Quantitative imaging reveals the role of MpARF proteasomal degradation during gemma germination.

The auxin signaling molecule controls a variety of growth and developmental processes in land plants. Auxin regulates gene expression through a nuclear auxin signaling pathway (NAP) consisting of a ubiquitin ligase auxin receptor TIR1/AFB, its Aux/IAA degradation substrate, and DNA-binding ARF transcription factors. While extensive qualitative understanding of the pathway and its interactions has been obtained, mostly by studying the flowering plant Arabidopsis thaliana, it is so far unknown how these translate to quantitative system behaviour in vivo, a problem that is confounded by large NAP gene families in most species. Here we used the minimal NAP of the liverwort Marchantia polymorpha to quantitatively map NAP protein accumulation and dynamics in vivo through the use of knock-in fluorescent fusion proteins. Beyond revealing the dynamic native accumulation profile of the entire NAP protein network, we discovered that the two central ARFs, MpARF1 and MpARF2, are proteasomally degraded. This auxin-independent degradation tunes ARF protein stoichiometry to favor gene activation, thereby reprogramming auxin response during developmental progression. Thus, quantitative analysis of the entire NAP allowed us to identify ARF degradation and stoichiometries of activator and repressor ARFs as a potential mechanism for controlling gemma germination.

A multifaceted kinase axis regulates plant organ abscission through conserved signaling mechanisms.

Plants have evolved mechanisms to abscise organs as they develop or when exposed to unfavorable conditions.1 Uncontrolled abscission of petals, fruits, or leaves can impair agricultural productivity.2,3,4,5 Despite its importance for abscission progression, our understanding of the IDA signaling pathway and its regulation remains incomplete. IDA is secreted to the apoplast, where it is perceived by the receptors HAESA (HAE) and HAESA-LIKE2 (HSL2) and somatic embryogenesis receptor kinase (SERK) co-receptors.6,7,8,9 These plasma membrane receptors activate an intracellular cascade of mitogen-activated protein kinases (MAPKs) by an unknown mechanism.10,11,12 Here, we characterize brassinosteroid signaling kinases (BSKs) as regulators of floral organ abscission in Arabidopsis. BSK1 localizes to the plasma membrane of abscission zone cells, where it interacts with HAESA receptors to regulate abscission. Furthermore, we demonstrate that YODA (YDA) has a leading role among other MAPKKKs in controlling abscission downstream of the HAESA/BSK complex. This kinase axis, comprising a leucine-rich repeat receptor kinase, a BSK, and an MAPKKK, is known to regulate stomatal patterning, early embryo development, and immunity.10,13,14,15,16 How specific cellular responses are obtained despite signaling through common effectors is not well understood. We show that the identified abscission-promoting allele of BSK1 also enhances receptor signaling in other BSK-mediated pathways, suggesting conservation of signaling mechanisms. Furthermore, we provide genetic evidence supporting independence of BSK1 function from its kinase activity in several developmental processes. Together, our findings suggest that BSK1 facilitates signaling between plasma membrane receptor kinases and MAPKKKs via conserved mechanisms across multiple facets of plant development.

Protein degradation in the auxin response.

The signaling molecule auxin sits at the nexus of plant biology and coordinates essentially all growth and developmental processes in plants. Auxin molecules are transported throughout plant tissues and are capable of evoking highly specific physiological responses in plant cells by inducing various molecular pathways. In many of these pathways, proteolysis plays a crucial role for correct physiological responses. This review provides a chronology of the discovery and characterisation of the auxin receptor, which is a fascinating example of separate research trajectories ultimately converging on the discovery of a core auxin signaling hub which relies on degradation of a family of transcriptional inhibitor proteins - the Aux/IAAs. Beyond describing the "classical" proteolysis-driven auxin response system, we explore more recent examples of the interconnection of proteolytic systems, which target a range of other auxin signaling proteins, and auxin response. By highlighting these emerging concepts, we provide potential future directions to further investigate the role of protein degradation within the framework of auxin response.

