The START domain potentiates HD-ZIPIII transcriptional activity.

The CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIPIII) transcription factors (TFs) were repeatedly deployed over 725 million years of evolution to regulate central developmental innovations. The START domain of this pivotal class of developmental regulators was recognized over 20 years ago, but its putative ligands and functional contributions remain unknown. Here, we demonstrate that the START domain promotes HD-ZIPIII TF homodimerization and increases transcriptional potency. Effects on transcriptional output can be ported onto heterologous TFs, consistent with principles of evolution via domain capture. We also show the START domain binds several species of phospholipids, and that mutations in conserved residues perturbing ligand binding and/or its downstream conformational readout abolish HD-ZIPIII DNA-binding competence. Our data present a model in which the START domain potentiates transcriptional activity and uses ligand-induced conformational change to render HD-ZIPIII dimers competent to bind DNA. These findings resolve a long-standing mystery in plant development and highlight the flexible and diverse regulatory potential coded within this widely distributed evolutionary module.

Evaluation of Peripapillary and Macular Optical Coherence Tomography Angiography Characteristics in Different Stages of Papilledema.

BACKGROUND: Prospective evaluation of optical coherence tomography (OCT) and OCT angiography (OCT-A) characteristics in different stages of papilledema in idiopathic intracranial hypertension (IIH). METHODS: In this prospective, observational study patients of IIH with papilledema were recruited and divided into 3 groups-early/established (Group 1), chronic (Group 2), and atrophic papilledema (Group 3). Peripapillary retinal nerve fiber layer (RNFL) and macular ganglion cell inner plexiform layer (GC-IPL) were recorded on OCT. Peripapillary and macular perfusion was documented at superficial retinal, deep retinal, and choriocapillary level using OCT-A. The investigations were repeated at 3 months. RESULTS: RNFL showed significant thinning in all groups on follow-up with the atrophic group showing maximum thinning ( P = 0.01-Group 3). GC-IPL was significantly reduced in all stages of papilledema at baseline compared with the controls. Thinnest GC-IPL was noted in the atrophic group (52.75 ± 7.44 µm; P = 0.00 in Group 3 vs controls) that showed further deterioration on follow-up. On Image J analysis, significant decrease was noted at various levels in the peripapillary and macular perfusion at baseline especially in the atrophic group which showed further deterioration noted on follow-up. The final visual acuity showed a statistically significant weak negative correlation with baseline RNFL (r = -0.306) and GC-IPL (r = -0.384) and moderately negative correlation with baseline superficial peripapillary retinal perfusion (r = -0.553). A significant negative correlation was seen between increasing grade of papilledema and superficial peripapillary retinal perfusion with both Image J and automated indices (r = -0.46; r = -0.61), respectively. CONCLUSIONS: GC-IPL may help identify early damage in papilledema even in the presence of thicker RNFL. Significant vascular changes can be observed on OCT-A that may help predict the final visual outcome in papilledema due to IIH.

Contractility kits promote assembly of the mechanoresponsive cytoskeletal network.

Cellular contractility is governed by a control system of proteins that integrates internal and external cues to drive diverse shape change processes. This contractility controller includes myosin II motors, actin crosslinkers and protein scaffolds, which exhibit robust and cooperative mechanoaccumulation. However, the biochemical interactions and feedback mechanisms that drive the controller remain unknown. Here, we use a proteomics approach to identify direct interactors of two key nodes of the contractility controller in the social amoeba Dictyostelium discoideum: the actin crosslinker cortexillin I and the scaffolding protein IQGAP2. We highlight several unexpected proteins that suggest feedback from metabolic and RNA-binding proteins on the contractility controller. Quantitative in vivo biochemical measurements reveal direct interactions between myosin II and cortexillin I, which form the core mechanosensor. Furthermore, IQGAP1 negatively regulates mechanoresponsiveness by competing with IQGAP2 for binding the myosin II-cortexillin I complex. These myosin II-cortexillin I-IQGAP2 complexes are pre-assembled into higher-order mechanoresponsive contractility kits (MCKs) that are poised to integrate into the cortex upon diffusional encounter coincident with mechanical inputs.This article has an associated First Person interview with the first author of the paper.

In Planta Single-Molecule Pull-Down Reveals Tetrameric Stoichiometry of HD-ZIPIII:LITTLE ZIPPER Complexes.

