein misfolding that leads to aggregation. Hsp70 is one of the versatile chaperones that interact with misfolded proteins and actively support their folding. Multifunctional Hsp70s are harnessed to specific roles by J-domain proteins (JDPs, also known as Hsp40s). Interaction with the J-domain of these cochaperones stimulates ATP hydrolysis in Hsp70, which stabilizes substrate binding. In eukaryotes, two classes of JDPs, Class A and Class B, engage Hsp70 in the reactivation of aggregated proteins. In most species, excluding metazoans, protein recovery also relies on an Hsp100 disaggregase. Although intensely studied, many mechanistic details of how the two JDP classes regulate protein disaggregation are still unknown. Here, we explore functional differences between the yeast Class A (Ydj1) and Class B (Sis1) JDPs at the individual stages of protein disaggregation. With real-time biochemical tools, we show that Ydj1 alone is superior to Sis1 in aggregate binding, yet it is Sis1 that recruits more Ssa1 molecules to the substrate. This advantage of Sis1 depends on its ability to bind to the EEVD motif of Hsp70, a quality specific to most of Class B JDPs. This second interaction also conditions the Hsp70-induced aggregate modification that boosts its subsequent dissolution by the Hsp104 disaggregase. Our results suggest that the Sis1-mediated chaperone assembly at the aggregate surface potentiates the entropic pulling, driven polypeptide disentanglement, while Ydj1 binding favors the refolding of the solubilized proteins. Such subspecialization of the JDPs across protein reactivation improves the robustness and efficiency of the disaggregation machinery.

Molecular chaperones are involved in the maintenance of protein homeostasis by aiding correct protein folding (1). Yet severe stress conditions induce excessive protein misfolding and aggregation (2). Upon stress relief, the return to the proteostasis is mediated by the Hsp70 chaperone with cochaperones, including J-domain proteins (JDPs/Hsp40s), which together restore the native state of misfolded polypeptides trapped in aggregates (3–5). The JDP–Hsp70 system acts alone in metazoans or in cooperation with an Hsp100 disaggregase in most other eukaryotes and bacteria (5, 6).Protein disaggregation and refolding starts with a recognition of misfolded polypeptides within an aggregate by a JDP, and then, its J-domain interacts with the nucleotide-binding domain of Hsp70, inducing ATP hydrolysis which triggers the closure of the Hsp70’s substrate-binding domain over the aggregated substrate (7, 8). The aggregate-bound Hsp70 interacts with an Hsp100 disaggregase, and this interaction allosterically activates Hsp100 and tethers it to the aggregate (9–16). Subsequently, in an ATP-driven process, Hsp100 disentangles and translocates polypeptides from aggregates (17–21), which enables their correct refolding, spontaneous or with an assistance of Hsp70 and its cochaperones (22, 23).JDPs are the major regulators of the Hsp70 activity and substrate specificity (3, 24, 25). In yeast Saccharomyces cerevisiae, a general Hsp70 chaperone, Ssa1, is recruited to protein disaggregation by two main cytosolic JDPs, Ydj1 and Sis1, assigned to the Class A and Class B, respectively (3, 4, 26). Both Ydj1 and Sis1 comprise a helical, highly conserved J-domain, a flexible, mostly unstructured G/F region, two beta-barrel peptide-binding domains, CTDI and CTDII, and a C-terminal dimerization domain (27–33). Ydj1 additionally features a Zn-binding domain located in the first part of the CTDI region of the protein, which is distinctive for the Class A JDPs (32, 34).Despite the structural similarities, the two JDPs are functionally nonredundant. Sis1 is essential, and Ydj1 is required for growth above 34 °C (26, 27, 35, 36). Overexpression of Sis1 suppresses the phenotype caused by the deletion of YDJ1, while Ydj1 overexpression is not sufficient to suppress the deletion of SIS1 (26, 27, 35–37). The two JDPs show different specificities toward amorphous and amyloid aggregates (35, 38) and different populations of amorphous aggregates formed in vitro (4, 24).Recent reports shed more light on the JDPs’ divergence. Both JDPs form homodimers, which differ in the structural orientation of the J-domain: In Sis1, the J-domain is restrained from Hsp70 binding by the interaction with the Helix 5 in the G/F region (26, 33, 39–41). Such autoinhibition, which also occurs in most human Class B JDPs, is released through the interaction with the C-terminal EEVD motif of Hsp70 (33, 42). This regulation is important for the disassembly of amyloid fibrils by the human JDP–Hsp70 system (43), but its role in the handling of stress-related, amorphous aggregates is not clear. Despite the breadth of data on Hsp70 mechanisms, we still lack understanding of how the disparate features of the JDPs impact Hsp70 functioning in protein disaggregation.Here, we investigate individual steps of protein disaggregation in the context of functional differences between Sis1 and Ydj1. Using various biochemical approaches, we show that the two JDPs drive different modes of Ssa1 binding to aggregated substrates, which dictate diverse kinetics of their disaggregation by Hsp104. The distinctive performance of Sis1 is associated with its interaction with the C terminus of Hsp70. Our results suggest that the bivalent interaction with the Class B JDP conditions aggregate remodeling by the Hsp70 system, resulting in enhanced Hsp104-dependent protein recovery. Our data indicate a mechanism by which the Class A and B JDPs contribute to the disaggregation efficacy in a complex and divergent manner.     