Revealing heterogeneity in correlation times of EGFP encapsulated in complex coacervate core micelles by analysis of fluorescence anisotropies.

Encapsulation of enhanced green fluorescent protein (EGFP) in complex coacervate core micelles (C3Ms) can be established by mixing EGFP with diblock polymers at equal charge ratio. It has previously been shown that this encapsulation system is highly dynamic, implying existence of different populations; GFP free in solution or complexed with polymers (small complexes) and EGFP encapsulated in C3Ms. We performed time resolved fluorescence anisotropy experiments to determine the relative populations of EGFP encapsulated in C3Ms using three different fluorescence anisotropy decay analysis methods. First, Maximum Entropy Method (MEM) data analysis was employed for five different EGFP concentrations in C3Ms that were mixed with dark fluorescent proteins (10, 20, 30, 40 and 50% EGFP, respectively). In all cases, correlation-time distributions between 0.1 and 100 ns (on a logarithmic timescale) are clearly visible showing bimodal distribution. The distribution between 0.1 and 2.0 ns is due to homo-FRET between EGFP molecules packed in micelles and the distribution between 8 and 30 ns coincides with the correlation-time distribution of free EGFP in solution. The fraction of homo-FRET distribution linearly increases with increase of relative micellar EGFP concentrations. These MEM results were corroborated by two different analysis methods: global population analysis of all five fluorescence anisotropy decays arising from EGFP in micelles together with the one of free EGFP (direct analysis of anisotropies) and global associative population analysis of anisotropies by fitting parallel and perpendicular fluorescence decay components. In contrast to global analyses approaches, the MEM method directly reveals distributions of correlation times without any prior information about the sample. However, global associative analysis of anisotropies by fitting parallel and perpendicular fluorescence decay components is the only method that allows to estimate accurately fractions of free fluorophores in solution and encapsulated fluorophores.

Enhanced stability of complex coacervate core micelles following different core-crosslinking strategies.

Complex coacervate core micelles (C3Ms) are formed by mixing aqueous solutions of a charged (bio)macromolecule with an oppositely charged-neutral hydrophilic diblock copolymer. The stability of these structures is dependent on the ionic strength of the solution; above a critical ionic strength, the micelles will completely disintegrate. This instability at high ionic strengths is the main drawback for their application in, e.g., drug delivery systems or protein protection. In addition, the stability of C3Ms composed of weak polyelectrolytes is pH-dependent as well. The aim of this study is to assess the effectiveness of covalent crosslinking of the complex coacervate core to improve the stability of C3Ms. We studied the formation of C3Ms using a quaternized and amine-functionalized cationic-neutral diblock copolymer, poly(2-vinylpyridine)-block-poly(ethylene oxide) (QP2VP-b-PEO), and an anionic homopolymer, poly(acrylic acid) (PAA). Two different core-crosslinking strategies were employed that resulted in crosslinks between both types of polyelectrolyte chains in the core (i.e., between QP2VP and PAA) or in crosslinks between polyelectrolyte chains of the same type only (i.e., QP2VP). For these two strategies we used the crosslinkers 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and dimethyl-3,3'-dithiopropionimidate dihydrochloride (DTBP), respectively. EDC provides permanent crosslinks, while DTBP crosslinks can be broken by a reducing agent. Dynamic light scattering showed that both approaches significantly improved the stability of C3Ms against salt and pH changes. Furthermore, reduction of the disulphide bridges in the DTBP core-crosslinked micelles largely restored the original salt-stability profile. Therefore, this feature provides an excellent starting point for the application of C3Ms in controlled release formulations.

Charged Polypeptide Tail Boosts the Salt Resistance of Enzyme-Containing Complex Coacervate Micelles.

