Regulatory myeloid cells paralyze T cells through cell-cell transfer of the metabolite methylglyoxal.

Regulatory myeloid immune cells, such as myeloid-derived suppressor cells (MDSCs), populate inflamed or cancerous tissue and block immune cell effector functions. The lack of mechanistic insight into MDSC suppressive activity and a marker for their identification has hampered attempts to overcome T cell inhibition and unleash anti-cancer immunity. Here, we report that human MDSCs were characterized by strongly reduced metabolism and conferred this compromised metabolic state to CD8+ T cells, thereby paralyzing their effector functions. We identified accumulation of the dicarbonyl radical methylglyoxal, generated by semicarbazide-sensitive amine oxidase, to cause the metabolic phenotype of MDSCs and MDSC-mediated paralysis of CD8+ T cells. In a murine cancer model, neutralization of dicarbonyl activity overcame MDSC-mediated T cell suppression and, together with checkpoint inhibition, improved the efficacy of cancer immune therapy. Our results identify the dicarbonyl methylglyoxal as a marker metabolite for MDSCs that mediates T cell paralysis and can serve as a target to improve cancer immune therapy.

Correction: A role for brassinosteroid signalling in decision-making processes in the Arabidopsis seedling.

[This corrects the article DOI: 10.1371/journal.pgen.1010541.].

A role for brassinosteroid signalling in decision-making processes in the Arabidopsis seedling.

Plants often adapt to adverse conditions via differential growth, whereby limited resources are discriminately allocated to optimize the growth of one organ at the expense of another. Little is known about the decision-making processes that underly differential growth. In this study, we developed a screen to identify decision making mutants by deploying two tools that have been used in decision theory: a well-defined yet limited budget, as well as conflict-of-interest scenarios. A forward genetic screen that combined light and water withdrawal was carried out. This identified BRASSINOSTEROID INSENSITIVE 2 (BIN2) alleles as decision mutants with "confused" phenotypes. An assessment of organ and cell length suggested that hypocotyl elongation occurred predominantly via cellular elongation. In contrast, root growth appeared to be regulated by a combination of cell division and cell elongation or exit from the meristem. Gain- or loss- of function bin2 mutants were most severely impaired in their ability to adjust cell geometry in the hypocotyl or cell elongation as a function of distance from the quiescent centre in the root tips. This study describes a novel paradigm for root growth under limiting conditions, which depends not only on hypocotyl-versus-root trade-offs in the allocation of limited resources, but also on an ability to deploy different strategies for root growth in response to multiple stress conditions.

TRIPP Is a Plant-Specific Component of the Arabidopsis TRAPPII Membrane Trafficking Complex with Important Roles in Plant Development.

How the membrane trafficking system spatially organizes intracellular activities and intercellular signaling networks in plants is not well understood. Transport Protein Particle (TRAPP) complexes play key roles in the selective delivery of membrane vesicles to various subcellular compartments in yeast and animals but remain to be fully characterized in plants. Here, we investigated TRAPP complexes in Arabidopsis (Arabidopsis thaliana) using immunoprecipitation followed by quantitative mass spectrometry analysis of AtTRS33, a conserved core component of all TRAPP complexes. We identified 14 AtTRS33-interacting proteins, including homologs of all 13 TRAPP components in mammals and a protein that has homologs only in multicellular photosynthetic organisms and is thus named TRAPP-Interacting Plant Protein (TRIPP). TRIPP specifically associates with the TRAPPII complex through binary interactions with two TRAPPII-specific subunits. TRIPP colocalized with a subset of TRS33 compartments and trans-Golgi network markers in a TRS33-dependent manner. Loss-of-function tripp mutants exhibited dwarfism, sterility, partial photomorphogenesis in the dark, reduced polarity of the auxin transporter PIN2, incomplete cross wall formation, and altered localization of a TRAPPII-specific component. Therefore, TRIPP is a plant-specific component of the TRAPPII complex with important functions in trafficking, plant growth, and development.

