Systems analysis of RNA trafficking in neural cells

In neural cells, certain RNAs are targeted to dendrites by a specific RNA trafficking pathway, termed the A2 pathway, mediated by the trans-acting trafficking factor, heterogeneous nuclear ribonucleoprotein (hnRNP) A2, which binds to an 11 nucleotide cis-acting trafficking sequence, termed the hnRNP A2 response element (A2RE). RNAs containing A2RE-like sequences are recognized by hnRNP A2 in the nucleus and exported to the cytoplasm where they assemble into trafficking intermediates, termed granules, which also contain components of the translation machinery and molecular motors (cytoplasmic dynein and conventional kinesin). RNA granules move along microtubules to the cell periphery where they become localized and where the encoded protein is translated. Intracellular trafficking of RNA molecules by the A2 pathway is mediated by a complex system consisting of five different subsystems, approximately 35 different molecules and approximately 45 different molecular interactions. Specificity in the A2 pathway is provided by specific interactions of hnRNP A2 with different molecular partners in different subsystems. Polarity of RNA trafficking is controlled by transitions of trafficking intermediates between different subsystems. Comprehensive understanding of the A2 RNA trafficking pathway will require quantitative analysis of concentrations and diffusion constants for each of the different molecules, on rates and off rates for each of the different interactions, relevant conditional operators controlling specific interactions, and interactions of different subsystems. Once the necessary quantitative data are available, mathematical models for the different RNA trafficking subsystems can be developed using computational platforms such as the 'Virtual Cell'. Here we describe how each of the subsystems in the A2 system functions and how the different subsystems interact to regulate RNA trafficking.     

Copper transport during lactation in transgenic mice expressing the human ATP7A protein

We investigated the membrane trafficking of AQP3 induced by epinephrine in Caco-2 cells to clarify the digestive absorption of glycerol permeated by AQP3. Epinephrine was found to promote within 60 min the translocation of AQP3 from the cytoplasmic fraction to the plasma membrane. This increased trafficking of AQP3 was suppressed by phospholipase C and protein kinase C (PKC) inhibitors and a phorbol ester accelerated the trafficking of AQP3 to the membrane fraction. In contrast, adenylyl cyclase (AC) and protein kinase A (PKA) inhibitors did not have any effect on the increased in trafficking of AQP3 by epinephrine and an AC activator did not affect the trafficking of AQP3. Phosphorylation of a threonine (514) residue in PKC was detected upon the treatment with epinephrine and the temporal transitional pattern of this phosphorylation paralleled that of the increased trafficking of AQP3. These results suggest that PKC modulates the trafficking of AQP3.     

Subcellular trafficking of the Arabidopsis auxin influx carrier AUX1 uses a novel pathway distinct from PIN1

The directional flow of the plant hormone auxin mediates multiple developmental processes, including patterning and tropisms. Apical and basal plasma membrane localization of AUXIN-RESISTANT1 (AUX1) and PIN-FORMED1 (PIN1) auxin transport components underpins the directionality of intercellular auxin flow in Arabidopsis thaliana roots. Here, we examined the mechanism of polar trafficking of AUX1. Real-time live cell analysis along with subcellular markers revealed that AUX1 resides at the apical plasma membrane of protophloem cells and at highly dynamic subpopulations of Golgi apparatus and endosomes in all cell types. Plasma membrane and intracellular pools of AUX1 are interconnected by actin-dependent constitutive trafficking, which is not sensitive to the vesicle trafficking inhibitor brefeldin A. AUX1 subcellular dynamics are not influenced by the auxin influx inhibitor NOA but are blocked by the auxin efflux inhibitors TIBA and PBA. Furthermore, auxin transport inhibitors and interference with the sterol composition of membranes disrupt polar AUX1 distribution at the plasma membrane. Compared with PIN1 trafficking, AUX1 dynamics display different sensitivities to trafficking inhibitors and are independent of the endosomal trafficking regulator ARF GEF GNOM. Hence, AUX1 uses a novel trafficking pathway in plants that is distinct from PIN trafficking, providing an additional mechanism for the fine regulation of auxin transport.     

