An atypical MAPK regulates translocation of a GATA transcription factor in response to chemoattractant stimulation.

The Dictyostelium atypical mitogen-activated protein kinase (MAPK) Erk2 is required for chemotactic responses to cAMP as amoeba undergo multicellular development. In this study, Erk2 was found to be essential for the cAMP-stimulated translocation of the GATA transcription factor GtaC as indicated by the distribution of a GFP-GtaC reporter. Erk2 was also found to be essential for the translocation of GtaC in response to external folate, a foraging signal that directs the chemotaxis of amoeba to bacteria. Erk1, the only other Dictyostelium MAPK, was not required for the GtaC translocation to either chemoattractant, indicating that GFP-GtaC is a kinase translocation reporter specific for atypical MAPKs. The translocation of GFP-GtaC in response to folate was absent in mutants lacking the folate receptor Far1 or the coupled G-protein subunit Gα4. Loss of GtaC function resulted in enhanced chemotactic movement to folate, suggesting that GtaC suppresses responses to folate. The alteration of four Erk2-preferred phosphorylation sites in GtaC impacted the translocation of GFP-GtaC in response to folate and the GFP-GtaC-mediated rescue of aggregation and development of gtaC- cells. The ability of different chemoattractants to stimulate Erk2-regulated GtaC translocation suggests that atypical MAPK-mediated regulation of transcription factors can contribute to different cell fates.

Oligopeptide transporter Slc15A modulates macropinocytosis in Dictyostelium by maintaining intracellular nutrient status.

Macropinocytosis mediates non-selective bulk uptake of extracellular fluid. It is the major route by which axenic Dictyostelium cells obtain nutrients and has emerged as a nutrient-scavenging pathway in mammalian cells. How environmental and cellular nutrient status modulates macropinocytic activity is not well understood. By developing a high-content imaging-based genetic screen in Dictyostelium discoideum we identified Slc15A, an oligopeptide transporter located at the plasma membrane and early macropinosome, as a novel macropinocytosis regulator. We show that deletion of slc15A but not two other related slc15 genes, leads to reduced macropinocytosis, reduced cell growth and aberrantly increased autophagy in cells grown in nutrient-rich medium. Expression of Slc15A protein or supplying cells with free amino acids rescues these defects. In contrast, expression of transport-defective Slc15A or supplying cells with amino acids in their di-peptide forms fails to rescue these defects. Therefore, Slc15A modulates the level of macropinocytosis by maintaining the intracellular availability of key amino acids through extraction of oligopeptides from the early macropinocytic pathway. We propose that Slc15A constitutes part of a positive feedback mechanism coupling cellular nutrient status and macropinocytosis. This article has an associated First Person interview with the first authors of the paper.

MicroRNA775 regulates intrinsic leaf size and reduces cell wall pectin levels by targeting a galactosyltransferase gene in Arabidopsis.

Plants possess unique primary cell walls made of complex polysaccharides that play critical roles in determining intrinsic cell and organ size. How genes responsible for synthesizing and modifying the polysaccharides in the cell wall are regulated by microRNAs (miRNAs) to control plant size remains largely unexplored. Here we identified 23 putative cell wall-related miRNAs, termed as CW-miRNAs, in Arabidopsis thaliana and characterized miR775 as an example. We showed that miR775 post-transcriptionally silences GALT9, which encodes an endomembrane-located galactosyltransferase belonging to the glycosyltransferase 31 family. Over-expression of miR775 and deletion of GALT9 led to significantly enlarged leaf-related organs, primarily due to increased cell size. Monosaccharide quantification, confocal Raman imaging, and immunolabeling combined with atomic force microscopy revealed that the MIR775A-GALT9 circuit modulates pectin levels and the elastic modulus of the cell wall. We also showed that MIR775A is directly repressed by the transcription factor ELONGATED HYPOCOTYL5 (HY5). Genetic analysis confirmed that HY5 is a negative regulator of leaf size that acts through the HY5-MIR775A-GALT9 repression cascade to control pectin levels. These findings demonstrate that miR775-regulated cell wall remodeling is an integral determinant of intrinsic leaf size in A. thaliana. Studying other CW-miRNAs would provide more insights into cell wall biology.

The structural basis for activation of the Rab Ypt1p by the TRAPP membrane-tethering complexes.

