Aspects Of Membrane Trafficking Research Articles

Regulators of the secretory pathway have distinct inputs into single-celled branching morphogenesis and seamless tube formation in the Drosophila trachea

Biological tubes serve as conduits through which gas, nutrients and other important fluids are delivered to tissues. Most biological tubes consist of multiple cells connected by epithelial junctions. Unlike these multicellular tubes, seamless tubes are unicellular and lack junctions. Seamless tubes are present in various organ systems, including the vertebrate vasculature, C.elegans excretory system, and Drosophila tracheal system. The Drosophila tracheal system is a network of air-filled tubes that delivers oxygen to all tissues. Specialized cells within the tracheal system, called terminal cells, branch extensively and form seamless tubes. Terminal tracheal tubes are polarized; the lumenal membrane has apical identity whereas the outer membrane exhibits basal characteristics. Although various aspects of membrane trafficking have been implicated in terminal cell morphogenesis, the precise secretory pathway requirements for basal and apical membrane growth have yet to be elucidated. In the present study, we demonstrate that anterograde trafficking, retrograde trafficking and Golgi-to-plasma membrane vesicle fusion are each required for the complex branched architecture of the terminal cell, but their inputs during seamless lumen formation are more varied. The COPII subunit, Sec31, and ER exit site protein, Sec16, are critical for subcellular tube architecture, whereas the SNARE proteins Syntaxin 5, Syntaxin 1 and Syntaxin 18 are more generally required for seamless tube growth and maintenance. These data suggest that distinct components of the secretory pathway have differential contributions to basal and apical membrane growth and maintenance during terminal cell morphogenesis.

Chemical Genetic Dissection of Membrane Trafficking.

The plant endomembrane system is an extensively connected functional unit for exchanging material between compartments. Secretory and endocytic pathways allow dynamic trafficking of proteins, lipids, and other molecules, regulating a myriad of biological processes. Chemical genetics-the use of compounds to perturb biological processes in a fast, tunable, and transient manner-provides elegant tools for investigating this system. Here, we review how chemical genetics has helped to elucidate different aspects of membrane trafficking. We discuss different strategies for uncovering the modes of action of such compounds and their use in unraveling membrane trafficking regulators. We also discuss how the bioactive chemicals that are currently used as probes to interrogate endomembrane trafficking were discovered and analyze the results regarding membrane trafficking and pathway crosstalk. The integration of different expertises and the rational implementation of chemical genetic strategies will improve the identification of molecular mechanisms that drive intracellular trafficking and our understanding of how trafficking interfaces with plant physiology and development.

0193 Practical application of the basic aspects of membrane trafficking and receptor-mediated signaling on issues related to animal agriculture

0193 Practical application of the basic aspects of membrane trafficking and receptor-mediated signaling on issues related to animal agriculture

Mon1-Ccz1 activates Rab7 only on late endosomes and dissociates from the lysosome in mammalian cells.

Rab GTPases act as molecular switches regulating various aspects of membrane trafficking. Among them, Rab5 and Rab7 play central roles in the endolysosomal network. Although many effectors downstream of Rab7 have been elucidated, our present understanding of the mechanism regulating Rab7 activity is extremely limited. It has only recently been accepted that the Mon1-Ccz1 complex is a Rab7 guanine nucleotide exchange factor, but it still remains unclear what the location where Mon1-Ccz1 works with Rab7 is. To address what kind of change or switch exists in the regulatory mechanism upstream of Rab7 during its transition from the late endosome to lysosome, we examined Rab7 activity in steady-state cells and during EGF-induced macropinocytosis using a newly developed FRET sensor. A combination of a Rab7 sensor and confocal FRET imaging techniques revealed that the activation of Rab7 on late endosomes depends on Mon1-Ccz1 and is implicated in late-endosome-lysosome fusion. In contrast, Rab7 activity on lysosomes was independent of Mon1-Ccz1 and active Rab7 played a role in perinuclear clustering of lysosomes.

Rab7 Is Functionally Required for Selective Cargo Sorting at the Early Endosome

The small GTPases of the Rab family act as a molecular switch regulating various aspects of membrane trafficking through the selective recruitment of effector proteins. Whereas Rab7 has been classically involved in the regulation of transport within the endolysosomal network, persistent controversy remains as to whether Rab7 also plays a role in earlier steps of endosomal trafficking. In this study, we show that Rab7 depletion or inactivation results in enlargement of both early and late endosomes. Rab7 depletion led to the retention of a significant fraction of internalized low-density lipoproteins (LDL) mainly in enlarged early endosomes (EE). As a result, LDL processing and the transcriptional regulation of sterol-sensitive genes were impaired. We found that Rab7 activity was also required for the sorting of the mannose-6-phosphate receptor, the interferon alpha-receptor and the Shiga toxin B-subunit. In contrast, epidermal growth factor (EGF) sorting at the EE or the recycling of transferrin and LDL-R were not affected by Rab7 depletion. Our findings demonstrate that in addition to regulating late endosomes (LE) to lysosomes transport, Rab7 plays a functional role in the selective sorting of distinct cargos at the EE and that the Rab5 to Rab7 exchange occurs early in the endosomal maturation process.

