DIX Domain Polymerization Drives Assembly of Plant Cell Polarity Complexes.

Cell polarity is fundamental for tissue morphogenesis in multicellular organisms. Plants and animals evolved multicellularity independently, and it is unknown whether their polarity systems are derived from a single-celled ancestor. Planar polarity in animals is conferred by Wnt signaling, an ancient signaling pathway transduced by Dishevelled, which assembles signalosomes by dynamic head-to-tail DIX domain polymerization. In contrast, polarity-determining pathways in plants are elusive. We recently discovered Arabidopsis SOSEKI proteins, which exhibit polar localization throughout development. Here, we identify SOSEKI as ancient polar proteins across land plants. Concentration-dependent polymerization via a bona fide DIX domain allows these to recruit ANGUSTIFOLIA to polar sites, similar to the polymerization-dependent recruitment of signaling effectors by Dishevelled. Cross-kingdom domain swaps reveal functional equivalence of animal and plant DIX domains. We trace DIX domains to unicellular eukaryotes and thus show that DIX-dependent polymerization is an ancient mechanism conserved between kingdoms and central to polarity proteins.

Exosomes.

Exosomes are small, single-membrane, secreted organelles of ∼30 to ∼200 nm in diameter that have the same topology as the cell and are enriched in selected proteins, lipids, nucleic acids, and glycoconjugates. Exosomes contain an array of membrane-associated, high-order oligomeric protein complexes, display pronounced molecular heterogeneity, and are created by budding at both plasma and endosome membranes. Exosome biogenesis is a mechanism of protein quality control, and once released, exosomes have activities as diverse as remodeling the extracellular matrix and transmitting signals and molecules to other cells. This pathway of intercellular vesicle traffic plays important roles in many aspects of human health and disease, including development, immunity, tissue homeostasis, cancer, and neurodegenerative diseases. In addition, viruses co-opt exosome biogenesis pathways both for assembling infectious particles and for establishing host permissiveness. On the basis of these and other properties, exosomes are being developed as therapeutic agents in multiple disease models.

Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds.

Liquid-liquid phase separation plays a key role in the assembly of diverse intracellular structures. However, the biophysical principles by which phase separation can be precisely localized within subregions of the cell are still largely unclear, particularly for low-abundance proteins. Here, we introduce an oligomerizing biomimetic system, "Corelets," and utilize its rapid and quantitative light-controlled tunability to map full intracellular phase diagrams, which dictate the concentrations at which phase separation occurs and the transition mechanism, in a protein sequence dependent manner. Surprisingly, both experiments and simulations show that while intracellular concentrations may be insufficient for global phase separation, sequestering protein ligands to slowly diffusing nucleation centers can move the cell into a different region of the phase diagram, resulting in localized phase separation. This diffusive capture mechanism liberates the cell from the constraints of global protein abundance and is likely exploited to pattern condensates associated with diverse biological processes. VIDEO ABSTRACT.

Ribosome-rescuer PELO catalyzes the oligomeric assembly of NOD-like receptor family proteins via activating their ATPase enzymatic activity.

NOD-like receptors (NLRs) are pattern recognition receptors for diverse innate immune responses. Self-oligomerization after engagement with a ligand is a generally accepted model for the activation of each NLR. We report here that a catalyzer was required for NLR self-oligomerization. PELO, a well-known surveillance factor in translational quality control and/or ribosome rescue, interacted with all cytosolic NLRs and activated their ATPase activity. In the case of flagellin-initiated NLRC4 inflammasome activation, flagellin-bound NAIP5 recruited the first NLRC4 and then PELO was required for correctly assembling the rest of NLRC4s into the NLRC4 complex, one by one, by activating the NLRC4 ATPase activity. Stoichiometric and functional data revealed that PELO was not a structural constituent of the NLRC4 inflammasome but a powerful catalyzer for its assembly. The catalytic role of PELO in the activation of cytosolic NLRs provides insight into NLR activation and provides a direction for future studies of NLR family members.

Centromere/kinetochore is assembled through CENP-C oligomerization.

