CiteULike: lechristophe's endocytosis

Glutamate receptor dynamics in dendritic microdomains.

TM Newpher — 2008-06-06T09:23:22-00:00

Neuron, Vol. 58, No. 4. (22 May 2008), pp. 472-497.

Among diverse factors regulating excitatory synaptic transmission, the abundance of postsynaptic glutamate receptors figures prominently in molecular memory and learning-related synaptic plasticity. To allow for both long-term maintenance of synaptic transmission and acute changes in synaptic strength, the relative rates of glutamate receptor insertion and removal must be tightly regulated. Interactions with scaffolding proteins control the targeting and signaling properties of glutamate receptors within the postsynaptic membrane. In addition, extrasynaptic receptor populations control the equilibrium of receptor exchange at synapses and activate distinct signaling pathways involved in plasticity. Here, we review recent findings that have shaped our current understanding of receptor mobility between synaptic and extrasynaptic compartments at glutamatergic synapses, focusing on AMPA and NMDA receptors. We also examine the cooperative relationship between intracellular trafficking and surface diffusion of glutamate receptors that underlies the expression of learning-related synaptic plasticity.

Dynamics of somatostatin type 2A receptor cargoes in living hippocampal neurons.

B Lelouvier — 2008-07-07T08:59:53-00:00

The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 28, No. 17. (23 April 2008), pp. 4336-4349.

Despite the large number of G-protein-coupled receptor (GPCR) types expressed in the CNS, little is known about their dynamics in neuronal cells. Dynamic properties of the somatostatin type 2A receptor were therefore examined in resting conditions and after agonist activation in living hippocampal neurons. Using fluorescence recovery after photobleaching experiments, we found that, in absence of ligand, the sst(2A) receptor is mobile and laterally and rapidly diffuse in neuronal membranes. We then observed by live-cell imaging that, after agonist activation, membrane-associated receptors induce the recruitment of beta-arrestin 1-enhanced green fluorescent protein (EGFP) and beta-arrestin 2-EGFP to the plasma membrane. In addition, beta-arrestin 1-EGFP translocate to the nucleus, suggesting that this protein could serve as a nuclear messenger for the sst(2A) receptor in neurons. Receptors are then recruited to preexisting clathrin coated pits, form clusters that internalize, fuse, and move to a perinuclear compartment that we identified as the trans-Golgi network (TGN), and recycle. Receptor cargoes are transported through a microtubule-dependent process directly from early endosomes/recycling endosomes to the TGN, bypassing the late endosomal compartment. Together, these results provide a comprehensive description of GPCR trafficking in living neurons and provide compelling evidence that GPCR cargoes can recycle through the TGN after endocytosis, a phenomenon that has not been anticipated from studies of non-neuronal cells.

An essential role for cortactin in the modulation of the potassium channel Kv1.2.

MR Williams — 2008-05-13T18:16:19-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 104, No. 44. (30 October 2007), pp. 17412-17417.

Ion channels are key determinants of membrane excitability. The actin cytoskeleton has a central role in morphology, migration, intracellular transport, and signaling. In this article, we show that the actin-binding protein cortactin regulates the potassium channel Kv1.2 and thereby provides a direct link between actin dynamics and membrane excitability. In previous reports, we showed that the tyrosine phosphorylation-mediated suppression of Kv1.2 ionic current occurs by endocytosis of the channel protein. Pull-down assays using recombinant-purified cortactin and Kv1.2 demonstrated that their interaction is direct and reduced by tyrosine phosphorylation of Kv1.2. This finding suggests a link between cortactin and Kv1.2 endocytosis. Here, we confirm that relationship and identify the molecular mechanisms involved. We use FRET to demonstrate that Kv1.2 and cortactin interact in vivo. By manipulating the cortactin-binding site within Kv1.2, we confirm that cortactin proximity influences channel function. We used flow cytometry in conjunction with cortactin gene replacement to identify C-terminal tyrosines, the fourth repeat actin-binding domain, and the N-terminal Arp2/3-binding region, as critical to Kv1.2 regulation. Surprisingly, cortactin's dynamin-binding Src homology 3 domain is not required for Kv1.2 endocytosis, despite that process being dynamin-dependent. These findings predict that cortactin-mediated actin remodeling in excitable cells is not only important for cell structure, but may directly impact membrane excitability.

Single-molecule diffusion study of activated EGFR implicates its endocytic pathway.

Z Xiao — 2008-05-12T15:32:56-00:00

Biochemical and biophysical research communications, Vol. 369, No. 2. (2 May 2008), pp. 730-734.

In this work, we have imaged the lateral diffusion of activated epidermal growth factor receptor (EGFR) on cell membrane for studying its internalization pathway. After EGF activation, the mobility of individual EGFR molecules was measured and compared with that in the cells disrupted of clathrin-coated pits and caveolae, the two endocytosis-competent membrane microdomains. The results implicated that activated EGFR molecules associated with clathrin-coated pits but not caveolae at low doses of EGF, whereas they were located in these two domains at high EGF doses. It provided supporting evidence for the occurrence of both clathrin-dependent and caveolae-dependent EGFR endocytosis.

Regulation of CB1 cannabinoid receptor internalization by a promiscuous phosphorylation-dependent mechanism

Tanya Daigle — 2008-04-14T04:41:21-00:00

Journal of Neurochemistry, Vol. 0, No. 0. (0), pp. ???-???.

Abstract Agonists stimulate cannabinoid 1 receptor (CB1R) internalization. Previous work suggests that the extreme carboxy-terminus of the receptor regulates this internalization - likely through the phosphorylation of serines and threonines clustered within this region. While truncation of the carboxy-terminus (V460Z CB1) and consequent removal of these putative phosphorylation sites prevents endocytosis in AtT20 cells, the residues necessary for CB1R internalization remain elusive. To determine the structural requirements for internalization, we evaluated endocytosis of carboxy-terminal mutant CB1Rs stably expressed in HEK293 cells. In contrast to AtT20 cells, V460Z CB1R expressed in HEK293 cells internalized to the same extent and with similar kinetics as the wild-type receptor. However, mutation of serine and/or threonine residues within the extreme carboxy-terminal attenuated internalization when these receptors were expressed in HEK293 cells. These results establish that the extreme carboxy-terminal phosphorylation sites are not required for internalization of truncated receptors, but are required for internalization of full-length receptors in HEK293 cells. Analysis of beta-arrestin-2 recruitment to mutant CB1R suggests that putative carboxy-terminal phosphorylation sites mediate beta-arrestin-2 translocation. This study indicates that the local cellular environment affects the structural determinants of CB1R internalization. Additionally, phosphorylation likely regulates the internalization of (full-length) CB1Rs.

Endocytosis as a Mechanism for Tyrosine Kinase-dependent Suppression of a Voltage-gated Potassium Channel

Edmund Nesti — 2008-05-12T15:21:40-00:00

Mol. Biol. Cell, Vol. 15, No. 9. (1 September 2004), pp. 4073-4088.

