Wetting of phase-separated droplets on plant vacuole membranes leads to a competition between tonoplast budding and nanotube formation.

Seeds of dicotyledonous plants store proteins in dedicated membrane-bounded organelles called protein storage vacuoles (PSVs). Formed during seed development through morphological and functional reconfiguration of lytic vacuoles in embryos [M. Feeney et al., Plant Physiol. 177, 241-254 (2018)], PSVs undergo division during the later stages of seed maturation. Here, we study the biophysical mechanism of PSV morphogenesis in vivo, discovering that micrometer-sized liquid droplets containing storage proteins form within the vacuolar lumen through phase separation and wet the tonoplast (vacuolar membrane). We identify distinct tonoplast shapes that arise in response to membrane wetting by droplets and derive a simple theoretical model that conceptualizes these geometries. Conditions of low membrane spontaneous curvature and moderate contact angle (i.e., wettability) favor droplet-induced membrane budding, thereby likely serving to generate multiple, physically separated PSVs in seeds. In contrast, high membrane spontaneous curvature and strong wettability promote an intricate and previously unreported membrane nanotube network that forms at the droplet interface, allowing molecule exchange between droplets and the vacuolar interior. Furthermore, our model predicts that with decreasing wettability, this nanotube structure transitions to a regime with bud and nanotube coexistence, which we confirmed in vitro. As such, we identify intracellular wetting [J. Agudo-Canalejo et al., Nature 591, 142-146 (2021)] as the mechanism underlying PSV morphogenesis and provide evidence suggesting that interconvertible membrane wetting morphologies play a role in the organization of liquid phases in cells.

Recent advances in the understanding of the aetiology and therapeutic strategies in burning mouth syndrome: Focus on the actions of cannabinoids.

Burning mouth syndrome (BMS) is a neuropathic pain disorder associated with a burning sensation on oral mucosal surfaces with frequently reported xerostomia, dysgeusia and tingling or paraesthetic sensations. However, patients present no clinically evident causative lesions. The poor classification of the disorder has resulted in a diagnostic challenge, particularly for the clinician/dentist evaluating these individuals. Major research developments have been made in the BMS field in recent years to address this concern, principally in terms of the pathophysiological mechanisms underlying the disorder, in addition to therapeutic advancements. For the purpose of this review, an update on the pathophysiological mechanisms will be discussed from a neuropathic, immunological, hormonal and psychological perspective. This review will also focus on the many therapeutic strategies that have been explored for BMS, including antidepressants/antipsychotics, non-steroidal anti-inflammatories, hormone replacement therapies, phytotherapeutic compounds and non-pharmacological interventions, overall highlighting the lack of controlled clinical studies to support the effectiveness of such therapeutic avenues. Particular focus is given to the cannabinoid system and the potential of cannabis-based therapeutics in managing BMS patients.

Elucidating connections between the strigolactone biosynthesis pathway, flavonoid production and root system architecture in Arabidopsis thaliana.

Strigolactones (SLs) are the most recently discovered phytohormones, and their roles in root architecture and metabolism are not fully understood. Here, we investigated four MORE AXILLARY GROWTH (MAX) SL mutants in Arabidopsis thaliana, max3-9, max4-1, max1-1 and max2-1, as well as the SL receptor mutant d14-1 and karrikin receptor mutant kai2-2. By characterising max2-1 and max4-1, we found that variation in SL biosynthesis modified multiple metabolic pathways in root tissue, including that of xyloglucan, triterpenoids, fatty acids and flavonoids. The transcription of key flavonoid biosynthetic genes, including TRANSPARENT TESTA4 (TT4) and TRANSPARENT TESTA5 (TT5) was downregulated in max2 roots and seedlings, indicating that the proposed MAX2 regulation of flavonoid biosynthesis has a widespread effect. We found an enrichment of BRI1-EMS-SUPPRESSOR 1 (BES1) targets amongst genes specifically altered in the max2 mutant, reflecting that the regulation of flavonoid biosynthesis likely occurs through the MAX2 degradation of BES1, a key brassinosteroid-related transcription factor. Finally, flavonoid accumulation decreased in max2-1 roots, supporting a role for MAX2 in regulating both SL and flavonoid biosynthesis.