Deciphering complex biological processes markedly benefits from approaches that directly assess the underlying biomolecular interactions. Most commonly used approaches to monitor protein-protein interactions typically provide nonquantitative readouts that lack statistical power and do not yield information on the heterogeneity or stoichiometry of protein complexes. Single-molecule pull-down (SiMPull) uses single-molecule fluorescence detection to mitigate these disadvantages and can quantitatively interrogate interactions between proteins and other compounds, such as nucleic acids, small molecule ligands, and lipids. Here, we establish SiMPull in plants using the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) and LITTLE ZIPPER (ZPR) interaction as proof-of-principle. Colocalization analysis of fluorophore-tagged HD-ZIPIII and ZPR proteins provides strong statistical evidence of complex formation. In addition, we use SiMPull to directly quantify YFP and mCherry maturation probabilities, showing these differ substantially from values obtained in mammalian systems. Leveraging these probabilities, in conjunction with fluorophore photobleaching assays on over 2000 individual complexes, we determined HD-ZIPIII:ZPR stoichiometry. Intriguingly, these complexes appear as heterotetramers, comprising two HD-ZIPIII and two ZPR molecules, rather than heterodimers as described in the current model. This surprising result raises new questions about the regulation of these key developmental factors and is illustrative of the unique contribution SiMPull is poised to make to in planta protein interaction studies.

BEND3 represses rDNA transcription by stabilizing a NoRC component via USP21 deubiquitinase.

Ribosome biogenesis dictates the translational capacity of cells. Several mechanisms establish and maintain transcriptional output from eukaryotic ribosomal DNA (rDNA) loci. rDNA silencing is one such mechanism that ensures the inactivity and hence the maintenance of a silenced state of a subset of rRNA gene copies. Whereas oncogenic agents stimulate rRNA gene transcription, tumor suppressors decrease rRNA gene transcription. We demonstrate in mammalian cells that BANP, E5R, and Nac1 (BEN) domain 3 (BEND3), a quadruple BEN domain-containing protein, localizes in nucleoli and binds to ribosomal RNA gene promoters to help repress rRNA genes. Loss of BEND3 increases histone H3K4 trimethylation and, correspondingly, decreases rDNA promoter DNA methylation, consistent with a role for BEND3 in rDNA silencing. BEND3 associates with the nucleolar-remodeling complex (NoRC), and SUMOylated BEND3 stabilizes NoRC component TTF-1-interacting protein 5 via association with ubiquitin specific protease 21 (USP21) debiquitinase. Our results provide mechanistic insights into how the novel rDNA transcription repressor BEND3 acts together with NoRC to actively coordinate the establishment of rDNA silencing.

An improved surface passivation method for single-molecule studies.

We report a surface passivation method based on dichlorodimethylsilane (DDS)-Tween-20 for in vitro single-molecule studies, which, under the conditions tested here, more efficiently prevented nonspecific binding of biomolecules than the standard poly(ethylene glycol) surface. The DDS-Tween-20 surface was simple and inexpensive to prepare and did not perturb the behavior and activities of tethered biomolecules. It can also be used for single-molecule imaging in the presence of high concentrations of labeled species in solution.

Stoichiometry and assembly of mTOR complexes revealed by single-molecule pulldown.

The mammalian target of rapamycin (mTOR) kinase is a master regulator of cellular, developmental, and metabolic processes. Deregulation of mTOR signaling is implicated in numerous human diseases including cancer and diabetes. mTOR functions as part of either of the two multisubunit complexes, mTORC1 and mTORC2, but molecular details about the assembly and oligomerization of mTORCs are currently lacking. We use the single-molecule pulldown (SiMPull) assay that combines principles of conventional pulldown assays with single-molecule fluorescence microscopy to investigate the stoichiometry and assembly of mTORCs. After validating our approach with mTORC1, confirming a dimeric assembly as previously reported, we show that all major components of mTORC2 exist in two copies per complex, indicating that mTORC2 assembles as a homodimer. Interestingly, each mTORC component, when free from the complexes, is present as a monomer and no single subunit serves as the dimerizing component. Instead, our data suggest that dimerization of mTORCs is the result of multiple subunits forming a composite surface. SiMPull also allowed us to distinguish complex disassembly from stoichiometry changes. Physiological conditions that abrogate mTOR signaling such as nutrient deprivation or energy stress did not alter the stoichiometry of mTORCs. On the other hand, rapamycin treatment leads to transient appearance of monomeric mTORC1 before complete disruption of the mTOR-raptor interaction, whereas mTORC2 stoichiometry is unaffected. These insights into assembly of mTORCs may guide future mechanistic studies and exploration of therapeutic potential.