The voltage-gated potassium channel Kv1.2 undergoes tyrosine phosphorylation-dependent suppression of its ionic current. However, little is known about the physical mechanism behind that process. We have found that the Kv1.2 alpha-subunit protein undergoes endocytosis in response to the same stimuli that evoke suppression of Kv1.2 ionic current. The process is tyrosine phosphorylation-dependent because the same tyrosine to phenylalanine mutation in the N-terminus of Kv1.2 that confers resistance to channel suppression (Y132F) also confers resistance to channel endocytosis. Overexpression of a dominant negative form of dynamin blocked stimulus-induced Kv1.2 endocytosis and also blocked suppression of Kv1.2 ionic current. These data indicate that endocytosis of Kv1.2 from the cell surface is a key mechanism for channel suppression by tyrosine kinases. 10.1091/mbc.E03-11-0788

Vaccinia Virus Uses Macropinocytosis and Apoptotic Mimicry to Enter Host Cells

Jason Mercer — 2008-04-25T16:18:21-00:00

Science, Vol. 320, No. 5875. (25 April 2008), pp. 531-535.

Viruses employ many different strategies to enter host cells. Vaccinia virus, a prototype poxvirus, enters cells in a pH-dependent fashion. Live cell imaging showed that fluorescent virus particles associated with and moved along filopodia to the cell body, where they were internalized after inducing the extrusion of large transient membrane blebs. p21-activated kinase 1 (PAK1) was activated by the virus, and the endocytic process had the general characteristics of macropinocytosis. The induction of blebs, the endocytic event, and infection were all critically dependent on the presence of exposed phosphatidylserine in the viral membrane, which suggests that vaccinia virus uses apoptotic mimicry to enter cells. 10.1126/science.1155164

Flotillin-dependent clustering of the amyloid precursor protein regulates its endocytosis and amyloidogenic processing in neurons.

A Schneider — 2008-04-23T08:17:58-00:00

The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 28, No. 11. (12 March 2008), pp. 2874-2882.

The flotillins/reggie proteins are associated with noncaveolar membrane microdomains and have been implicated in the regulation of a clathrin- and caveolin-independent endocytosis pathway. Endocytosis is required for the amyloidogenic processing of the amyloid precursor protein (APP) and thus to initiate the release of the neurotoxic beta-amyloid peptide (Abeta), the major component of extracellular plaques found in the brains of Alzheimer's disease patients. Here, we report that small interference RNA-mediated downregulation of flotillin-2 impairs the endocytosis of APP, in both neuroblastoma cells and primary cultures of hippocampal neurons, and reduces the production of Abeta. Similar to tetanus neurotoxin endocytosis, but unlike the internalization of transferrin, clathrin-dependent endocytosis of APP requires cholesterol and adaptor protein-2 but is independent of epsin1 function. Moreover, on a nanoscale resolution using stimulated emission depletion microscopy and by Förster resonance energy transfer with fluorescence lifetime imaging microscopy, we provide evidence that flotillin-2 promotes the clustering of APP at the cell surface. We show that the interaction of flotillin-2 with APP is dependent on cholesterol and that clustering of APP enhances its endocytosis rate. Together, our data suggest that cholesterol/flotillin-dependent clustering of APP may stimulate the internalization into a specialized clathrin-dependent endocytosis pathway to promote amyloidogenic processing.

Endosomal trafficking of AMPA-type glutamate receptors

H Hirling — 2008-04-17T08:12:05-00:00

Neuroscience, Vol. In Press, Corrected Proof

Many different forms of synaptic plasticity have been shown to ultimately modulate the number of AMPA-type glutamate receptors at the synapse. This trafficking involves lateral movements between synaptic and extrasynaptic sites at the neuron surface, as well as vesicular transport between the plasma membrane and intracellular compartments. Several new studies have shed light on the location and regulation of AMPA-type receptor (AMPAR) endocytosis, their intracellular sorting to divergent pathways at the level of endosomes, and the mechanism and sites of receptor recycling. This review summarizes this recent data on the trafficking along the endocytic pathway, and follows the path of internalized AMPAR from endocytosis up to sites of recycling.

Type-1 cannabinoid receptors colocalize with caveolin-1 in neuronal cells.

M Bari — 2008-04-01T10:20:38-00:00

Neuropharmacology, Vol. 54, No. 1. (January 2008), pp. 45-50.

Type-1 (CB1) and type-2 (CB2) cannabinoid receptors belong to the rhodopsin family of G protein-coupled receptors, and are activated by endogenous lipids termed "endocannabinoids". Recent reports have demonstrated that CB1R, unlike CB2R and other receptors and metabolic enzymes of endocannabinoids, functions in the context of lipid rafts, i.e. plasma membrane microdomains which may be important in modulating signal transduction. Here, we present novel data based on cell subfractionation, immunoprecipitation and confocal microscopy studies, that show that in C6 cells CB1R co-localizes almost entirely with caveolin-1. We also show that trafficking of CB1R in response to the raft disruptor methyl-beta-cyclodextrin (MCD) is superimposable on that of caveolin-1, and that MCD treatment increases the accessibility of CB1R to its specific antibodies. These findings may be relevant for the manifold CB1R-dependent activities of endocannabinoids, like the regulation of apoptosis and of neurodegenerative diseases.

Integrating molecular and network biology to decode endocytosis

Eva Schmid — 2007-08-23T14:27:13-00:00

Nature, Vol. 448, No. 7156. (2007), pp. 883-888.

The strength of network biology lies in its ability to derive cell biological information without a priori mechanistic or molecular knowledge. It is shown here how a careful understanding of a given biological pathway can refine an interactome approach. This permits the elucidation of additional design principles and of spatio-temporal dynamics behind pathways, and aids in experimental design and interpretation.

Postsynaptic Positioning of Endocytic Zones and AMPA Receptor Cycling by Physical Coupling of Dynamin-3 to Homer

Jiuyi Lu — 2007-12-13T23:42:15-00:00

Neuron, Vol. 55, No. 6. (20 September 2007), pp. 874-889.

Summary Endocytosis of AMPA receptors and other postsynaptic cargo occurs at endocytic zones (EZs), stably positioned sites of clathrin adjacent to the postsynaptic density (PSD). The tight localization of postsynaptic endocytosis is thought to control spine composition and regulate synaptic transmission. However, the mechanisms that situate the EZ near the PSD and the role of spine endocytosis in synaptic transmission are unknown. Here, we report that a physical link between dynamin-3 and the postsynaptic adaptor Homer positions the EZ near the PSD. Disruption of dynamin-3 or its interaction with Homer uncouples the PSD from the EZ, resulting in synapses lacking postsynaptic clathrin. Loss of the EZ leads to a loss of synaptic AMPA receptors and reduced excitatory synaptic transmission that corresponds with impaired synaptic recycling. Thus, a physical link between the PSD and the EZ ensures localized endocytosis and recycling by recapturing and maintaining a proximate pool of cycling AMPA receptors.

A Role for ARF6 and ARNO in the Regulation of Endosomal Dynamics in Neurons

Hernandez-Deviez — 2007-11-10T19:04:05-00:00

Traffic, Vol. 8, No. 12. (December 2007), pp. 1750-1764.

Cholesterol Level Regulates Endosome Mobility via Rab Proteins.