Kinetochore is an essential protein complex required for accurate chromosome segregation. The constitutive centromere-associated network (CCAN), a subcomplex of the kinetochore, associates with centromeric chromatin and provides a platform for the kinetochore assembly. The CCAN protein CENP-C is thought to be a central hub for the centromere/kinetochore organization. However, the role of CENP-C in CCAN assembly needs to be elucidated. Here, we demonstrate that both the CCAN-binding domain and the C-terminal region that includes the Cupin domain of CENP-C are necessary and sufficient for chicken CENP-C function. Structural and biochemical analyses reveal self-oligomerization of the Cupin domains of chicken and human CENP-C. We find that the CENP-C Cupin domain oligomerization is vital for CENP-C function, centromeric localization of CCAN, and centromeric chromatin organization. These results suggest that CENP-C facilitates the centromere/kinetochore assembly through its oligomerization.

Integration of light and temperature sensing by liquid-liquid phase separation of phytochrome B.

Light and temperature in plants are perceived by a common receptor, phytochrome B (phyB). How phyB distinguishes these signals remains elusive. Here, we report that phyB spontaneously undergoes phase separation to assemble liquid-like droplets. This capacity is driven by its C terminus through self-association, whereas the intrinsically disordered N-terminal extension (NTE) functions as a biophysical modulator of phase separation. Light exposure triggers a conformational change to subsequently alter phyB condensate assembly, while temperature sensation is directly mediated by the NTE to modulate the phase behavior of phyB droplets. Multiple signaling components are selectively incorporated into phyB droplets to form concentrated microreactors, allowing switch-like control of phyB signaling activity through phase transitions. Therefore, light and temperature cues are separately read out by phyB via allosteric changes and spontaneous phase separation, respectively. We provide a conceptual framework showing how the distinct but highly correlated physical signals are interpreted and sorted by one receptor.

A pro-metastatic tRNA fragment drives Nucleolin oligomerization and stabilization of its bound metabolic mRNAs.

Stress-induced cleavage of transfer RNAs (tRNAs) into tRNA-derived fragments (tRFs) occurs across organisms from yeast to humans; yet, its mechanistic underpinnings and pathological consequences remain poorly defined. Small RNA profiling revealed increased abundance of a cysteine tRNA fragment (5'-tRFCys) during breast cancer metastatic progression. 5'-tRFCys was required for efficient breast cancer metastatic lung colonization and cancer cell survival. We identified Nucleolin as the direct binding partner of 5'-tRFCys. 5'-tRFCys promoted the oligomerization of Nucleolin and its bound metabolic transcripts Mthfd1l and Pafah1b1 into a higher-order transcript stabilizing ribonucleoprotein complex, which protected these transcripts from exonucleolytic degradation. Consistent with this, Mthfd1l and Pafah1b1 mediated pro-metastatic and metabolic effects downstream of 5'-tRFCys-impacting folate, one-carbon, and phosphatidylcholine metabolism. Our findings reveal that a tRF can promote oligomerization of an RNA-binding protein into a transcript stabilizing ribonucleoprotein complex, thereby driving specific metabolic pathways underlying cancer progression.

Demystifying heparan sulfate-protein interactions.

Numerous proteins, including cytokines and chemokines, enzymes and enzyme inhibitors, extracellular matrix proteins, and membrane receptors, bind heparin. Although they are traditionally classified as heparin-binding proteins, under normal physiological conditions these proteins actually interact with the heparan sulfate chains of one or more membrane or extracellular proteoglycans. Thus, they are more appropriately classified as heparan sulfate-binding proteins (HSBPs). This review provides an overview of the various modes of interaction between heparan sulfate and HSBPs, emphasizing biochemical and structural insights that improve our understanding of the many biological functions of heparan sulfate.

Interaction of tau with HNRNPA2B1 and N6-methyladenosine RNA mediates the progression of tauopathy.

The microtubule-associated protein tau oligomerizes, but the actions of oligomeric tau (oTau) are unknown. We have used Cry2-based optogenetics to induce tau oligomers (oTau-c). Optical induction of oTau-c elicits tau phosphorylation, aggregation, and a translational stress response that includes stress granules and reduced protein synthesis. Proteomic analysis identifies HNRNPA2B1 as a principle target of oTau-c. The association of HNRNPA2B1 with endogenous oTau was verified in neurons, animal models, and human Alzheimer brain tissues. Mechanistic studies demonstrate that HNRNPA2B1 functions as a linker, connecting oTau with N6-methyladenosine (m6A) modified RNA transcripts. Knockdown of HNRNPA2B1 prevents oTau or oTau-c from associating with m6A or from reducing protein synthesis and reduces oTau-induced neurodegeneration. Levels of m6A and the m6A-oTau-HNRNPA2B1 complex are increased up to 5-fold in the brains of Alzheimer subjects and P301S tau mice. These results reveal a complex containing oTau, HNRNPA2B1, and m6A that contributes to the integrated stress response of oTau.