Single-Molecule Analysis of Lipid-Protein Interactions in Crude Cell Lysates.

Recognition of signaling phospholipids by proteins is a critical requirement for the targeting and initiation of many signaling cascades. Most biophysical methods for measuring protein interactions with signaling phospholipids use purified proteins, which do not take into account the effect of post-translational modifications and other cellular components on these interactions. To potentially circumvent these problems, we have developed a single-molecule fluorescence approach to analyzing lipid-protein interactions in crude cell extracts. As a proof of principle for this assay, we show that a variety of lipid-binding domains (LBDs) can be recruited from cell lysates specifically onto their target phospholipids. With single-molecule analysis in real-time, our assay allows direct determination of binding kinetics for transient lipid-protein interactions and has revealed unique assembly properties and multiple binding modes of different LBDs. Whereas single-copy LBDs display transient interaction with lipid vesicles, tandem-repeat LBDs, often used as lipid biosensors, tend to form stable interactions that accumulate over time. We have extended the assay to study a cellular protein, Akt, and discovered marked differences in the lipid binding properties of the full-length protein compared to its PH domain. Importantly, we have found that phosphorylation of Akt at T308 and S473 does not affect the lipid binding behaviors of Akt, contrary to the long-standing model of Akt regulation. Overall, this work establishes the single-molecule lipid pulldown assay as a simple and highly sensitive approach to interrogating lipid-protein interactions in a setting that at least partly mimics the cellular environment.

Single-molecule pull-down (SiMPull) for new-age biochemistry: methodology and biochemical applications of single-molecule pull-down (SiMPull) for probing biomolecular interactions in crude cell extracts.

Macromolecular interactions play a central role in many biological processes. Protein-protein interactions have mostly been studied by co-immunoprecipitation, which cannot provide quantitative information on all possible molecular connections present in the complex. We will review a new approach that allows cellular proteins and biomolecular complexes to be studied in real-time at the single-molecule level. This technique is called single-molecule pull-down (SiMPull), because it integrates principles of conventional immunoprecipitation with the powerful single-molecule fluorescence microscopy. SiMPull is used to count how many of each protein is present in the physiological complexes found in cytosol and membranes. Concurrently, it serves as a single-molecule biochemical tool to perform functional studies on the pulled-down proteins. In this review, we will focus on the detailed methodology of SiMPull, its salient features and a wide range of biological applications in comparison with other biosensing tools.

Ligand-modulated parallel mechanical unfolding pathways of maltose-binding proteins.

Protein folding and unfolding are complex phenomena, and it is accepted that multidomain proteins generally follow multiple pathways. Maltose-binding protein (MBP) is a large (a two-domain, 370-amino acid residue) bacterial periplasmic protein involved in maltose uptake. Despite the large size, it has been shown to exhibit an apparent two-state equilibrium unfolding in bulk experiments. Single-molecule studies can uncover rare events that are masked by averaging in bulk studies. Here, we use single-molecule force spectroscopy to study the mechanical unfolding pathways of MBP and its precursor protein (preMBP) in the presence and absence of ligands. Our results show that MBP exhibits kinetic partitioning on mechanical stretching and unfolds via two parallel pathways: one of them involves a mechanically stable intermediate (path I) whereas the other is devoid of it (path II). The apoMBP unfolds via path I in 62% of the mechanical unfolding events, and the remaining 38% follow path II. In the case of maltose-bound MBP, the protein unfolds via the intermediate in 79% of the cases, the remaining 21% via path II. Similarly, on binding to maltotriose, a ligand whose binding strength with the polyprotein is similar to that of maltose, the occurrence of the intermediate is comparable (82% via path I) with that of maltose. The precursor protein preMBP also shows a similar behavior upon mechanical unfolding. The percentages of molecules unfolding via path I are 53% in the apo form and 68% and 72% upon binding to maltose and maltotriose, respectively, for preMBP. These observations demonstrate that ligand binding can modulate the mechanical unfolding pathways of proteins by a kinetic partitioning mechanism. This could be a general mechanism in the unfolding of other large two-domain ligand-binding proteins of the bacterial periplasmic space.

