Concurrent depletion of Vps37 proteins evokes ESCRT-I destabilization and profound cellular stress responses.

Molecular details of how endocytosis contributes to oncogenesis remain elusive. Our in silico analysis of colorectal cancer (CRC) patients revealed stage-dependent alterations in the expression of 112 endocytosis-related genes. Among them, transcription of the endosomal sorting complex required for transport (ESCRT)-I component VPS37B was decreased in the advanced stages of CRC. Expression of other ESCRT-I core subunits remained unchanged in the investigated dataset. We analyzed an independent cohort of CRC patients, which also showed reduced VPS37A mRNA and protein abundance. Transcriptomic profiling of CRC cells revealed non-redundant functions of Vps37 proteins. Knockdown of VPS37A and VPS37B triggered p21 (CDKN1A)-mediated inhibition of cell proliferation and sterile inflammatory response driven by the nuclear factor (NF)-κB transcription factor and associated with mitogen-activated protein kinase signaling. Co-silencing of VPS37C further potentiated activation of these independently induced processes. The type and magnitude of transcriptional alterations correlated with the differential ESCRT-I stability upon individual and concurrent Vps37 depletion. Our study provides novel insights into cancer cell biology by describing cellular stress responses that are associated with ESCRT-I destabilization.

Endosomal "sort" of signaling control: The role of ESCRT machinery in regulation of receptor-mediated signaling pathways.

The endosomal sorting complexes required for transport (ESCRTs) machinery consists of four protein assemblies (ESCRT-0 to -III subcomplexes) which mediate various processes of membrane remodeling in the cell. In the endocytic pathway, ESCRTs sort cargo destined for degradation into intraluminal vesicles (ILVs) of endosomes. Cargos targeted by ESCRTs include various signaling molecules, mainly internalized cell-surface receptors but also some cytosolic proteins. It is therefore expected that aberrant trafficking caused by ESCRT dysfunction affects different signaling pathways. Here we review how perturbation of ESCRT activity alters intracellular transport of membrane receptors, causing their accumulation on endocytic compartments, decreased degradation and/or altered recycling to the plasma membrane. We further describe how perturbed trafficking of receptors impacts the activity of their downstream signaling pathways, with or without changes in transcriptional responses. Finally, we present evidence that ESCRT components can also control activity and intracellular distribution of cytosolic signaling proteins (kinases, other effectors and soluble receptors). The underlying mechanisms involve sequestration of such proteins in ILVs, their sorting for degradation or towards non-lysosomal destinations, and regulating their availability in various cellular compartments. All these ESCRT-mediated processes can modulate final outputs of multiple signaling pathways.

Reformulation of spray-dried apple concentrate and honey for the enhancement of drying process performance and the physicochemical properties of powders.

BACKGROUND: Apple concentrate and honey were spray-dried with traditional carriers (maltodextrin, Nutriose®, Kleptose®) and with skimmed milk powder as a carrier substitute offering added value for the final product. RESULTS: Skimmed milk powder, although having the lowest glass transition temperature, was the best carrier in terms of the highest powder recovery (higher than 80% was achieved) and with powder physical properties comparable to those obtained with other carriers. Thus, skimmed milk powder was used to produce honey powder of reduced carrier content (to 25% solids). The physical properties of the powders obtained and lower powder recovery indicated more problematic drying of AC. Powders produced with skimmed milk were characterized by enhanced nutritional value compared with variants with traditional carriers. CONCLUSION: Thus, skimmed milk is proposed as a natural carrier for spray-drying, offering high processing yield, the possibility to reduce carrier content in honey powder, and to create products of nutritional added value. © 2020 Society of Chemical Industry.

Electron impact induced anion production in acetylene.

