Arabidopsis CaLB1 undergoes phase separation with the ESCRT protein ALIX and modulates autophagosome maturation.

Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require a complex choreography of numerous factors. The endosomal sorting complex required for transport (ESCRT) is implicated in the final step of autophagosomal maturation by sealing of the phagophore membrane. ESCRT-III components were shown to mediate membrane scission by forming filaments that interact with cellular membranes. However, the molecular mechanisms underlying the recruitment of ESCRTs to non-endosomal membranes remain largely unknown. Here we focus on the ESCRT-associated protein ALG2-interacting protein X (ALIX) and identify Ca2+-dependent lipid binding protein 1 (CaLB1) as its interactor. Our findings demonstrate that CaLB1 interacts with AUTOPHAGY8 (ATG8) and PI(3)P, a phospholipid found in autophagosomal membranes. Moreover, CaLB1 and ALIX localize with ATG8 on autophagosomes upon salt treatment and assemble together into condensates. The depletion of CaLB1 impacts the maturation of salt-induced autophagosomes and leads to reduced delivery of autophagosomes to the vacuole. Here, we propose a crucial role of CaLB1 in augmenting phase separation of ALIX, facilitating the recruitment of ESCRT-III to the site of phagophore closure thereby ensuring efficient maturation of autophagosomes.

Role of the dynamin-related protein 2 family and SH3P2 in clathrin-mediated endocytosis in Arabidopsis thaliana.

Clathrin-mediated endocytosis (CME) is vital for the regulation of plant growth and development through controlling plasma membrane protein composition and cargo uptake. CME relies on the precise recruitment of regulators for vesicle maturation and release. Homologues of components of mammalian vesicle scission are strong candidates to be part of the scission machinery in plants, but the precise roles of these proteins in this process are not fully understood. Here, we characterised the roles of the plant dynamin-related protein 2 (DRP2) family (hereafter DRP2s) and SH3-domain containing protein 2 (SH3P2), the plant homologue to recruiters of dynamins, such as endophilin and amphiphysin, in CME by combining high-resolution imaging of endocytic events in vivo and characterisation of the purified proteins in vitro. Although DRP2s and SH3P2 arrive similarly late during CME and physically interact, genetic analysis of the sh3p123 triple mutant and complementation assays with non-SH3P2-interacting DRP2 variants suggest that SH3P2 does not directly recruit DRP2s to the site of endocytosis. These observations imply that, despite the presence of many well-conserved endocytic components, plants have acquired a distinct mechanism for CME.

Measuring the DUB Activity of Arabidopsis Deubiquitylating Enzymes.

Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here we show methods to analyze DUB activity using immunodetection, Coomassie brilliant blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.

Lipid-mediated activation of plasma membrane-localized deubiquitylating enzymes modulate endosomal trafficking.

The abundance of plasma membrane-resident receptors and transporters has to be tightly regulated by ubiquitin-mediated endosomal degradation for the proper coordination of environmental stimuli and intracellular signaling. Arabidopsis OVARIAN TUMOR PROTEASE (OTU) 11 and OTU12 are plasma membrane-localized deubiquitylating enzymes (DUBs) that bind to phospholipids through a polybasic motif in the OTU domain. Here we show that the DUB activity of OTU11 and OTU12 towards K63-linked ubiquitin is stimulated by binding to lipid membranes containing anionic lipids. In addition, we show that the DUB activity of OTU11 against K6- and K11-linkages is also stimulated by anionic lipids, and that OTU11 and OTU12 can modulate the endosomal degradation of a model cargo and the auxin efflux transporter PIN2-GFP in vivo. Our results suggest that the catalytic activity of OTU11 and OTU12 is tightly connected to their ability to bind membranes and that OTU11 and OTU12 are involved in the fine-tuning of plasma membrane proteins in Arabidopsis.

Patterns of Domain-Specific Learning Among Medical Undergraduate Students in Relation to Confidence in Their Physiology Knowledge: Insights From a Pre-post Study.