Structural insights into the ubiquitylation strategy of the oligomeric CRL2FEM1B E3 ubiquitin ligase.

Cullin-RING E3 ubiquitin ligase (CRL) family members play critical roles in numerous biological processes and diseases including cancer and Alzheimer's disease. Oligomerization of CRLs has been reported to be crucial for the regulation of their activities. However, the structural basis for its regulation and mechanism of its oligomerization are not fully known. Here, we present cryo-EM structures of oligomeric CRL2FEM1B in its unneddylated state, neddylated state in complex with BEX2 as well as neddylated state in complex with FNIP1/FLCN. These structures reveal that asymmetric dimerization of N8-CRL2FEM1B is critical for the ubiquitylation of BEX2 while FNIP1/FLCN is ubiquitylated by monomeric CRL2FEM1B. Our data present an example of the asymmetric homo-dimerization of CRL. Taken together, this study sheds light on the ubiquitylation strategy of oligomeric CRL2FEM1B according to substrates with different scales.

Structural insights into the activation mechanism of antimicrobial GBP1.

The dynamin-related human guanylate-binding protein 1 (GBP1) mediates host defenses against microbial pathogens. Upon GTP binding and hydrolysis, auto-inhibited GBP1 monomers dimerize and assemble into soluble and membrane-bound oligomers, which are crucial for innate immune responses. How higher-order GBP1 oligomers are built from dimers, and how assembly is coordinated with nucleotide-dependent conformational changes, has remained elusive. Here, we present cryo-electron microscopy-based structural data of soluble and membrane-bound GBP1 oligomers, which show that GBP1 assembles in an outstretched dimeric conformation. We identify a surface-exposed helix in the large GTPase domain that contributes to the oligomerization interface, and we probe its nucleotide- and dimerization-dependent movements that facilitate the formation of an antimicrobial protein coat on a gram-negative bacterial pathogen. Our results reveal a sophisticated activation mechanism for GBP1, in which nucleotide-dependent structural changes coordinate dimerization, oligomerization, and membrane binding to allow encapsulation of pathogens within an antimicrobial protein coat.

Loss of TDP-43 oligomerization or RNA binding elicits distinct aggregation patterns.

Aggregation of the RNA-binding protein TAR DNA-binding protein 43 (TDP-43) is the key neuropathological feature of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). In physiological conditions, TDP-43 is predominantly nuclear, forms oligomers, and is contained in biomolecular condensates assembled by liquid-liquid phase separation (LLPS). In disease, TDP-43 forms cytoplasmic or intranuclear inclusions. How TDP-43 transitions from physiological to pathological states remains poorly understood. Using a variety of cellular systems to express structure-based TDP-43 variants, including human neurons and cell lines with near-physiological expression levels, we show that oligomerization and RNA binding govern TDP-43 stability, splicing functionality, LLPS, and subcellular localization. Importantly, our data reveal that TDP-43 oligomerization is modulated by RNA binding. By mimicking the impaired proteasomal activity observed in ALS/FTLD patients, we found that monomeric TDP-43 forms inclusions in the cytoplasm, whereas its RNA binding-deficient counterpart aggregated in the nucleus. These differentially localized aggregates emerged via distinct pathways: LLPS-driven aggregation in the nucleus and aggresome-dependent inclusion formation in the cytoplasm. Therefore, our work unravels the origins of heterogeneous pathological species reminiscent of those occurring in TDP-43 proteinopathy patients.

An updated view of the kinetochore architecture.

The kinetochore is a supramolecular complex that facilitates faithful chromosome segregation by bridging the centromere and spindle microtubules. Recent functional and structural studies on the inner kinetochore subcomplex, constitutive centromere-associated network (CCAN) have updated our understanding of kinetochore architecture. While the CCAN core establishes a stable interface with centromeric chromatin, CCAN organization is dynamically altered and coupled with cell cycle progression. Furthermore, the CCAN components, centromere protein (CENP)-C and CENP-T, mediate higher-order assembly of multiple kinetochore units on the regional centromeres of vertebrates. This review highlights new insights into kinetochore rigidity, plasticity, and clustering, which are key to understanding temporal and spatial regulatory mechanisms of chromosome segregation.

