Nbr1 Is a Novel Inhibitor of Ligand-Mediated Receptor Tyrosine Kinase Degradation

endocytic trafficking and selective autophagy. However, the exact function of Nbr1 in these contexts has not been studied in detail. Here we investigated the role of Nbr1 in the trafficking of receptor tyrosine kinases (RTKs). We report that ectopic Nbr1 expression inhibits the ligand-mediated lysosomal degradation of RTKs, and this is probably done via the inhibition of receptor internalization. Conversely, the depletion of endogenous NBR1 enhances RTK degradation. Analyses of truncation mutations demonstrated that the C terminus of Nbr1 is essential but not sufficient for this activity. Moreover, the C terminus of Nbr1 is essential but not sufficient for the localization of the protein to late endosomes. We demonstrate that the C terminus of Nbr1 contains a novel membrane-interacting amphipathic -helix, which is essential for the late endocytic localization of the protein but not for its effect on RTK degradation. Finally, autophagic and late endocytic localizations of Nbr1 are independent of one another, suggesting that the roles of Nbr1 in each context might be distinct. Our results define Nbr1 as a negative regulator of ligand-mediated RTK degradation and reveal the interplay between its various regions for protein localization and function

GC/MSn analysis of the crude reaction mixtures from Friedel–Crafts acylation: Unambiguous identification and differentiation of 3-aroylbenzofurans from their 4- and 6-regioisomers

Rationale: 3-Aroylbenzofurans and their 2-nitrophenyl derivatives constitute fundamental intermediates for the synthesis of target compounds with pharmaceutical properties. However, their preparation via the Friedel–Crafts acylation of 2-phenylbenzofurans, using Lewis acid as catalyst, often leads to mixtures of regioisomeric aroylbenzofurans that can be challenging to distinguish, thus preventing the reaction characterization. Method: We report a method for the unambiguous identification and differentiation of the desired 3-benzoyl isomers from their 4- and 6-regioisomers in a crude reaction mixture using gas chromatography coupled to multiple-stage mass spectrometric (GC/MSn) analysis performed in collision-induced dissociation (CID) mode. Results: Upon electron ionization, each set of isomers displayed nearly identical mass spectra. MSn revealed fragmentation patterns that varied in the location of the benzoyl group on the benzofuran scaffold: CID experiments performed on the molecular ion allowed the distinction of the 3-acyl isomers from the 4- and 6-regioisomers; CID experiments on the [M − Ar]+ ion allowed the distinction of the 4-benzoyl from the 6-benzoyl regioisomer, when the nitro group is located on the 2-phenyl ring. Moreover, the unusual loss of OH• radical allowed ascertaining the position of the nitro group in 3-acyl regioisomers bearing the NO2 group. The origin of the diagnostic OH• loss was investigated through MSn experiments using 18O-labelled 3-benzoyl derivatives. Conclusions: The method allows the rapid characterization of crude reaction mixtures of benzoylbenzofurans using solely GC/MSn analysis, simplifying the workflow of extensive isolation and purification for structure elucidationThe present work was partially supported by FIR (Fondo Integrativo per la Ricerca – annualità 2018 and 2019), University of CagliariS

Dynamics of Dynamin during Clathrin Mediated Endocytosis in PC12 Cells

Members of the dynamin super-family of GTPases are involved in disparate cellular pathways. Dynamin1 and dynamin2 have been implicated in clathrin-mediated endocytosis. While some models suggest that dynamin functions specifically at the point of vesicle fission, evidence also exists for a role prior to fission during vesicle formation and it is unknown if there is a role for dynamin after vesicle fission. Although dynamin2 is ubiquitously expressed, dynamin1 is restricted to the nervous system. These two structurally similar endocytic accessory proteins have not been studied in cells that endogenously express both.The present study quantitatively assesses the dynamics of dynamin1 and dynamin2 during clathrin-mediated endocytosis in PC12 cells, which endogenously express both proteins. Both dynamin isoforms co-localized with clathrin and showed sharp increases in fluorescence intensity immediately prior to internalization of the nascent clathrin-coated vesicle. The fluorescence intensity of both proteins then decreased with two time constants. The slower time constant closely matched the time constant for the decrease of clathrin intensity and likely represents vesicle movement away from the membrane. The faster rate may reflect release of dynamin at the neck of nascent vesicle following GTP hydrolysis.This study analyses the role of dynamin in clathrin-mediated endocytosis in a model for cellular neuroscience and these results may provide direct evidence for the existence of two populations of dynamin associated with nascent clathrin-coated vesicles