A detailed experimental investigation of electron induced anion production in acetylene, C2H2, in the energy range between 1 and 90 eV is presented. The anions are formed by two processes in this energy range: dissociative electron attachment (DEA) and dipolar dissociation (DD). DEA in C2H2 is found to lead to the formation of H(-) and C2(-)/C2H(-) through excitation of resonances in the electron energy range 1-15 eV. These anionic fragments are formed with super thermal kinetic energy and reveal no anisotropy in the angular distributions. DD in C2H2 leads to the formation of H(-), C(-)/CH(-) and C2(-)/C2H(-) with threshold energies of 15.7, 20.0 and 16.5 eV respectively. The measured anion yields have been used to calculate anion production rates for H(-), C(-)/CH(-) and C2(-)/C2H(-) in Titan's ionosphere.

Quantum superposition of target and product states in reactive electron scattering from CF4 revealed through velocity slice imaging.

Exploiting the technique of velocity slice imaging, we have performed a detailed study of reactive electron scattering with CF4. We have measured the electron impact energy dependence of both the angular and kinetic energy distributions of the channels yielding F- and CF3(-) anions. These data provide an unprecedented insight into the quantum superposition of the target state and product channels, respectively, of Td and C3v symmetry, and shed new light on the dissociation dynamics.

Dissociative electron attachment to acetaldehyde, CH3CHO. A laboratory study using the velocity map imaging technique.

A detailed experimental investigation of the dissociative electron attachment (DEA) process to acetaldehyde, CH(3)CHO is presented. To investigate this process we use a time of flight spectrometer coupled with the velocity slice imaging technique. DEA in CH(3)CHO is found to lead to the formation of CH(3)(-), O(-), OH(-), C(2)H(-), C(2)HO(-) and CH(3)CO(-) anionic products produced through scattering resonances in the electron energy range of 6 to 13 eV. Of these product ions only O(-) is formed with any measurable kinetic energy distribution indicating a two-body dissociation process. CH(3)CO(-), although formed with very low kinetic energy, shows anisotropy in the velocity slice image, indicating ejection of the H atom in the 180° direction with respect to the electron beam. The low kinetic energy distributions and absence of any anisotropy in the angular distributions of the other product ions indicate that they are formed through multiple fragmentation of the transient molecular negative ion. The angular distribution of O(-) is analysed in terms of the various partial waves.

ESCRT-I fuels lysosomal degradation to restrict TFEB/TFE3 signaling via the Rag-mTORC1 pathway.

Within the endolysosomal pathway in mammalian cells, ESCRT complexes facilitate degradation of proteins residing in endosomal membranes. Here, we show that mammalian ESCRT-I restricts the size of lysosomes and promotes degradation of proteins from lysosomal membranes, including MCOLN1, a Ca2+ channel protein. The altered lysosome morphology upon ESCRT-I depletion coincided with elevated expression of genes annotated to biogenesis of lysosomes due to prolonged activation of TFEB/TFE3 transcription factors. Lack of ESCRT-I also induced transcription of cholesterol biosynthesis genes, in response to inefficient delivery of cholesterol from endolysosomal compartments. Among factors that could possibly activate TFEB/TFE3 signaling upon ESCRT-I deficiency, we excluded lysosomal cholesterol accumulation and Ca2+-mediated dephosphorylation of TFEB/TFE3. However, we discovered that this activation occurs due to the inhibition of Rag GTPase-dependent mTORC1 pathway that specifically reduced phosphorylation of TFEB at S112. Constitutive activation of the Rag GTPase complex in cells lacking ESCRT-I restored S112 phosphorylation and prevented TFEB/TFE3 activation. Our results indicate that ESCRT-I deficiency evokes a homeostatic response to counteract lysosomal nutrient starvation, that is, improper supply of nutrients derived from lysosomal degradation.

Contribution of PIP-5 kinase Ialpha to raft-based FcgammaRIIA signaling.