AAA + ATPase Thorase inhibits mTOR signaling through the disassembly of the mTOR complex 1.

The mechanistic target of rapamycin (mTOR) signals through the mTOR complex 1 (mTORC1) and the mTOR complex 2 to maintain cellular and organismal homeostasis. Failure to finely tune mTOR activity results in metabolic dysregulation and disease. While there is substantial understanding of the molecular events leading mTORC1 activation at the lysosome, remarkably little is known about what terminates mTORC1 signaling. Here, we show that the AAA + ATPase Thorase directly binds mTOR, thereby orchestrating the disassembly and inactivation of mTORC1. Thorase disrupts the association of mTOR to Raptor at the mitochondria-lysosome interface and this action is sensitive to amino acids. Lack of Thorase causes accumulation of mTOR-Raptor complexes and altered mTORC1 disassembly/re-assembly dynamics upon changes in amino acid availability. The resulting excessive mTORC1 can be counteracted with rapamycin in vitro and in vivo. Collectively, we reveal Thorase as a key component of the mTOR pathway that disassembles and thus inhibits mTORC1.

ORCA/LRWD1 Regulates Homologous Recombination at ALT-Telomeres by Modulating Heterochromatin Organization.

Telomeres are maintained by telomerase or in a subset of cancer cells by a homologous recombination (HR)-based mechanism, Alternative Lengthening of Telomeres (ALT). The mechanisms regulating telomere-homeostasis in ALT cells remain unclear. We report that a replication initiator protein, Origin Recognition Complex-Associated (ORCA/LRWD1), by localizing at the ALT-telomeres, modulates HR activity. ORCA's localization to the ALT-telomeres is facilitated by its interaction to SUMOylated shelterin components. The loss of ORCA in ALT-positive cells elevates the levels of two mediators of HR, RPA and RAD51, and consistent with this, we observe increased ALT-associated promyelocytic leukemia body formation and telomere sister chromatid exchange. ORCA binds to RPA and modulates the association of RPA to telomeres. Finally, the loss of ORCA causes global chromatin decondensation, including at the telomeres. Our results demonstrate that ORCA acts as an inhibitor of HR by modulating RPA binding to ssDNA and inducing chromatin compaction.

Single-molecule fluorescence microscopy of native macromolecular complexes.

Macromolecular complexes consisting of proteins, lipids, and/or nucleic acids are ubiquitous in biological processes. Their composition, stoichiometry, order of assembly, and conformations can be heterogeneous or can change dynamically, making single-molecule studies best suited to measure these properties accurately. Recent single-molecule pull-down and other related approaches have combined the principles of conventional co-immunoprecipitation assay with single-molecule fluorescence microscopy to probe native macromolecular complexes. In this review, we present the advances in single-molecule pull-down methods and biological systems that have been investigated in such semi vivo manner.

The preRC protein ORCA organizes heterochromatin by assembling histone H3 lysine 9 methyltransferases on chromatin.

Heterochromatic domains are enriched with repressive histone marks, including histone H3 lysine 9 methylation, written by lysine methyltransferases (KMTs). The pre-replication complex protein, origin recognition complex-associated (ORCA/LRWD1), preferentially localizes to heterochromatic regions in post-replicated cells. Its role in heterochromatin organization remained elusive. ORCA recognizes methylated H3K9 marks and interacts with repressive KMTs, including G9a/GLP and Suv39H1 in a chromatin context-dependent manner. Single-molecule pull-down assays demonstrate that ORCA-ORC (Origin Recognition Complex) and multiple H3K9 KMTs exist in a single complex and that ORCA stabilizes H3K9 KMT complex. Cells lacking ORCA show alterations in chromatin architecture, with significantly reduced H3K9 di- and tri-methylation at specific chromatin sites. Changes in heterochromatin structure due to loss of ORCA affect replication timing, preferentially at the late-replicating regions. We demonstrate that ORCA acts as a scaffold for the establishment of H3K9 KMT complex and its association and activity at specific chromatin sites is crucial for the organization of heterochromatin structure.