Hongtao Chen — 2008-01-03T10:38:14-00:00

Biophys J (2 November 2007)

The role of cholesterol in regulation of endosome motility was investigated by monitoring the intracellular trafficking of endocytosed folate receptors (FRs) labeled with fluorescent folate conjugates. Real-time fluorescence imaging of HeLa cells transfected with GFP-tubulin revealed that FR-containing endosomes migrate along microtubules. Moreover, microinjection with antibodies that inhibit microtubule-associated motor proteins demonstrated that dynein and kinesin I participate in delivery of FR-containing endosomes to the perinuclear area and plasma membrane, respectively. Further, single particle tracking analysis revealed bidirectional motions of FR endosomes, mediated by dynein and kinesin motors associated with the same endosome. These experimental tools allowed us to use FR-containing endosomes to evaluate the impact of cholesterol on intracellular membrane trafficking. Lowering plasma membrane cholesterol by metabolic depletion or MbetaCD extraction was found to both increase FR-containing endosome motility and change endosome distribution from colocalization with Rab7 to colocalization with Rab4. These data provide evidence that cholesterol regulates intracellular membrane trafficking via modulation of the distribution of low molecular weight G-proteins that are adaptors for microtubule motors.

Role of receptor internalization in the agonist-induced desensitization of cannabinoid type 1 receptors

Dai Wu — 2007-11-19T00:32:33-00:00

Journal of Neurochemistry, Vol. 0, No. 0. (0), pp. ???-???.

Abstract Agonist-induced internalization of G protein-coupled receptors (GPCRs) is an important mechanism for regulating signaling transduction of functional receptors at the plasma membrane. We demonstrate here that both caveolae/lipid-rafts- and clathrin-coated-pits-mediated pathways were involved in agonist-induced endocytosis of the cannabinoid type 1 receptor (CB1R) in stably transfected human embryonic kidney (HEK) 293 cells and that the internalized receptors were predominantly sorted into recycling pathway for reactivation. The treatment of CB1 receptors with the low endocytotic agonist Delta9-THC induced a faster receptor desensitization and slower resensitization than the high endocytotic agonist WIN 55,212-2. In addition, the blockade of receptor endocytosis or recycling pathway markedly enhanced agonist-induced CB1 receptor desensitization. Furthermore, co-expression of phospholipase D2, an enhancer of receptor endocytosis, reduced CB1 receptor desensitization, whereas co-expression of a phospholipase D2 negative mutant significantly increased the desensitization after WIN 55,212-2 treatment. These findings provide evidences for the importance of receptor endocytosis in counteracting CB1 receptor desensitization by facilitating receptor reactivation. Moreover, in primary cultured neurons, the low endocytotic agonist Delta9-THC or anandamide exhibited a greater desensitization of endogenous CB1 receptors than the high endocytotic agonist WIN 55,212-2, CP 55940 or 2-arachidonoyl glycerol, indicating that cannabinoids with high endocytotic efficacy might cause reduced development of cannabinoid tolerance to some kind cannabinoid-mediated effects.

Regulation of dendritic excitability by activity-dependent trafficking of the A-type K+ channel subunit Kv4.2 in hippocampal neurons.

J Kim — 2008-01-03T10:19:57-00:00

Neuron, Vol. 54, No. 6. (21 June 2007), pp. 933-947.

Voltage-gated A-type K+ channel Kv4.2 subunits are highly expressed in the dendrites of hippocampal CA1 neurons. However, little is known about the subcellular distribution and trafficking of Kv4.2-containing channels. Here we provide evidence for activity-dependent trafficking of Kv4.2 in hippocampal spines and dendrites. Live imaging and electrophysiological recordings showed that Kv4.2 internalization is induced rapidly upon glutamate receptor stimulation. Kv4.2 internalization was clathrin mediated and required NMDA receptor activation and Ca2+ influx. In dissociated hippocampal neurons, mEPSC amplitude depended on functional Kv4.2 expression level and was enhanced by stimuli that induced Kv4.2 internalization. Long-term potentiation (LTP) induced by brief glycine application resulted in synaptic insertion of GluR1-containing AMPA receptors along with Kv4.2 internalization. We also found evidence of Kv4.2 internalization upon synaptically evoked LTP in CA1 neurons of hippocampal slice cultures. These results present an additional mechanism for synaptic integration and plasticity through the activity-dependent regulation of Kv4.2 channel surface expression.

SNX4 coordinates endosomal sorting of TfnR with dynein-mediated transport into the endocytic recycling compartment.

Colin J Traer — 2007-11-14T09:38:28-00:00

Nat Cell Biol (11 November 2007)

SNX-BAR proteins are a sub-family of sorting nexins implicated in endosomal sorting. Here, we establish that through its phox homology (PX) and Bin-Amphiphysin-Rvs (BAR) domains, sorting nexin-4 (SNX4) is associated with tubular and vesicular elements of a compartment that overlaps with peripheral early endosomes and the juxtanuclear endocytic recycling compartment (ERC). Suppression of SNX4 perturbs transport between these compartments and causes lysosomal degradation of the transferrin receptor (TfnR). Through an interaction with KIBRA, a protein previously shown to bind dynein light chain 1, we establish that SNX4 associates with the minus end-directed microtubule motor dynein. Although suppression of KIBRA and dynein perturbs early endosome-to-ERC transport, TfnR sorting is maintained. We propose that by driving membrane tubulation, SNX4 coordinates iterative, geometric-based sorting of the TfnR with the long-range transport of carriers from early endosomes to the ERC. Finally, these data suggest that by associating with molecular motors, SNX-BAR proteins may coordinate sorting with carrier transport between donor and recipient membranes.

Trafficking and Cellular Distribution of Voltage-Gated Sodium Channels

Cusdin — 2007-12-14T06:30:25-00:00

Traffic, Vol. 9, No. 1. (January 2008), pp. 17-26.

The Tumor Suppressor eIF3e Mediates Calcium-Dependent Internalization of the L-Type Calcium Channel Ca(V)1.2.

EM Green — 2007-08-21T13:08:20-00:00

Neuron, Vol. 55, No. 4. (16 August 2007), pp. 615-632.

Voltage-gated calcium channels (VGCCs) convert electrical activity into calcium (Ca(2+)) signals that regulate cellular excitability, differentiation, and connectivity. The magnitude and kinetics of Ca(2+) signals depend on the number of VGCCs at the plasma membrane, but little is known about the regulation of VGCC surface expression. We report that electrical activity causes internalization of the L-type Ca(2+) channel (LTC) Ca(V)1.2 and that this is mediated by binding to the tumor suppressor eIF3e/Int6 (eukaryotic initiation factor 3 subunit e). Using total internal reflection microscopy, we identify a population of Ca(V)1.2 containing endosomes whose rapid trafficking is strongly regulated by Ca(2+). We define a domain in the II-III loop of Ca(V)1.2 that binds eIF3e and is essential for the activity dependence of both channel internalization and endosomal trafficking. These findings provide a mechanism for activity-dependent internalization and trafficking of Ca(V)1.2 and provide a tantalizing link between Ca(2+) homeostasis and a mammalian oncogene.

Rab conversion as a mechanism of progression from early to late endosomes.

J Rink — 2005-11-09T22:33:31-00:00

Cell, Vol. 122, No. 5. (9 September 2005), pp. 735-749.