The regulation of vesicle trafficking by small GTPases and phospholipids during pollen tube growth

Polarized and directional growth of pollen tubes is the only means by which immotile sperm of flowering plants reach the deeply embedded female gametes for fertilization. Vesicle trafficking is among the most critical cellular activities for pollen tube growth. Vesicle trafficking maintains membrane homeostasis during rapid tube growth and provides polarity information by regulating protein/lipid compositions of different membrane compartments. In this review, we will focus on two classes of factors that orchestrate vesicle trafficking, small GTPases and phospholipids. We discuss the features of small GTPases and phospholipids that make them ideal components to regulate vesicle trafficking, review recent advances in understanding their involvement in vesicle trafficking, and propose directions for future research.     

Alcohol consumption impairs hepatic protein trafficking: mechanisms and consequences

Alcoholic liver disease is a major biomedical health concern in the United States. Despite considerable research efforts aimed at understanding the progression of the disease, the specific mechanisms leading to alcohol-induced damage remain elusive. Numerous proteins are known to have alcohol-induced alterations in their dynamics. Defining these defects in protein trafficking is an active area of research. In general, two trafficking pathways are affected: transport of newly synthesized secretory or membrane glycoproteins from the Golgi to the basolateral membrane and clathrin-mediated endocytosis from the sinusoidal surface. Both impaired secretion and internalization require ethanol metabolism and are likely mediated by acetaldehyde. Although the mechanisms by which ethanol exposure impairs protein trafficking are not fully understood, recent work implicates alcohol-induced modifications on tubulin or components of the clathrin machinery as potential mediators. Furthermore, the physiological ramifications of impaired protein trafficking are not fully understood. In this review, we will list and discuss the proteins whose trafficking patterns are known to be impaired by ethanol exposure. We will then describe what is known about the possible mechanisms leading to impaired protein trafficking and how disrupted protein trafficking alters liver function and may explain clinical features of the alcoholic patient.     

Control of vesicular trafficking by Rho GTPases

Although vesicular trafficking is essential for a large variety of cellular processes, the regulation of vesicular trafficking is still poorly understood. Members of the Rho family of small GTPases have recently emerged as important control elements of many stages of vesicular trafficking, providing new insight into the regulation of these events. We will discuss the diverse roles played by Rho proteins in membrane trafficking and focus on the biological implications of these functions.     

Tied up: Does altering phosphoinositide-mediated membrane trafficking influence neurodegenerative disease phenotypes?

Phosphoinositides are a class of membrane lipids that are found on several intracellular compartments and play diverse roles inside cells, such as vesicle formation, protein trafficking, endocytosis etc. Intracellular distribution and levels of phosphoinositides are regulated by enzymes that generate and breakdown these lipids as well as other proteins that associate with phosphoinositides. These events lead to differing levels of specific phosphoinositides on different intracellular compartments. At these intracellular locations, phosphoinositides and their associated proteins, such as Rab GTPases, dynamin and BAR domain-containing proteins, regulate a variety of membrane trafficking pathways. Neurodegenerative phenotypes in disorders such as Parkinson’s disease (PD) can arise as a consequence of altered or hampered intracellular trafficking. Altered trafficking can cause proteins such as \(\upalpha \)-synuclein to aggregate intracellularly. Several trafficking pathways are regulated by master regulators such as LRRK2, which is known to regulate the activity of phosphoinositide effector proteins. Perturbing either the levels of phosphoinositides or their interactions with different proteins disrupts intracellular trafficking pathways, contributing to phenotypes often observed in disorders such as Alzheimer’s or PDs. Thus, studying phosphoinositide regulation and its role in trafficking can give us a deeper understanding of the contribution of disrupted trafficking to neurodegenerative phenotypes.     