Receptor FcgammaIIA (FcgammaRIIA) associates with plasma membrane rafts upon activation to trigger signaling cascades leading to actin polymerization. We examined whether compartmentalization of PI(4,5)P(2) and PI(4,5)P(2)-synthesizing PIP5-kinase Ialpha to rafts contributes to FcgammaRIIA signaling. A fraction of PIP5-kinase Ialpha was detected in raft-originating detergent-resistant membranes (DRM) isolated from U937 monocytes and other cells. The DRM of U937 monocytes contained also a major fraction of PI(4,5)P(2). PIP5-kinase Ialpha bound PI(4,5)P(2), and depletion of the lipid displaced PIP5-kinase Ialpha from the DRM. Activation of FcgammaRIIA in BHK transfectants led to recruitment of the kinase to the plasma membrane and enrichment of DRM in PI(4,5)P(2). Immunofluorescence studies revealed that in resting cells the kinase was associated with the plasma membrane, cytoplasmic vesicles and the nucleus. After FcgammaRIIA activation, PIP5-kinase Ialpha and PI(4,5)P(2) co-localized transiently with the activated receptor at distinct cellular locations. Immunoelectron microscopy studies revealed that PIP5-kinase Ialpha and PI(4,5)P(2) were present at the edges of electron-dense assemblies containing activated FcgammaRIIA in their core. The data suggest that activation of FcgammaRIIA leads to membrane rafts coalescing into signaling platforms containing PIP5-kinase Ialpha and PI(4,5)P(2).

Synthetic lethality between VPS4A and VPS4B triggers an inflammatory response in colorectal cancer.

Somatic copy number alterations play a critical role in oncogenesis. Loss of chromosomal regions containing tumor suppressors can lead to collateral deletion of passenger genes. This can be exploited therapeutically if synthetic lethal partners of such passenger genes are known and represent druggable targets. Here, we report that VPS4B gene, encoding an ATPase involved in ESCRT-dependent membrane remodeling, is such a passenger gene frequently deleted in many cancer types, notably in colorectal cancer (CRC). We observed downregulation of VPS4B mRNA and protein levels from CRC patient samples. We identified VPS4A paralog as a synthetic lethal interactor for VPS4B in vitro and in mouse xenografts. Depleting both proteins profoundly altered the cellular transcriptome and induced cell death accompanied by the release of immunomodulatory molecules that mediate inflammatory and anti-tumor responses. Our results identify a pair of novel druggable targets for personalized oncology and provide a rationale to develop VPS4 inhibitors for precision therapy of VPS4B-deficient cancers.

Impaired dynamin 2 function leads to increased AP-1 transcriptional activity through the JNK/c-Jun pathway.

Activation of AP-1 transcription factors, composed of the Jun and Fos proteins, regulates cellular fates, such as proliferation, differentiation or apoptosis. Among other stimuli, the AP-1 pathway can be initiated by extracellular ligands, such as growth factors or cytokines, which undergo internalization in complex with their receptors. Endocytosis has been implicated in the regulation of several signaling pathways; however its possible impact on AP-1 signaling remains unknown. Here we show that inhibition of dynamin 2 (Dyn2), a major regulator of endocytic internalization, strongly stimulates the AP-1 pathway. Specifically, expression of a dominant-negative Dyn2 K44A mutant increases the total levels of c-Jun, its phosphorylation on Ser63/73 and transcription of AP-1 target genes. Interestingly, DNM2 mutations implicated in human neurological disorders exhibit similar effects on AP-1 signaling. Mechanistically, Dyn2 K44A induces AP-1 by increasing phosphorylation of several receptor tyrosine kinases. Their activation is required to initiate a Src- and JNK-dependent signaling cascade converging on c-Jun and stimulating expression of AP-1 target genes. Cumulatively, our data uncover a link between the Dyn2 function and JNK signaling which leads to AP-1 induction.

Be Healthy as a Fish Educational Program at the International Institute of Molecular and Cell Biology in Warsaw, Poland.