Independent yet overlapping pathways ensure the robustness and responsiveness of trans-Golgi network functions in Arabidopsis.

The trans-Golgi-network (TGN) has essential housekeeping functions in secretion, endocytosis and protein sorting, but also more specialized functions in plant development. How the robustness of basal TGN function is ensured while specialized functions are differentially regulated is poorly understood. Here, we investigate two key regulators of TGN structure and function, ECHIDNA and the Transport Protein Particle II (TRAPPII) tethering complex. An analysis of physical, network and genetic interactions suggests that two network communities are implicated in TGN function and that ECHIDNA and TRAPPII belong to distinct yet overlapping pathways. Whereas ECHIDNA and TRAPPII colocalized at the TGN in interphase cells, their localization diverged in dividing cells. Moreover, ECHIDNA and TRAPPII localization patterns were mutually independent. TGN structure, endocytosis and sorting decisions were differentially impacted in echidna and trappii mutants. Our analyses point to a partitioning of specialized TGN functions, with ECHIDNA being required for cell elongation and TRAPPII for cytokinesis. Two independent pathways able to compensate for each other might contribute to the robustness of TGN housekeeping functions and to the responsiveness and fine tuning of its specialized functions.

Interactions between Transport Protein Particle (TRAPP) complexes and Rab GTPases in Arabidopsis.

Transport Protein Particle II (TRAPPII) is essential for exocytosis, endocytosis, protein sorting and cytokinesis. In spite of a considerable understanding of its biological role, little information is known about Arabidopsis TRAPPII complex topology and molecular function. In this study, independent proteomic approaches initiated with TRAPP components or Rab-A GTPase variants converge on the TRAPPII complex. We show that the Arabidopsis genome encodes the full complement of 13 TRAPPC subunits, including four previously unidentified components. A dimerization model is proposed to account for binary interactions between TRAPPII subunits. Preferential binding to dominant negative (GDP-bound) versus wild-type or constitutively active (GTP-bound) RAB-A2a variants discriminates between TRAPPII and TRAPPIII subunits and shows that Arabidopsis complexes differ from yeast but resemble metazoan TRAPP complexes. Analyzes of Rab-A mutant variants in trappii backgrounds provide genetic evidence that TRAPPII functions upstream of RAB-A2a, allowing us to propose that TRAPPII is likely to behave as a guanine nucleotide exchange factor (GEF) for the RAB-A2a GTPase. GEFs catalyze exchange of GDP for GTP; the GTP-bound, activated, Rab then recruits a diverse local network of Rab effectors to specify membrane identity in subsequent vesicle fusion events. Understanding GEF-Rab interactions will be crucial to unravel the co-ordination of plant membrane traffic.

Cell cycle-regulated PLEIADE/AtMAP65-3 links membrane and microtubule dynamics during plant cytokinesis.

Cytokinesis, the partitioning of the cytoplasm following nuclear division, requires extensive coordination between cell cycle cues, membrane trafficking and microtubule dynamics. Plant cytokinesis occurs within a transient membrane compartment known as the cell plate, to which vesicles are delivered by a plant-specific microtubule array, the phragmoplast. While membrane proteins required for cytokinesis are known, how these are coordinated with microtubule dynamics and regulated by cell cycle cues remains unclear. Here, we document physical and genetic interactions between Transport Protein Particle II (TRAPPII) tethering factors and microtubule-associated proteins of the PLEIADE/AtMAP65 family. These interactions do not specifically affect the recruitment of either TRAPPII or MAP65 proteins to the cell plate or midzone. Rather, and based on single versus double mutant phenotypes, it appears that they are required to coordinate cytokinesis with the nuclear division cycle. As MAP65 family members are known to be targets of cell cycle-regulated kinases, our results provide a conceptual framework for how membrane and microtubule dynamics may be coordinated with each other and with the nuclear cycle during plant cytokinesis.

Regulation of adaptive growth decisions via phosphorylation of the TRAPPII complex in Arabidopsis.