Encapsulation of proteins can have advantages for their protection, stability, and delivery purposes. One of the options to encapsulate proteins is to incorporate them in complex coacervate core micelles (C3Ms). This can easily be achieved by mixing aqueous solutions of the protein and an oppositely charged neutral-hydrophilic diblock copolymer. However, protein-containing C3Ms often suffer from salt-inducible disintegration due to the low charge density of proteins. The aim of this study is to improve the salt stability of protein-containing C3Ms by increasing the net charge of the protein by tagging it with a charged polypeptide. As a model protein, we used CotA laccase and generated variants with 10, 20, 30, and 40 glutamic acids attached at the C-terminus of CotA using genetic engineering. Micelles were obtained by mixing the five CotA variants with poly(N-methyl-2-vinyl-pyridinium)-block-poly(ethylene oxide) (PM2VP128-b-PEO477) at pH 10.8. Hydrodynamic radii of the micelles of approximately 31, 27, and 23 nm for native CotA, CotA-E20, and CotA-E40, respectively, were determined using dynamic light scattering (DLS) and fluorescence correlation spectroscopy (FCS). The encapsulation efficiency was not affected using enzymes with a polyglutamic acid tail but resulted in more micelles with a smaller number of enzyme molecules per micelle. Furthermore, it was shown that the addition of a polyglutamic acid tail to CotA indeed resulted in improved salt stability of enzyme-containing C3Ms. Interestingly, the polyglutamic acid CotA variants showed an enhanced enzyme activity. This study demonstrates that increasing the net charge of enzymes through genetic engineering is a promising strategy to improve the practical applicability of C3Ms as enzyme delivery systems.

Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells.

Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application in living cells, and we were able to distinguish GFP, diGFP, as well as ligand-induced dimerization of FKBP12 (FK506 binding protein 12)-GFP. While other analysis methods have only sporadically been used to study dimerization in living cells and may be prone to errors, this paper provides a robust approach for the investigation of any cytosolic protein using single-color fluorescence fluctuation spectroscopy.

Auxin Response by the Numbers.

Auxin is fundamental to the growth and development of land plants, and acts in large part through the control of gene activity. Genetic and biochemical analysis of the nuclear auxin signaling pathway (NAP) has led to the establishment of a generic model for auxin-dependent gene regulation. To understand how this dynamic system operates in living cells, quantitative data are needed. For this, the liverwort Marchantia polymorpha provides a useful model system. Its limited number of NAP components, combined with experimental approaches to determine concentrations, binding affinities, and turnover rates, will enable a new, quantitative view on the mechanisms that allow auxin to control plant growth and development.

Hypoxia-inducible lipid droplet-associated induces DGAT1 and promotes lipid storage in hepatocytes.

OBJECTIVE: Storage of triglycerides in lipid droplets is governed by a set of lipid droplet-associated proteins. One of these lipid droplet-associated proteins, hypoxia-inducible lipid droplet-associated (HILPDA), was found to impair lipid droplet breakdown in macrophages and cancer cells by inhibiting adipose triglyceride lipase. Here, we aimed to better characterize the role and mechanism of action of HILPDA in hepatocytes. METHODS: We performed studies in HILPDA-deficient and HILPDA-overexpressing liver cells, liver slices, and mice. The functional role and physical interactions of HILPDA were investigated using a variety of biochemical and microscopic techniques, including real-time fluorescence live-cell imaging and Förster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM). RESULTS: Levels of HILPDA were markedly induced by fatty acids in several hepatoma cell lines. Hepatocyte-specific deficiency of HILPDA in mice modestly but significantly reduced hepatic triglycerides in mice with non-alcoholic steatohepatitis. Similarly, deficiency of HILPDA in mouse liver slices and primary hepatocytes reduced lipid storage and accumulation of fluorescently-labeled fatty acids in lipid droplets, respectively, which was independent of adipose triglyceride lipase. Fluorescence microscopy showed that HILPDA partly colocalizes with lipid droplets and with the endoplasmic reticulum, is especially abundant in perinuclear areas, and mainly associates with newly added fatty acids. Real-time fluorescence live-cell imaging further revealed that HILPDA preferentially localizes to lipid droplets that are being remodeled. Overexpression of HILPDA in liver cells increased the activity of diacylglycerol acyltransferases (DGAT) and DGAT1 protein levels, concurrent with increased lipid storage. Confocal microscopy coupled to FRET-FLIM analysis demonstrated that HILPDA physically interacts with DGAT1 in living liver cells. The stimulatory effect of HILPDA on lipid storage via DGAT1 was corroborated in adipocytes. CONCLUSIONS: Our data indicate that HILPDA physically interacts with DGAT1 and increases DGAT activity. Our findings suggest a novel regulatory mechanism by which fatty acids promote triglyceride synthesis and storage.