Rab GTPases are required for early orientation of the left–right axis in Xenopus

The earliest steps of left–right (LR) patterning in Xenopus embryos are driven by biased intracellular transport that ensures a consistently asymmetric localization of maternal ion channels and pumps in the first 2–4 blastomeres. The subsequent differential net efflux of ions by these transporters generates a bioelectrical asymmetry; this LR voltage gradient redistributes small signaling molecules along the LR axis that later regulate transcription of the normally left-sided Nodal. This system thus amplifies single cell chirality into a true left–right asymmetry across multi-cellular fields. Studies using molecular-genetic gain- and loss-of-function reagents have characterized many of the steps involved in this early pathway in Xenopus. Yet one key question remains: how is the chiral cytoskeletal architecture interpreted to localize ion transporters to the left or right side? Because Rab GTPases regulate nearly all aspects of membrane trafficking, we hypothesized that one or more Rab proteins were responsible for the directed, asymmetric shuttling of maternal ion channel or pump proteins. After performing a screen using dominant negative and wildtype (overexpressing) mRNAs for four different Rabs, we found that alterations in Rab11 expression randomize both asymmetric gene expression and organ situs. We also demonstrated that the asymmetric localization of two ion transporter subunits requires Rab11 function, and that Rab11 is closely associated with at least one of these subunits. Yet, importantly, we found that endogenous Rab11 mRNA and protein are expressed symmetrically in the early embryo. We conclude that Rab11-mediated transport is responsible for the movement of cargo within early blastomeres, and that Rab11 expression is required throughout the early embryo for proper LR patterning.

Mutational analysis of the yeast TRAPP subunit Trs20p identifies roles in endocytic recycling and sporulation.

Trs20p is a subunit of the evolutionarily conserved TRAPP (TRAnsport Protein Particle) complex that mediates various aspects of membrane trafficking. Three TRAPP complexes have been identified in yeast with roles in ER-to-Golgi trafficking, post-Golgi and endosomal-to-Golgi transport and in autophagy. The role of Trs20p, which is essential for viability and a component of all three complexes, and how it might function within each TRAPP complex, has not been clarified to date. To begin to address the role of Trs20p we generated different mutants by random mutagenesis but, surprisingly, no defects were observed in diverse anterograde transport pathways or general secretion in Trs20 temperature-sensitive mutants. Instead, mutation of Trs20 led to defects in endocytic recycling and a block in sporulation/meiosis. The phenotypes of different mutants appear to be separable suggesting that the mutations affect the function of Trs20 in different TRAPP complexes.

Vacuolar H+-ATPase (V-ATPase) Promotes Vacuolar Membrane Permeabilization and Nonapoptotic Death in Stressed Yeast

Stress in the endoplasmic reticulum caused by tunicamycin, dithiothreitol, and azole-class antifungal drugs can induce nonapoptotic cell death in yeasts that can be blocked by the action of calcineurin (Cn), a Ca(2+)-dependent serine/threonine protein phosphatase. To identify additional factors that regulate nonapoptotic cell death in yeast, a collection of gene knock-out mutants was screened for mutants exhibiting altered survival rates. The screen revealed an endocytic protein (Ede1) that can function upstream of Ca(2+)/calmodulin-dependent protein kinase 2 (Cmk2) to suppress cell death in parallel to Cn. The screen also revealed the vacuolar H(+)-ATPase (V-ATPase), which acidifies the lysosome-like vacuole. The V-ATPase performed its death-promoting functions very soon after imposition of the stress and was not required for later stages of the cell death program. Cn did not inhibit V-ATPase activities but did block vacuole membrane permeabilization (VMP), which occurred at late stages of the cell death program. All of the other nondying mutants identified in the screens blocked steps before VMP. These findings suggest that VMP is the lethal event in dying yeast cells and that fungi may employ a mechanism of cell death similar to the necrosis-like cell death of degenerating neurons.