Harnessing the power of the endosome to regulate neural development

Endocytosis and endosomal trafficking play a multitude of roles in cellular function beyond regulating entry of essential nutrients. In this review, we discuss the cell biological principles of endosomal trafficking, the neuronal adaptations to endosomal organization, and the role of endosomal trafficking in neural development. In particular, we consider how cell fate decisions, polarity, migration, and axon outgrowth and guidance are influenced by five endosomal tricks: dynamic modulation of receptor levels by endocytosis and recycling, cargo-specific responses via cargo-specific endocytic regulators, cell-type-specific endocytic regulation, ligand-specific endocytic regulation, and endosomal regulation of ligand processing and trafficking.     

Plasmid DNA induces increased lymphocyte trafficking: a specific role for CpG motifs

Bacterial DNA, primarily through immunostimulatory cytosine-guanine (CpG) motifs, induces the secretion of cytokines and activates a variety of effector cells. We investigated the possibility that CpG motifs might also modulate immunosurveillance by altering cell trafficking through a regional lymph node. Intradermal injection of plasmid DNA induced rapid and prolonged increases in the number of lymphocytes collected in efferent lymph. This effect on cell trafficking was not dependent on the expression of an encoded reporter gene but varied with plasmid construct and required a circular form. Injection of synthetic oligodeoxyribonucleotides containing CpG motifs did not alter lymphocyte trafficking but CpG-enhanced plasmid induced a dose-dependent increase in cell trafficking. Phenotypic analyses revealed that the increase in cell trafficking involved all lymphocyte subpopulations and represented a mass movement of cells. These observations reveal that bacterial DNA, through immunostimulatory CpG motifs, alters immunosurveillance by increasing cell recruitment to a regional lymph node.     

Na+/H+ Exchangers and RhoA Regulate Acidic Extracellular pH-Induced Lysosome Trafficking in Prostate Cancer Cells

Acidic extracellular pH (pHe) is a common feature of the tumor microenvironment and has been implicated in tumor invasion through the induction of protease secretion. Since lysosomes constitute the major storehouse of cellular proteases, the trafficking of lysosomes to the cell periphery may be required in order to secrete proteases. We demonstrate that a pHe of 6.4-6.8 induced the trafficking of lysosomes to membrane protrusions in the cell periphery. This trafficking event depended upon the PI3K pathway, the GTPase RhoA and sodium-proton exchange activity, resulting in lysosomal exocytosis. Acidic pHe induced a cytoplasmic acidification (although cytoplasmic acidification was not sufficient for acidic pHe-induced lysosome trafficking and exocytosis) and inhibition of NHE activity with the amiloride derivative, EIPA or the anti-diabetic agent troglitazone prevented lysosome trafficking to the cell periphery. Interestingly, using the more specific NHE1 and NHE3 inhibitors, cariporide and s3226 respectively, we show that multiple NHE isoforms are involved in acidic pHe-induced lysosome trafficking and exocytosis. Moreover, in cells expressing NHE1 shRNA, although basal NHE activity was decreased, lysosomes still underwent acidic pHe-induced trafficking, suggesting compensation by other NHE family members. Together these data implicate proton exchangers, especially NHE1 and NHE3, in acidic pHe-induced lysosome trafficking and exocytosis.     

AMPA receptor trafficking pathways and links to dendritic spine morphogenesis

Alterations in synaptic strength are proposed to underlie learning and memory, and two major mechanisms utilised by neurons to bring about such changes involve regulating the number of AMPARs found at the synaptic plasma membrane, and altering the size and/or shape of the specialised postsynaptic compartment, the dendritic spine. It is now well-established that control of receptor number is brought about by precise modulation of vesicle trafficking, although the details of this crucial process are still far from clear. Regulation of spine size involves dynamic alterations in the spine actin cytoskeleton, but recent studies suggest that trafficking events may also play a role. In this review, I will summarise some recent findings about AMPAR trafficking pathways, and highlight the idea that membrane trafficking events not only regulate the complement of AMPARs at the synaptic plasma membrane, but also contribute to spine morphogenesis, either by regulating the plasma membrane content of spines, or as a result of changes in AMPAR trafficking.     