Hierarchical self-assembly of polydisperse colloidal bananas into a two-dimensional vortex phase

Self-assembly of microscopic building blocks into highly ordered and functional structures is ubiquitous in nature and found at all length scales. Hierarchical structures formed by colloidal building blocks are typically assembled from monodisperse particles interacting via engineered directional interactions. Here, we show that polydisperse colloidal bananas self-assemble into a complex and hierarchical quasi–two-dimensional structure, called the vortex phase, only due to excluded volume interactions and polydispersity in the particle curvature. Using confocal microscopy, we uncover the remarkable formation mechanism of the vortex phase and characterize its exotic structure and dynamics at the single-particle level. These results demonstrate that hierarchical self-assembly of complex materials can be solely driven by entropy and shape polydispersity of the constituting particles.

Self-assembly of microscopic building blocks is a powerful route for preparing materials with predesigned structure and engineered properties (1–7). Nature provides a fascinating range of self-assembled architectures offering insight into how structural organization can emerge at different length scales (8–13). In the biological world, for instance, tobacco mosaic virus coat proteins self-organize into sophisticated capsids around viral RNA strands (11, 14). In molecular systems, lipid molecules, such as fatty acids, form a range of self-assembled structures as relevant as cell membranes and vesicles (15, 16). At the colloidal scale, a rich variety of crystals with remarkable optical properties, such as opal and other gemstones, also assembles from a range of colloidal constituents (12, 17–20). The structural complexity of self-assembled materials is typically dictated by the combination of the type of interactions between the constituent building blocks and their shape (2, 3, 5, 6). Colloids are ideal systems to independently study the role of these key parameters, as their shape and interactions can be systematically tuned and rationally designed (5, 18, 21–23).In colloidal systems interacting solely via excluded volume interactions, the shape of the particles can already lead to the assembly of complex structures (24–28). For instance, binary colloidal crystals (25) are obtained from spherical particles, complex dodecagonal quasicrystals are formed by tetrahedrons (26), and exotic banana-shaped liquid crystals are assembled from colloidal bananas (28). Introducing complex interactions between the colloidal building blocks—on the top of their shape—leads to their assembly into hierarchical materials with structural order at multiple length scales (3, 29–31). Examples include colloidal diamond structures assembled by patchy tetrahedrons functionalized with DNA strands (20) and superlattice structures formed by octapod-like particles functionalized with hydrophobic molecules (32). The successful hierarchical self-assembly of these structures relies not only on the directionality of the particle interactions but also, on the uniformity in size of the constituent building blocks, as polydispersity typically disrupts ordering via the formation of defects (33, 34).In this work, however, we show that a colloidal suspension of polydisperse banana-shaped particles interacting only via simple excluded volume interactions (28) self-assembles into remarkably ordered concentric structures, which we term colloidal vortices. At high packing fractions, these structures form a quasi–two-dimensional (quasi-2D) hierarchical material, which we term the vortex phase. Using confocal microscopy, we uncover the formation mechanism of this tightly packed phase and characterize its exotic structure and dynamics at the single-particle level.     