RESEARCH FOCUS: The promotion of domain-specific knowledge is a central goal of higher education and, in the field of medicine, it is particularly essential to promote global health. Domain-specific knowledge on its own is not exhaustive; confidence regarding the factual truth of this knowledge content is also required. An increase in both knowledge and confidence is considered a necessary prerequisite for making professional decisions in the clinical context. Especially the knowledge of human physiology is fundamental and simultaneously critical to medical decision-making. However, numerous studies have shown difficulties in understanding and misconceptions in this area of knowledge. Therefore, we investigate (i) how preclinical medical students acquire knowledge in physiology over the course of their studies and simultaneously gain confidence in the correctness of this knowledge as well as (ii) the interrelations between these variables, and (iii) how they affect the development of domain-specific knowledge. METHOD: In a pre-post study, 169 medical students' development of physiology knowledge and their confidence related to this knowledge were assessed via paper-pencil questionnaires before and after attending physiology seminars for one semester. Data from a longitudinal sample of n = 97 students were analyzed using mean comparisons, regression analyses, and latent class analyses (LCAs). In addition, four types of item responses were formed based on confidence and correctness in the knowledge test. RESULTS: We found a significant and large increase in the students' physiology knowledge, with task-related confidence being the strongest predictor (apart from learning motivation). Moreover, a significantly higher level of confidence at t2 was confirmed, with the level of prior confidence being a strong predictor (apart from knowledge at t2). Furthermore, based on the students' development of knowledge and confidence levels between measurement points, three empirically distinct groups were distinguished: knowledge gainers, confidence gainers, and overall gainers. The students whose confidence in incorrect knowledge increased constituted one particularly striking group. Therefore, the training of both knowledge and the ability to critically reflect on one's knowledge and skills as well as an assessment of their development in education is required, especially in professions such as medicine, where knowledge-based decisions made with confidence are of vital importance.

Ubiquitin recognition in endocytic trafficking - with or without ESCRT-0.

The ability to sense and adapt to the constantly changing environment is important for all organisms. Cell surface receptors and transporters are key for the fast response to extracellular stimuli and, thus, their abundance on the plasma membrane has to be strictly controlled. Heteromeric endosomal sorting complexes required for transport (ESCRTs) are responsible for mediating the post-translational degradation of endocytosed plasma membrane proteins in eukaryotes and are essential both in animals and plants. ESCRTs bind and sort ubiquitylated cargoes for vacuolar degradation. Although many components that comprise the multi-subunit ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III complexes are conserved in eukaryotes, plant and animal ESCRTs have diverged during the course of evolution. Homologues of ESCRT-0, which recognises ubiquitylated cargo, have emerged in metazoan and fungi but are not found in plants. Instead, the Arabidopsis genome encodes plant-specific ubiquitin adaptors and a greater number of target of Myb protein 1 (TOM1) homologues than in mammals. In this Review, we summarise and discuss recent findings on ubiquitin-binding proteins in Arabidopsis that could have equivalent functions to ESCRT-0. We further hypothesise that SH3 domain-containing proteins might serve as membrane curvature-sensing endophilin and amphiphysin homologues during plant endocytosis.

Transient Expression of ESCRT Components in Arabidopsis Root Cell Suspension Culture-Derived Protoplasts.

Localization studies are important to understand the function of diverse proteins. The endosomal trafficking pathway is very complex, and a lot of proteins function in this pathway, primarily the endosomal sorting complexes required for transport (ESCRTs). Some of the ESCRT-related proteins or mutant variants cannot be stably expressed in planta due to the toxicity of their expression. Therefore, a transient expression system is necessary to study their function. Transient expression in protoplasts from Arabidopsis root cell-derived culture serves as a fast and reliable method for the expression and cell biological and biochemical analyses of otherwise toxic constructs.

The Arabidopsis receptor kinase STRUBBELIG undergoes clathrin-dependent endocytosis.