The mechanisms of endosome biogenesis and maintenance are largely unknown. The small GTPases Rab 5 and Rab 7 are key determinants of early and late endosomes, organizing effector proteins into specific membrane subdomains. Whether such Rab machineries are indefinitely maintained on membranes or can disassemble in the course of cargo transport is an open question. Here, we combined novel image-analysis algorithms with fast live-cell imaging. We found that the level of Rab 5 dynamically fluctuates on individual early endosomes, linked by fusion and fission events into a network in time. Within it, degradative cargo concentrates in progressively fewer and larger endosomes that migrate from the cell periphery to the center where Rab 5 is rapidly replaced with Rab 7. The class C VPS/HOPS complex, an established GEF for Rab 7, interacts with Rab 5 and is required for Rab 5-to-Rab 7 conversion. Our results reveal unexpected dynamics of Rab domains and suggest Rab conversion as the mechanism of cargo progression between early and late endosomes.

Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomes.

A Vonderheit — 2007-08-07T13:15:30-00:00

PLoS Biol, Vol. 3, No. 7. (July 2005)

Semliki forest virus (SFV) is internalized by clathrin-mediated endocytosis, and transported via early endosomes to late endosomes and lysosomes. The intracellular pathway taken by individual fluorescently labeled SFV particles was followed using immunofluorescence in untransfected cells, and by video-enhanced, triple-color fluorescence microscopy in live cells transfected with GFP- and RFP-tagged Rab5, Rab7, Rab4, and Arf1. The viruses progressed from Rab5-positive early endosomes to a population of early endosomes (about 10% of total) that contained both Rab5 and Rab7. SFV were sequestered in the Rab7 domains, and they were sorted away from the early endosomes when these domains detached as separate transport carriers devoid of Rab5, Rab4, EEA1, Arf1, and transferrin. The process was independent of Arf1 and the acidic pH in early endosomes. Nocodazole treatment showed that the release of transport carriers was assisted by microtubules. Expression of constitutively inactive Rab7T22N resulted in accumulation of SFV in early endosomes. We concluded that Rab7 is recruited to early endosomes, where it forms distinct domains that mediate cargo sorting as well as the formation of late-endosome-targeted transport vesicles.

Meeting Report: Seventh Annaberg EMBO Workshop Membrane Traffic in the Secretory Pathway, Goldegg, Austria, 914 January 2007

Morvan — 2007-07-13T21:31:46-00:00

Traffic, Vol. 8, No. 8. (August 2007), pp. 1111-1119.

Trafficking and regulation of neuronal voltage-gated calcium channels.

Scott E Jarvis — 2007-08-06T10:45:35-00:00

Curr Opin Cell Biol (10 July 2007)

The importance of voltage-gated calcium channels is underscored by the multitude of intracellular processes that depend on calcium, notably gene regulation and neurotransmission. Given their pivotal roles in calcium (and hence, cellular) homeostasis, voltage-gated calcium channels have been the subject of intense research, much of which has focused on channel regulation. While ongoing research continues to delineate the myriad of interactions that govern calcium channel regulation, an increasing amount of work has focused on the trafficking of voltage-gated calcium channels. This includes the mechanisms by which calcium channels are targeted to the plasma membrane, and, more specifically, to their appropriate loci within a given cell. In addition, we are beginning to gain some insights into the mechanisms by which calcium channels can be removed from the plasma membrane for recycling and/or degradation. Here we highlight recent advances in our understanding of these fundamentally important mechanisms.

Pathways of clathrin-independent endocytosis

Satyajit Mayor — 2007-07-23T11:30:08-00:00

Nature Reviews Molecular Cell Biology, Vol. 8, No. 8., pp. 603-612.

There are numerous ways that endocytic cargo molecules may be internalized from the surface of eukaryotic cells. In addition to the classical clathrin-dependent mechanism of endocytosis, several pathways that do not use a clathrin coat are emerging. These pathways transport a diverse array of cargoes and are sometimes hijacked by bacteria and viruses to gain access to the host cell. Here, we review our current understanding of various clathrin-independent mechanisms of endocytosis and propose a classification scheme to help organize the data in this complex and evolving field.

Identification of Sequence Motifs That Target Neuronal Nicotinic Receptors to Dendrites and Axons

Jian Xu — 2007-08-06T10:03:44-00:00

J. Neurosci., Vol. 26, No. 38. (20 September 2006), pp. 9780-9793.

Neuronal nicotinic acetylcholine receptors (nAChRs) belong to a family of ligand-gated ion channels that play important roles in central and peripheral nervous systems. The subcellular distribution of neuronal nAChRs has important implications for function and is not well understood. Here, we analyzed the targeting of two major types of neuronal nAChRs by expressing epitope-tagged subunits in cultured hippocampal neurons. Surprisingly, the alpha7 nAChR (alpha7) and alpha4/[beta]2 nAChR (alpha4[beta]2) displayed distinct patterns of expression, with alpha7 targeted preferentially to the somatodendritic compartments, whereas alpha4[beta]2 was localized to both axonal and dendritic domains. When fused to CD4 or IL2RA (interleukin 2 receptor alpha subunit) proteins, which are normally distributed ubiquitously, the M3-M4 intracellular loop from the alpha7 subunit promoted dendritic expression, whereas the homologous M3-M4 loop from the alpha4 subunit led to surface axonal expression. Systemic screening and alanine substitution further identified a 25-residue leucine motif ([DE]XXXL[LI]) containing an axonal targeting sequence within the alpha4 loop and a 48-residue dileucine and tyrosine motif (YXXO) containing a dendritic targeting sequence from the alpha7 loop. These results provide valuable information in understanding diverse roles of neuronal nAChRs in mediating and modulating synaptic transmission, synaptic plasticity, and nicotine addiction. 10.1523/JNEUROSCI.0840-06.2006

Endocytosis Optimizes the Dynamic Localization of Membrane Proteins that Regulate Cortical Polarity

Eugenio Marco — 2007-04-26T10:12:12-00:00

Cell, Vol. 129, No. 2. (20 April 2007), pp. 411-422.

Summary Diverse cell types require the ability to maintain dynamically polarized membrane-protein distributions through balancing transport and diffusion. However, design principles underlying dynamically maintained cortical polarity are not well understood. Here we constructed a mathematical model for characterizing the morphology of dynamically polarized protein distributions. We developed analytical approaches for measuring all model parameters from single-cell experiments. We applied our methods to a well-characterized system for studying polarized membrane proteins: budding yeast cells expressing activated Cdc42. We found that a balance of diffusion, directed transport, and endocytosis was sufficient for accurately describing polarization morphologies. Surprisingly, the model predicts that polarized regions are defined with a precision that is nearly optimal for measured endocytosis rates and that polarity can be dynamically stabilized through positive feedback with directed transport. Our approach provides a step toward understanding how biological systems shape spatially precise, unambiguous cortical polarity domains using dynamic processes.

Rapidly inducible changes in phosphatidylinositol 4,5-bisphosphate levels influence multiple regulatory functions of the lipid in intact living cells.

P Varnai — 2007-03-28T08:11:43-00:00

J Cell Biol, Vol. 175, No. 3. (6 November 2006), pp. 377-382.