The spectrin–ankyrin–4.1–adducin membrane skeleton: adapting eukaryotic cells to the demands of animal life

The cells in animals face unique demands beyond those encountered by their unicellular eukaryotic ancestors. For example, the forces engendered by the movement of animals places stresses on membranes of a different nature than those confronting free-living cells. The integration of cells into tissues, as well as the integration of tissue function into whole animal physiology, requires specialisation of membrane domains and the formation of signalling complexes. With the evolution of mammals, the specialisation of cell types has been taken to an extreme with the advent of the non-nucleated mammalian red blood cell. These and other adaptations to animal life seem to require four proteins--spectrin, ankyrin, 4.1 and adducin--which emerged during eumetazoan evolution. Spectrin, an actin cross-linking protein, was probably the earliest of these, with ankyrin, adducin and 4.1 only appearing as tissues evolved. The interaction of spectrin with ankyrin is probably a prerequisite for the formation of tissues; only with the advent of vertebrates did 4.1 acquires the ability to bind spectrin and actin. The latter activity seems to allow the spectrin complex to regulate the cell surface accumulation of a wide variety of proteins. Functionally, the spectrin-ankyrin-4.1-adducin complex is implicated in the formation of apical and basolateral domains, in aspects of membrane trafficking, in assembly of certain signalling and cell adhesion complexes and in providing stability to otherwise mechanically fragile cell membranes. Defects in this complex are manifest in a variety of hereditary diseases, including deafness, cardiac arrhythmia, spinocerebellar ataxia, as well as hereditary haemolytic anaemias. Some of these proteins also function as tumor suppressors. The spectrin-ankyrin-4.1-adducin complex represents a remarkable system that underpins animal life; it has been adapted to many different functions at different times during animal evolution.

Gyrating Clathrin: Highly Dynamic Clathrin Structures Involved in Rapid Receptor Recycling

We report here detection of novel intracellular clathrin-coated structures revealed by continuous high-speed imaging of cells expressing green fluorescent protein fusion proteins. These structures, which we operationally term 'gyrating clathrin' (G-clathrin), are characterized by localized but extremely rapid movement, leading to the hypothesis that they are coated buds on waving membrane tubules. G-clathrin structures have structurally and functionally distinct features. They lack detectable adaptor proteins AP-1 and AP-2 but contain GGA1 [Golgi-localized, gamma-ear-containing, Arf (ADP-ribosylation factor)-binding protein] as well as the cation-dependent mannose-6-phosphate receptor. While they accumulate internalized transferrin (Tf), they do not contain detectable levels of cargos targeted for the late endosome/lysosome pathway such as EGF and dextran. Pulse-chase studies indicate that Tf appears in G-clathrin structures in the cell periphery after sorting endosomes (SEs), but before filling of the perinuclear endocytic recycling compartment. Furthermore, the inhibitors LY294002 and wortmannin, which inhibit direct recycling of Tf from SEs to the plasma membrane, also block its appearance in G-clathrin. These observations suggest that peripheral G-clathrin contributes to rapid recycling, a kinetically defined compartment that has largely eluded structural identification. More generally, the rapid continuous live cell imaging reported here reveals new aspects of membrane trafficking.

Regulation of membrane trafficking by a novel Cdc42-related protein in Caenorhabditis elegans epithelial cells.

Rho GTPases are mainly known for their implication in cytoskeleton remodeling. They have also been recently shown to regulate various aspects of membrane trafficking. Here, we report the identification and the characterization of a novel Caenorhabditis elegans Cdc42-related protein, CRP-1, that shows atypical enzymatic characteristics in vitro. Expression in mouse fibroblasts revealed that, in contrast with CDC-42, CRP-1 was unable to reorganize the actin cytoskeleton and mainly localized to trans-Golgi network and recycling endosomes. This subcellular localization, as well as its expression profile restricted to a subset of epithelial-like cells in C. elegans, suggested a potential function for this protein in polarized membrane trafficking. Consistent with this hypothesis, alteration of CRP-1 expression affected the apical trafficking of CHE-14 in vulval and rectal epithelial cells and sphingolipids (C(6)-NBD-ceramide) uptake and/or trafficking in intestinal cells. However, it did not affect basolateral trafficking of myotactin in the pharynx and the targeting of IFB-2 and AJM-1, two cytosolic apical markers of intestine epithelial cells. Hence, our data demonstrate a function for CRP-1 in the regulation of membrane trafficking in a subset of cells with epithelial characteristics.

Dynamin Isoform-specific Interaction with the Shank/ProSAP Scaffolding Proteins of the Postsynaptic Density and Actin Cytoskeleton

Dynamin is a GTPase involved in endocytosis and other aspects of membrane trafficking. A critical function in the presynaptic compartment attributed to the brain-specific dynamin isoform, dynamin-1, is in synaptic vesicle recycling. We report that dynamin-2 specifically interacts with members of the Shank/ProSAP family of postsynaptic density scaffolding proteins and present evidence that dynamin-2 is specifically associated with the postsynaptic density. These data are consistent with a role for this otherwise broadly distributed form of dynamin in glutamate receptor down-regulation and other aspects of postsynaptic membrane turnover.

GGA proteins: new players in the sorting game.