On the question of whether lubricants fluidize in stick–slip friction

Intermittent sliding (stick–slip motion) between solids is commonplace (e.g., squeaking hinges), even in the presence of lubricants, and is believed to occur by shear-induced fluidization of the lubricant film (slip), followed by its resolidification (stick). Using a surface force balance, we measure how the thickness of molecularly thin, model lubricant films (octamethylcyclotetrasiloxane) varies in stick–slip sliding between atomically smooth surfaces during the fleeting (ca. 20 ms) individual slip events. Shear fluidization of a film of five to six molecular layers during an individual slip event should result in film dilation of 0.4–0.5 nm, but our results show that, within our resolution of ca. 0.1 nm, slip of the surfaces is not correlated with any dilation of the intersurface gap. This reveals that, unlike what is commonly supposed, slip does not occur by such shear melting, and indicates that other mechanisms, such as intralayer slip within the lubricant film, or at its interface with the confining surfaces, may be the dominant dissipation modes.Intermittent sliding (stick–slip) of solids in contact is an everyday effect, such as in the squeak of hinges or the music of violins, when the bow slides past the strings, or, at a different scale, in earthquakes (where tectonic plates slide past each other). Such solid sliding is a major cause of frictional dissipation, and can persist even in the presence of lubricants (1). At a nanotribological level, surface force balance (SFB) measurements, supported by theory and computer simulations, have shown that when simple organic liquids are confined between atomically smooth, solid (mica) surfaces to films thinner than some six to eight molecular layers, they may become solid-like, and are often layered (2–14). Subsequent sliding of the surfaces across such films when they are subjected to shear may then take place via stick–slip motion (15, 16). During the stick part, the surfaces are in rigid contact until the shear force between them exceeds the static friction, at which point they slip rapidly past each other (relaxing the shear stress) and then stick again, in a repeating cycle. The issue of how the confined (lubricant) layer progressively yields and then becomes rigid again during such stick–slip sliding has been intensely studied over the past several decades, not least because a better understanding may result in improved lubrication approaches.The molecular basis of the stick–slip cycle in sheared solid-like lubricant films as described above is not well understood (17–28). This is at least in part because, experimentally, it is very challenging to capture what happens to the lubricant layer during the fleeting, individual slip events taking place in the nanometrically confined film. Even when measured under controlled conditions, as in the SFB, these slip events are not only of very short duration [ca. 20 ms (18)] but generally occupy only a tiny fraction of the stick–slip cycle, with the surfaces in nonsliding contact (stick) for almost the entire cycle period. For this reason, much of our understanding has been derived from theoretical modeling and computer simulation studies (17, 19–25, 27–29). Classically, these almost all suggest that the stick–slip motion involves periodic shear melting transitions and resolidification of the film as it undergoes transition between solid-like and liquid-like phases during sliding. Even where there is some disagreement in the model details [for example, on the precise mechanism by which the films solidify at the end of the slip (22, 25)], they maintain the essential idea of fluidization of the lubricant layers during the slip part of the stick–slip cycle. In the shear-induced solid to liquid transition (fluidization), a density change is also expected because the fluidized phase is less dense than the solid phase. This leads to a volumetric expansion and contraction cycle (corresponding respectively to slip and stick), with a dilation of the thin lubricant film during the slip event (17, 23, 25, 27). Some more recent simulations suggest that slip may occur at the wall–fluid interfaces or via interlayer slip within the film rather than via film melting (19, 27, 28), although the scenario of lubricant fluidization during slip is the generally accepted mechanism.There have been few experimental studies on individual slips during stick–slip sliding across lubricant films, and none where the film thickness in such fleeting events has been examined (15, 16, 18, 30–32). Clues may also be extracted from stick–slip motion of confined granular systems under shear, where numerical simulations (33, 34) and some experiments (35–37) suggest that fluidization and dilation may play a role in the stick–slip instability. While this is suggestive, differences between granular layers and lubricant films include not only five orders of magnitude between size of grains and of molecules but, in particular, the issue of molecular interactions, negligible in granular shear but all-important when shearing lubricants.In the present study, we examine directly the individual slip events during stick–slip sliding across thin lubricant films, and in particular the issue of film dilation during the fleeting slip motion itself. This is done to provide “smoking gun” evidence concerning the issue of film fluidization, where such dilation is expected to be a clear signature. We confine a thin (few nanometers) model liquid film between smooth solid surfaces in an SFB, shear it, and monitor the film thickness during stick–slip sliding via fast video microscopy. To overcome the major challenge presented by the shortness of the slip events, which occupy only some 1% of the stick–slip cycle over which a subnanometer dilation needs to be detected against a comparable level of noise, we analyze our data using tools from classical signal detection theory to correlate the slip events with the instantaneous value of the film thickness.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Synaptotagmin-1 is a bidirectional Ca2+ sensor for neuronal endocytosis

Exocytosis and endocytosis are tightly coupled. In addition to initiating exocytosis, Ca²⁺ plays critical roles in exocytosis–endocytosis coupling in neurons and nonneuronal cells. Both positive and negative roles of Ca²⁺ in endocytosis have been reported; however, Ca²⁺ inhibition in endocytosis remains debatable with unknown mechanisms. Here, we show that synaptotagmin-1 (Syt1), the primary Ca²⁺ sensor initiating exocytosis, plays bidirectional and opposite roles in exocytosis–endocytosis coupling by promoting slow, small-sized clathrin-mediated endocytosis but inhibiting fast, large-sized bulk endocytosis. Ca²⁺-binding ability is required for Syt1 to regulate both types of endocytic pathways, the disruption of which leads to inefficient vesicle recycling under mild stimulation and excessive membrane retrieval following intense stimulation. Ca²⁺-dependent membrane tubulation may explain the opposite endocytic roles of Syt1 and provides a general membrane-remodeling working model for endocytosis determination. Thus, Syt1 is a primary bidirectional Ca²⁺ sensor facilitating clathrin-mediated endocytosis but clamping bulk endocytosis, probably by manipulating membrane curvature to ensure both efficient and precise coupling of endocytosis to exocytosis.

Endocytosis and subsequent vesicle recycling are spatiotemporally coupled to exocytosis, which is critical for neurons and endocrinal cells to maintain the integrity of plasma membrane architecture, intracellular homeostasis, and sustained neurotransmission (1–3). In addition to triggering vesicular exocytosis, neural activity/Ca²⁺ also play an executive role in the coupling of endocytosis to exocytosis (1, 2, 4–6). Following a pioneering study 40 y ago (7), extensive studies have been conducted and showed that Ca²⁺ triggers and facilitates vesicle endocytosis in neurons and nonneuronal secretory cells (1, 8–11). Accumulating evidence also shows that intracellular Ca²⁺ may inhibit endocytosis (12–15), which has been challenged greatly due to the apparently lower occurrences in few preparations and the missing underlining mechanisms, making the endocytic role of Ca²⁺ a four-decades–long dispute (1, 2, 4, 6).Machineries and regulators involved in exocytosis–endocytosis coupling have been extensively studied for over 30 y. The soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) and synaptophysin play critical dual roles in exocytosis and endocytosis during neurotransmission (2, 3, 16, 17). Calmodulin and synaptotagmin-1 (Syt1) are currently known primary Ca²⁺ sensors facilitating endocytosis (1, 9, 16, 18, 19). Ca²⁺/calmodulin activate calcineurin, which dephosphorylates endocytic proteins (e.g., dynamin, synaptojanin, and amphiphysin) to facilitate clathrin-mediated endocytosis (CME) and clathrin-independent fast endocytosis (1, 2). Syt1 is a dual Ca²⁺ sensor for both exocytosis and endocytosis (5, 16, 18–20). It promotes CME through binding with the endocytic adaptors adaptor protein-2 (AP-2) and stonin-2 (21–24). In contrast to the well-established Ca²⁺ sensors that promote endocytosis, the mechanism of Ca²⁺-dependent inhibition in endocytosis remains unknown.CME is the classical but slow endocytosis pathway for vesicle retrieval under resting conditions or in response to mild stimulation, while the accumulated Ca²⁺ also triggers calmodulin/calcineurin-dependent bulk endocytosis, which takes up a large area of plasma membrane to fulfill the urgent requirement for high-speed vesicle exocytosis (1–3). They cooperate with kiss-and-run and ultrafast endocytosis to ensure both sufficient and precise membrane retrieval following exocytosis (3, 25–27). These endocytic pathways are all initiated from membrane invagination and are critically controlled by neural activity. However, how the switch between different endocytic modes is precisely determined remains largely unknown.Here, by combining electrophysiological recordings, confocal live imaging, superresolution stimulated emission depletion (STED) imaging, in vitro liposome manipulation, and electron microscope imaging of individual endocytic vesicles, we define Syt1 as a primary and bidirectional Ca²⁺ sensor for endocytosis, which promotes CME but inhibits bulk endocytosis, probably by mediating membrane remodeling. The balance between the facilitatory and inhibitory effects of Syt1 on endocytosis offers a fine-tuning mechanism to ensure both efficient and precise coupling of endocytosis to exocytosis. By including a non-Ca²⁺–binding Syt as the constitutive brake, this work also explains the four-decades–long puzzle about the positive and negative Ca²⁺ effects on endocytosis.     