Rapamycin (rapa)-induced heterodimerization of the FRB domain of the mammalian target of rapa and FKBP12 was used to translocate a phosphoinositide 5-phosphatase (5-ptase) enzyme to the plasma membrane (PM) to evoke rapid changes in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) levels. Rapa-induced PM recruitment of a truncated type IV 5-ptase containing only the 5-ptase domain fused to FKBP12 rapidly decreased PM PtdIns(4,5)P(2) as monitored by the PLCdelta1PH-GFP fusion construct. This decrease was paralleled by rapid termination of the ATP-induced Ca(2+) signal and the prompt inactivation of menthol-activated transient receptor potential melastatin 8 (TRPM8) channels. Depletion of PM PtdIns(4,5)P(2) was associated with a complete blockade of transferrin uptake and inhibition of epidermal growth factor internalization. None of these changes were observed upon rapa-induced translocation of an mRFP-FKBP12 fusion protein that was used as a control. These data demonstrate that rapid inducible depletion of PM PtdIns(4,5)P(2) is a powerful tool to study the multiple regulatory roles of this phospholipid and to study differential sensitivities of various processes to PtdIns(4,5)P(2) depletion.

Loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate.

R Zoncu — 2007-03-28T08:08:07-00:00

Proc Natl Acad Sci U S A, Vol. 104, No. 10. (6 March 2007), pp. 3793-3798.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], a phosphoinositide concentrated predominantly in the plasma membrane, binds endocytic clathrin adaptors, many of their accessory factors, and a variety of actin-regulatory proteins. Here we have used fluorescent fusion proteins and total internal reflection fluorescence microscopy to investigate the effect of acute PI(4,5)P(2) breakdown on the dynamics of endocytic clathrin-coated pit components and of the actin regulatory complex, Arp2/3. PI(4,5)P(2) breakdown was achieved by the inducible recruitment to the plasma membrane of an inositol 5-phosphatase module through the rapamycin/FRB/FKBP system or by treatment with ionomycin. PI(4,5)P(2) depletion resulted in a dramatic loss of clathrin puncta, which correlated with a massive dissociation of endocytic adaptors from the plasma membrane. Remaining clathrin spots at the cell surface had only weak fluorescence and were static over time. Dynamin and the p20 subunit of the Arp2/3 actin regulatory complex, which were concentrated at late-stage clathrin-coated pits and in lamellipodia, also dissociated from the plasma membrane, and these changes correlated with an arrest of motility at the cell edge. These findings demonstrate the critical importance of PI(4,5)P(2) in clathrin coat dynamics and Arp2/3-dependent actin regulation.

Cargo regulates clathrin-coated pit dynamics.

MA Puthenveedu — 2006-10-30T16:10:33-00:00

Cell, Vol. 127, No. 1. (6 October 2006), pp. 113-124.

Clathrin-coated pits (CCPs) are generally considered a uniform population of endocytic machines containing mixed constitutive and regulated membrane cargo. Contrary to this view, we show that regulated endocytosis of G protein-coupled receptors (GPCRs) occurs preferentially through a subset of CCPs. Significantly, GPCR-containing CCPs are also functionally distinct, as their surface residence time is regulated locally by GPCR cargo via PDZ-dependent linkage to the actin cytoskeleton. Such cargo-regulated CCPs show delayed recruitment of dynamin and can undergo an abortive event in which clathrin coats separate from the plasma membrane without concomitant receptor endocytosis. Segregation of cargo into CCP subsets, combined with cargo-dependent control of CCP dynamics, suggests a simple kinetic mechanism to generate functional specialization early in the endocytic pathway and reduce competition between diverse endocytic cargo.

Phospho-dependent binding of the clathrin AP2 adaptor complex to GABAA receptors regulates the efficacy of inhibitory synaptic transmission.

JT Kittler — 2006-10-30T16:05:13-00:00

Proc Natl Acad Sci U S A, Vol. 102, No. 41. (11 October 2005), pp. 14871-14876.

The efficacy of synaptic inhibition depends on the number of gamma-aminobutyric acid type A receptors (GABA(A)Rs) expressed on the cell surface of neurons. The clathrin adaptor protein 2 (AP2) complex is a critical regulator of GABA(A)R endocytosis and, hence, surface receptor number. Here, we identify a previously uncharacterized atypical AP2 binding motif conserved within the intracellular domains of all GABA(A)R beta subunit isoforms. This AP2 binding motif (KTHLRRRSSQLK in the beta3 subunit) incorporates the major sites of serine phosphorylation within receptor beta subunits, and phosphorylation within this site inhibits AP2 binding. Furthermore, by using surface plasmon resonance, we establish that a peptide (pepbeta3) corresponding to the AP2 binding motif in the GABA(A)R beta3 subunit binds to AP2 with high affinity only when dephosphorylated. Moreover, the pepbeta3 peptide, but not its phosphorylated equivalent (pepbeta3-phos), enhanced the amplitude of miniature inhibitory synaptic current and whole cell GABA(A)R current. These effects of pepbeta3 on GABA(A)R current were occluded by inhibitors of dynamin-dependent endocytosis supporting an action of pepbeta3 on GABA(A)R endocytosis. Therefore phospho-dependent regulation of AP2 binding to GABA(A)Rs provides a mechanism to specify receptor cell surface number and the efficacy of inhibitory synaptic transmission.

Rab5 and Rab7 Control Endocytic Sorting along the Axonal Retrograde Transport Pathway

Katrin Deinhardt — 2006-10-19T16:11:48-00:00

Neuron, Vol. 52, No. 2. (19 October 2006), pp. 293-305.

SummaryVesicular pathways coupling the neuromuscular junction with the motor neuron soma are essential for neuronal function and survival. To characterize the organelles responsible for this long-distance crosstalk, we developed a purification strategy based on a fragment of tetanus neurotoxin (TeNT HC) conjugated to paramagnetic beads. This approach enabled us to identify, among other factors, the small GTPase Rab7 as a functional marker of a specific pool of axonal retrograde carriers, which transport neurotrophins and their receptors. Furthermore, Rab5 is essential for an early step in TeNT HC sorting but is absent from axonally transported vesicles. Our data demonstrate that TeNT HC uses a retrograde transport pathway shared with p75NTR, TrkB, and BDNF, which is strictly dependent on the activities of both Rab5 and Rab7. Therefore, Rab7 plays an essential role in axonal retrograde transport by controlling a vesicular compartment implicated in neurotrophin traffic.

Distinct Clathrin-Coated Pits Sort Different G Protein-Coupled Receptor Cargo.

Stuart J Mundell — 2006-08-14T08:51:55-00:00

Traffic (10 August 2006)

Upon activation, many G protein-coupled receptors (GPCRs) internalize by clathrin-mediated endocytosis and are subsequently sorted to undergo recycling or lysosomal degradation. Here we observe that sorting can take place much earlier than previously thought, by entry of different GPCRs into distinct populations of clathrin-coated pit (CCP). These distinct populations were revealed by analysis of two purinergic GPCRs, P2Y(1) and P2Y(12), which enter two populations of CCPs in a mutually exclusive manner. The mechanisms underlying early GPCR sorting involve differential kinase-dependent processes because internalization of P2Y(12) is mediated by GPCR kinases (GRKs) and arrestin, whereas P2Y(1) internalization is GRK- and arrestin-independent but requires protein kinase C. Importantly, the beta(2) adrenoceptor which also internalizes in a GRK-dependent manner also traffics exclusively to P2Y(12)-containing CCPs. Our data therefore reveal distinct populations of CCPs that sort GPCR cargo at the plasma membrane using different kinase-dependent mechanisms.