Complete microviscosity maps of living plant cells and tissues with a toolbox of targeting mechanoprobes.

Mechanical patterns control a variety of biological processes in plants. The microviscosity of cellular structures effects the diffusion rate of molecules and organelles, thereby affecting processes such as metabolism and signaling. Spatial variations in local viscosity are also generated during fundamental events in the cell life cycle. While crucial to a complete understanding of plant mechanobiology, resolving subcellular microviscosity patterns in plants has remained an unsolved challenge. We present an imaging microviscosimetry toolbox of molecular rotors that yield complete microviscosity maps of cells and tissues, specifically targeting the cytosol, vacuole, plasma membrane, and wall of plant cells. These boron-dipyrromethene (BODIPY)-based molecular rotors are rigidochromic by means of coupling the rate of an intramolecular rotation, which depends on the mechanics of their direct surroundings, with their fluorescence lifetime. This enables the optical mapping of fluidity and porosity patterns in targeted cellular compartments. We show how apparent viscosity relates to cell function in the root, how the growth of cellular protrusions induces local tension, and how the cell wall is adapted to perform actuation surrounding leaf pores. These results pave the way to the noninvasive micromechanical mapping of complex tissues.

Balancing Enzyme Encapsulation Efficiency and Stability in Complex Coacervate Core Micelles.

Encapsulation of charged proteins into complex coacervate core micelles (C3Ms) can be accomplished by mixing them with oppositely charged diblock copolymers. However, these micelles tend to disintegrate at high ionic strength. Previous research showed that the addition of a homopolymer with the same charge sign as the protein improved the stability of protein-containing C3Ms. In this research, we used fluorescence correlation spectroscopy (FCS) and dynamic light scattering (DLS) to study how the addition of the homopolymer affects the encapsulation efficiency and salt stability of the micelles. We studied the encapsulation of laccase spore coat protein A (CotA), a multicopper oxidase, using a strong cationic-neutral diblock copolymer, poly(N-methyl-2-vinyl-pyridinium iodide)-block-poly(ethylene oxide) (PM2VP128-b-PEO477), and a negatively charged homopolymer, poly(4-styrenesulfonate) (PSS215). DLS indeed showed an improved stability of this three-component C3M system against the addition of salt compared to a two-component system. Remarkably, FCS showed that the release of CotA from a three-component C3M system occurred at a lower salt concentration and over a narrower concentration range than the dissociation of C3Ms. In conclusion, although the addition of the homopolymer to the system leads to micelles with a higher salt stability, CotA is excluded from the C3Ms already at lower ionic strengths because the homopolymer acts as a competitor of the enzyme for encapsulation.

Design principles of a minimal auxin response system.

Auxin controls numerous growth processes in land plants through a gene expression system that modulates ARF transcription factor activity1-3. Gene duplications in families encoding auxin response components have generated tremendous complexity in most land plants, and neofunctionalization enabled various unique response outputs during development1,3,4. However, it is unclear what fundamental biochemical principles underlie this complex response system. By studying the minimal system in Marchantia polymorpha, we derive an intuitive and simple model where a single auxin-dependent A-ARF activates gene expression. It is antagonized by an auxin-independent B-ARF that represses common target genes. The expression patterns of both ARF proteins define developmental zones where auxin response is permitted, quantitatively tuned or prevented. This fundamental design probably represents the ancestral system and formed the basis for inflated, complex systems.

HILPDA Uncouples Lipid Droplet Accumulation in Adipose Tissue Macrophages from Inflammation and Metabolic Dysregulation.