SARS-CoV-2 spreads through cell-to-cell transmission

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein, we provide evidence that SARS-CoV-2 spreads through cell–cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than is SARS-CoV spike, which reflects, in part, their differential cell–cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While angiotensin-converting enzyme 2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccinee sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis.

SARS-CoV-2 is a novel beta-coronavirus that is closely related to two other highly pathogenic human coronaviruses, SARS-CoV and MERS-CoV (1). The spike (S) proteins of SARS-CoV-2 and SARS-CoV mediate entry into target cells, and both use angiotensin-converting enzyme 2 (ACE2) as the primary receptor (2–6). The spike protein of SARS-CoV-2 is also responsible for induction of neutralizing antibodies, thus playing a critical role in host immunity to viral infection (7–10).Similar to HIV and other class I viral fusion proteins, SARS-CoV-2 spike is synthesized as a precursor that is subsequently cleaved and highly glycosylated; these properties are critical for regulating viral fusion activation, native spike structure, and evasion of host immunity (11–15). However, distinct from SARS-CoV, yet similar to MERS-CoV, the spike protein of SARS-CoV-2 is cleaved by furin into S1 and S2 subunits during the maturation process in producer cells (6, 16, 17). S1 is responsible for binding to the ACE2 receptor, whereas S2 mediates viral membrane fusion (18, 19). SARS-CoV-2 spike can also be cleaved by additional host proteases, including transmembrane serine protease 2 (TMPRSS2) on the plasma membrane and several cathepsins in the endosome, which facilitate viral membrane fusion and entry into host cells (20–22).Enveloped viruses spread in cultured cells and tissues via two routes: by cell-free particles and through cell–cell contact (23–26). The latter mode of viral transmission normally involves tight cell–cell contacts, sometimes forming virological synapses, where local viral particle density increases (27), resulting in efficient transfer of virus to neighboring cells (24). Additionally, cell-to-cell transmission has the ability to evade antibody neutralization, accounting for efficient virus spread and pathogenesis, as has been shown for HIV and hepatitis C virus (HCV) (28–32). Low levels of neutralizing antibodies, as well as a deficiency in type I IFNs, have been reported for SARS-CoV-2 (18, 33–37) and may have contributed to the COVID-19 pandemic and disease progression (38–43).In this work, we evaluated cell-to-cell transmission of SARS-CoV-2 in the context of cell-free infection and in comparison with SARS-CoV. Results from this in vitro study reveal the heretofore unrecognized role of cell-to-cell transmission that potentially impacts SARS-CoV-2 spread, pathogenesis, and shielding from antibodies in vivo.     

Reciprocal antagonistic regulation of E3 ligases controls ACC synthase stability and responses to stress

Ethylene influences plant growth, development, and stress responses via crosstalk with other phytohormones; however, the underlying molecular mechanisms are still unclear. Here, we describe a mechanistic link between the brassinosteroid (BR) and ethylene biosynthesis, which regulates cellular protein homeostasis and stress responses. We demonstrate that as a scaffold, 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACS), a rate-limiting enzyme in ethylene biosynthesis, promote the interaction between Seven-in-Absentia of Arabidopsis (SINAT), a RING-domain containing E3 ligase involved in stress response, and ETHYLENE OVERPRODUCER 1 (ETO1) and ETO1-like (EOL) proteins, the E3 ligase adaptors that target a subset of ACS isoforms. Each E3 ligase promotes the degradation of the other, and this reciprocally antagonistic interaction affects the protein stability of ACS. Furthermore, 14–3-3, a phosphoprotein-binding protein, interacts with SINAT in a BR-dependent manner, thus activating reciprocal degradation. Disrupted reciprocal degradation between the E3 ligases compromises the survival of plants in carbon-deficient conditions. Our study reveals a mechanism by which plants respond to stress by modulating the homeostasis of ACS and its cognate E3 ligases.