A nuclear localization signal targets tail-anchored membrane proteins to the inner nuclear envelope in plants.

Protein targeting to the inner nuclear membrane (INM) is one of the least understood protein targeting pathways. INM proteins are important for chromatin organization, nuclear morphology and movement, and meiosis, and have been implicated in human diseases. In opisthokonts, one mechanism for INM targeting is transport factor-mediated trafficking, in which nuclear localization signals (NLSs) function in nuclear import of transmembrane proteins. To explore whether this pathway exists in plants, we fused the SV40 NLS to a plant ER tail-anchored protein and showed that the GFP-tagged fusion protein was significantly enriched at the nuclear envelope (NE) of leaf epidermal cells. Airyscan subdiffraction limited confocal microscopy showed that this protein displays a localization consistent with an INM protein. Nine different monopartite and bipartite NLSs from plants and opisthokonts, fused to a chimeric tail-anchored membrane protein, were all sufficient for NE enrichment, and both monopartite and bipartite NLSs were sufficient for trafficking to the INM. Tolerance for different linker lengths and protein conformations suggests that INM trafficking rules might differ from those in opisthokonts. The INM proteins developed here can be used to target new functionalities to the plant nuclear periphery. This article has an associated First Person interview with the first author of the paper.

Identification and characterization of genes encoding the nuclear envelope LINC complex in the monocot species Zea mays.

The linker of nucleoskeleton to cytoskeleton (LINC) complex is an essential multi-protein structure spanning the nuclear envelope. It connects the cytoplasm to the nucleoplasm, functions to maintain nuclear shape and architecture and regulates chromosome dynamics during cell division. Knowledge of LINC complex composition and function in the plant kingdom is primarily limited to Arabidopsis, but critically missing from the evolutionarily distant monocots, which include grasses, the most important agronomic crops worldwide. To fill this knowledge gap, we identified and characterized 22 maize genes, including a new grass-specific KASH gene family. By using bioinformatic, biochemical and cell biological approaches, we provide evidence that representative KASH candidates localize to the nuclear periphery and interact with Zea mays (Zm)SUN2 in vivo FRAP experiments using domain deletion constructs verified that this SUN-KASH interaction was dependent on the SUN but not the coiled-coil domain of ZmSUN2. A summary working model is proposed for the entire maize LINC complex encoded by conserved and divergent gene families. These findings expand our knowledge of the plant nuclear envelope in a model grass species, with implications for both basic and applied cellular research.This article has an associated First Person interview with the first author of the paper.

The Arabidopsis leucine-rich repeat receptor kinase MIK2/LRR-KISS connects cell wall integrity sensing, root growth and response to abiotic and biotic stresses.

Plants actively perceive and respond to perturbations in their cell walls which arise during growth, biotic and abiotic stresses. However, few components involved in plant cell wall integrity sensing have been described to date. Using a reverse-genetic approach, we identified the Arabidopsis thaliana leucine-rich repeat receptor kinase MIK2 as an important regulator of cell wall damage responses triggered upon cellulose biosynthesis inhibition. Indeed, loss-of-function mik2 alleles are strongly affected in immune marker gene expression, jasmonic acid production and lignin deposition. MIK2 has both overlapping and distinct functions with THE1, a malectin-like receptor kinase previously proposed as cell wall integrity sensor. In addition, mik2 mutant plants exhibit enhanced leftward root skewing when grown on vertical plates. Notably, natural variation in MIK2 (also named LRR-KISS) has been correlated recently to mild salt stress tolerance, which we could confirm using our insertional alleles. Strikingly, both the increased root skewing and salt stress sensitivity phenotypes observed in the mik2 mutant are dependent on THE1. Finally, we found that MIK2 is required for resistance to the fungal root pathogen Fusarium oxysporum. Together, our data identify MIK2 as a novel component in cell wall integrity sensing and suggest that MIK2 is a nexus linking cell wall integrity sensing to growth and environmental cues.

Fast-slow analysis of the Integrated Oscillator Model for pancreatic ß-cells.

