Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in BR signaling share this mechanism, we used coimmunoprecipitation of epitope-tagged proteins to show that in vivo association of BRI1 and BAK1 was affected by endogenous and exogenous BR levels and that phosphorylation of both BRI1 and BAK1 on Thr residues was BR dependent. Immunoprecipitation of epitope-tagged BRI1 from Arabidopsis thaliana followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) identified S-838, S-858, T-872, and T-880 in the juxtamembrane region, T-982 in the kinase domain, and S-1168 in C-terminal region as in vivo phosphorylation sites of BRI1. MS analysis also strongly suggested that an additional two residues in the juxtamembrane region and three sites in the activation loop of kinase subdomain VII/VIII were phosphorylated in vivo. We also identified four specific BAK1 autophosphorylation sites in vitro using LC/MS/MS. Sitedirected mutagenesis of identified and predicted BRI1 phosphorylation sites revealed that the highly conserved activation loop residue T-1049 and either S-1044 or T-1045 were essential for kinase function in vitro and normal BRI1 signaling in planta. Mutations in the juxtamembrane or C-terminal regions had only small observable effects on autophosphorylation and in planta signaling but dramatically affected phosphorylation of a peptide substrate in vitro. These findings are consistent with many aspects of the animal receptor kinase model in which ligand-dependent autophosphorylation of the activation loop generates a functional kinase, whereas phosphorylation of noncatalytic intracellular domains is required for recognition and/or phosphorylation of downstream substrates.

Song-learning birds and humans share independently evolved similarities in brain pathways for vocal learning that are essential for song and speech and are not found in most other species. Comparisons of brain transcriptomes of song-learning birds and humans relative to vocal nonlearners identified convergent gene expression specializations in specific song and speech brain regions of avian vocal learners and humans. The strongest shared profiles relate bird motor and striatal song-learning nuclei, respectively, with human laryngeal motor cortex and parts of the striatum that control speech production and learning. Most of the associated genes function in motor control and brain connectivity. Thus, convergent behavior and neural connectivity for a complex trait are associated with convergent specialized expression of multiple genes.

Inhibitory synapses dampen neuronal activity through postsynaptic hyperpolarization. The composition of the inhibitory postsynapse and the mechanistic basis of its regulation, however, remains poorly understood. We used an in vivo chemico-genetic proximity-labeling approach to discover inhibitory postsynaptic proteins. Quantitative mass spectrometry not only recapitulated known inhibitory postsynaptic proteins, but also revealed a large network of new proteins, many of which are either implicated in neurodevelopmental disorders or are of unknown function. CRISPR-depletion of one of these previously uncharacterized proteins, InSyn1, led to decreased postsynaptic inhibitory sites, reduced frequency of miniature inhibitory currents, and increased excitability in the hippocampus. Our findings uncover a rich and functionally diverse assemblage of previously unknown proteins that regulate postsynaptic inhibition and might contribute to developmental brain disorders.

Because proteins are the major functional components of cells, knowledge of their cellular localization is crucial to gaining an understanding of the biology of multicellular organisms. We have generated a protein expression map of the Arabidopsis root providing the identity and cell type-specific localization of nearly 2,000 proteins. Grouping proteins into functional categories revealed unique cellular functions and identified cell type-specific biomarkers. Cellular colocalization provided support for numerous protein-protein interactions. With a binary comparison, we found that RNA and protein expression profiles are weakly correlated. We then performed peak integration at cell type-specific resolution and found an improved correlation with transcriptome data using continuous values. We performed GeLC-MS/MS (in-gel tryptic digestion followed by liquid chromatography-tandem mass spectrometry) proteomic experiments on mutants with ectopic and no root hairs, providing complementary proteomic data. Finally, among our root hair-specific proteins we identified two unique regulators of root hair development.plant proteome | cell-type expression | FACS | RNA-protein correlation | root hair mutant M ulticellular organisms use specialized cell types to perform activities that are integral to their function. Cellular tasks are usually achieved by proteins, which act in signaling cascades, provide structural support, and catalyze enzymatic reactions vital to growth and metabolism. Knowledge of protein cellular localization and abundance using proteomic approaches is thus crucial to our understanding of biological systems (1, 2). Proteome data can be visually represented in a map, which highlights the spatial relationships of proteins at the level of cell type, tissue, or organ. Proteome maps are useful representations of the complex "building plan" of a biological system and also serve as valuable tools for the discovery of new cellular functions (2, 3). Proteomic studies of single cell populations isolated from a variety of multicellular organisms have recently been achieved, including the oocytes of worms and mice (4-6); pollen grains (consisting of two sperm and one vegetative cell) and stomatal guard cells of plants (7,8); and sperm cells of mice and flies (9, 10). These cell types were relatively accessible because they either reside on the surface and can be purified in large quantities using biochemical fractionation (e.g., guard cells) or are large and can easily be collected (e.g., Caenorhabditis elegans oocytes). However, similar proteomic studies of internal cell populations have been more difficult and are usually only partially represented in proteomes of whole organs owing to signal dilution (e.g., refs. 11-16).The Arabidopsis thaliana root is an excellent model for investigating cellular functions internal to an organ because it is transparent, radially symmetric, and cell types can be isolated by FACS to allow molecular profiling (17). The goal of this study was to investigate cell-type function by genera...