The purpose of the Be Healthy as a Fish educational program that is organized by the International Institute of Molecular and Cell Biology (IIMCB) in Warsaw, Poland, is to educate children about the ways in which zebrafish can be used as a model organism to help scientists understand the way the human body works. We introduce Be Healthy as a Fish workshops to children in fourth to sixth grades of primary school (9-11 years old), together with two kinds of materials under the same title: a book and a movie. We focus on the field of biology in a way that complements the children's classroom curriculum and encourages them to broaden their interests in biology in the future. The Be Healthy as a Fish educational program was inaugurated in 2014 at the Warsaw Science Festival. As of October 31, 2015, 526 primary school students participated in 27 workshops. Approximately 2000 people have received the book and nearly 1700 people have watched the movie. Be Healthy as a Fish: Origin of the Title There is a popular saying in Poland that someone is "healthy as a fish" meaning that one enjoys good health. Does this imply that fish are really that healthy? Obviously, some fish may not be healthy. Just like other animals and humans, they can and do get sick. However, this common and deceptive impression of "healthy fish" results from the fact that people hardly ever have an opportunity to observe a fish that is sick. Why does our educational program have such a possibly misleading title that may not always be true? We took advantage of this provocative title and commonly known expression and assigned to it a completely new meaning: fish can get sick, but they are important for human health. Notably, this catchy sentence intrinsically combines two keywords-health and fish-which, in our opinion, makes it a good title for a successful educational program.

ESCRT proteins restrict constitutive NF-κB signaling by trafficking cytokine receptors.

Because signaling mediated by the transcription factor nuclear factor κB (NF-κB) is initiated by ligands and receptors that can undergo internalization, we investigated how endocytic trafficking regulated this key physiological pathway. We depleted all of the ESCRT (endosomal sorting complexes required for transport) subunits, which mediate receptor trafficking and degradation, and found that the components Tsg101, Vps28, UBAP1, and CHMP4B were essential to restrict constitutive NF-κB signaling in human embryonic kidney 293 cells. In the absence of exogenous cytokines, depletion of these proteins led to the activation of both canonical and noncanonical NF-κB signaling, as well as the induction of NF-κB-dependent transcriptional responses in cultured human cells, zebrafish embryos, and fat bodies in flies. These effects depended on cytokine receptors, such as the lymphotoxin ß receptor (LTßR) and tumor necrosis factor receptor 1 (TNFR1). Upon depletion of ESCRT subunits, both receptors became concentrated on and signaled from endosomes. Endosomal accumulation of LTßR induced its ligand-independent oligomerization and signaling through the adaptors TNFR-associated factor 2 (TRAF2) and TRAF3. These data suggest that ESCRTs constitutively control the distribution of cytokine receptors in their ligand-free state to restrict their signaling, which may represent a general mechanism to prevent spurious activation of NF-κB.

APPL1 endocytic adaptor as a fine tuner of Dvl2-induced transcription.

APPL1 is a multifunctional endocytic adaptor which acts at different steps of various signaling pathways. Here we report that APPL1 interacts with Dvl2, a protein known to activate the canonical and non-canonical Wnt pathways. APPL1 synergizes with Dvl2 and potentiates transcription driven by AP-1 transcription factors, specifically by c-Jun, in non-canonical Wnt signaling. This function of APPL1 requires its endosomal recruitment. Overproduction of APPL1 increases Dvl2-mediated expression of AP-1 target gene encoding metalloproteinase 1 (MMP1) in a JNK-dependent manner. Collectively, we propose a novel role of APPL1 as a positive regulator of Dvl2-dependent transcriptional activity of AP-1.

Endocytic Adaptor Protein Tollip Inhibits Canonical Wnt Signaling.