Plants often adapt to adverse or stress conditions via differential growth. The trans-Golgi network (TGN) has been implicated in stress responses, but it is not clear in what capacity it mediates adaptive growth decisions. In this study, we assess the role of the TGN in stress responses by exploring the previously identified interactome of the Transport Protein Particle II (TRAPPII) complex required for TGN structure and function. We identified physical and genetic interactions between AtTRAPPII and shaggy-like kinases (GSK3/AtSKs) and provided in vitro and in vivo evidence that the TRAPPII phosphostatus mediates adaptive responses to abiotic cues. AtSKs are multifunctional kinases that integrate a broad range of signals. Similarly, the AtTRAPPII interactome is vast and considerably enriched in signaling components. An AtSK-TRAPPII interaction would integrate all levels of cellular organization and instruct the TGN, a central and highly discriminate cellular hub, as to how to mobilize and allocate resources to optimize growth and survival under limiting or adverse conditions.

Regulation of adaptive growth decisions via phosphorylation of the TRAPPII complex in Arabidopsis.

Plants often adapt to adverse or stress conditions via differential growth. The trans-Golgi Network (TGN) has been implicated in stress responses, but it is not clear in what capacity it mediates adaptive growth decisions. In this study, we assess the role of the TGN in stress responses by exploring the interactome of the Transport Protein Particle II (TRAPPII) complex, required for TGN structure and function. We identified physical and genetic interactions between TRAPPII and shaggy-like kinases (GSK3/AtSKs). Kinase assays and pharmacological inhibition provided in vitro and in vivo evidence that AtSKs target the TRAPPII-specific subunit AtTRS120/TRAPPC9. GSK3/AtSK phosphorylation sites in AtTRS120/TRAPPC9 were mutated, and the resulting AtTRS120 phosphovariants subjected to a variety of single and multiple stress conditions in planta . The non-phosphorylatable TRS120 mutant exhibited enhanced adaptation to multiple stress conditions and to osmotic stress whereas the phosphomimetic version was less resilient. Higher order inducible trappii atsk mutants had a synthetically enhanced defect in root gravitropism. Our results suggest that the TRAPPII phosphostatus mediates adaptive responses to abiotic cues. AtSKs are multifunctional kinases that integrate a broad range of signals. Similarly, the TRAPPII interactome is vast and considerably enriched in signaling components. An AtSK-TRAPPII interaction would integrate all levels of cellular organization and instruct the TGN, a central and highly discriminate cellular hub, as to how to mobilize and allocate resources to optimize growth and survival under limiting or adverse conditions.

Closely related receptor complexes differ in their ABA selectivity and sensitivity.

The recent discovery of a variety of receptors has led to new models for hormone perception in plants. In the case of the hormone abscisic acid (ABA), which regulates plant responses to abiotic stress, perception seems to occur both at the plasma membrane and in the cytosol. The cytosolic receptors for ABA have recently been identified as complexes between protein phosphatases 2C (PP2C) and regulatory components (RCAR/PYR/PYL) that bind ABA. Binding of ABA to the receptor complexes inactivates the PP2Cs, thereby activating the large variety of physiological processes regulated by ABA. The Arabidopsis genome encodes 13 homologues of RCAR1 and approximately 80 PP2Cs, of which six in clade A have been identified as negative regulators of ABA responses. In this study we characterize a novel member of the RCAR family, RCAR3. RCAR3 was identified in a screen for interactors of the PP2Cs ABI1 and ABI2, which are key regulators of ABA responses. RCAR3 was shown to repress ABI1 and ABI2 in vitro, and to stimulate ABA signalling in protoplast cells. RCAR3 conferred greater ABA sensitivity to the PP2C regulation than RCAR1, whereas stereo-selectivity for (S)-ABA was less stringent with RCAR3 as compared with RCAR1. In addition, regulation of the protein phosphatase activity by RCAR1 and RCAR3 was more sensitive to ABA for ABI1 than for ABI2. Based on the differences we have observed in transcriptional regulation and biochemical properties, we propose a model whereby differential expression of the co-receptors and combinatorial assembly of the receptor complexes act in concert to modulate and fine-tune ABA responses.