Visualizing odorant receptor trafficking in living cells down to the single-molecule level.

V Jacquier — 2006-09-20T08:27:46-00:00

Proc Natl Acad Sci U S A (15 September 2006)

Despite the importance of trafficking for regulating G protein-coupled receptor signaling, for many members of the seven transmembrane helix protein family, such as odorant receptors, little is known about this process in live cells. Here, the complete life cycle of the human odorant receptor OR17-40 was directly monitored in living cells by ensemble and single-molecule imaging, using a double-labeling strategy. While the overall, intracellular trafficking of the receptor was visualized continuously by using a GFP tag, selective imaging of cell surface receptors was achieved by pulse-labeling an acyl carrier protein tag. We found that OR17-40 efficiently translocated to the plasma membrane only at low expression, whereas at higher biosynthesis the receptor accumulated in intracellular compartments. Receptors in the plasma membrane showed high turnover resulting from constitutive internalization along the clathrin pathway, even in the absence of ligand. Single-molecule microscopy allowed monitoring of the early, dynamic processes in odorant receptor signaling. Although mobile receptors initially diffused either freely or within domains of various sizes, binding of an agonist or an antagonist increased partitioning of receptors into small domains of approximately 190 nm, which likely are precursors of clathrin-coated pits. The binding of a ligand, therefore, resulted in modulation of the continuous, constitutive internalization. After endocytosis, receptors were directed to early endosomes for recycling. This unique mechanism of continuous internalization and recycling of OR17-40 might be instrumental in allowing rapid recovery of odor perception.

Quantum Dots Monitor TrkA Receptor Dynamics in the Interior of Neural PC12 Cells.

S Sundara Rajan — 2006-09-20T11:31:08-00:00

Nano Lett, Vol. 6, No. 9. (September 2006), pp. 2049-2059.

Can quantum dots (QDs) serve as physiologically relevant receptor probes in the interior of cells? We directly visualize endocytosis, redistribution, and shuttling of QD bound-TrkA receptors to PC12 neural processes and far-reaching growth cone tips. Internalized QDs are contained in microtubule-associated vesicles and possess transport properties that reflect TrkA receptor dynamics. This opens up new possibilities for the development of QD platforms as molecular tools to image biochemical signaling and transport cargo in the cell interior.

Dendrite-selective redistribution of the chemokine receptor CXCR4 following agonist stimulation.

Stéphane J Baudouin — 2006-09-11T08:18:34-00:00

Mol Cell Neurosci (2 September 2006)

The chemokine SDF-1 is a secreted protein that plays a critical role in several aspects of neuron development through interaction with its unique receptor CXCR4. A key mechanism that controls neuron responsiveness to extracellular signals during neuronal growth is receptor endocytosis. Since we previously reported that SDF-1 regulates axon development without affecting the other neurites, we asked whether this could correlate with a compartment-selective trafficking of CXCR4. We thus studied CXCR4 behavior upon SDF-1 exposure in rat hippocampus slices and in transfected neuron cultures. A massive agonist-induced redistribution of CXCR4 in endosomes was observed in dendrites whereas no modification was evidenced in axons. Our data suggest that CXCR4 trafficking may play a role in mediating selective effects of SDF-1 on distinct neuronal membrane subdomains.

RNA Interference Screen Reveals an Essential Role of Nedd4-2 in Dopamine Transporter Ubiquitination and Endocytosis

Tatiana Sorkina — 2006-08-03T09:49:11-00:00

J. Neurosci., Vol. 26, No. 31. (2 August 2006), pp. 8195-8205.

The function of the dopamine transporter (DAT) to terminate dopamine neurotransmission is regulated by endocytic trafficking of DAT. To elucidate the mechanisms of DAT endocytosis, we generated a fully functional mutant of the human DAT in which a hemagglutinin epitope (HA) was incorporated into the second extracellular loop. The endocytosis assay, based on the uptake of an HA antibody, was designed to study constitutive- and protein kinase C (PKC)-dependent internalization of HA-DAT expressed in non-neuronal cells and rat dopaminergic neurons. Large-scale RNA interference analysis of PKC-dependent endocytosis of HA-DAT revealed the essential and specific role of an E3 ubiquitin ligase, Nedd4-2 (neural precursor cell expressed, developmentally downregulated 4-2), as well as the involvement of adaptor proteins present in clathrin-coated pits, such as epsin, Eps15 (epidermal growth factor pathway substrate clone 15), and Eps15R (Eps15-related protein). Depletion of Nedd4-2 resulted in a dramatic reduction of PKC-dependent ubiquitination of DAT. Endogenous Nedd4-2, epsin, and Eps15 were coimmunoprecipitated with heterologously expressed human HA-DAT and endogenous DAT isolated from rat striatum. A new mechanistic model of DAT endocytosis is proposed whereby the PKC-induced ubiquitination of DAT mediated by Nedd4-2 leads to interaction of DAT with adaptor proteins in coated pits and acceleration of DAT endocytosis. 10.1523/JNEUROSCI.1301-06.2006

Tetanus toxin is internalized by a sequential clathrin-dependent mechanism initiated within lipid microdomains and independent of epsin1

Katrin Deinhardt — 2006-08-01T09:30:40-00:00

J. Cell Biol., Vol. 174, No. 3. (31 July 2006), pp. 459-471.

Ligand-receptor complexes are internalized by a variety of endocytic mechanisms. Some are initiated within clathrin-coated membranes, whereas others involve lipid microdomains of the plasma membrane. In neurons, where alternative targeting to short- or long-range trafficking routes underpins the differential processing of synaptic vesicle components and neurotrophin receptors, the mechanism giving access to the axonal retrograde pathway remains unknown. To investigate this sorting process, we examined the internalization of a tetanus neurotoxin fragment (TeNT HC), which shares axonal carriers with neurotrophins and their receptors. Previous studies have shown that the TeNT HC receptor, which comprises polysialogangliosides, resides in lipid microdomains. We demonstrate that TeNT HC internalization also relies on a specialized clathrin-mediated pathway, which is independent of synaptic vesicle recycling. Moreover, unlike transferrin uptake, this AP-2-dependent process is independent of epsin1. These findings identify a pathway for TeNT, beginning with the binding to a lipid raft component (GD1b) and followed by dissociation from GD1b as the toxin internalizes via a clathrin-mediated mechanism using a specific subset of adaptor proteins. 10.1083/jcb.200508170

Knockin mice expressing fluorescent delta-opioid receptors uncover G protein-coupled receptor dynamics in vivo

Gregory Scherrer — 2006-07-24T09:11:16-00:00

PNAS, Vol. 103, No. 25. (20 June 2006), pp. 9691-9696.