PINK1 Phosphorylates MIC60/Mitofilin to Control Structural Plasticity of Mitochondrial Crista Junctions.

Mitochondrial crista structure partitions vital cellular reactions and is precisely regulated by diverse cellular signals. Here, we show that, in Drosophila, mitochondrial cristae undergo dynamic remodeling among distinct subcellular regions and the Parkinson's disease (PD)-linked Ser/Thr kinase PINK1 participates in their regulation. Mitochondria increase crista junctions and numbers in selective subcellular areas, and this remodeling requires PINK1 to phosphorylate the inner mitochondrial membrane protein MIC60/mitofilin, which stabilizes MIC60 oligomerization. Expression of MIC60 restores crista structure and ATP levels of PINK1-null flies and remarkably rescues their behavioral defects and dopaminergic neurodegeneration. In an extension to human relevance, we discover that the PINK1-MIC60 pathway is conserved in human neurons, and expression of several MIC60 coding variants in the mitochondrial targeting sequence found in PD patients in Drosophila impairs crista junction formation and causes locomotion deficits. These findings highlight the importance of maintenance and plasticity of crista junctions to cellular homeostasis in vivo.

RIG-I Uses an ATPase-Powered Translocation-Throttling Mechanism for Kinetic Proofreading of RNAs and Oligomerization.

RIG-I has a remarkable ability to specifically select viral 5'ppp dsRNAs for activation from a pool of cytosolic self-RNAs. The ATPase activity of RIG-I plays a role in RNA discrimination and activation, but the underlying mechanism was unclear. Using transient-state kinetics, we elucidated the ATPase-driven "kinetic proofreading" mechanism of RIG-I activation and RNA discrimination, akin to DNA polymerases, ribosomes, and T cell receptors. Even in the autoinhibited state of RIG-I, the C-terminal domain kinetically discriminates against self-RNAs by fast off rates. ATP binding facilitates dsRNA engagement but, interestingly, makes RIG-I promiscuous, explaining the constitutive signaling by Singleton-Merten syndrome-linked mutants that bind ATP without hydrolysis. ATP hydrolysis dissociates self-RNAs faster than 5'ppp dsRNA but, more importantly, drives RIG-I oligomerization through translocation, which we show to be regulated by helicase motif IVa. RIG-I translocates directionally from the dsRNA end into the stem region, and the 5'ppp end "throttles" translocation to provide a mechanism for threading and building a signaling-active oligomeric complex.

Oligomer-to-monomer transition underlies the chaperone function of AAGAB in AP1/AP2 assembly.

Assembly of protein complexes is facilitated by assembly chaperones. Alpha and gamma adaptin-binding protein (AAGAB) is a chaperone governing the assembly of the heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) involved in clathrin-mediated membrane trafficking. Here, we found that before AP1/2 binding, AAGAB exists as a homodimer. AAGAB dimerization is mediated by its C-terminal domain (CTD), which is critical for AAGAB stability and is missing in mutant proteins found in patients with the skin disease punctate palmoplantar keratoderma type 1 (PPKP1). We solved the crystal structure of the dimerization-mediating CTD, revealing an antiparallel dimer of bent helices. Interestingly, AAGAB uses the same CTD to recognize and stabilize the γ subunit in the AP1 complex and the α subunit in the AP2 complex, forming binary complexes containing only one copy of AAGAB. These findings demonstrate a dual role of CTD in stabilizing resting AAGAB and binding to substrates, providing a molecular explanation for disease-causing AAGAB mutations. The oligomerization state transition mechanism may also underlie the functions of other assembly chaperones.

Activation of caspase-9 on the apoptosome as studied by methyl-TROSY NMR.