Protein homeostasis is a core mechanism for maintaining cellular function, which enables organisms to rapidly respond to environmental stress in a specific manner. The ubiquitin–proteasome-mediated degradation pathway is one of the main pathways that govern protein homeostasis in cells and has been linked to diverse functions in plants, including hormone signaling, plant defense response, photomorphogenesis, and stress response (1–6).The function of the gaseous hormone ethylene is largely regulated by the ubiquitin–proteasome system (7). In ethylene signaling, the abundance of positive regulators ETHYLENE-INSENSITIVE 2 (EIN2) and EIN3 is regulated by E3 ubiquitin ligases, EIN2-TARGETING PROTEIN 1 (ETP1) and ETP2 or EIN3-BINDING F-BOX PROTEIN 1 (EBF1) and EBF2, respectively (3, 8). Similar to the signaling pathway, in ethylene biosynthesis, the abundance of a subset of 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACS), the rate-limiting enzymes in the pathway, is specifically regulated by ETHYLENE OVERPRODUCER 1 (ETO1) and its two paralogs, ETO1-like 1 (EOL1) and EOL2. ETO1/EOLs are components of a CULLIN3 E3 ligase, which specifically recognize type-2 ACS isoforms for rapid degradation via the 26S proteasome (9, 10). E3 ligase substrate-specificity subunits such as ETO1/EOLs determine the accessibility of E3 ligase complex to the substrate; thus, the abundance of ETO1/EOLs is an important regulatory factor for determining ACS stability in plants (11). The stability of EOL2 has been shown to be negatively regulated by 14–3-3, a family of phosphoprotein-binding proteins, though the underlying mechanism remains elusive (12).Arabidopsis contains eight functional ACS isoforms that can be grouped into three types, namely type-1 (ACS2, 6), type-2 (ACS4, 5, 8, 9, and 11), and type-3 (ACS7), based on the presence or absence of phosphorylation sites in the C-terminal domains (9, 13, 14). The stability of different ACS is differentially regulated by diverse stimuli, including phytohormones (14–16). Most plant hormones regulate the protein stability of ACS with distinct effects on different ACS isoforms (15, 16). Brassinosteroid (BR) is one such hormone that regulates the protein stability of ACS. However, the underlying mechanisms and molecular components involved in the process are unknown (15). A component that likely plays a role in BR–ethylene crosstalk is the 14–3-3 proteins. The 14–3-3 proteins are evolutionally well-conserved dimeric proteins in all eukaryotic organisms and are involved in varied biological processes via phosphorylation-dependent protein–protein interactions (17–19). The Arabidopsis 14–3-3 family consists of 13 isoforms, and their roles have been suggested in a diverse range of physiological processes including BR signaling (20, 21), ethylene biosynthesis (12, 22), abiotic stress response (22–24), and light signaling (25). In the BR signaling pathway, 14–3-3 proteins interact with multiple BR signaling molecules, including BRASSINOID INSENSITIVE 1 (BRI1), BRI1 KINASE INHIBITOR 1 (BKI1), and BRI1-EMS SUPPRESSOR 1 (BES1)/BRASSINAZOLE-RESISTANT 1 (BZR1), resulting in the regulation of the BR signaling pathway (17). The role of 14–3-3 proteins has been implicated in ethylene biosynthesis via their interaction with ACS in rice, barley, and Arabidopsis (26–29). In Arabidopsis, 14–3-3 positively regulates the protein stability of ACS5 through increased turnover of EOL2 or through an ETO1/EOLs-independent mechanism (12); however, the detailed mechanism such as the stimuli triggering the 14–3-3–mediated regulation of ACS and ETO1/EOL stability or other regulatory components in the process remains unknown. Given the roles of 14–3-3 in ethylene biosynthesis and BR signaling, 14–3-3 could be a crosstalk point that integrates the interaction between ethylene biosynthesis and BR signaling in a phosphorylation-dependent manner.SEVEN-IN-ABSENTIA (SINA) is a RING-type E3 ligase that has been linked to protein degradation and stress response in Drosophila, Mammalian, and plants (30–36). Arabidopsis contains a family of five SINA of Arabidopsis (SINAT) genes that encode two distinct clades of SINAT proteins (SINAT1 and 2; SINAT3, 4, and 5) based on sequence similarities (23). Several recent studies in different plant species have demonstrated that SINA family members play a role in response to abiotic and biotic stresses, including cold, drought, and pathogen invasion; some of these are linked to abscisic acid (ABA) or BR hormone signaling and autophagy, a highly conserved cellular degradation process linked to stress response (23, 33, 37). An autophagy receptor, DOMINANT SUPPRESSOR OF KAR 2 (DSK2), controls the degradation of BES1, a positive regulator for BR signaling, and SINAT2 participates in targeting BES1 for degradation via the DSK2 autophagy receptor under drought and fixed carbon starvation (33). SINAT E3 ligases also regulate the activity and/or stability of a subset of AUTOPHAGY-RELATED PROTEINS (ATGs) (30, 38). They also regulate the stability of FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1), the endosomal sorting complex required for the transport (ESCRT) component, thus controlling autophagy (30, 39–41). Intriguingly, SINAT2 has been identified as a putative 14–3-3–interacting protein along with EOL2 and several ACS isoforms through the proteomic profiling of purified complexes from Arabidopsis (27).In this study, we investigated the regulatory mechanism for ACS5 protein stability through SINAT E3 ligases. Strikingly, ACS5 acts as a scaffold that tethers SINAT2 and EOL2 in a functional complex, increasing the stability of ACS5 via the reciprocal degradation of SINAT2 and EOL2. 14–3-3 activates the reciprocal degradation of SINAT2 and EOL2 through direct interaction with SINAT2 only in the presence of BR, thereby linking ethylene biosynthesis and BR signaling. Our results reveal a regulatory mechanism that allows the simultaneous fine-tuning of the protein abundance of ACS and its cognate E3 ligases, which is critical for stress response via autophagy.     