Tethering factors required for cytokinesis in Arabidopsis.

At the end of the cell cycle, the nascent cross wall is laid down within a transient membrane compartment referred to as the cell plate. Tethering factors, which act by capturing vesicles and holding them in the vicinity of their target membranes, are likely to play an important role in the first stages of cell plate assembly. Factors required for cell plate biogenesis, however, remain to be identified. In this study, we used a reverse genetic screen to isolate tethering factors required for cytokinesis in Arabidopsis (Arabidopsis thaliana). We focused on the TRAPPI and TRAPPII (for transport protein particle) tethering complexes, which are thought to be required for the flow of traffic through the Golgi and for trans-Golgi network function, as well as on the GARP complex, thought to be required for the tethering of endocytotic vesicles to the trans-Golgi network. We found weak cytokinesis defects in some TRAPPI mutants and strong cytokinesis defects in all the TRAPPII lines we surveyed. Indeed, four insertion lines at the TRAPPII locus AtTRS120 had canonical cytokinesis-defective seedling-lethal phenotypes, including cell wall stubs and incomplete cross walls. Confocal and electron microscopy showed that in trs120 mutants, vesicles accumulated at the equator of dividing cells yet failed to assemble into a cell plate. This shows that AtTRS120 is required for cell plate biogenesis. In contrast to the TRAPP complexes, we found no conclusive evidence for cytokinesis defects in seven GARP insertion lines. We discuss the implications of these findings for the origin and identity of cell plate membranes.

The timely deposition of callose is essential for cytokinesis in Arabidopsis.

The primary plant cell wall is laid down over a brief period of time during cytokinesis. Initially, a membrane network forms at the equator of a dividing cell. The cross-wall is then assembled and remodeled within this membrane compartment. Callose is the predominant luminal component of the nascent cross-wall or cell plate, but is not a component of intact mature cell walls, which are composed primarily of cellulose, pectins and xyloglucans. Widely accepted models postulate that callose comprises a transient, rapid spreading force for the expansion of membrane networks during cytokinesis. In this study, we clone and characterize an Arabidopsis gene, MASSUE/AtGSL8, which encodes a putative callose synthase. massue mutants are seedling-lethal and have a striking cytokinesis-defective phenotype. Callose deposition was delayed in the cell plates of massue mutants. Mutant cells were occasionally bi- or multi-nucleate, with cell-wall stubs, and we frequently observed gaps at the junction between cross-walls and parental cell walls. The results suggest that the timely deposition of callose is essential for the completion of plant cytokinesis. Surprisingly, confocal analysis revealed that the cell-plate membrane compartment forms and expands, seemingly as far as the parental wall, prior to the appearance of callose. We discuss the possibility that callose may be required to establish a lasting connection between the nascent cross-wall and the parental cell wall.

A putative TRAPPII tethering factor is required for cell plate assembly during cytokinesis in Arabidopsis.

*At the end of the cell cycle, the plant cell wall is deposited within a membrane compartment referred to as the cell plate. Little is known about the biogenesis of this transient membrane compartment. *We have positionally cloned and characterized a novel Arabidopsis gene, CLUB, identified by mutation. *CLUB/AtTRS130 encodes a putative TRAPPII tethering factor. club mutants are seedling-lethal and have a canonical cytokinesis-defective phenotype, characterized by the appearance of bi- or multinucleate cells with cell wall stubs, gaps and floating walls. Confocal microscopy showed that in club mutants, KNOLLE-positive vesicles formed and accumulated at the cell equator throughout cytokinesis, but failed to assemble into a cell plate. Similarly, electron micrographs showed large vesicles loosely connected as patchy, incomplete cell plates in club root tips. Neither the formation of KNOLLE-positive vesicles nor the delivery of these vesicles to the cell equator appeared to be perturbed in club mutants. Thus, the primary defect in club mutants appears to be an impairment in cell plate assembly. *As a putative tethering factor required for cell plate biogenesis, CLUB/AtTRS130 helps to define the identity of this membrane compartment and comprises an important handle on the regulation of cell plate assembly.