Signaling mediated by cell surface receptor kinases is central to the coordination of growth patterns during organogenesis. Receptor kinase signaling is in part controlled through endocytosis and subcellular distribution of the respective receptor kinase. For the majority of plant cell surface receptors, the underlying trafficking mechanisms are not characterized. In Arabidopsis, tissue morphogenesis requires the atypical receptor kinase STRUBBELIG (SUB). Here, we studied the endocytic mechanism of SUB. Our data revealed that a functional SUB-enhanced green fluorescent protein (EGFP) fusion is ubiquitinated in vivo. We further showed that plasma membrane-bound SUB:EGFP becomes internalized in a clathrin-dependent fashion. We also found that SUB:EGFP associates with the trans-Golgi network and accumulates in multivesicular bodies and the vacuole. Co-immunoprecipitation experiments revealed that SUB:EGFP and clathrin are present within the same protein complex. Our genetic analysis showed that SUB and CLATHRIN HEAVY CHAIN (CHC) 2 regulate root hair patterning. By contrast, genetic reduction of CHC activity ameliorates the floral defects of sub mutants. Taken together, the data indicate that SUB undergoes clathrin-mediated endocytosis, that this process does not rely on stimulation of SUB signaling by an exogenous agent, and that SUB genetically interacts with clathrin-dependent pathways in a tissue-specific manner.

Preparation of Clathrin-Coated Vesicles From Arabidopsis thaliana Seedlings.

Clathrin coated vesicles (CCVs) mediate endocytosis of plasma membrane proteins and deliver their content to the endosomes for either subsequent recycling to the plasma membrane or transport to the vacuole for degradation. CCVs assemble also at the trans-Golgi network (TGN) and is responsible for the transport of proteins to other membranes. Oligomerization of clathrin and recruitment of adaptor protein complexes promote the budding and the release of CCVs. However, many of the details during plant CCV formation are not completely elucidated. The analysis of isolated CCVs is therefore important to better understand the formation of plant CCVs, their cargos and the regulation of clathrin-mediated transport processes. In this article, we describe an optimized method to isolate CCVs from Arabidopsis thaliana seedlings.

Arabidopsis SH3P2 is an ubiquitin-binding protein that functions together with ESCRT-I and the deubiquitylating enzyme AMSH3.

Clathrin-mediated endocytosis of plasma membrane proteins is an essential regulatory process that controls plasma membrane protein abundance and is therefore important for many signaling pathways, such as hormone signaling and biotic and abiotic stress responses. On endosomal sorting, plasma membrane proteins maybe recycled or targeted for vacuolar degradation, which is dependent on ubiquitin modification of the cargos and is driven by the endosomal sorting complexes required for transport (ESCRTs). Components of the ESCRT machinery are highly conserved among eukaryotes, but homologs of ESCRT-0 that are responsible for recognition and concentration of ubiquitylated proteins are absent in plants. Recently several ubiquitin-binding proteins have been identified that serve in place of ESCRT-0; however, their function in ubiquitin recognition and endosomal trafficking is not well understood yet. In this study, we identified Src homology-3 (SH3) domain-containing protein 2 (SH3P2) as a ubiquitin- and ESCRT-I-binding protein that functions in intracellular trafficking. SH3P2 colocalized with clathrin light chain-labeled punctate structures and interacted with clathrin heavy chain in planta, indicating a role for SH3P2 in clathrin-mediated endocytosis. Furthermore, SH3P2 cofractionates with clathrin-coated vesicles (CCVs), suggesting that it associates with CCVs in planta Mutants of SH3P2 and VPS23 genetically interact, suggesting that they could function in the same pathway. Based on these results, we suggest a role of SH3P2 as an ubiquitin-binding protein that binds and transfers ubiquitylated proteins to the ESCRT machinery.

Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes.

Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here, we show methods to analyze DUB activity using immunodetection, Coomassie Brilliant Blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.

The importin α/ß-specific inhibitor Ivermectin affects HIF-dependent hypoxia response pathways.