Mitochondrial apoptotic signaling cascades lead to the formation of the apoptosome, a 1.1-MDa heptameric protein scaffold that recruits and activates the caspase-9 protease. Once activated, caspase-9 cleaves and activates downstream effector caspases, triggering the onset of cell death through caspase-mediated proteolysis of cellular proteins. Failure to activate caspase-9 enables the evasion of programmed cell death, which occurs in various forms of cancer. Despite the critical apoptotic function of caspase-9, the structural mechanism by which it is activated on the apoptosome has remained elusive. Here, we used a combination of methyl-transverse relaxation-optimized NMR spectroscopy, protein engineering, and biochemical assays to study the activation of caspase-9 bound to the apoptosome. In the absence of peptide substrate, we observed that both caspase-9 and its isolated protease domain (PD) only very weakly dimerize with dissociation constants in the millimolar range. Methyl-NMR spectra of isotope-labeled caspase-9, within the 1.3-MDa native apoptosome complex or an engineered 480-kDa apoptosome mimic, reveal that the caspase-9 PD remains monomeric after recruitment to the scaffold. Binding to the apoptosome, therefore, organizes caspase-9 PDs so that they can rapidly and extensively dimerize only when substrate is present, providing an important layer in the regulation of caspase-9 activation. Our work highlights the unique role of NMR spectroscopy to structurally characterize protein domains that are flexibly tethered to large scaffolds, even in cases where the molecular targets are in excess of 1 MDa, as in the present example.

Structural analysis of the SAM domain of the Arabidopsis mitochondrial tRNA import receptor.

Mitochondria are membrane-bound organelles of endosymbiotic origin with limited protein-coding capacity. The import of nuclear-encoded proteins and nucleic acids is required and essential for maintaining organelle mass, number, and activity. As plant mitochondria do not encode all the necessary tRNA types required, the import of cytosolic tRNA is vital for organelle maintenance. Recently, two mitochondrial outer membrane proteins, named Tric1 and Tric2, for tRNA import component, were shown to be involved in the import of cytosolic tRNA. Tric1/2 binds tRNAalavia conserved residues in the C-terminal Sterile Alpha Motif (SAM) domain. Here we report the X-ray crystal structure of the Tric1 SAM domain. We identified the ability of the SAM domain to form a helical superstructure with six monomers per helical turn and key amino acid residues responsible for its formation. We determined that the oligomerization of the Tric1 SAM domain may play a role in protein function whereby mutation of Gly241 introducing a larger side chain at this position disrupted the oligomer and resulted in the loss of RNA binding capability. Furthermore, complementation of Arabidopsis thaliana Tric1/2 knockout lines with a mutated Tric1 failed to restore the defective plant phenotype. AlphaFold2 structure prediction of both the SAM domain and Tric1 support a cyclic pentameric or hexameric structure. In the case of a hexameric structure, a pore of sufficient dimensions to transfer tRNA across the mitochondrial membrane is observed. Our results highlight the importance of oligomerization of Tric1 for protein function.

Transmembrane helix interactions regulate oligomerization of the receptor tyrosine kinase EphA2.

The transmembrane helices of receptor tyrosine kinases (RTKs) have been proposed to switch between two different dimeric conformations, one associated with the inactive RTK and the other with the active RTK. Furthermore, recent work has demonstrated that some full-length RTKs are associated into oligomers that are larger than dimers, raising questions about the roles of the TM helices in the assembly and function of these oligomers. Here we probe the roles of the TM helices in the assembly of EphA2 RTK oligomers in the plasma membrane. We employ mutagenesis to evaluate the relevance of a published NMR dimeric structure of the isolated EphA2 TM helix in the context of the full-length EphA2 in the plasma membrane. We use two fluorescence methods, Förster Resonance Energy Transfer and Fluorescence Intensity Fluctuations spectrometry, which yield complementary information about the EphA2 oligomerization process. These studies reveal that the TM helix mutations affect the stability, structure, and size of EphA2 oligomers. However, the effects are multifaceted and point to a more complex role of the TM helix than the one expected from the "TM dimer switch" model.

Mechanistic insights into the C-type lectin receptor CLEC12A-mediated immune recognition of monosodium urate crystal.

CLEC12A, a member of the C-type lectin receptor family involved in immune homeostasis, recognizes MSU crystals released from dying cells. However, the molecular mechanism underlying the CLEC12A-mediated recognition of MSU crystals remains unclear. Herein, we reported the crystal structure of the human CLEC12A-C-type lectin-like domain (CTLD) and identified a unique "basic patch" site on CLEC12A-CTLD that is necessary for the binding of MSU crystals. Meanwhile, we determined the interaction strength between CLEC12A-CTLD and MSU crystals using single-molecule force spectroscopy. Furthermore, we found that CLEC12A clusters at the cell membrane and seems to serve as an internalizing receptor of MSU crystals. Altogether, these findings provide mechanistic insights for understanding the molecular mechanisms underlying the interplay between CLEC12A and MSU crystals.