The combination of fluorescent genetically encoded proteins with mouse engineering provides a fascinating means to study dynamic biological processes in mammals. At present, green fluorescent protein (GFP) mice were mainly developed to study gene expression patterns or cell morphology and migration. Here we used enhanced GFP (EGFP) to achieve functional imaging of a G protein-coupled receptor (GPCR) in vivo. We created mice where the delta-opioid receptor (DOR) is replaced by an active DOR-EGFP fusion. Confocal imaging revealed detailed receptor neuroanatomy throughout the nervous system of knockin mice. Real-time imaging in primary neurons allowed dynamic visualization of drug-induced receptor trafficking. In DOR-EGFP animals, drug treatment triggered receptor endocytosis that correlated with the behavioral response. Mice with internalized receptors were insensitive to subsequent agonist administration, providing evidence that receptor sequestration limits drug efficacy in vivo. Direct receptor visualization in mice is a unique approach to receptor biology and drug design. 10.1073/pnas.0603359103

Receptor recycling mediates plasma membrane recovery of dopamine D1 receptors in dendrites and axons after agonist-induced endocytosis in primary cultures of striatal neurons.

ML Martin-Negrier — 2006-07-24T09:09:48-00:00

Synapse, Vol. 60, No. 3. (September 2006), pp. 194-204.

The pharmacological stimulation of G-protein-coupled receptor induces receptor internalization. Receptor's fate after the step of internalization remains poorly characterized despite its incidence on the neuronal responsiveness. In this context, we studied the dopamine (DA) D1 receptor (D1R) trafficking in a model of striatal neuronal culture that endogenously express the D1R. We first characterized by immunohistochemistry the spatial distribution of the compartments involved in the endocytic pathways and then the D1R trafficking in dendrites and axons. In dendrites, immunohistochemical analysis showed that acute stimulation by the D1R agonist SKF 82958 (1 muM) induces an internalization of D1R in early endosomes labeled with Alexa-488-conjugated transferrin. We show that, 20 min after removal of the agonist, the D1R immunolabeling pattern returns to the basal state in dendrites and in axons. Recovery was unaffected by cycloheximide (70 muM) but was prevented by monensin (100 muM) that inhibits endosomal acidification and receptor recycling. These data suggest that dendritic and axonal D1Rs are internalized after agonist stimulation and targeted to the recycling pathway demonstrating that the machinery involved in GPCR endocytosis and recycling is functional both in dendrites and in axons. Temporal characteristics observed for the recovery of D1R density to the basal state and those observed for the resensitization process strongly suggest that D1R recycling supports the receptor resensitization. Synapse 60:194-204, 2006. (c) 2006 Wiley-Liss, Inc.

Exocytotic and endocytotic membrane traffic in neurons.

WB Huttner — 2006-07-21T17:46:15-00:00

Curr Opin Neurobiol, Vol. 1, No. 3. (October 1991), pp. 388-392.

Because neurons are highly polarized and capable of various modes of neurosecretion the exocytotic and endocytotic membrane traffic in these cells is more complex than in other eukaryotic cells. Progress in our understanding of neuronal membrane traffic and organelle biogenesis has come from recently discovered analogies to epithelial and endocrine cells.

Axonal and dendritic endocytic pathways in cultured neurons.

RG Parton — 2006-07-21T17:45:37-00:00

J Cell Biol, Vol. 119, No. 1. (October 1992), pp. 123-137.

The endocytic pathways from the axonal and dendritic surfaces of cultured polarized hippocampal neurons were examined. The dendrites and cell body contained extensive networks of tubular early endosomes which received endocytosed markers from the somatodendritic domain. In axons early endosomes were confined to presynaptic terminals and to varicosities. The somatodendritic but not the presynaptic early endosomes were labeled by internalized transferrin. In contrast to early endosomes, late endosomes and lysosomes were shown to be predominantly located in the cell body. Video microscopy was used to follow the transport of internalized markers from the periphery of axons and dendrites back to the cell body. Labeled structures in both domains moved unidirectionally by retrograde fast transport. Axonally transported organelles were sectioned for EM after video microscopic observation and shown to be large multivesicular body-like structures. Similar structures accumulated at the distal side of an axonal lesion. Multivesicular bodies therefore appear to be the major structures mediating transport of endocytosed markers between the nerve terminals and the cell body. Late endocytic structures were also shown to be highly mobile and were observed moving within the cell body and proximal dendritic segments. The results show that the organization of the endosomes differs in the axons and dendrites of cultured rat hippocampal neurons and that the different compartments or stages of the endocytic pathways can be resolved spatially.

Cell biology of neuronal endocytosis.

RG Parton — 2006-07-21T17:44:13-00:00

J Neurosci Res, Vol. 36, No. 1. (1 September 1993), pp. 1-9.

Endocytosis is the process by which cells take in fluid and components of the plasma membrane. In this way cells obtain nutrients and trophic factors, retrieve membrane proteins for degradation, and sample their environment. In neuronal cells endocytosis is essential for the recycling of membrane after neurotransmitter release and plays a critical role during early developmental stages. Moreover, alterations of the endocytic pathway have been attributed a crucial role in the pathophysiology of certain neurological diseases. Although well characterized at the ultrastructural level, little is known of the dynamics and molecular organization of the neuronal endocytic pathways. In this respect most of our knowledge comes from studies of non-neuronal cells. In this review we will examine the endocytic pathways in neurons from a cell biological viewpoint by making comparisons with non-neuronal cells and in particular with another polarized cell, the epithelial cell.

The involvement of the small GTP-binding protein Rab5a in neuronal endocytosis.

MJ de Hoop — 2006-07-21T17:41:58-00:00

Neuron, Vol. 13, No. 1. (July 1994), pp. 11-22.

Rab5a is a small GTPase that regulates fusion of endocytic vesicles to early endosomes. We investigated whether Rab5a is involved in early endocytic traffic in both the axonal and the somatodendritic domains of polarized neurons. Using immunofluorescence, endogenous Rab5a was detected in axons and dendrites. Its localization in axons strongly overlapped that of the synaptic vesicle protein synaptophysin. Indeed, Rab5a co-immunoisolated with synaptophysin-containing vesicles, and antibodies against Rab5a labeled synaptic vesicle-like structures in nerve terminals. The functional association of Rab5a with dendritic and axonal early endosomes was assayed by electron microscopy after overexpression of wild-type and mutant Rab5a in cultured hippocampal neurons. This induced the formation of abnormal endosomes in both the somatodendritic and the axonal domains. These results show a role for Rab5a in axonal and dendritic endocytosis, and the presence of Rab5a on synaptic vesicles indicates that the axonal endosomes participate in the biogenesis of these vesicles.

EEA1, a tethering protein of the early sorting endosome, shows a polarized distribution in hippocampal neurons, epithelial cells, and fibroblasts.

JM Wilson — 2006-07-21T17:27:05-00:00

Mol Biol Cell, Vol. 11, No. 8. (August 2000), pp. 2657-2671.

EEA1 is an early endosomal Rab5 effector protein that has been implicated in the docking of incoming endocytic vesicles before fusion with early endosomes. Because of the presence of complex endosomal pathways in polarized and nonpolarized cells, we have examined the distribution of EEA1 in diverse cell types. Ultrastructural analysis demonstrates that EEA1 is present on a subdomain of the early sorting endosome but not on clathrin-coated vesicles, consistent with a role in providing directionality to early endosomal fusion. Furthermore, EEA1 is associated with filamentous material that extends from the cytoplasmic surface of the endosomal domain, which is also consistent with a tethering/docking role for EEA1. In polarized cells (Madin-Darby canine kidney cells and hippocampal neurons), EEA1 is present on a subset of "basolateral-type" endosomal compartments, suggesting that EEA1 regulates specific endocytic pathways. In both epithelial cells and fibroblastic cells, EEA1 and a transfected apical endosomal marker, endotubin, label distinct endosomal populations. Hence, there are at least two distinct sets of early endosomes in polarized and nonpolarized mammalian cells. EEA1 could provide specificity and directionality to fusion events occurring in a subset of these endosomes in polarized and nonpolarized cells.

