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

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

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

Cellular entry of nanoparticles via serum sensitive clathrin-mediated endocytosis, and plasma membrane permeabilization

Increasing production and application of nanomaterials raises significant questions regarding the potential for cellular entry and toxicity of nanoparticles. It was observed that the presence of serum reduces the cellular association of 20 nm carboxylate-modified fluorescent polystyrene beads up to 20-fold, relative to cells incubated in serum-free media. Analysis by confocal microscopy demonstrated that the presence of serum greatly reduces the cell surface association of nanoparticles, as well as the potential for internalization. However, both in the presence and absence of serum, nanoparticle entry depends upon clathrin-mediated endocytosis. Finally, experiments performed with cells cooled to 4°C suggest that a proportion of the accumulation of nanoparticles in cells was likely due to direct permeabilization of the plasma membrane

Podocytic PKC-Alpha Is Regulated in Murine and Human Diabetes and Mediates Nephrin Endocytosis

Background: Microalbuminuria is an early lesion during the development of diabetic nephropathy. The loss of high molecular weight proteins in the urine is usually associated with decreased expression of slit diaphragm proteins. Nephrin, is the major component of the glomerular slit diaphragm and loss of nephrin has been well described in rodent models of experimental diabetes as well as in human diabetic nephropathy. Methodology/Principal Findings: In this manuscript we analyzed the role of PKC-alpha (PKCa) on endocytosis of nephrin in podocytes. We found that treatment of diabetic mice with a PKCa-inhibitor (GÖ6976) leads to preserved nephrin expression and reduced proteinuria. In vitro, we found that high glucose stimulation would induce PKCa protein expression in murine and human podocytes. We can demonstrate that PKCa mediates nephrin endocytosis in podocytes and that overexpression of PKCa leads to an augmented endocytosis response. After PKC-activation, we demonstrate an inducible association of PKCa, PICK1 and nephrin in podocytes. Moreover, we can demonstrate a strong induction of PKCa in podocytes of patients with diabetic nephropathy. Conclusions/Significance: We therefore conclude that activation of PKCa is a pathomechanistic key event during the development of diabetic nephropathy. PKCa is involved in reduction of nephrin surface expression and therefore PKC

EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling.

Purpose: Progenitor cells of the limbal epithelium reside in a discrete area peripheral to the more differentiated corneal epithelium and maintain tissue homeostasis. What regulates the limbal-corneal epithelial boundary is a major unanswered question. Ephrin-A1 ligand is enriched in the limbal epithelium, whereas EphA2 receptor is concentrated in the corneal epithelium. This reciprocal pattern led us to assess the role of ephrin-A1 and EphA2 in limbal-corneal epithelial boundary organization. Methods: EphA2-expressing corneal epithelial cells engineered to express ephrin-A1 were used to study boundary formation in vitro in a manner that mimicked the relative abundance of these juxtamembrane signaling proteins in the limbal and corneal epithelium in vivo. Interaction of these two distinct cell populations following initial seeding into discrete culture compartments was assessed by live cell imaging. Immunofluoresence and immunoblotting was used to evaluate the contribution of downstream growth factor signaling and cell-cell adhesion systems to boundary formation at sites of heterotypic contact between ephrin-A1 and EphA2 expressing cells. Results: Ephrin-A1-expressing cells impeded and reversed the migration of EphA2-expressing corneal epithelial cells upon heterotypic contact formation leading to coordinated migration of the two cell populations in the direction of an ephrin-A1-expressing leading front. Genetic silencing and pharmacologic inhibitor studies demonstrated that the ability of ephrin-A1 to direct migration of EphA2-expressing cells depended on an a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and epidermal growth factor receptor (EGFR) signaling pathway that limited E-cadherin-mediated adhesion at heterotypic boundaries. Conclusions: Ephrin-A1/EphA2 signaling complexes play a key role in limbal-corneal epithelial compartmentalization and the response of these tissues to injury