The GGA proteins are a novel family of proteins that were discovered nearly simultaneously by several labs studying very different aspects of membrane trafficking. Since then, several studies have described the GGA proteins and their functions in yeast and mammalian cells. Four protein domains are present in all GGA proteins, as defined by sequence homology and function. These different domains interact directly with ARF proteins, cargo and clathrin. Alteration of the levels of GGA proteins by gene knockout or overexpression affects specific trafficking events between the trans-Golgi network and endosomes. These data suggest that GGAs function as ARF-dependent, monomeric clathrin adaptors to facilitate cargo sorting and vesicle formation at the trans-Golgi network.

HIV-1 Nef Plays an Essential Role in Two Independent Processes in CD4 Down-regulation: Dissociation of the CD4–p56lckComplex and Targeting of CD4 to Lysosomes

Human immunodeficiency virus type 1 (HIV-1) Nef down-regulates CD4 by triggering rapid endocytosis of cell surface CD4. To better understand how Nef induces CD4 down-regulation, we generated a series of Nef mutants with small in-frame deletions in the coding region. Three classes of mutants were obtained. The first class produces neither CD4 down-regulation nor dissociation of the CD4–p56lckcomplex. The second class induces CD4 down-regulation in cells lacking p56lckexpression, but not in cells with p56lck; these mutants fail to dissociate CD4 from p56lck. These results show that Nef-mediated CD4 dissociation from p56lckis important for CD4 down-regulation. The third class of mutants is able to dissociate the CD4–p56lckcomplex but fails to down-regulate surface CD4; internalized CD4 molecules are recycled back to the cell surface. This result suggests that Nef diverts the CD4 recycling pathway to a degradative pathway. We also demonstrate that Nef associates with phosphatidylinositol-3-kinase (PI3K) activity, which is known to be involved in several aspects of membrane trafficking. However, Nef mutants that cause internalized CD4 to be recycled do not associate with PI3K activity; thus Nef-associated PI3K activity might be involved in the latter process of targeting CD4 to a degradative pathway. We conclude that HIV-1 Nef plays a critical role in multiple processes in CD4 down-regulation: (i) disrupting the CD4–p56lckcomplex on the cell surface to allow CD4 internalization and (ii) diverting the internalized CD4 to a lysosomal pathway for its degradation, likely through a PI3K activity.

Microtubule-dependent vesicle transport: modulation of channel and transporter activity in liver and kidney.

Microtubule-based vesicle transport driven by kinesin and cytoplasmic dynein motor proteins facilitates several membrane-trafficking steps including elements of endocytosis and exocytosis in many different cell types. Most early studies on the role of microtubule-dependent vesicle transport in membrane trafficking focused either on neurons or on simple cell lines. More recently, other work has considered the role of microtubule-based vesicle transport in other physiological systems, including kidney and liver. Investigation of the role of microtubule-based vesicle transport in membrane trafficking in cells of the kidney and liver suggests a major role for microtubule-based vesicle transport in the rapid and directed movement of ion channels and transporters to and from the apical plasma membranes, events essential for kidney and liver function and homeostasis. This review discusses the evidence supporting a role for microtubule-based vesicle transport and the motor proteins, kinesin and cytoplasmic dynein, in different aspects of membrane trafficking in cells of the kidney and liver, with emphasis on those functions such as maintenance of ion channel and transporter composition in apical membranes that are specialized functions of these organs. Evidence that defects in microtubule-based transport contribute to diseases of the kidney and liver is also discussed.

A Requirement for ARF6 in Fcγ Receptor-mediated Phagocytosis in Macrophages

Phagocytosis requires extension of F-actin-rich pseudopods and is accompanied by membrane fusion events. Members of the ARF family of GTPases are essential for many aspects of membrane trafficking. To test a role for this family of proteins in Fcgamma receptor-mediated phagocytosis, we utilized the fungal metabolite brefeldin A (BFA). The addition of 100 microM BFA to a subclone of RAW 264.7 macrophages disrupted the appearance and function of the Golgi apparatus as indicated by altered immunofluorescent distribution of beta-COP and reduced efflux of BODIPY C5-ceramide, a phospholipid that normally accumulates in the Golgi apparatus. In contrast, BFA had no effect on phagocytosis of IgG-coated erythrocytes. These results suggested that activation of BFA-sensitive ARFs is not required for phagocytosis. ARF6 is unique among members of the ARF family in that its membrane association is unaffected by BFA. Expression of ARF6 mutants defective in either GTP hydrolysis (Q67L) or binding (T27N) inhibited phagocytosis of IgG-coated erythrocytes and attenuated the focal accumulation of F-actin beneath the test particles. These results indicate a requirement for ARF6 in Fcgamma receptor-mediated phagocytosis and suggest that ARF6 is an important mediator of cytoskeletal alterations after Fcgamma receptor activation.