Knot theory realizations in nematic colloids

Nematic braids are reconfigurable knots and links formed by the disclination loops that entangle colloidal particles dispersed in a nematic liquid crystal. We focus on entangled nematic disclinations in thin twisted nematic layers stabilized by 2D arrays of colloidal particles that can be controlled with laser tweezers. We take the experimentally assembled structures and demonstrate the correspondence of the knot invariants, constructed graphs, and surfaces associated with the disclination loop to the physically observable features specific to the geometry at hand. The nematic nature of the medium adds additional topological parameters to the conventional results of knot theory, which couple with the knot topology and introduce order into the phase diagram of possible structures. The crystalline order allows the simplified construction of the Jones polynomial and medial graphs, and the steps in the construction algorithm are mirrored in the physics of liquid crystals.From the invention of ropes and textiles, up to the present day, knots have played a prominent role in everyday life, essential crafts, and artistic expression. Beyond the simple tying of strings, the intriguing irreducibility of knots has led to Kelvin’s vortex model of atoms, and, subsequently, a more systematic study of knots and links in the context of knot theory (1–3). As a branch of topology, knot theory is a developing field, with many unresolved questions, including the ongoing search for an algorithm that will provide an exact identification of arbitrary knots.As knots cannot be converted one into another without the crossing of the strands––a discrete singular event––knotting topologically stabilizes the structure. In physical fields, this coexistence of discrete and continuous phenomena leads to the stabilization of geometrically and topologically nontrivial high-energy excitations (4, 5). Examples of strand-like objects in physics that can be knotted include vortices in fluids (6–9), synthetic molecules (10, 11), DNA, polymer strands and proteins (12–14), electromagnetic field lines (15, 16), zero-intensity loci in optical interference patterns (17), wave functions in topological insulators (18), cosmic strings (19), and defects in a broad selection of ordered media (20–23).Nematic liquid crystals (NLC) are liquids with a local apolar orientational order of rod-like molecules. The director field, which describes the spatial variation of the local alignment axis, supports topological point and line defects, making it an interesting medium for the observation of topological phenomena (20, 24). Defect structures in NLC and their colloidal composites (25) have been extensively studied for their potential in self-assembly and light control (26), but also to further the theoretical understanding of topological phenomena in director fields (23, 27). Objects of interest include chiral solitons (28, 29), fields around knotted particles (30–35), and knotted defects in nematic colloids (36–42). Each of these cases is unique, as the rules of knot theory interact with the rules and restrictions of each underlying material and confinement. The investigation of knotted fields is thus a specialized topic where certain theoretical aspects of knot theory emerge in a physical context.In nematic colloids––dispersions of spherical particles confined in a twisted nematic (TN) cell––disclination lines entangle arrays of particles into “nematic braids,” which can be finely controlled by laser tweezers to form various linked and knotted structures (38, 39, 43). In this paper, we focus on the diverse realizations of knot theory in such nematic colloidal structures. We complement and extend the classification and analysis of knotted disclinations from refs. 32, 38, 40 with the direct application of graph and knot theory to polarized optical micrographs. We further analyze the nematic director with constructed Pontryagin–Thom surfaces and polynomial knot invariants, which enables a comprehensive topological characterization of the knotted nematic field based on experimental data and analytical tools. We use a λ-retardation plate to observe and distinguish differently twisted domains in the optical micrograph, which correspond to medial graphs of the represented knots and contribute to the Pontryagin–Thom construction of the nematic director. Finally, we explore the organization of the space of possible configurations on a selected rectangular particle array and discuss the observed hierarchy of entangled and knotted structures.     

Self-catalytic growth of one-dimensional materials within dislocations in gold

Dislocations in metals affect their properties on the macro- and the microscales. For example, they increase a metal’s hardness and strength. Dislocation outcrops exist on the surfaces of such metals, and atoms in the proximity of these outcrops are more loosely bonded, facilitating local chemical corrosion and reactivity. In this study, we present a unique autocatalytic mechanism by which a system of inorganic semiconducting gold(I) cyanide nanowires forms within preexisting dislocation lines in a plastically deformed Au-Ag alloy. The formation occurs during the classical selective dealloying process that forms nanoporous Au. Nucleation of the nanowire originates at the surfaces of the catalytic dislocation outcrops. The nanowires are single crystals that spontaneously undergo layer-by-layer one-dimensional growth. The continuous growth of nanowires is achieved when the dislocation density exceeds a critical value evaluated on the basis of a kinetic model that we developed.