Hypoxia-inducible transcription factors (HIFs) regulate hundreds of genes involved in cellular adaptation to reduced oxygen availability. HIFs consist of an O2-labile α-subunit (primarily HIF-1α and HIF-2α) and a constitutive HIF-1ß subunit. In normoxia the HIF-α subunit is hydroxylated by members of a family of prolyl-4-hydroxylase domain (PHD) proteins, PHD1-3, resulting in recognition by von Hippel-Lindau protein, ubiquitination and proteasomal degradation. In contrast, reduced oxygen availability inhibits PHD activity resulting in HIF-1α stabilisation and nuclear accumulation. Nuclear import of HIF-1α mainly depends on classical nuclear localisation signals (NLS) and involves importin α/ß heterodimers. Recently, a specific inhibitor of nuclear import has been identified that inhibits importin α/ß-dependent import with no effects on a range of other nuclear transport pathways involving members of the importin protein family. In this study we evaluated the physiological activity of this importin α/ß-inhibitor (Ivermectin) in the hypoxia response pathway. Treatment with Ivermectin decreases binding activity of HIF-1α to the importin α/ß-heterodimer. Moreover, HIF-1α nuclear localisation, nuclear HIF-1α protein levels, HIF-target gene expression, as well as HIF-transcriptional activity are reduced upon Ivermectin treatment. For the first time, we demonstrate the effect of specific importin α/ß-inhibition on the hypoxic response on the molecular level.

Arabidopsis ALIX is required for the endosomal localization of the deubiquitinating enzyme AMSH3.

Ubiquitination is a signal for various cellular processes, including for endocytic degradation of plasma membrane cargos. Ubiquitinating as well as deubiquitinating enzymes (DUBs) can regulate these processes by modifying the ubiquitination status of target protein. Although accumulating evidence points to the important regulatory role of DUBs, the molecular basis of their regulation is still not well understood. Associated molecule with the SH3 domain of signal transduction adaptor molecule (STAM) (AMSH) is a conserved metalloprotease DUB in eukaryotes. AMSH proteins interact with components of the endosomal sorting complex required for transport (ESCRT) and are implicated in intracellular trafficking. To investigate how the function of AMSH is regulated at the cellular level, we carried out an interaction screen for the Arabidopsis AMSH proteins and identified the Arabidopsis homolog of apoptosis-linked gene-2 interacting protein X (ALIX) as a protein interacting with AMSH3 in vitro and in vivo. Analysis of alix knockout mutants in Arabidopsis showed that ALIX is essential for plant growth and development and that ALIX is important for the biogenesis of the vacuole and multivesicular bodies (MVBs). Cell biological analysis revealed that ALIX and AMSH3 colocalize on late endosomes. Although ALIX did not stimulate AMSH3 activity in vitro, in the absence of ALIX, AMSH3 localization on endosomes was abolished. Taken together, our data indicate that ALIX could function as an important regulator for AMSH3 function at the late endosomes.

High Modulus Biodegradable Polyurethanes for Vascular Stents: Evaluation of Accelerated in vitro Degradation and Cell Viability of Degradation Products.

We have recently reported the mechanical properties and hydrolytic degradation behavior of a series of NovoSorb™ biodegradable polyurethanes (PUs) prepared by varying the hard segment (HS) weight percentage from 60 to 100. In this study, the in vitro degradation behavior of these PUs with and without extracellular matrix (ECM) coating was investigated under accelerated hydrolytic degradation (phosphate buffer saline; PBS/70°C) conditions. The mass loss at different time intervals and the effect of aqueous degradation products on the viability and growth of human umbilical vein endothelial cells (HUVEC) were examined. The results showed that PUs with HS 80% and below completely disintegrated leaving no visual polymer residue at 18 weeks and the degradation medium turned acidic due to the accumulation of products from the soft segment (SS) degradation. As expected the PU with the lowest HS was the fastest to degrade. The accumulated degradation products, when tested undiluted, showed viability of about 40% for HUVEC cells. However, the viability was over 80% when the solution was diluted to 50% and below. The growth of HUVEC cells is similar to but not identical to that observed with tissue culture polystyrene standard (TCPS). The results from this in vitro study suggested that the PUs in the series degraded primarily due to the SS degradation and the cell viability of the accumulated acidic degradation products showed poor viability to HUVEC cells when tested undiluted, however particles released to the degradation medium showed cell viability over 80%.