The Arabidopsis HINKEL gene encodes a kinesin-related protein involved in cytokinesis and is expressed in a cell cycle-dependent manner.

Plant cytokinesis starts in the center of the division plane, with vesicle fusion generating a new membrane compartment, the cell plate, that subsequently expands laterally by continuous fusion of newly arriving vesicles to its margin. Targeted delivery of vesicles is assisted by the dynamic reorganization of a plant-specific cytoskeletal array, the phragmoplast, from a solid cylinder into an expanding ring-shaped structure. This lateral translocation is brought about by depolymerization of microtubules in the center, giving way to the expanding cell plate, and polymerization of microtubules along the edge. Whereas several components are known to mediate cytokinetic vesicle fusion [8-10], no gene function involved in phragmoplast dynamics has been identified by mutation. Mutations in the Arabidopsis HINKEL gene cause cytokinesis defects, such as enlarged cells with incomplete cell walls and multiple nuclei. Proper targeting of the cytokinesis-specific syntaxin KNOLLE [8] and lateral expansion of the phragmoplast are not affected. However, the phragmoplast microtubules appear to persist in the center, where vesicle fusion should result in cell plate formation. Molecular analysis reveals that the HINKEL gene encodes a plant-specific kinesin-related protein with a putative N-terminal motor domain and is expressed in a cell cycle-dependent manner similar to the KNOLLE gene. Our results suggest that HINKEL plays a role in the reorganization of phragmoplast microtubules during cell plate formation.

The PEN1 syntaxin defines a novel cellular compartment upon fungal attack and is required for the timely assembly of papillae.

Attack by the host powdery mildew Erysiphe cichoracearum usually results in successful penetration and rapid proliferation of the fungus on Arabidopsis. By contrast, the nonhost barley powdery mildew Blumeria graminis f. sp. hordei (Bgh) typically fails to penetrate Arabidopsis epidermal cells. In both instances the plant secretes cell wall appositions or papillae beneath the penetration peg of the fungus. Genetic screens for mutations that result in increased penetration of Bgh on Arabidopsis have recently identified the PEN1 syntaxin. Here we examine the role of PEN1 and of its closest homologue, SYP122, identified as a syntaxin whose expression is responsive to infection. pen1 syp122 double mutants are both dwarfed and necrotic, suggesting that the two syntaxins have overlapping functions. Although syp122-1 and the cell wall mur mutants have considerably more pronounced primary cell wall defects than pen1 mutants, these have relatively subtle or no effects on penetration resistance. Upon fungal attack, PEN1 appears to be actively recruited to papillae, and there is a 2-h delay in papillae formation in the pen1-1 mutant. We conclude that SYP122 may have a general function in secretion, including a role in cell wall deposition. By contrast, PEN1 appears to have a basal function in secretion and a specialized defense-related function, being required for the polarized secretion events that give rise to papilla formation.

Multisubunit tethering complexes in higher plants.

Tethering complexes mediate the initial, specific contact between donor and acceptor membranes. This review focuses on the modularity and function of multisubunit tethering complexes (MTCs) in higher plants. One emphasis is on molecular interactions of plant MTCs. Here, a number of insights have been gained concerning interactions between different tethering complexes, and between tethers and microtubule-associated proteins. The roles of tethering complexes in abiotic stress responses appear indirect, but in the context of biotic stress responses it has been suggested that some tethers are direct targets of pathogen effectors or virulence factors. In light of the central roles tethering complexes play in plant development, an emerging concept is that tethers may be co-opted for plant adaptive responses.

Plant Cytokinesis: Terminology for Structures and Processes.