Summary Perisynaptic astrocyte processes are an integral part of central nervous system synapses 1 , 2 ; however, the molecular mechanisms governing astrocyte-synapse adhesions and how astrocyte contacts control synapse formation and function are largely unknown. Here we develop an in vivo chemico-genetic approach, Split-TurboID, that uses a cell surface fragment complementation strategy. We thus identify a proteome enriched at astrocyte-neuron junctions in vivo , including Neuronal Cell Adhesion Molecule (NrCAM). We find that NrCAM is expressed in cortical astrocytes, localized to perisynaptic contacts and is required to restrict neuropil infiltration by astrocytic processes. Furthermore, we show that astrocytic NrCAM transcellularly interacts with neuronal NrCAM that is coupled to gephyrin at inhibitory postsynapses. Depletion of astrocytic NrCAM significantly reduces inhibitory synapse numbers without altering glutamatergic synaptic density. Moreover, loss of astrocytic NrCAM dramatically reduces inhibitory synaptic function with minor effects on excitation. Thus, our results present a proteomic framework for how astrocytes interface with neurons, and reveal how astrocytes control GABAergic synapse formation and function.

Calcineurin governs stress survival, sexual differentiation, and virulence of the human fungal pathogen Cryptococcus neoformans. Calcineurin is activated by increased Ca2+ levels caused by stress, and transduces signals by dephosphorylating protein substrates. Herein, we identified and characterized calcineurin substrates in C. neoformans by employing phosphoproteomic TiO2 enrichment and quantitative mass spectrometry. The identified targets include the transactivator Crz1 as well as novel substrates whose functions are linked to P-bodies/stress granules (PBs/SGs) and mRNA translation and decay, such as Pbp1 and Puf4. We show that Crz1 is a bona fide calcineurin substrate, and Crz1 localization and transcriptional activity are controlled by calcineurin. We previously demonstrated that thermal and other stresses trigger calcineurin localization to PBs/SGs. Several calcineurin targets localized to PBs/SGs, including Puf4 and Pbp1, contribute to stress resistance and virulence individually or in conjunction with Crz1. Moreover, Pbp1 is also required for sexual development. Genetic epistasis analysis revealed that Crz1 and the novel targets Lhp1, Puf4, and Pbp1 function in a branched calcineurin pathway that orchestrates stress survival and virulence. These findings support a model whereby calcineurin controls stress and virulence, at the transcriptional level via Crz1, and post-transcriptionally by localizing to PBs/SGs and acting on targets involved in mRNA metabolism. The calcineurin targets identified in this study share little overlap with known calcineurin substrates, with the exception of Crz1. In particular, the mRNA binding proteins and PBs/SGs residents comprise a cohort of novel calcineurin targets that have not been previously linked to calcineurin in mammals or in Saccharomyces cerevisiae. This study suggests either extensive evolutionary rewiring of the calcineurin pathway, or alternatively that these novel calcineurin targets have yet to be characterized as calcineurin targets in other organisms. These findings further highlight C. neoformans as an outstanding model to define calcineurin-responsive virulence networks as targets for antifungal therapy.