FYVE1 is essential for vacuole biogenesis and intracellular trafficking in Arabidopsis.

The plant vacuole is a central organelle that is involved in various biological processes throughout the plant life cycle. Elucidating the mechanism of vacuole biogenesis and maintenance is thus the basis for our understanding of these processes. Proper formation of the vacuole has been shown to depend on the intracellular membrane trafficking pathway. Although several mutants with altered vacuole morphology have been characterized in the past, the molecular basis for plant vacuole biogenesis has yet to be fully elucidated. With the aim to identify key factors that are essential for vacuole biogenesis, we performed a forward genetics screen in Arabidopsis (Arabidopsis thaliana) and isolated mutants with altered vacuole morphology. The vacuolar fusion defective1 (vfd1) mutant shows seedling lethality and defects in central vacuole formation. VFD1 encodes a Fab1, YOTB, Vac1, and EEA1 (FYVE) domain-containing protein, FYVE1, that has been implicated in intracellular trafficking. FYVE1 localizes on late endosomes and interacts with Src homology-3 domain-containing proteins. Mutants of FYVE1 are defective in ubiquitin-mediated protein degradation, vacuolar transport, and autophagy. Altogether, our results show that FYVE1 is essential for plant growth and development and place FYVE1 as a key regulator of intracellular trafficking and vacuole biogenesis.

Endoplasmic reticulum KDEL-tailed cysteine endopeptidase 1 of Arabidopsis (AtCEP1) is involved in pathogen defense.

Programmed cell death (PCD) is a genetically determined process in all multicellular organisms. Plant PCD is effected by a unique group of papain-type cysteine endopeptidases (CysEP) with a C-terminal KDEL endoplasmic reticulum (ER) retention signal (KDEL CysEP). KDEL CysEPs can be stored as pro-enzymes in ER-derived endomembrane compartments and are released as mature CysEPs in the final stages of organelle disintegration. KDEL CysEPs accept a wide variety of amino acids at the active site, including the glycosylated hydroxyprolines of the extensins that form the basic scaffold of the cell wall. In Arabidopsis, three KDEL CysEPs (AtCEP1, AtCEP2, and AtCEP3) are expressed. Cell- and tissue-specific activities of these three genes suggest that KDEL CysEPs participate in the abscission of flower organs and in the collapse of tissues in the final stage of PCD as well as in developmental tissue remodeling. We observed that AtCEP1 is expressed in response to biotic stress stimuli in the leaf. atcep1 knockout mutants showed enhanced susceptibility to powdery mildew caused by the biotrophic ascomycete Erysiphe cruciferarum. A translational fusion protein of AtCEP1 with a three-fold hemaglutinin-tag and the green fluorescent protein under control of the endogenous AtCEP1 promoter (PCEP1::pre-pro-3xHA-EGFP-AtCEP1-KDEL) rescued the pathogenesis phenotype demonstrating the function of AtCEP1 in restriction of powdery mildew. The spatiotemporal AtCEP1-reporter expression during fungal infection together with microscopic inspection of the interaction phenotype suggested a function of AtCEP1 in controlling late stages of compatible interaction including late epidermal cell death. Additionally, expression of stress response genes appeared to be deregulated in the interaction of atcep1 mutants and E. cruciferarum. Possible functions of AtCEP1 in restricting parasitic success of the obligate biotrophic powdery mildew fungus are discussed.

Deubiquitylating enzymes and their emerging role in plant biology.

Ubiquitylation is a reversible post-translational modification that is involved in various cellular pathways and that thereby regulates various aspects of plant biology. For a long time, functional studies of ubiquitylation have focused on the function of ubiquitylating enzymes, especially the E3 ligases, rather than deubiquitylating enzymes (DUBs) or ubiquitin isopeptidases, enzymes that hydrolyze ubiquitin chains. One reason may be the smaller number of DUBs in comparison to E3 ligases, implying the broader substrate specificities of DUBs and the difficulties to identify the direct targets. However, recent studies have revealed that DUBs also actively participate in controlling cellular events and thus play pivotal roles in plant development and growth. DUBs are also essential for processing ubiquitin precursors and are important for recycling ubiquitin molecules from target proteins prior to their degradation and thereby maintaining the free ubiquitin pool in the cell. Here, we will discuss the five different DUB families (USP/UBP, UCH, JAMM, OTU, and MJD) and their known biochemical and physiological roles in plants.

