A phosphoinositide map at the shoot apical meristem in Arabidopsis thaliana.
ABSTRACT: In plants, the shoot apical meristem (SAM) has two main functions, involving the production of all aerial organs on the one hand and self-maintenance on the other, allowing the production of organs during the entire post-embryonic life of the plant. Transcription factors, microRNA, hormones, peptides and forces have been involved in meristem function. Whereas phosphatidylinositol phosphates (PIPs) have been involved in almost all biological functions, including stem cell maintenance and organogenesis in animals, the processes in meristem biology to which PIPs contribute still need to be delineated.Using biosensors for PI4P and PI(4,5)P2, the two most abundant PIPs at the plasma membrane, we reveal that meristem functions are associated with a stereotypical PIP tissue-scale pattern, with PI(4,5)P2 always displaying a more clear-cut pattern than PI4P. Using clavata3 and pin-formed1 mutants, we show that stem cell maintenance is associated with reduced levels of PIPs. In contrast, high PIP levels are signatures for organ-meristem boundaries. Interestingly, this pattern echoes that of cortical microtubules and stress anisotropy at the meristem. Using ablations and pharmacological approaches, we further show that PIP levels can be increased when the tensile stress pattern is altered. Conversely, we find that katanin mutant meristems, with increased isotropy of microtubule arrays and slower response to mechanical perturbations, exhibit reduced PIP gradients within the SAM. Comparable PIP pattern defects were observed in phospholipase A3? overexpressor lines, which largely phenocopy katanin mutants at the whole plant level.Using phospholipid biosensors, we identified a stereotypical PIP accumulation pattern in the SAM that negatively correlates with stem cell maintenance and positively correlates with organ-boundary establishment. While other cues are very likely to contribute to the final PIP pattern, we provide evidence that the patterns of PIP, cortical microtubules and mechanical stress are positively correlated, suggesting that the PIP pattern, and its reproducibility, relies at least in part on the mechanical status of the SAM.
Project description:Phosphatidylinositolphosphates (PIPs) are phospholipids that contain a phosphorylated inositol head group. PIPs represent a minor fraction of total phospholipids, but are involved in many regulatory processes, such as cell signalling and intracellular trafficking. Membrane compartments are enriched or depleted in specific PIPs, providing a unique composition for these compartments and contributing to their identity. The precise subcellular localization and dynamics of most PIP species is not fully understood in plants. Here, we designed genetically encoded biosensors with distinct relative affinities and expressed them stably in Arabidopsis thaliana. Analysis of this multi-affinity 'PIPline' marker set revealed previously unrecognized localization of various PIPs in root epidermis. Notably, we found that PI(4,5)P2 is able to localize PIP2 -interacting protein domains to the plasma membrane in non-stressed root epidermal cells. Our analysis further revealed that there is a gradient of PI4P, with the highest concentration at the plasma membrane, intermediate concentration in post-Golgi/endosomal compartments, and the lowest concentration in the Golgi. Finally, we also found a similar gradient of PI3P from high in late endosomes to low in the tonoplast. Our library extends the range of available PIP biosensors, and will allow rapid progress in our understanding of PIP dynamics in plants.
Project description:Phosphatidylinositol phosphates (PIPs) are involved in many cellular events as important secondary messengers. In Entamoeba histolytica, a human intestinal protozoan parasite, virulence-associated mechanisms such as cell motility, vesicular traffic, trogo- and phagocytosis are regulated by PIPs. It has been well established that PI3P, PI4P, and PI(3,4,5)P3 play specific roles during amoebic trogo- and phagocytosis. In the present study, we demonstrated the nuclear localization of PI4P in E. histolytica trophozoites in steady state with immunofluorescence imaging and immunoelectron microscopy, using anti-PI4P antibodies and PI4P biosensors [substrate of the Icm/ Dot type IV secretion system (SidM)]. We further showed that the nuclear PI4P decreased after a co-culture with human erythrocytes or Chinese hamster ovary (CHO) cells. However, concomitant changes in the localization and the amount of PI(4,5)P2, which is the expected major metabolized (phosphorylated) product of PI4P, were not observed. This phenomenon was specifically caused by whole or ghost erythrocytes and CHO cells, but not artificial beads. The amount of PIP2 and PIP, biochemically estimated by [32P]-phosphate metabolic labeling and thin layer chromatography, was decreased upon erythrocyte adherence. Altogether, our data indicate for the first time in eukaryotes that erythrocyte attachment leads to the metabolism of nuclear PIPs, and metabolites other than PI(4,5)P2 may be involved in the regulation of downstream cellular events such as cytoskeleton rearrangement or transcriptional regulation.