IntroductionSickle (homozygous hemoglobin S, SS) RBC-based adhesion and vaso-occlusive events likely initiate and/or exacerbate the profound vasculopathy present in patients with sickle cell disease (SCD). 1,2 SS RBCs possess unusually active signaling pathways that contribute to a panoply of abnormalities, including RBC adhesion to the endothelium and vaso-occlusion. [2][3][4] Cell adhesion is a multistep cellular process that is regulated by complex extracellular and intracellular signals that may differ from one cell type to another. We have previously shown that abnormal SS RBC interaction with the endothelium and with leukocytes can be induced via ␤ 2 adrenergic receptor (␤ 2 AR) activation by the stress hormone epinephrine. [4][5][6] Such stimulation activates the intracellular cAMP/protein kinase A (PKA) pathway. 4 ␤ 2 ARs are prototypic G-coupled receptors whose signaling properties are in part mediated by the activation of stimulatory GTP-binding proteins (G s proteins), which in turn activate adenylate cyclase (AC), leading to the generation of cAMP and the subsequent activation of PKA. The cAMP/PKA pathway can modulate the MAPK/ERKs cascade both directly and indirectly. 7-9 PKA has been reported to stimulate B-Raf, while inhibiting c-Raf. Therefore, the activity of downstream signaling proteins, such as MEKs and ERKs, could be either enhanced or inhibited depending on the balance of c-Raf and B-Raf activation. 10,11 The cellular functions mediated by ␤ 2 ARs can also be independent of adenylyl cyclase activation and involve other mediators instead. [12][13][14][15] The functions attributed to ERK1/2 at both the cellular and physiologic levels are diverse, including modulation of proliferation, differentiation, apoptosis, migration, and cell adhesion. [16][17][18][19] Physiologically, ERK1/2 is required for immune system development, homeostasis and antigen activation, memory formation, development of the heart, and responses to many hormones, growth factors, and insulin. Most of these previous studies have involved only nucleated cells, including erythroid cells, in which erythropoietin is the primary regulatory cytokine of this pathway. 20 However, aberrations in ERK1/2 signaling are known to occur in a wide range of pathologies, including cancer, diabetes, viral infection, and cardiovascular disease. 21,22 In preliminary studies, authors have indicated that ERK1/2 is highly abundant in both SS and normal RBCs. Yet, whether this kinase remains functional in normal or SS RBCs is unknown, and an extremely critical question in the study of SCD pathophysiology. Such a mechanism of action could represent a novel target for the treatment of SCD. Methods Endothelial cellsPrimary HUVECs were grown as monolayers in EBM2 medium (Lonza Walkersville) supplemented with EGM2 (Lonza Walkersville) as described previously. 4 All experiments were approved by the Duke University institutional review board. AntibodiesAbs used included the following monoclonal and polyclonal Abs (as purified Ig unless otherwise noted): B...

Chemical cross-linking combined with mass spectrometry is a viable approach to study the low-resolution structure of protein and protein complexes. However, unambiguous identification of the residues involved in a cross-link remains analytically challenging. To enable a more effective analysis across various MS platforms, we have developed a novel set of collision-induced dissociative cross-linking reagents and methodology for chemical cross-linking experiments using tandem mass spectrometry (CID-CXL-MS/MS). These reagents incorporate a single gas-phase cleavable bond within their linker region that can be selectively fragmented within the in-source region of the mass spectrometer, enabling independent MS/MS analysis for each peptide. Initial design concepts were characterized using a synthesized cross-linked peptide complex. Following verification and subsequent optimization of cross-linked peptide complex dissociation, our reagents were applied to homodimeric glutathione S-transferase and monomeric bovine serum albumin. Cross-linked residues identified by our CID-CXL-MS/MS method were in agreement with published crystal structures and previous cross-linking studies using conventional approaches. Common LC/MS/MS acquisition approaches such as data-dependent acquisition experiments using ion trap mass spectrometers and product ion spectral analysis using SEQUEST were shown to be compatible with our CID-CXL-MS/MS reagents, obviating the requirement for high resolution and high mass accuracy measurements to identify both intra- and interpeptide cross-links.