Obesity leads to a state of chronic, low-grade inflammation that features the accumulation of lipid-laden macrophages in adipose tissue. Here, we determined the role of macrophage lipid-droplet accumulation in the development of obesity-induced adipose-tissue inflammation, using mice with myeloid-specific deficiency of the lipid-inducible HILPDA protein. HILPDA deficiency markedly reduced intracellular lipid levels and accumulation of fluorescently labeled fatty acids. Decreased lipid storage in HILPDA-deficient macrophages can be rescued by inhibition of adipose triglyceride lipase (ATGL) and is associated with increased oxidative metabolism. In diet-induced obese mice, HILPDA deficiency does not alter inflammatory and metabolic parameters, despite markedly reducing lipid accumulation in macrophages. Overall, we find that HILPDA is a lipid-inducible, physiological inhibitor of ATGL-mediated lipolysis in macrophages and uncouples lipid storage in adipose tissue macrophages from inflammation and metabolic dysregulation. Our data question the contribution of lipid droplet accumulation in adipose tissue macrophages in obesity-induced inflammation and metabolic dysregulation.

Time-gated confocal microscopy reveals accumulation of exocyst subunits at the plant-pathogen interface.

Polarized exocytosis is essential for plant development and defence. The exocyst, an octameric protein complex that tethers exocytotic vesicles to the plasma membrane, targets exocytosis. Upon pathogen attack, secreted materials form papillae to halt pathogen penetration. To determine if the exocyst is directly involved in targeting exocytosis to infection sites, information about its localization is instrumental. Here, we investigated exocyst subunit localization in the moss Physcomitrella patens upon pathogen attack and infection by Phytophthora capsici. Time-gated confocal microscopy was used to eliminate autofluorescence of deposited material around infection sites, allowing the visualization of the subcellular localization of exocyst subunits and of v-SNARE Vamp72A1-labelled exocytotic vesicles during infection. This showed that exocyst subunits Sec3a, Sec5b, Sec5d, and Sec6 accumulated at sites of attempted pathogen penetration. Upon pathogen invasion, the exocyst subunits accumulated on the membrane surrounding papilla-like structures and hyphal encasements. Vamp72A1-labelled vesicles were found to localize in the cytoplasm around infection sites. The re-localization of exocyst subunits to infection sites suggests that the exocyst is directly involved in facilitating polarized exocytosis during pathogenesis.

Analysis and modeling of enhanced green fluorescent protein diffusivity in whey protein gels.

During gastric digestion, hydrolysis of proteins by pepsin contributes largely to the breakdown of protein-rich food. We hypothesized that the effect of pepsin is limited by its diffusivity, which is co-determined by the food structure and the local pH in the food during digestion. To investigate the principle mechanism of enzyme diffusion in food matrices, we used enhanced green fluorescent protein (EGFP) as probe to study the diffusivity of proteins in whey protein isolate gels, using fluorescence correlation spectroscopy (FCS). Gels made with different ionic strength showed distinctive elastic moduli but did not show differences in diffusivity of EGFP. Some models for diffusion in hydrogels yield good description of the obtained data, and can approximate the enzyme diffusion in diverse food matrices. However, the enzyme pepsin is more complicated than the probe EGFP, to yield more accurate predictions, electrostatic and enzyme-substrate interaction also need to be considered.

FRET Reveals the Formation and Exchange Dynamics of Protein-Containing Complex Coacervate Core Micelles.

The encapsulation of proteins into complex coacervate core micelles (C3Ms) is of potential interest for a wide range of applications. To address the stability and dynamic properties of these polyelectrolyte complexes, combinations of cyan, yellow, and blue fluorescent proteins were encapsulated with cationic-neutral diblock copolymer poly(2-methyl-vinyl-pyridinium)128- b-poly(ethylene-oxide)477. Förster resonance energy transfer (FRET) allowed us to determine the kinetics of C3M formation and of protein exchange between C3Ms. Both processes follow first-order kinetics with relaxation times of ±100 s at low ionic strength ( I = 2.5 mM). Stability studies revealed that 50% of FRET was lost at I = 20 mM, pointing to the disintegration of the C3Ms. On the basis of experimental and theoretical considerations, we propose that C3Ms relax to their final state by association and dissociation of near-neutral soluble protein-polymer complexes. To obtain protein-containing C3Ms suitable for applications, it is necessary to improve the rigidity and salt stability of these complexes.