Project description:Phosphatidylinositol phosphates (PIPs) are lipid signaling molecules that play key roles in many cellular processes. PIP5K1A kinase catalyzes phosphorylation of PI4P to form PIP2, which in turn interacts with membrane and membrane-associated proteins. We explore the mechanism of membrane binding by the PIP5K1A kinase using a multiscale molecular dynamics approach. Coarse-grained simulations show binding of monomeric PIP5K1A to a model cell membrane containing PI4P. PIP5K1A did not bind to zwitterionic or anionic membranes lacking PIP molecules. Initial encounter of kinase and bilayer was followed by reorientation to enable productive binding to the PI4P-containing membrane. The simulations suggest that unstructured regions may be important for the preferred orientation for membrane binding. Atomistic simulations indicated that the dimeric kinase could not bind to the membrane via both active sites at the same time, suggesting a conformational change in the protein and/or bilayer distortion may be needed for dual-site binding to occur.
Project description:In Schizosaccharomyces pombe, loss of the plasma membrane PI4-kinase scaffold Efr3 leads to sliding of the cytokinetic ring (CR) away from the cell center during anaphase, implicating phosphoinositides (PIPs) in CR anchoring. However, whether other PIP regulators contribute to CR anchoring has not been investigated. Here we report that mutants of other PIP kinases and their regulators divide with off-center septa, similar to efr3?. Using new biosensors for S. pombe PIPs, we confirm that these mutants have disrupted PIP composition. We extend a previous finding that a mutant known to decrease PI(3,5)P2 levels indirectly affects CR positioning by increasing vacuole size which disrupts nuclear position at the onset of mitosis. Indeed, we found that other mutants with increased vacuole size also disrupt medial division via this mechanism. Although elevated plasma membrane PI(4,5)P2 levels do not affect medial cytokinesis, mutants with decreased levels display CR sliding events indicating a specific role for PI(4,5)P2 in CR anchoring.
Project description:Members of the genus Monophyllaea are unique in that they produce no new organ during the vegetative phase in the shoot; instead, one of the cotyledons grows indeterminately. The mechanism of this unique trait is unclear, in part because of the lack of suitable assessment techniques. We therefore established a whole-mount in situ hybridization technique, a powerful means of examining spatial patterns in gene expression, for Monophyllaea glabra. By using this, we examined the expression pattern of a SHOOT MERISTEMLESS (STM) ortholog, which is indispensable for the formation and maintenance of the shoot apical meristem (SAM) in typical angiosperms. Expression was confined to the groove meristem (GM), which corresponds to the SAM. We also assessed the expression pattern of ANGUSTIFOLIA3 (AN3), a key promoter for cell division in the leaf meristem. It was expressed not only in the basal meristem (BM) tissue with active cell division in the basal part of the growing cotyledon but also in the GM. The findings suggest that the unusual gene expression pattern of the GM underpins the fuzzy morphogenesis of Monophyllaea.
Project description:The control of cell position and division act in concert to dictate multicellular organization in tissues and organs. How these processes shape global order and molecular movement across organs is an outstanding problem in biology. Using live 3D imaging and computational analyses, we extracted networks capturing cellular connectivity dynamics across the Arabidopsis shoot apical meristem (SAM) and topologically analyzed the local and global properties of cellular architecture. Locally generated cell division rules lead to the emergence of global tissue-scale organization of the SAM, facilitating robust global communication. Cells that lie upon more shorter paths have an increased propensity to divide, with division plane placement acting to limit the number of shortest paths their daughter cells lie upon. Cell shape heterogeneity and global cellular organization requires KATANIN, providing a multiscale link between cell geometry, mechanical cell-cell interactions, and global tissue order.
Project description:Kir2.1 channels are uniquely activated by phosphoinositide 4,5-bisphosphate (PI(4,5)P2) and can be inhibited by other phosphoinositides (PIPs). Using biochemical and computational approaches, we assess PIP-channel interactions and distinguish residues that are energetically critical for binding from those that alter PIP sensitivity by shifting the open-closed equilibrium. Intriguingly, binding of each PIP is disrupted by a different subset of mutations. In silico ligand docking indicates that PIPs bind to two sites. The second minor site may correspond to the secondary anionic phospholipid site required for channel activation. However, 96-99% of PIP binding localizes to the first cluster, which corresponds to the general PI(4,5)P2 binding location in recent Kir crystal structures. PIPs can encompass multiple orientations; each di- and triphosphorylated species binds with comparable energies and is favored over monophosphorylated PIPs. The data suggest that selective activation by PI(4,5)P2 involves orientational specificity and that other PIPs inhibit this activation through direct competition.