The multimeric membrane-tethering complexes TRAPPI and TRAPPII share seven subunits, of which four (Bet3p, Bet5p, Trs23p, and Trs31p) are minimally needed to activate the Rab GTPase Ypt1p in an event preceding membrane fusion. Here, we present the structure of a heteropentameric TRAPPI assembly complexed with Ypt1p. We propose that TRAPPI facilitates nucleotide exchange primarily by stabilizing the nucleotide-binding pocket of Ypt1p in an open, solvent-accessible form. Bet3p, Bet5p, and Trs23p interact directly with Ypt1p to stabilize this form, while the C terminus of Bet3p invades the pocket to participate in its remodeling. The Trs31p subunit does not interact directly with the GTPase but allosterically regulates the TRAPPI interface with Ypt1p. Our findings imply that TRAPPII activates Ypt1p by an identical mechanism. This view of a multimeric membrane-tethering assembly complexed with a Rab provides a framework for understanding events preceding membrane fusion at the molecular level.

Laccase3-based extracellular domain provides possible positional information for directing Casparian strip formation in Arabidopsis.

The Casparian strip (CS) is a tight junction-like structure formed by lignin impregnation on the walls of endodermal cells in plant roots. The CS membrane domain (CSDM), demarked by the CASP proteins, is important for orienting lignification enzymes. Here, we report that an endodermis-expressed multicopper oxidase, LACCASE3 (LAC3) in Arabidopsis, locates to the interface between lignin domains and the cell wall during early CS development prior to CASP1 localizing to CSDM and eventually flanks the mature CS. Pharmacological perturbation of LAC3 causes dispersed localization of CASP1 and compensatory ectopic lignification. These results support the existence of a LAC3-based CS wall domain which coordinates with CSDM to provide bidirectional positional information that guides precise CS lignification.

Statin-induced GGPP depletion blocks macropinocytosis and starves cells with oncogenic defects.

Cancer cells display novel characteristics which can be exploited for therapeutic advantage. Isolated studies have shown that 1) the mevalonate pathway and 2) increased macropinocytosis are important in tumorigenesis, but a connection between these two observations has not been envisioned. A library screen for compounds that selectively killed Dictyostelium pten- cells identified pitavastatin. Pitavastatin also killed human breast epithelial MCF10A cells lacking PTEN or expressing K-RasG12V, as well as mouse tumor organoids. The selective killing of cells with oncogenic defects was traced to GGPP (geranylgeranyl diphosphate) depletion. Disruption of GGPP synthase in Dictyostelium revealed that GGPP is needed for pseudopod extension and macropinocytosis. Fluid-phase uptake through macropinocytosis is lower in PTEN-deleted cells and, as reported previously, higher in cells expressing activated Ras. Nevertheless, uptake was more sensitive to pitavastatin in cells with either of these oncogenic mutations than in wild-type cells. Loading the residual macropinosomes after pitavastatin with high concentrations of protein mitigated the cell death, indicating that defective macropinocytosis leads to amino acid starvation. Our studies suggest that the dependence of cancer cells on the mevalonate pathway is due to the role of GGPP in macropinocytosis and the reliance of these cells on macropinocytosis for nutrient uptake. Thus, inhibition of the networks mediating these processes is likely to be effective in cancer intervention.

The carboxy terminal transmembrane domain of SPL7 mediates interaction with RAN1 at the endoplasmic reticulum to regulate ethylene signalling in Arabidopsis.

In Arabidopsis, copper (Cu) transport to the ethylene receptor ETR1 mediated using RAN1, a Cu transporter located at the endoplasmic reticulum (ER), and Cu homeostasis mediated using SPL7, the key Cu-responsive transcription factor, are two deeply conserved vital processes. However, whether and how the two processes interact to regulate plant development remain elusive. We found that its C-terminal transmembrane domain (TMD) anchors SPL7 to the ER, resulting in dual compartmentalisation of the transcription factor. Immunoprecipitation coupled mass spectrometry, yeast-two-hybrid assay, luciferase complementation imaging and subcellular co-localisation analyses indicate that SPL7 interacts with RAN1 at the ER via the TMD. Genetic analysis revealed that the ethylene-induced triple response was significantly compromised in the spl7 mutant, a phenotype rescuable by RAN1 overexpression but not by SPL7 without the TMD. The genetic interaction was corroborated by molecular analysis showing that SPL7 modulates RAN1 abundance in a TMD-dependent manner. Moreover, SPL7 is feedback regulated by ethylene signalling via EIN3, which binds the SPL7 promoter and represses its transcription. These results demonstrate that ER-anchored SPL7 constitutes a cellular mechanism to regulate RAN1 in ethylene signalling and lay the foundation for investigating how Cu homeostasis conditions ethylene sensitivity in the developmental context.