Regulation of fibroblast growth factor receptor signalling and trafficking by Src and Eps8

Fibroblast growth factor receptors (FGFRs) mediate a wide spectrum of cellular responses that are crucial for development and wound healing. However, aberrant FGFR activity leads to cancer. Activated growth factor receptors undergo stimulated endocytosis, but can continue to signal along the endocytic pathway. Endocytic trafficking controls the duration and intensity of signalling, and growth factor receptor signalling can lead to modifications of trafficking pathways. We have developed live-cell imaging methods for studying FGFR dynamics to investigate mechanisms that coordinate the interplay between receptor trafficking and signal transduction. Activated FGFR enters the cell following recruitment to pre-formed clathrin-coated pits (CCPs). However, FGFR activation stimulates clathrin-mediated endocytosis; FGF treatment increases the number of CCPs, including those undergoing endocytosis, and this effect is mediated by Src and its phosphorylation target Eps8. Eps8 interacts with the clathrin-mediated endocytosis machinery and depletion of Eps8 inhibits FGFR trafficking and immediate Erk signalling. Once internalized, FGFR passes through peripheral early endosomes en route to recycling and degredative compartments, through an Src- and Eps8-dependent mechanism. Thus Eps8 functions as a key coordinator in the interplay between FGFR signalling and trafficking. This work provides the first detailed mechanistic analysis of growth factor receptor clustering at the cell surface through signal transduction and endocytic trafficking. As we have characterised the Src target Eps8 as a key regulator of FGFR signalling and trafficking, and identified the early endocytic system as the site of Eps8-mediated effects, this work provides novel mechanistic insight into the reciprocal regulation of growth factor receptor signalling and trafficking

SNX4 in Complex with Clathrin and Dynein: Implications for Endosome Movement

BACKGROUND:Sorting nexins (SNXs) constitute a family of proteins classified by their phosphatidylinositol (PI) binding Phox homology (PX) domain. Some members regulate intracellular trafficking. We have here investigated mechanisms underlying SNX4 mediated endosome to Golgi transport. METHODOLOGY/PRINCIPAL FINDINGS:We show that SNX4 forms complexes with clathrin and dynein. The interactions were inhibited by wortmannin, a PI3-kinase inhibitor, suggesting that they form when SNX4 is associated with PI(3)P on endosomes. We further localized the clathrin interacting site on SNX4 to a clathrin box variant. A short peptide containing this motif was sufficient to pull down both clathrin and dynein. Knockdown studies demonstrated that clathrin is not required for the SNX4/dynein interaction. Moreover, clathrin knockdown led to increased Golgi transport of the toxin ricin, as well as redistribution of endosomes. CONCLUSIONS/SIGNIFICANCE:We discuss the possibility of clathrin serving as a regulator of SNX4-dependent transport. Upon clathrin release, dynein may bind SNX4 and mediate retrograde movement

Microtubules Regulate Migratory Polarity through Rho/ROCK Signaling in T Cells

Background: Migrating leukocytes normally have a polarized morphology with an actin-rich lamellipodium at the front and a uropod at the rear. Microtubules (MTs) are required for persistent migration and chemotaxis, but how they affect cell polarity is not known.Methodology/Principal Findings: Here we report that T cells treated with nocodazole to disrupt MTs are unable to form a stable uropod or lamellipodium, and instead often move by membrane blebbing with reduced migratory persistence. However, uropod-localized receptors and ezrin/radixin/moesin proteins still cluster in nocodazole-treated cells, indicating that MTs are required specifically for uropod stability. Nocodazole stimulates RhoA activity, and inhibition of the RhoA target ROCK allows nocodazole-treated cells to re-establish lamellipodia and uropods and persistent migratory polarity. ROCK inhibition decreases nocodazole-induced membrane blebbing and stabilizes MTs. The myosin inhibitor blebbistatin also stabilizes MTs, indicating that RhoA/ROCK act through myosin II to destabilize MTs.Conclusions/Significance: Our results indicate that RhoA/ROCK signaling normally contributes to migration by affecting both actomyosin contractility and MT stability. We propose that regulation of MT stability and RhoA/ROCK activity is a mechanism to alter T-cell migratory behavior from lamellipodium-based persistent migration to bleb-based migration with frequent turning