Insulin-secreting pancreatic ß-cells are electrically excitable cells that are unusual because their electrical activity is influenced directly by metabolism via ATP-sensitive K+ channels. At the same time, changes in the intracellular Ca2+concentration that result from the cell's electrical activity influence metabolism in several ways. Thus, there is bidirectional coupling between the electrical dynamics and the metabolic dynamics in ß-cells. A mathematical model has been previously developed, called the Integrated. Oscillator Model (IOM), to highlight the bidirectional coupling involved in the oscillation mechanism. In this study, we show how this coupling can produce oscillations in ß-cell activity. These oscillations have period similar to that of insulin secretion pulses observed in rats, mice, dogs, and humans, which has been shown to facilitate the action of the liver in maintaining glucose homeostasis. In a companion paper we show that the IOM can produce oscillations using two distinct mechanisms, depending on the values of electrical and metabolic parameters. In the present article, we use fast-slow analysis to understand the mechanisms underlying each of these oscillations. In particular, we show why a key variable in the glycolytic pathway generates a pulsatile time course in one type of oscillation, while it generates a sawtooth time course in the other type. The significance of these patterns is that the time course is a reflection of whether an intrinsic glycolytic oscillator is active, or whether the oscillations are a direct consequence of Ca2+ feedback onto glycolysis.

Plasma IL-8 signature correlates with pain and depressive symptomatology in patients with burning mouth syndrome: Results from a pilot study.

BACKGROUND: Burning mouth syndrome (BMS) is a neuropathic orofacial pain condition of unknown aetiology that encompasses intra-oral burning pain without abnormal clinical findings. Psychological, neural and inflammatory processes are associated with BMS pathogenesis. Currently, studies characterising plasma cytokine/chemokine profiles with pain and depression in patients with BMS are lacking. Considering that inflammation is associated with the pathophysiology of BMS, and that inflammation is closely associated with pain and depression, we aimed to correlate depressive symptomatology and oral cavity pain with plasma cytokine/chemokine signatures in a cohort of patients with BMS. METHODS: In this study, plasma protein levels of Th1 cytokines (IFN-γ, IL-2, IL-12p70, TNF-α), Th2 cytokines (IL-4, IL-10, IL-6, IL-13) and the chemokine IL-8 were assessed in patients with BMS (n = 10) and healthy volunteers (n = 10), using pro-inflammatory-10-plex assays. Clinical histories, alongside self-rated oral cavity pain intensities and depressive symptomatology were assessed using a visual analogue scale and the 16-item Quick Inventory of Depressive Symptomatology questionnaires, respectively. RESULTS: We present evidence that BMS is associated with increased depressive symptomatology and enhanced oral cavity pain. Plasma isolated from BMS patients display enhanced expression of the pro-inflammatory chemokine IL-8, when compared to plasma from healthy individuals. Plasma IL-8 signature correlates with pain and depressive symptomatology in the study cohort. CONCLUSIONS: Overall, these findings indicate that plasma IL-8 profiles are dysregulated in BMS and that modulation of IL-8 production in the disorder may be a tool in the management of BMS symptomatology.

Plasma N-acylethanolamine and endocannabinoid levels in burning mouth syndrome: Potential role in disease pathogenesis.

OBJECTIVE: The objective was to measure endocannabinoid (eCB) ligands and non-cannabinoid N-acylethanolamine (NAE) molecules in plasma from individuals with burning mouth syndrome (BMS) and to determine whether plasma eCB/NAE levels correlated with pain, inflammation and depressive symptomatology in this cohort. STUDY DESIGN: Plasma content of the eCBs, anandamide (AEA) and 2-arachidonoyl-glycerol (2-AG), and the NAE molecules, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) were assessed in healthy subjects (n = 8) and in a cohort of newly diagnosed BMS patients (n = 9) using liquid chromatography-tandem mass spectrometry. Plasma eCBs and NAE profiles were correlated with self-rated oral cavity pain intensities, depressive symptomatology and plasma IL-8 levels. RESULTS: Plasma levels of PEA, but not OEA, AEA or 2-AG, were significantly elevated in patients with BMS, when compared to plasma from healthy individuals. Plasma PEA, OEA and AEA levels correlated with depressive symptomatology. CONCLUSIONS: This is the first evidence to indicate that circulating eCB/NAE levels are altered in BMS.

The cell wall of Arabidopsis thaliana influences actin network dynamics.