Insight from the maximal activation of the signal transduction excitable network in Dictyostelium discoideum.

Cell migration requires the coordination of an excitable signal transduction network involving Ras and PI3K pathways with cytoskeletal activity. We show that expressing activated Ras GTPase-family proteins in cells lacking PTEN or other mutations which increase cellular protrusiveness transforms cells into a persistently activated state. Leading- and trailing-edge markers were found exclusively at the cell perimeter and the cytosol, respectively, of the dramatically flattened cells. In addition, the lifetimes of dynamic actin puncta were increased where they overlapped with actin waves, suggesting a mechanism for the coupling between these two networks. All of these phenotypes could be reversed by inhibiting signal transduction. Strikingly, maintaining cells in this state of constant activation led to a form of cell death by catastrophic fragmentation. These findings provide insight into the feedback loops that control excitability of the signal transduction network, which drives migration.

Gß Regulates Coupling between Actin Oscillators for Cell Polarity and Directional Migration.

For directional movement, eukaryotic cells depend on the proper organization of their actin cytoskeleton. This engine of motility is made up of highly dynamic nonequilibrium actin structures such as flashes, oscillations, and traveling waves. In Dictyostelium, oscillatory actin foci interact with signals such as Ras and phosphatidylinositol 3,4,5-trisphosphate (PIP3) to form protrusions. However, how signaling cues tame actin dynamics to produce a pseudopod and guide cellular motility is a critical open question in eukaryotic chemotaxis. Here, we demonstrate that the strength of coupling between individual actin oscillators controls cell polarization and directional movement. We implement an inducible sequestration system to inactivate the heterotrimeric G protein subunit Gß and find that this acute perturbation triggers persistent, high-amplitude cortical oscillations of F-actin. Actin oscillators that are normally weakly coupled to one another in wild-type cells become strongly synchronized following acute inactivation of Gß. This global coupling impairs sensing of internal cues during spontaneous polarization and sensing of external cues during directional motility. A simple mathematical model of coupled actin oscillators reveals the importance of appropriate coupling strength for chemotaxis: moderate coupling can increase sensitivity to noisy inputs. Taken together, our data suggest that Gß regulates the strength of coupling between actin oscillators for efficient polarity and directional migration. As these observations are only possible following acute inhibition of Gß and are masked by slow compensation in genetic knockouts, our work also shows that acute loss-of-function approaches can complement and extend the reach of classical genetics in Dictyostelium and likely other systems as well.

MicroRNA408 is critical for the HY5-SPL7 gene network that mediates the coordinated response to light and copper.

Light and copper are important environmental determinants of plant growth and development. Despite the wealth of knowledge on both light and copper signaling, the molecular mechanisms that integrate the two pathways remain poorly understood. Here, we use Arabidopsis thaliana to demonstrate an interaction between SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7) and ELONGATED HYPOCOTYL5 (HY5), which mediate copper and light signaling, respectively. Through whole-genome chromatin immunoprecipitation and RNA sequencing analyses, we elucidated the SPL7 regulon and compared it with that of HY5. We found that the two transcription factors coregulate many genes, including those involved in anthocyanin accumulation and photosynthesis. Moreover, SPL7 and HY5 act coordinately to transcriptionally regulate MIR408, which results in differential expression of microRNA408 (miR408) and its target genes in response to changing light and copper conditions. We demonstrate that this regulation is tied to copper allocation to the chloroplast and plastocyanin levels. Finally, we found that constitutively activated miR408 rescues the distinct developmental defects of the hy5, spl7, and hy5 spl7 mutants. These findings revealed the existence of crosstalk between light and copper, mediated by a HY5-SPL7 network. Furthermore, integration of transcriptional and posttranscriptional regulation is critical for governing proper metabolism and development in response to combined copper and light signaling.

Leep2A and Leep2B function as a RasGAP complex to regulate macropinosome formation.

Macropinocytosis mediates the non-selective bulk uptake of extracellular fluid, enabling cells to survey the environment and obtain nutrients. A conserved set of signaling proteins orchestrates the actin dynamics that lead to membrane ruffling and macropinosome formation across various eukaryotic organisms. At the center of this signaling network are Ras GTPases, whose activation potently stimulates macropinocytosis. However, how Ras signaling is initiated and spatiotemporally regulated during macropinocytosis is not well understood. By using the model system Dictyostelium and a proteomics-based approach to identify regulators of macropinocytosis, we uncovered Leep2, consisting of Leep2A and Leep2B, as a RasGAP complex. The Leep2 complex specifically localizes to emerging macropinocytic cups and nascent macropinosomes, where it modulates macropinosome formation by regulating the activities of three Ras family small GTPases. Deletion or overexpression of the complex, as well as disruption or sustained activation of the target Ras GTPases, impairs macropinocytic activity. Our data reveal the critical role of fine-tuning Ras activity in directing macropinosome formation.