Many adaptor proteins involved in endocytic cargo transport exhibit additional functions in other cellular processes which may be either related to or independent from their trafficking roles. The endosomal adaptor protein Tollip is an example of such a multitasking regulator, as it participates in trafficking and endosomal sorting of receptors, but also in interleukin/Toll/NF-κB signaling, bacterial entry, autophagic clearance of protein aggregates and regulation of sumoylation. Here we describe another role of Tollip in intracellular signaling. By performing a targeted RNAi screen of soluble endocytic proteins for their additional functions in canonical Wnt signaling, we identified Tollip as a potential negative regulator of this pathway in human cells. Depletion of Tollip potentiates the activity of ß-catenin/TCF-dependent transcriptional reporter, while its overproduction inhibits the reporter activity and expression of Wnt target genes. These effects are independent of dynamin-mediated endocytosis, but require the ubiquitin-binding CUE domain of Tollip. In Wnt-stimulated cells, Tollip counteracts the activation of ß-catenin and its nuclear accumulation, without affecting its total levels. Additionally, under conditions of ligand-independent signaling, Tollip inhibits the pathway after the stage of ß-catenin stabilization, as observed in human cancer cell lines, characterized by constitutive ß-catenin activity. Finally, the regulation of Wnt signaling by Tollip occurs also during early embryonic development of zebrafish. In summary, our data identify a novel function of Tollip in regulating the canonical Wnt pathway which is evolutionarily conserved between fish and humans. Tollip-mediated inhibition of Wnt signaling may contribute not only to embryonic development, but also to carcinogenesis. Mechanistically, Tollip can potentially coordinate multiple cellular pathways of trafficking and signaling, possibly by exploiting its ability to interact with ubiquitin and the sumoylation machinery.

Electron induced chemistry: a new frontier in astrochemistry.

The commissioning of the ALMA array and the next generation of space telescopes heralds the dawn of a new age of Astronomy, in which the role of chemistry in the interstellar medium and in star and planet formation may be quantified. A vital part of these studies will be to determine the molecular complexity in these seemingly hostile regions and explore how molecules are synthesised and survive. The current hypothesis is that many of these species are formed within the ice mantles on interstellar dust grains with irradiation by UV light or cosmic rays stimulating chemical reactions. However, such irradiation releases many secondary electrons which may themselves induce chemistry. In this article we discuss the potential role of such electron induced chemistry and demonstrate, through some simple experiments, the rich molecular synthesis that this may lead to.

Expression of PI(4,5)P2-binding proteins lowers the PI(4,5)P2level and inhibits FcgammaRIIA-mediated cell spreading and phagocytosis.

We found that FcgammaRII-mediated cell spreading and phagocytosis were correlated with an increase of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] level in cells. During the spreading, a long-lasting elevation of PI(4,5)P(2) and concomitant actin polymerization occurred. Filopodia and lamellae of spreading cells were enriched in phosphatidylinositol 4-phosphate 5-kinase Ialpha (PIP5-kinase Ialpha) that colocalized with PI(4,5)P(2 )and actin filaments. Both spreading and phagocytosis were inhibited by expression of the C(374-440) fragment of PIP5-kinase Ialpha or the pleckstrin homology domain of phospholipase Cdelta(1 )(PLCdelta(1)-PH), two probes binding PI(4,5)P(2). These probes reduced the amount of PI(4,5)P(2) in the cells, evoked reorganization of the actin cytoskeleton and abolished PI(4,5)P(2) elevation during phagocytosis. Simultaneously, PLCdelta(1)-PH-GFP reduced the amount of PIP5-kinase Ialpha associated with the plasma membrane. In vitro studies demonstrated that PIP5-kinase Ialpha-GST bound PI(4,5)P(2), phosphatidylinositol 4-monophosphate, and less efficiently, phosphatidic acid. The data suggest that the PLCdelta(1)-PH domain, and possibly also the C(374-440) fragment, when expressed in cells, can compete with endogenous PIP5-kinase Ialpha for PI(4,5)P(2 )binding in the plasma membrane leading eventually to PI(4,5)P(2) depletion.