Properties and in vitro evaluation of high modulus biodegradable polyurethanes for applications in cardiovascular stents.

This study examined the suitability of a family of biodegradable polyurethanes (PUs) NovoSorb developed for the vascular stent application. These segmented PUs are formulated to be biodegradable using degradable polyester and PU blocks. A series of PUs comprising different hard segment weight percentage ranging from 60 to 100 were investigated. The mechanical properties of the PUs were evaluated before and after gamma sterilization to assess their suitability for vascular implants. The real-time (PBS/37°C/pH 7.4) hydrolytic degradation studies were carried out under sterile conditions and PU glass transition temperature, molecular weight, and mass loss at 3, 6, and 9 months were determined. The viability and growth of Human Umbilical Vein Endothelial Cells (HUVEC) on PU surfaces were determined to assess the effect of PU degradation. The effect of coating of extracellular matrix (ECM) components on cell viability was also investigated. The study showed that the PUs possess excellent mechanical properties exhibiting high tensile strength (41-56 MPa) and tensile modulus (897-1496 MPa). The PU films maintained mechanical strength during the early phase of the degradation but lost strength at latter stages. The unmodified polymer surface of each PU promotes endothelial cell growth and proliferation, with a HUVEC retention rate of >70%.

Long-term effects of a neonatal low-protein diet in rats on the number of macrophages in culture and the expression/production of fusion proteins.

AIM: To investigate the effects of a neonatal low-protein diet on the number of macrophages in culture and the expression/production of proteins that regulate macrophage fusion in young and adult rats. METHODS: Male Wistar rats (n = 18) were suckled by mothers fed diets containing 17 % protein (controls, C) or 8 % protein (undernourished, UN). All rats were fed a normal protein diet after weaning. Bronchoalveolar lavage was collected from 42-, 60- and 90-day-old rats. Alveolar macrophages were cultured for 4 days to assess the number of cells and the expression of cadherins, key proteins involved in macrophage fusion, by western blotting. IL-4 and IFN-γ levels in culture supernatants were measured by ELISA. RESULTS: Offspring from mothers fed a low-protein diet showed a lower body weight gain. The number of cells in cultured macrophages from UN was reduced at 42 and 60 days and increased at 90 days. IL-4 production was increased in the supernatants from UN group at 60 days but did not affect the expression of cadherins. IFN-γ production was increased in the supernatants from UN group at 42 and 60 days and reduced at 90 days. CONCLUSIONS: This study thus demonstrated that dietary restriction during lactation altered the number of alveolar macrophages in culture and the production of fusion proteins of offspring aged 42, 60 or 90 days but did not modify the expression of adhesion molecules important for the fusion of these cells.

Collagen type I together with fibronectin provide a better support for endothelialization.

Endothelialization of vascular implants is limited by the inability of cells to retain adhesion when exposed to flow. Extracellular matrix proteins, including fibronectin and collagen, enhance cell adherence on materials. This study investigated the behaviour of Human Umbilical Vein Endothelial Cells (HUVEC) on extracellular matrix coated polystyrene. Collagen and fibronectin were coated as single and double layers to analyse differences in cell proliferation, morphology, and cell-protein interactions. Significantly higher endothelial cell proliferation and migration rates were observed on the collagen and collagen+fibronectin coating compared to the uncoated or fibronectin-coated sample. Immmunofluorescent microscopy showed evidence of extracellular matrix remodelling in the double, collagen+fibronectin coating. These results strongly suggest that a double coating of collagen+fibronectin provides a better support structure for endothelial cell growth and contributes to improve the ability of vascular implants to become and remain endothelialized.