Stay in touch with the endoplasmic reticulum.

The endoplasmic reticulum (ER), which is composed of a continuous network of tubules and sheets, forms the most widely distributed membrane system in eukaryotic cells. As a result, it engages a variety of organelles by establishing membrane contact sites (MCSs). These contacts regulate organelle positioning and remodeling, including fusion and fission, facilitate precise lipid exchange, and couple vital signaling events. Here, we systematically review recent advances and converging themes on ER-involved organellar contact. The molecular basis, cellular influence, and potential physiological functions for ER/nuclear envelope contacts with mitochondria, Golgi, endosomes, lysosomes, lipid droplets, autophagosomes, and plasma membrane are summarized.

A transcription factor complex in Dictyostelium enables adaptive changes in macropinocytosis during the growth-to-development transition.

Macropinocytosis, an evolutionarily conserved endocytic pathway, mediates nonselective bulk uptake of extracellular fluid. It is the primary route for axenic Dictyostelium cells to obtain nutrients and has also emerged as a nutrient-scavenging pathway for mammalian cells. How cells adjust macropinocytic activity in various physiological or developmental contexts remains to be elucidated. We discovered that, in Dictyostelium cells, the transcription factors Hbx5 and MybG form a functional complex in the nucleus to maintain macropinocytic activity during the growth stage. In contrast, during starvation-induced multicellular development, the transcription factor complex undergoes nucleocytoplasmic shuttling in response to oscillatory cyclic adenosine 3',5'-monophosphate (cAMP) signals, which leads to increased cytoplasmic retention of the complex and progressive downregulation of macropinocytosis. Therefore, by coupling macropinocytosis-related gene expression to the cAMP oscillation system, which facilitates long-range cell-cell communication, the dynamic translocation of the Hbx5-MybG complex orchestrates a population-level adjustment of macropinocytic activity to adapt to changing environmental conditions.

IqgC is a potent regulator of macropinocytosis in the presence of NF1 and its loading to macropinosomes is dependent on RasG.

RasG is a major regulator of macropinocytosis in Dictyostelium discoideum. Its activity is under the control of an IQGAP-related protein, IqgC, which acts as a RasG-specific GAP (GTPase activating protein). IqgC colocalizes with the active Ras at the macropinosome membrane during its formation and for some time after the cup closure. However, the loss of IqgC induces only a minor enhancement of fluid uptake in axenic cells that already lack another RasGAP, NF1. Here, we show that IqgC plays an important role in the regulation of macropinocytosis in the presence of NF1 by restricting the size of macropinosomes. We further provide evidence that interaction with RasG is indispensable for the recruitment of IqgC to forming macropinocytic cups. We also demonstrate that IqgC interacts with another small GTPase from the Ras superfamily, Rab5A, but is not a GAP for Rab5A. Since mammalian Rab5 plays a key role in early endosome maturation, we hypothesized that IqgC could be involved in macropinosome maturation via its interaction with Rab5A. Although an excessive amount of Rab5A reduces the RasGAP activity of IqgC in vitro and correlates with IqgC dissociation from endosomes in vivo, the physiological significance of the Rab5A-IqgC interaction remains elusive.

Moving in the right direction: how eukaryotic cells migrate along chemical gradients.

Many cells have the ability to grow or migrate towards chemical cues. Oriented growth and movement require detection of the external chemical gradient, transduction of signals, and reorganization of the cytoskeleton. Recent studies in Dictyostelium discoideum and mammalian neutrophils have revealed a complex signaling network that enables cells to migrate in chemical gradients.

TRAPPI tethers COPII vesicles by binding the coat subunit Sec23.

The budding of endoplasmic reticulum (ER)-derived vesicles is dependent on the COPII coat complex. Coat assembly is initiated when Sar1-GTP recruits the cargo adaptor complex, Sec23/Sec24, by binding to its GTPase-activating protein (GAP) Sec23 (ref. 2). This leads to the capture of transmembrane cargo by Sec24 (refs 3, 4) before the coat is polymerized by the Sec13/Sec31 complex. The initial interaction of a vesicle with its target membrane is mediated by tethers. We report here that in yeast and mammalian cells the tethering complex TRAPPI (ref. 7) binds to the coat subunit Sec23. This event requires the Bet3 subunit. In vitro studies demonstrate that the interaction between Sec23 and Bet3 targets TRAPPI to COPII vesicles to mediate vesicle tethering. We propose that the binding of TRAPPI to Sec23 marks a coated vesicle for fusion with another COPII vesicle or the Golgi apparatus. An implication of these findings is that the intracellular destination of a transport vesicle may be determined in part by its coat and its associated cargo.