Plant cytokinesis is orchestrated by a specialized structure, the phragmoplast. The phragmoplast first occurred in representatives of Charophyte algae and then became the main division apparatus in land plants. Major cellular activities, including cytoskeletal dynamics, vesicle trafficking, membrane assembly, and cell wall biosynthesis, cooperate in the phragmoplast under the guidance of a complex signaling network. Furthermore, the phragmoplast combines plant-specific features with the conserved cytokinetic processes of animals, fungi, and protists. As such, the phragmoplast represents a useful system for understanding both plant cell dynamics and the evolution of cytokinesis. We recognize that future research and knowledge transfer into other fields would benefit from standardized terminology. Here, we propose such a lexicon of terminology for specific structures and processes associated with plant cytokinesis.

The Membrane-Associated Sec1/Munc18 KEULE is Required for Phragmoplast Microtubule Reorganization During Cytokinesis in Arabidopsis.

Cytokinesis, the partitioning of the cytoplasm following nuclear division, requires extensive coordination between membrane trafficking and cytoskeletal dynamics. In plants, the onset of cytokinesis is characterized by the assembly of a bipolar microtubule array, the phragmoplast, and of a transient membrane compartment, the cell plate. Little is known about the coordination between membrane deposition at the cell plate and the dynamics of phragmoplast microtubules. In this study, we monitor the localization dynamics of microtubule and membrane markers throughout cytokinesis. Our spatiotemporal resolution is consistent with the general view that microtubule dynamics drive membrane movements. Nonetheless, we provide evidence for active sorting at the cell plate and show that this is, at least in part, mediated by the TRAPPII tethering complex. We also characterize phragmoplast microtubule organization and cell plate formation in a suite of cytokinesis-defective mutants. Of four mutant lines with defects in phragmoplast microtubule organization, only mor1 microtubule-associated mutants exhibited aberrant cell plates. Conversely, the mutants with the strongest impairment in phragmoplast microtubule reorganization are keule alleles, which have a primary defect in membrane fusion. Our findings identify the SEC1/Munc18 protein KEULE as a central regulatory node in the coordination of membrane and microtubule dynamics during plant cytokinesis.

Plant cytokinesis is orchestrated by the sequential action of the TRAPPII and exocyst tethering complexes.

Plant cytokinesis is initiated in a transient membrane compartment, the cell plate, and completed by a process of maturation during which the cell plate becomes a cross wall. How the transition from juvenile to adult stages occurs is poorly understood. In this study, we monitor the Arabidopsis transport protein particle II (TRAPPII) and exocyst tethering complexes throughout cytokinesis. We show that their appearance is predominantly sequential, with brief overlap at the onset and end of cytokinesis. The TRAPPII complex is required for cell plate biogenesis, and the exocyst is required for cell plate maturation. The TRAPPII complex sorts plasma membrane proteins, including exocyst subunits, at the cell plate throughout cytokinesis. We show that the two tethering complexes physically interact and propose that their coordinated action may orchestrate not only plant but also animal cytokinesis.

Cytokinesis-defective mutants of Arabidopsis.

We have identified mutations in six previously uncharacterized genes of Arabidopsis, named club, bublina, massue, rod, bloated, and bims, that are required for cytokinesis. The mutants are seedling lethal, have morphological abnormalities, and are characterized by cell wall stubs, gapped walls, and multinucleate cells. In these and other respects, the new mutants are phenotypically similar to knolle, keule, hinkel, and pleiade mutants. The mutants display a gradient of stomatal phenotypes, correlating roughly with the severity of their cytokinesis defect. Similarly, the extent to which the different mutant lines were capable of growing in tissue culture correlated well with the severity of the cytokinesis defect. Phenotypic analysis of the novel and previously characterized loci indicated that the secondary consequences of a primary defect in cytokinesis include anomalies in body organization, organ number, and cellular differentiation, as well as organ fusions and perturbations of the nuclear cycle. Two of the 10 loci are required for both cytokinesis and root hair morphogenesis. The results have implications for the identification of novel cytokinesis genes and highlight the mechanistic similarity between cytokinesis and root hair morphogenesis, two processes that result in a rapid deposition of new cell walls via polarized secretion.