Dislocations in metals have been known for almost a century, ever since edge dislocations were postulated in 1934 by Orowan (1), Polanyi (2), and Taylor (3). Dislocations can form during crystal growth as well as due to plastic deformation. Dislocations formed in metals lead to an increase in their strength, hardness, and brittleness (4). Dislocation outcrops on the metal surfaces can be easily etched by corrosive chemicals to form dissolution etch pits (5, 6). Atoms at the dislocation outcrops are more reactive and more loosely bonded than other atoms on the surface and therefore demonstrate enhanced chemical reactivity (7–9). It was shown several decades ago that plastically deformed copper (10) and silver (11) are more catalytically active than their annealed counterparts, which have lower dislocation densities. More recently, it was also shown that the chemical properties of strained metal surfaces differ considerably from those of unstrained surfaces (12) and in particular that catalysis can be enhanced near the points of intersection of edge dislocations with the surface (13). Here, we demonstrate that dislocations formed upon plastic deformation in a Au-Ag alloy can serve as catalytic centers for the nucleation of one-dimensional (1D) semiconducting gold(I) cyanide nanowires (AuCN NWs). We further show that the dislocation lines serve as templates for the formation of nanowires (NWs).We subjected an Au₅₀Ag₅₀ alloy to varying degrees of plastic deformation, followed by the classical free-corrosion selective dealloying of Ag, a procedure known to form nanoporous (np) Au (14–19). The latter exhibits multiple properties, such as high surface area (20–22), chemical and thermal stability (23, 24), and excellent catalytic activity (23, 25–28). Owing to its wide range of properties, np-Au has been used extensively in sensors (29, 30), catalysis (15, 27, 31–33), rechargeable batteries (34, 35), actuators (36, 37), electrodes (38–40), and more.Here, we present a method for the formation of AuCN NWs along dislocation lines, which provide a template for their growth and are integrated in a classical np-Au structure. We did this by instigating an autocatalytic reaction on the dislocation outcrops of plastically deformed Au-Ag alloys during selective dissolution of the Ag. This work reports the layer-by-layer 1D growth of AuCN NWs by means of an autocatalytic reaction at dislocation outcrops. We developed a kinetic model that demonstrates the correlation between AuCN NWs formation and the dislocation density in the predeformed alloy.     

Active topolectrical circuits

The transfer of topological concepts from the quantum world to classical mechanical and electronic systems has opened fundamentally different approaches to protected information transmission and wave guidance. A particularly promising emergent technology is based on recently discovered topolectrical circuits that achieve robust electric signal transduction by mimicking edge currents in quantum Hall systems. In parallel, modern active matter research has shown how autonomous units driven by internal energy reservoirs can spontaneously self-organize into collective coherent dynamics. Here, we unify key ideas from these two previously disparate fields to develop design principles for active topolectrical circuits (ATCs) that can self-excite topologically protected global signal patterns. Realizing autonomous active units through nonlinear Chua diode circuits, we theoretically predict and experimentally confirm the emergence of self-organized protected edge oscillations in one- and two-dimensional ATCs. The close agreement between theory, simulations, and experiments implies that nonlinear ATCs provide a robust and versatile platform for developing high-dimensional autonomous electrical circuits with topologically protected functionalities.

Information transfer and storage in natural and man-made active systems, from sensory organs (1–3) to the internet, rely on the robust exchange of electrical signals between a large number of autonomous units that balance local energy uptake and dissipation (4, 5). Major advances in the understanding of photonic (6–9), acoustic (10–12), and mechanical (13–16) metamaterials have shown that topological protection (17–24) enables the stabilization and localization of signal propagation in passive and active (25–27) dynamical systems that violate time-reversal and/or other symmetries. Recent studies have successfully applied these ideas to realize topolectrical circuits (28) in the passive linear (29–34) and passive nonlinear (35, 36) regimes. However, despite substantial progress in the development of topological wave guides (37), lasers (38, 39), and transmission lines (40–43), the transfer of these concepts to active (44, 45) nonlinear circuits made from autonomously acting units still poses an unsolved challenge. From a broader perspective, not only does harnessing topological protection in nonlinear active circuits promise a new generation of autonomous devices, but also understanding their design and self-organization principles may offer insights into information storage and processing mechanisms in living systems, which integrate cellular activity, electrical signaling, and nonlinear feedback to coordinate essential biological functions (46, 47).Exploiting a mathematical analogy with active Brownian particle systems (26), we theoretically develop and experimentally demonstrate general design principles for active topolectrical circuits (ATCs) that achieve self-organized, self-sustained, topologically protected electric current patterns. The main building blocks of ATCs are nonlinear dissipative elements that exhibit an effectively negative resistance over a certain voltage range. Negative resistances can be realized using van der Pol (vdP) circuits (48), tunnel diodes, unijunction transistors, solid-state thyristors (49), or operational amplifiers set as negative impedance converters through current inversion (50), and the design principles described below are applicable to all these systems. Indeed, we expect them to apply to an even broader class of nonlinear systems, as similar dynamics also describe electromagnetic resonators with Kerr-type nonlinearities (51–53).     

Wnt signaling recruits KIF2A to the spindle to ensure chromosome congression and alignment during mitosis

Canonical Wnt signaling plays critical roles in development and tissue renewal by regulating β-catenin target genes. Recent evidence showed that β-catenin–independent Wnt signaling is also required for faithful execution of mitosis. However, the targets and specific functions of mitotic Wnt signaling still remain uncharacterized. Using phosphoproteomics, we identified that Wnt signaling regulates the microtubule depolymerase KIF2A during mitosis. We found that Dishevelled recruits KIF2A via its N-terminal and motor domains, which is further promoted upon LRP6 signalosome formation during cell division. We show that Wnt signaling modulates KIF2A interaction with PLK1, which is critical for KIF2A localization at the spindle. Accordingly, inhibition of basal Wnt signaling leads to chromosome misalignment in somatic cells and pluripotent stem cells. We propose that Wnt signaling monitors KIF2A activity at the spindle poles during mitosis to ensure timely chromosome alignment. Our findings highlight a function of Wnt signaling during cell division, which could have important implications for genome maintenance, notably in stem cells.