Trs85 directs a Ypt1 GEF, TRAPPIII, to the phagophore to promote autophagy.

Macroautophagy (hereafter autophagy) is a ubiquitous process in eukaryotic cells that is integrally involved in various aspects of cellular and organismal physiology. The morphological hallmark of autophagy is the formation of double-membrane cytosolic vesicles, autophagosomes, which sequester cytoplasmic cargo and deliver it to the lysosome or vacuole. Thus, autophagy involves dynamic membrane mobilization, yet the source of the lipid that forms the autophagosomes and the mechanism of membrane delivery are poorly characterized. The TRAPP complexes are multimeric guanine nucleotide exchange factors (GEFs) that activate the Rab GTPase Ypt1, which is required for secretion. Here we describe another form of this complex (TRAPPIII) that acts as an autophagy-specific GEF for Ypt1. The Trs85 subunit of the TRAPPIII complex directs this Ypt1 GEF to the phagophore assembly site (PAS) that is involved in autophagosome formation. Consistent with the observation that a Ypt1 GEF is directed to the PAS, we find that Ypt1 is essential for autophagy. This is an example of a Rab GEF that is specifically targeted for canonical autophagosome formation.

GxcM-Fbp17/RacC-WASP signaling regulates polarized cortex assembly in migrating cells via Arp2/3.

The actin-rich cortex plays a fundamental role in many cellular processes. Its architecture and molecular composition vary across cell types and physiological states. The full complement of actin assembly factors driving cortex formation and how their activities are spatiotemporally regulated remain to be fully elucidated. Using Dictyostelium as a model for polarized and rapidly migrating cells, we show that GxcM, a RhoGEF localized specifically in the rear of migrating cells, functions together with F-BAR protein Fbp17, a small GTPase RacC, and the actin nucleation-promoting factor WASP to coordinately promote Arp2/3 complex-mediated cortical actin assembly. Overactivation of this signaling cascade leads to excessive actin polymerization in the rear cortex, whereas its disruption causes defects in cortical integrity and function. Therefore, apart from its well-defined role in the formation of the protrusions at the cell front, the Arp2/3 complex-based actin carries out a previously unappreciated function in building the rear cortical subcompartment in rapidly migrating cells.

Coats, tethers, Rabs, and SNAREs work together to mediate the intracellular destination of a transport vesicle.

Tethering factors have been shown to interact with Rabs and SNAREs and, more recently, with coat proteins. Coat proteins are required for cargo selection and membrane deformation to bud a transport vesicle from a donor compartment. It was once thought that a vesicle must uncoat before it recognizes its target membrane. However, recent findings have revealed a role for the coat in directing a vesicle to its correct intracellular destination. In this review we will discuss the literature that links coat proteins to vesicle targeting events.

Gradients of PI(4,5)P2 and PI(3,5)P2 Jointly Participate in Shaping the Back State of Dictyostelium Cells.

Polarity, which refers to the molecular or structural asymmetry in cells, is essential for diverse cellular functions. Dictyostelium has proven to be a valuable system for dissecting the molecular mechanisms of cell polarity. Previous studies in Dictyostelium have revealed a range of signaling and cytoskeletal proteins that function at the leading edge to promote pseudopod extension and migration. In contrast, how proteins are localized to the trailing edge is not well understood. By screening for asymmetrically localized proteins, we identified a novel trailing-edge protein we named Teep1. We show that a charged surface formed by two pleckstrin homology (PH) domains in Teep1 is necessary and sufficient for targeting it to the rear of cells. Combining biochemical and imaging analyses, we demonstrate that Teep1 interacts preferentially with PI(4,5)P2 and PI(3,5)P2 in vitro and simultaneous elimination of these lipid species in cells blocks the membrane association of Teep1. Furthermore, a leading-edge localized myotubularin phosphatase likely mediates the removal of PI(3,5)P2 from the front, as well as the formation of a back-to-front gradient of PI(3,5)P2. Together our data indicate that PI(4,5)P2 and PI(3,5)P2 on the plasma membrane jointly participate in shaping the back state of Dictyostelium cells.