A Mutational Analysis of the Endophilin-A N-BAR Domain Performed in Living Flies

BACKGROUND: Endophilin is a cytoplasmic protein with an important function in clathrin-dependent endocytosis at synapses and elsewhere. Endophilin has a BAR (Bin/Amphiphysin/Rvs-homology) domain, which is implicated in the sensing and induction of membrane curvature. Previous structure-function studies of the endophilin-A BAR domain have almost exclusively been made in reduced systems, either in vitro or ex vivo in cultured cells. To extend and complement this work, we have analyzed the role played by the structural features of the endophilin-A BAR domain in Drosophila in vivo. METHODOLOGY/PRINCIPAL FINDINGS: The study is based on genetic rescue of endophilin-A (endoA) null mutants with wild type or mutated endoA transgenes. We evaluated the viability of the rescuants, the locomotor behavior in adult flies and the neurotransmission at the larval neuromuscular junction. Whereas mutating the endophilin BAR domain clearly affected adult flies, larval endophilin function was surprisingly resistant to mutagenesis. Previous reports have stressed the importance of a central appendage on the convex BAR surface, which forms a hydrophobic ridge able to directly insert into the lipid bilayer. We found that the charge-negative substitution A66D, which targets the hydrophobic ridge and was reported to completely disrupt the ability of endophilin-BAR to tubulate liposomes in vitro, rescued viability and neurotransmission with the same efficiency as wild type endoA transgenes, even in adults. A similar discrepancy was found for the hydrophilic substitutions A63S/A66S and A63S/A66S/M70Q. The A66W mutation, which introduces a bulky hydrophobic side chain and induces massive vesiculation of liposomes in vitro, strongly impeded eye development, even in presence of the endogenous endoA gene. Substantial residual function was observed in larvae rescued with the EndoA(Arf) transgene, which encodes a form of endophilin-A that completely lacks the central appendage. Whereas a mutation (D151P) designed to increase the BAR curvature was functional, another mutation (P143A, DeltaLEN) designed to decrease the curvature was not. CONCLUSIONS/SIGNIFICANCE: Our results provide novel insight into the structure/function relationship of the endophilin-A BAR domain in vivo, especially with relation to synaptic function

Decoupling Internalization, Acidification and Phagosomal-Endosomal/lysosomal Fusion during Phagocytosis of InlA Coated Beads in Epithelial Cells

BACKGROUND: Phagocytosis has been extensively examined in 'professional' phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established. In addition, very little work has been done to systematically examine phagosomal maturation in 'non-professional' phagocytic cells. Therefore, in this study, we developed a simple method to measure and decouple particle internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK) and Caco-2 epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA), a membrane surface protein from Listeria monocytogenes known to trigger receptor-mediated phagocytosis. We were able to independently measure the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads) with antibody quenching, a pH sensitive dye and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales for each. In a separate set of experiments, we exploited the phagosomal acidification process to demonstrate an additional, real-time method for tracking bead binding, internalization and phagosomal acidification. CONCLUSIONS/SIGNIFICANCE: Using this method, we found that the time scales for internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion ranged from 23-32 min, 3-4 min and 74-120 min, respectively, for MDCK and Caco-2 epithelial cells. Both the static and real-time methods developed here are expected to be readily and broadly applicable, as they simply require fluorophore conjugation to a particle of interest, such as a pathogen or mimetic, in combination with common cell labeling dyes. As such, these methods hold promise for future measurements of receptor-mediated internalization in other cell systems, e.g. pathogen-host systems