The canonical Wnt signaling pathway plays essential roles in embryonic development and tissue homeostasis (1, 2). In particular, Wnt signaling governs stem cell maintenance and proliferation in many tissues, and its misregulation is a common cause of tumor initiation (3, 4).Wnt ligands bind Frizzled (FZD) receptors and the coreceptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) (5). The activated receptor complexes cluster on Dishevelled (DVL) platforms and are internalized via caveolin into endosomes termed LRP6 signalosomes, which triggers sequential phosphorylation of LRP6 by GSK3β and CK1γ (6–10). LRP6 signalosomes recruit the β-catenin destruction complex, which contains the scaffold proteins AXIN1 and adenomatous polyposis coli, the kinases CK1α and GSK3β, and the E3 ubiquitin ligase β-TrCP (11). This recruitment inhibits GSK3β and releases β-TrCP, which leads to β-catenin stabilization and nuclear translocation in a IFT-A/KIF3A–dependent manner (12–16). LRP6 signalosomes mature into multivesicular bodies, sequestering the Wnt receptors together with GSK3β, thereby maintaining long-term activation of the Wnt pathway and promoting macropinocytosis (14, 17–21). In contrast to Wnt ligands, the Wnt inhibitor Dickkopf-related protein 1 (DKK1) induces the clathrin-dependent internalization and turnover of LRP5/6 and thereby abrogates canonical Wnt signaling (22).LRP6 signalosome formation peaks in mitosis (23, 24). On the one hand, the LRP6 competence to respond to Wnt ligands is promoted during G2/M by a priming phosphorylation at its intracellular domain by CDK14/16 and CCNY/CCNYL1 (24, 25). On the other hand, CDK1 phosphorylates and recruits B-cell CLL/lymphoma 9 (BCL9) to the mitotic LRP6 signalosomes (23). BCL9 protects the signalosome from clathrin-dependent turnover, thereby sustaining basal Wnt activity on the onset of mitosis.Mitotic Wnt signaling not only modulates β-catenin (24) but increasing evidence suggests that it promotes a complex posttranslational program during mitosis (26). For instance, we have shown that mitotic Wnt signaling promotes stabilization of proteins (Wnt/STOP), which is required for cell growth and ensures chromosome segregation in somatic and embryonic cells (23, 26–31). Particularly, basal Wnt/STOP activity maintains proper microtubule plus-end polymerization rates during mitosis, and its misregulation leads to whole chromosome missegregation (31, 32). Furthermore, mitotic Wnt signaling controls the orientation of the spindle (33) and promotes asymmetric division in stem cells through components of the LRP6 signalosome (34). Accordingly, several Wnt components functionally associate with centrosomes, kinetochores, and the spindle during mitosis (25, 33, 35, 36). Consequently, both aberrant up-regulation or down-regulation of Wnt signaling have been associated with chromosome instability (CIN) (31, 32, 35, 37), which is a hallmark of cancer (38). Despite the importance of these processes for tissue renewal and genome maintenance, the targets and specific functions of mitotic Wnt signaling remain largely uncharacterized.Kinesin family member 2A (KIF2A) is a member of the kinesin-13 group (KIF2A,B,C) of minus-end microtubule depolymerases (39–41). KIF2A is essential for the scaling of the spindle during early development (42) and plays critical roles in neurogenesis by modulating both cilium disassembly and neuronal wiring (43–47). In dividing cells, KIF2A was thought to be required for the assembly of a bipolar spindle due to a small interfering RNA (siRNA) off-target effect (48, 49). Current evidence supports a role of KIF2A in microtubule depolymerization at the spindle poles, which can generate pulling forces on attached kinetochores, thereby ensuring the congression, alignment, and segregation of chromosomes (50–56). Genetic depletion of KIF2A in mouse leads to neonatal lethality and to severe brain malformations, including microcephaly (43, 44, 57). KIF2A recruitment to microtubules is tightly coordinated by several protein kinases (45, 47, 50–52, 58–60). For instance, phosphorylation of KIF2A at several sites by Polo-like kinase 1 (PLK1) stimulates its recruitment to and activity at the spindle (45, 58, 61). On the other hand, Aurora kinase A and B inhibit KIF2A activity and restrict its subcellular localization during mitosis (50, 58, 60).Here, we show that mitotic Wnt signaling promotes chromosome congression and alignment in prometaphase by recruiting KIF2A to the spindle in both somatic cells and pluripotent stem cells. We found that KIF2A is recruited by the LRP6 signalosome during mitosis. Mechanistically, we identified that KIF2A clusters with DVL via the N-terminal and motor domains of the depolymerase. We show that Wnt signaling controls KIF2A interaction with PLK1, which is critical for KIF2A localization at the spindle poles. We propose that basal Wnt signaling ensures timely chromosome congression and alignment prior cell division by modulating the spindle minus-end depolymerization dynamics through KIF2A.