In plant cells, molecular connections link the cell wall-plasma membrane-actin cytoskeleton to form a continuum. It is hypothesized that the cell wall provides stable anchor points around which the actin cytoskeleton remodels. Here we use live cell imaging of fluorescently labelled marker proteins to quantify the organization and dynamics of the actin cytoskeleton and to determine the impact of disrupting connections within the continuum. Labelling of the actin cytoskeleton with green fluorescent protein (GFP)-fimbrin actin-binding domain 2 (FABD2) resulted in a network composed of fine filaments and thicker bundles that appeared as a highly dynamic remodelling meshwork. This differed substantially from the GFP-Lifeact-labelled network that appeared much more sparse with thick bundles that underwent 'simple movement', in which the bundles slightly change position, but in such a manner that the structure of the network was not substantially altered during the time of observation. Label-dependent differences in actin network morphology and remodelling necessitated development of two new image analysis techniques. The first of these, 'pairwise image subtraction', was applied to measurement of the more rapidly remodelling actin network labelled with GFP-FABD2, while the second, 'cumulative fluorescence intensity', was used to measure bulk remodelling of the actin cytoskeleton when labelled with GFP-Lifeact. In each case, these analysis techniques show that the actin cytoskeleton has a decreased rate of bulk remodelling when the cell wall-plasma membrane-actin continuum is disrupted either by plasmolysis or with isoxaben, a drug that specifically inhibits cellulose deposition. Changes in the rate of actin remodelling also affect its functionality, as observed by alteration in Golgi body motility.

Glucose Oscillations Can Activate an Endogenous Oscillator in Pancreatic Islets.

Pancreatic islets manage elevations in blood glucose level by secreting insulin into the bloodstream in a pulsatile manner. Pulsatile insulin secretion is governed by islet oscillations such as bursting electrical activity and periodic Ca2+ entry in ß-cells. In this report, we demonstrate that although islet oscillations are lost by fixing a glucose stimulus at a high concentration, they may be recovered by subsequently converting the glucose stimulus to a sinusoidal wave. We predict with mathematical modeling that the sinusoidal glucose signal's ability to recover islet oscillations depends on its amplitude and period, and we confirm our predictions by conducting experiments with islets using a microfluidics platform. Our results suggest a mechanism whereby oscillatory blood glucose levels recruit non-oscillating islets to enhance pulsatile insulin output from the pancreas. Our results also provide support for the main hypothesis of the Dual Oscillator Model, that a glycolytic oscillator endogenous to islet ß-cells drives pulsatile insulin secretion.

Phase Analysis of Metabolic Oscillations and Membrane Potential in Pancreatic Islet ß-Cells.

Metabolism in islet ß-cells displays oscillations that can trigger pulses of electrical activity and insulin secretion. There has been a decades-long debate among islet biologists about whether metabolic oscillations are intrinsic or occur in response to oscillations in intracellular Ca(2+) that result from bursting electrical activity. In this article, the dynamics of oscillatory metabolism were investigated using five different optical reporters. Reporter activity was measured simultaneously with membrane potential bursting to determine the phase relationships between the metabolic oscillations and electrical activity. Our experimental findings suggest that Ca(2+) entry into ß-cells stimulates the rate of mitochondrial metabolism, accounting for the depletion of glycolytic intermediates during each oscillatory burst. We also performed Ca(2+) clamp tests in which we clamped membrane potential with the KATP channel-opener diazoxide and KCl to fix Ca(2+) at an elevated level. These tests confirm that metabolic oscillations do not require Ca(2+) oscillations, but show that Ca(2+) plays a larger role in shaping metabolic oscillations than previously suspected. A dynamical picture of the mechanisms of oscillations emerged that requires the restructuring of contemporary mathematical ß-cell models, including our own dual oscillator model. In the companion article, we modified our model to account for these new data.

Ca2+ Effects on ATP Production and Consumption Have Regulatory Roles on Oscillatory Islet Activity.