Distinct Roles of Non-Overlapping Surface Regions of the Coiled-Coil Domain in the Potato Immune Receptor Rx1.

The intracellular immune receptor Rx1 of potato (Solanum tuberosum), which confers effector-triggered immunity to Potato virus X, consists of a central nucleotide-binding domain (NB-ARC) flanked by a carboxyl-terminal leucine-rich repeat (LRR) domain and an amino-terminal coiled-coil (CC) domain. Rx1 activity is strictly regulated by interdomain interactions between the NB-ARC and LRR, but the contribution of the CC domain in regulating Rx1 activity or immune signaling is not fully understood. Therefore, we used a structure-informed approach to investigate the role of the CC domain in Rx1 functionality. Targeted mutagenesis of CC surface residues revealed separate regions required for the intramolecular and intermolecular interaction of the CC with the NB-ARC-LRR and the cofactor Ran GTPase-activating protein2 (RanGAP2), respectively. None of the mutant Rx1 proteins was constitutively active, indicating that the CC does not contribute to the autoinhibition of Rx1 activity. Instead, the CC domain acted as a modulator of downstream responses involved in effector-triggered immunity. Systematic disruption of the hydrophobic interface between the four helices of the CC enabled the uncoupling of cell death and disease resistance responses. Moreover, a strong dominant negative effect on Rx1-mediated resistance and cell death was observed upon coexpression of the CC alone with full-length Rx1 protein, which depended on the RanGAP2-binding surface of the CC. Surprisingly, coexpression of the N-terminal half of the CC enhanced Rx1-mediated resistance, which further indicated that the CC functions as a scaffold for downstream components involved in the modulation of disease resistance or cell death signaling.

Alcohol Acetyltransferase Eat1 Is Located in Yeast Mitochondria.

Eat1 is a recently discovered alcohol acetyltransferase responsible for bulk ethyl acetate production in yeasts such as Wickerhamomyces anomalus and Kluyveromyces lactis These yeasts have the potential to become efficient bio-based ethyl acetate producers. However, some fundamental features of Eat1 are still not understood, which hampers the rational engineering of efficient production strains. The cellular location of Eat1 in yeast is one of these features. To reveal its location, Eat1 was fused with yeast-enhanced green fluorescent protein (yEGFP) to allow intracellular tracking. Despite the current assumption that bulk ethyl acetate production occurs in the yeast cytosol, most of Eat1 localized to the mitochondria of Kluyveromyces lactis CBS 2359 Δku80 We then compared five bulk ethyl acetate-producing yeasts in iron-limited chemostats with glucose as the carbon source. All yeasts produced ethyl acetate under these conditions. This strongly suggests that the mechanism and location of bulk ethyl acetate synthesis are similar in these yeast strains. Furthermore, an in silico analysis showed that Eat1 proteins from various yeasts were mostly predicted as mitochondrial. Altogether, it is concluded that Eat1-catalyzed ethyl acetate production occurs in yeast mitochondria. This study has added new insights into bulk ethyl acetate synthesis in yeast, which is relevant for developing efficient production strains.IMPORTANCE Ethyl acetate is a common bulk chemical that is currently produced from petrochemical sources. Several Eat1-containing yeast strains naturally produce large amounts of ethyl acetate and are potential cell factories for the production of bio-based ethyl acetate. Rational design of the underlying metabolic pathways may result in improved production strains, but it requires fundamental knowledge on the function of Eat1. A key feature is the location of Eat1 in the yeast cell. The precursors for ethyl acetate synthesis can be produced in multiple cellular compartments through different metabolic pathways. The location of Eat1 determines the relevance of each pathway, which will provide future targets for the metabolic engineering of bulk ethyl acetate production in yeast.