CRMP-2 regulates polarized Numb-mediated endocytosis for axon growth.

T Nishimura — 2006-07-21T17:08:48-00:00

Nat Cell Biol, Vol. 5, No. 9. (September 2003), pp. 819-826.

Axon growth during neural development is highly dependent on both cytoskeletal re-organization and polarized membrane trafficking. Previously, we demonstrated that collapsin response mediator protein-2 (CRMP-2) is critical for specifying axon/dendrite fate and axon growth in cultured hippocampal neurons, possibly by interacting with tubulin heterodimers and promoting microtubule assembly. Here, we identify Numb as a CRMP-2-interacting protein. Numb has been shown to interact with alpha-adaptin and to be involved in endocytosis. We found that Numb was associated with L1, a neuronal cell adhesion molecule that is endocytosed and recycled at the growth cone, where CRMP-2 and Numb were colocalized. Furthermore, expression of dominant-negative CRMP-2 mutants or knockdown of CRMP-2 message with small-interfering (si) RNA inhibited endocytosis of L1 at axonal growth cones and suppressed axon growth. These results suggest that in addition to regulating microtubule assembly, CRMP-2 is involved in polarized Numb-mediated endocytosis of proteins such as L1.

Mécanisme de la fusion membranaire [Mechanisms of membrane fusion]

T Galli — 2006-07-21T11:39:50-00:00

Medecine/Sciences, Vol. 18, No. 11. (November 2002), pp. 1113-1119.

Les cellules eucaryotes possedent un reseau complexe de membranes intracellulaires. Les differents compartiments (reticulum endoplasmique, appareil de Golgi, endosomes, lysosomes, membrane plasmique) communiquent entre eux grace a des vesicules qui bourgeonnent d' un compartiment donneur et fusionnent avec un compartiment accepteur. Au cours de ces transports vectoriels qui definissent le trafic membranaire, les proteines SNARE ont un role cle. Dans cet article, nous evoquerons les proteines qui interviennent dans les differentes etapes de la fusion membranaire: l' arrimage des vesicules a la membrane cible, l' amorcage de la reaction, la fusion membranaire elle-meme et, enfin, le recyclage des proteines SNARE. [Eukaryotic cells have a complex network of intracellular membranes. The different compartments (endoplasmic reticulum, Golgi apparatus, endosomes, lysosomes, plasma membrane) are connected by vesicles that bud from a donor compartment and fuse with an acceptor compartment thus defining membrane trafficking as a dynamic phenomenon. Membrane fusion depends on the SNARE family of proteins, the function of which can be recapitulated in vitro. In addition, a large number of proteins, that is discussed in the present paper, control the tethering of the membranes, the priming of the reaction, the regulation of fusion itself and the recycling of SNARE proteins.]

Les compartiments membranaires de la cellule eucaryote [Membrane Compartments in Eukaryotic Cells]

A Tixier-Vidal — 2006-07-21T11:19:24-00:00

Medecine/Sciences, Vol. 18, No. 10. (October 2002), pp. 1004-1011.

Les donnees issues du decryptage du genome humain puis de la proteomique naissante doivent etre integrees dans les structures de la cellule vivante si l' on veut parvenir a une meilleure comprehension de son fonctionnement normal et pathologique. L' organisation compartimentee de la cellule eucaryote, pressentie des la fin du XIX" siecle, est maintenant definitivement etablie dans l' ensemble du monde vivant. Cet article situe dans une perspective historique les donnees de base indispensables a la comprehension des recherches modernes sur les mecanismes du transport intracellulaire. [The existence of an intracellular reticulum in eukaryotic cells was revealed at the middle of the XXth century by electron microscopic observations performed on living fibroblasts and then on cell thin sections. Two compartments were morphologically distinguished in this reticulum: the rough endoplasmic reticulum and the smooth endoplasmic reticulum which was identified to the "apparatus" previously described, by the end of the XIXth century, at the light microscope by Golgi. An important progress was then accomplished due to functional studies that demonstrated that these compartments are in continuous communication and that they are the site of the intracellular transport of exported proteins. This led to the concept of membrane traffic and to the hypothesis put forward by Palade of the existence of intracellular transport vesicles, budding and pinching off from donor membranes and fusing with acceptor membranes. Biochemical, molecular and genetic studies performed during the last quarter of the XXth century aimed at elucidating the structural features and functions of the various intracellular compartments. At the same time they have revealed the unicity of organization of all eukaryotic cells. The present chapter surveys the main properties of the intracellular membrane compartments and subcompartments involved in the exportation of proteins (the biosynthetic pathway) or in their importation (endocytic pathway). This knowledge represents necessary basis to the understanding of the most recent studies on the molecular mechanisms of intracellular transport.]

Inhibition of sphingolipid synthesis affects kinetics but not fidelity of L1/NgCAM transport along direct but not transcytotic axonal pathways.

MC Chang — 2006-07-21T09:15:17-00:00

Mol Cell Neurosci, Vol. 31, No. 3. (March 2006), pp. 525-538.

Glycosphingolipids are constituents of lipid rafts which might function in sorting apical and axonal cargoes in the trans-Golgi network. In fact, two GPI-linked proteins, Thy1 and PrPC, require lipid raft lipids for sorting to the axon. It was previously shown that inhibition of glycosphingolipid synthesis by FumonisinB1 (FB1) impairs axon outgrowth but not axon specification, leading to the hypothesis that formation of axonally-targeted vesicles is coupled to sphingolipid synthesis. Since the axonal cell adhesion molecule L1/NgCAM can partition into membrane rafts biochemically, we asked whether correct targeting to the axon is FB1-sensitive, similarly to GPI-linked proteins. We previously showed that cultured hippocampal neurons use more than one trafficking pathway to the axon: a transcytotic pathway and a direct pathway. We show here that reducing raft lipid levels does not disrupt axonal targeting of L1/NgCAM along either pathway. Unexpectedly, FB1 selectively slowed the kinetics of surface expression of a truncated NgCAM using the direct pathway, but not of NgCAM using the transcytotic pathway. Therefore, the formation and/or transport of a subset of axonally-targeted vesicles are coupled to sphingolipid synthesis. Our results yield a mechanism for the axon outgrowth defect observed in FB1.

Scientiae forum / models and speculations pathways for axonal targeting of membrane proteins.

B Winckler — 2006-07-21T09:14:00-00:00

Biol Cell, Vol. 96, No. 9. (December 2004), pp. 669-674.

The distribution of proteins to the correct domains of neurons is crucial for neuronal functioning. Here I discuss possible mechanisms underlying sorting to the axonal plasma membrane which differ with respect to the steps providing selectivity for axonal vs. somatodendritic cargo. Selectivity can be executed at one or more than one step, including sorting into distinct carriers in the Golgi, preferential transport along axonal microtubules, fusion with the plasma membrane, and importantly retrieval by endocytosis. A protein might in fact be sorted multiple times at different places to achieve axonal localization.