Pancreatic islets respond to elevated blood glucose by secreting pulses of insulin that parallel oscillations in ß-cell metabolism, intracellular Ca(2+) concentration, and bursting electrical activity. The mechanisms that maintain an oscillatory response are not fully understood, yet several models have been proposed. Only some can account for experiments supporting that metabolism is intrinsically oscillatory in ß-cells. The dual oscillator model (DOM) implicates glycolysis as the source of oscillatory metabolism. In the companion article, we use recently developed biosensors to confirm that glycolysis is oscillatory and further elucidate the coordination of metabolic and electrical signals in the insulin secretory pathway. In this report, we modify the DOM by incorporating an established link between metabolism and intracellular Ca(2+) to reconcile model predictions with experimental observations from the companion article. With modification, we maintain the distinguishing feature of the DOM, oscillatory glycolysis, but introduce the ability of Ca(2+) influx to reshape glycolytic oscillations by promoting glycolytic efflux. We use the modified model to explain measurements from the companion article and from previously published experiments with islets.

The ubiquitin ligase HUWE1 enhances WNT signaling by antagonizing destruction complex-mediated ß-catenin degradation and through a mechanism independent of ß-catenin stability.

WNT/ß-catenin signaling is mediated by the transcriptional coactivator ß-catenin (CTNNB1). CTNNB1 abundance is regulated by phosphorylation and proteasomal degradation promoted by a destruction complex composed of the scaffold proteins APC and AXIN1 or AXIN2, and the kinases CSNK1A1 and GSK3A or GSK3B. Loss of CSNK1A1 increases CTNNB1 abundance, resulting in hyperactive WNT signaling. Previously, we demonstrated that the HECT domain ubiquitin ligase HUWE1 is necessary for hyperactive WNT signaling in HAP1 haploid human cells lacking CSNK1A1. Here, we investigate the mechanism underlying this requirement. In the absence of CSNK1A1, GSK3A/GSK3B still phosphorylated a fraction of CTNNB1, promoting its degradation. HUWE1 loss enhanced GSK3A/GSK3B-dependent CTNNB1 phosphorylation, further reducing CTNNB1 abundance. However, the reduction in CTNNB1 caused by HUWE1 loss was disproportionately smaller than the reduction in WNT target gene transcription. To test if the reduction in WNT signaling resulted from reduced CTNNB1 abundance alone, we engineered the endogenous CTNNB1 locus in HAP1 cells to encode a CTNNB1 variant insensitive to destruction complex-mediated phosphorylation and degradation. HUWE1 loss in these cells reduced WNT signaling with no change in CTNNB1 abundance. Genetic interaction and overexpression analyses revealed that the effects of HUWE1 on WNT signaling were not only mediated by GSK3A/GSK3B, but also by APC and AXIN1. Regulation of WNT signaling by HUWE1 required its ubiquitin ligase activity. These results suggest that in cells lacking CSNK1A1, a destruction complex containing APC, AXIN1 and GSK3A/GSK3B downregulates WNT signaling by phosphorylating and targeting CTNNB1 for degradation. HUWE1 enhances WNT signaling by antagonizing this activity. Therefore, HUWE1 enhances WNT/CTNNB1 signaling through two mechanisms, one that regulates CTNNB1 abundance and another that is independent of CTNNB1 stability. Coordinated regulation of CTNNB1 abundance and an independent signaling step by HUWE1 would be an efficient way to control WNT signaling output, enabling sensitive and robust activation of the pathway.

Osmosensitive changes of carbohydrate metabolism in response to cellulose biosynthesis inhibition.

Cellulose is the most abundant biopolymer in the world, the main load-bearing element in plant cell walls, and represents a major sink for carbon fixed during photosynthesis. Previous work has shown that photosynthetic activity is partially regulated by carbohydrate sinks. However, the coordination of cellulose biosynthesis with carbohydrate metabolism and photosynthesis is not well understood. Here, we demonstrate that cellulose biosynthesis inhibition (CBI) leads to reductions in transcript levels of genes involved in photosynthesis, the Calvin cycle, and starch degradation in Arabidopsis (Arabidopsis thaliana) seedlings. In parallel, we show that CBI induces changes in carbohydrate distribution and influences Rubisco activase levels. We find that the effects of CBI on gene expression and carbohydrate metabolism can be neutralized by osmotic support in a concentration-dependent manner. However, osmotic support does not suppress CBI-induced metabolic changes in seedlings impaired in mechanoperception (mid1 complementing activity1 [mca1]) and osmoperception (cytokinin receptor1 [cre1]) or reactive oxygen species production (respiratory burst oxidase homolog DF [rbohDF]). These results show that carbohydrate metabolism is responsive to changes in cellulose biosynthesis activity and turgor pressure. The data suggest that MCA1, CRE1, and RBOHDF-derived reactive oxygen species are involved in the regulation of osmosensitive metabolic changes. The evidence presented here supports the notion that cellulose and carbohydrate metabolism may be coordinated via an osmosensitive mechanism.