Project description:A fundamental question in developmental biology is how spatial patterns are self-organized from homogeneous structures. In 1952, Turing proposed the reaction-diffusion model in order to explain this issue. Experimental evidence of reaction-diffusion patterns in living organisms was first provided by the pigmentation pattern on the skin of fishes in 1995. However, whether or not this mechanism plays an essential role in developmental events of living organisms remains elusive. Here we show that a reaction-diffusion model can successfully explain the shoot apical meristem (SAM) development of plants. SAM of plants resides in the top of each shoot and consists of a central zone (CZ) and a surrounding peripheral zone (PZ). SAM contains stem cells and continuously produces new organs throughout the lifespan. Molecular genetic studies using Arabidopsis thaliana revealed that the formation and maintenance of the SAM are essentially regulated by the feedback interaction between WUSHCEL (WUS) and CLAVATA (CLV). We developed a mathematical model of the SAM based on a reaction-diffusion dynamics of the WUS-CLV interaction, incorporating cell division and the spatial restriction of the dynamics. Our model explains the various SAM patterns observed in plants, for example, homeostatic control of SAM size in the wild type, enlarged or fasciated SAM in clv mutants, and initiation of ectopic secondary meristems from an initial flattened SAM in wus mutant. In addition, the model is supported by comparing its prediction with the expression pattern of WUS in the wus mutant. Furthermore, the model can account for many experimental results including reorganization processes caused by the CZ ablation and by incision through the meristem center. We thus conclude that the reaction-diffusion dynamics is probably indispensable for the SAM development of plants.
Project description:Abstract The role of an innovative Pharmacist Independent Prescriber (PIP) for care homes to optimise medications has not been examined. We explored stakeholders’ views on issues and barriers that the PIP might address to inform a service specification for the PIP intervention in older people's care homes. Focus groups (n = 72 participants) and semi?structured interviews (n = 13) undertaken in 2015 across four sites in the United Kingdom captured the views of doctors, pharmacists, care?home managers and staff, residents and relatives. Stakeholders identified their expectations of what service should be provided by PIPs, what might affect their support for the role, and barriers and enablers to providing the service. Transcripts were analysed using the Theoretical Domains Framework to identify key components, which were reviewed by stakeholders in 2016. A PIP service was envisaged offering benefits for residents, care homes and doctors but stakeholders raised challenges including agreement on areas where PIPs might prescribe, contextual barriers in chronic disease management, PIPs’ knowledge of older people's medicine, and implementation barriers in integrated team?working and ensuring role clarity. Introducing a PIP was welcomed in principle but conditional on: a clearly defined PIP role communicated to stakeholders; collaboration across doctors, PIPs and care?home staff; dialogue about developing the service with residents and relatives, based on trust and effective communication. To embed a PIP service within increasingly complex care?homes provision, the overarching theme from this research was that everyone must “understand each other's systems”.
Project description:KEY MESSAGE:CLV3p-mediated phosphorylation of MPK3 and MPK6 occurs via CLV1 and BAM1 receptors to regulate the maintenance of SAM development. The CLAVATA peptide-receptor (CLV3p-CLV1) pathway modulates a homeodomain master regulator WUSCHEL (WUS) transcription factor in the shoot apical meristem (SAM) with poorly defined signaling mechanisms. Here, we report that mitogen-activated protein kinases (MAPKs, also known as MPKs in plants) act in an intracellular signaling cascade to play an important role in the maintenance of SAM development. Interestingly, the application of exogenous CLV3p triggers rapid signaling in the SAM via dynamic activation of MPK3 and MPK6, which are positively regulated by both CLV1 and BARELY ANY MERISTEM 1 (BAM1) receptors. Surprisingly, the timing of MAPK activation is tightly correlated with the transcriptional repression of WUS expression in the SAM, indicating a fast CLV3p-CLV1/BAM1 signaling event. Furthermore, conditional mpk3,6 double mutants exhibited CLV3p insensitivity in stem cell maintenance manifested by the persistent SAM growth in the presence of exogenous CLV3p signals, as well as elevated WUS expression and repressed WUS-specific target genes. Taken together, these results suggest that MPK3 and MPK6 activated by CLV3p signals through mainly CLV1 and BAM1 receptors are key regulators controlling stem cell homeostasis in the SAM.