Studying Nuclear Dynamics in Response to Actin Disruption in Planta.

The plant nucleus and the actin cytoskeleton are intimately connected. The actin cytoskeleton is pivotal for nuclear positioning, shape, and dynamics. These properties of the nucleus are important for its functions during normal development and in response to external cues such as biotic and abiotic stresses. Moreover, we know that there is a direct physical connection between the actin cytoskeleton and the nucleus which spans the double-membraned nuclear envelope into the nuclear lamina, and this connection is called the linker of nucleoskeleton and cytoskeleton (LINC) complex. Recently a role for actin in regulating inter-nuclear organization via the control of nuclear invaginations has emerged. Therefore, a detailed understanding of nuclear shape, organization, and dynamics and the techniques used to measure and quantify these metrics will allow us to determine and further understand the contribution made by actin to these parameters. The protocols described here will allow researchers to determine the circularity index of a nucleus, quantify nuclear deformations, and determine dynamics of nuclei within plant cells.

Optimization of a Class of Dihydrobenzofurane Analogs toward Orally Efficacious YAP-TEAD Protein-Protein Interaction Inhibitors.

The inhibition of the YAP-TEAD protein-protein interaction constitutes a promising therapeutic approach for the treatment of cancers linked to the dysregulation of the Hippo signaling pathway. The identification of a class of small molecules which potently inhibit the YAP-TEAD interaction by binding tightly to the Ω-loop pocket of TEAD has previously been communicated. This report details the further multi-parameter optimization of this class of compounds resulting in advanced analogs combining nanomolar cellular potency with a balanced ADME and off-target profile, and efficacy of these compounds in tumor bearing mice is demonstrated for the first time.

Cell wall damage-induced lignin biosynthesis is regulated by a reactive oxygen species- and jasmonic acid-dependent process in Arabidopsis.

The plant cell wall is a dynamic and complex structure whose functional integrity is constantly being monitored and maintained during development and interactions with the environment. In response to cell wall damage (CWD), putatively compensatory responses, such as lignin production, are initiated. In this context, lignin deposition could reinforce the cell wall to maintain functional integrity. Lignin is important for the plant's response to environmental stress, for reinforcement during secondary cell wall formation, and for long-distance water transport. Here, we identify two stages and several components of a genetic network that regulate CWD-induced lignin production in Arabidopsis (Arabidopsis thaliana). During the early stage, calcium and diphenyleneiodonium-sensitive reactive oxygen species (ROS) production are required to induce a secondary ROS burst and jasmonic acid (JA) accumulation. During the second stage, ROS derived from the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D and JA-isoleucine generated by JASMONIC ACID RESISTANT1, form a negative feedback loop that can repress each other's production. This feedback loop in turn seems to influence lignin accumulation. Our results characterize a genetic network enabling plants to regulate lignin biosynthesis in response to CWD through dynamic interactions between JA and ROS.

Quantifying the Organization and Dynamics of the Plant Plasma Membrane Across Scales Using Light Microscopy.

The plant cell surface continuum is composed of the cell wall, plasma membrane, and cytoskeleton. Plasmodesmata are specialized channels in the cell wall allowing intercellular communication and resource distribution. Proteins within these organelles play fundamental roles in development, perception of the external environment, and resource acquisition. Therefore, an understanding of protein dynamics and organization within the membrane and plasmodesmata is of fundamental importance to understanding both how plants develop as well as perceive the myriad of external stimuli they experience and initiate appropriate downstream responses. In this chapter, I will describe protocols for quantifying the dynamics and organization of the plasma membrane and plasmodesmata proteins across scales. The protocols described below allow researchers to determine bulk protein mobility within the membrane using fluorescence recovery after photobleaching (FRAP), imaging, and quantification of nanodomain size (with Airyscan confocal microscopy) and determining the dynamics of these nanodomains at the single particle level using total internal reflection (TIRF) single particle imaging.