Ral: mediator of membrane trafficking

Ral is a multifunctional small GTPase involved in tumorigenesis and in controlling intracellular membrane trafficking. It is mainly activated by factors downstream of Ras, or independently of these factors and operates by protein-protein interactions with an expanding repertoire of partners. RalA is a positive regulator of calcium-evoked exocytosis via binding phospholipase D and is involved in G protein coupled receptor signalling by binding phospholipase C-delta1. The binding of Ral to calmodulin links to intracellular trafficking events. Another link is direct binding of activated Ral (Ral-GTP) to the endocytic and exocytic machineries. Ral-GTP binds RalBP1, which connects to receptor-mediated endocytosis via AP-2. Alternatively, Ral-GTP binds the exocyst complex, which controls secretory vesicle trafficking in regulated secretion and filopodia formation. Thus, Ral-GTP "chooses" between different membrane trafficking pathways. Other Ral partners are still being uncovered that may provide further mechanistic insights into how Ral controls diverse membrane trafficking pathways.     

Sphingosine kinases and their metabolites modulate endolysosomal trafficking in photoreceptors

Internalized membrane proteins are either transported to late endosomes and lysosomes for degradation or recycled to the plasma membrane. Although proteins involved in trafficking and sorting have been well studied, far less is known about the lipid molecules that regulate the intracellular trafficking of membrane proteins. We studied the function of sphingosine kinases and their metabolites in endosomal trafficking using Drosophila melanogaster photoreceptors as a model system. Gain- and loss-of-function analyses show that sphingosine kinases affect trafficking of the G protein-coupled receptor Rhodopsin and the light-sensitive transient receptor potential (TRP) channel by modulating the levels of dihydrosphingosine 1 phosphate (DHS1P) and sphingosine 1 phosphate (S1P). An increase in DHS1P levels relative to S1P leads to the enhanced lysosomal degradation of Rhodopsin and TRP and retinal degeneration in wild-type photoreceptors. Our results suggest that sphingosine kinases and their metabolites modulate photoreceptor homeostasis by influencing endolysosomal trafficking of Rhodopsin and TRP.     

腺病毒感染及胞内运输途径的研究进展

目前关于腺病毒感染及胞内运输的分子机制研究主要来源于C亚群腺病毒在肿瘤细胞系中的研究结果。腺病毒对靶细胞的感染及胞内运输大致分为几步:病毒与细胞表面受体的特异结合,胞吞介导的病毒内化,病毒逃脱胞内体进入细胞质,病毒沿着微管运输至核孔,病毒基因组入核。病毒胞内运输效率极高,感染后1 h,80%以上的病毒基因组被送至核内。但是腺病毒胞内的运输方式会因以下几个因素变化而产生差异:靶细胞类型,细胞生理状态,病毒血清型。文中对腺病毒感染靶细胞及胞内运输的已有分子机制进行综述,为临床基因治疗用途的病毒载体研发提供思路。     

Mutant huntingtin impairs axonal trafficking in mammalian neurons in vivo and in vitro

Recent data in invertebrates demonstrated that huntingtin (htt) is essential for fast axonal trafficking. Here, we provide direct and functional evidence that htt is involved in fast axonal trafficking in mammals. Moreover, expression of full-length mutant htt (mhtt) impairs vesicular and mitochondrial trafficking in mammalian neurons in vitro and in whole animals in vivo. Particularly, mitochondria become progressively immobilized and stop more frequently in neurons from transgenic animals. These defects occurred early in development prior to the onset of measurable neurological or mitochondrial abnormalities. Consistent with a progressive loss of function, wild-type htt, trafficking motors, and mitochondrial components were selectively sequestered by mhtt in human Huntington's disease-affected brain. Data provide a model for how loss of htt function causes toxicity; mhtt-mediated aggregation sequesters htt and components of trafficking machinery leading to loss of mitochondrial motility and eventual mitochondrial dysfunction.     

溶酶体相关细胞器生物发生复合体相互作用组构成胞内体运输网络

随着高等生物中十几个新的参与囊泡运输的 Hermansky-Pudlak 综合征(HPS)蛋白质的发现, 认为可能存在一类新的囊泡运输通路。该通路主要由新近鉴定的 3 个被称为溶酶体相关细胞器生物发生复合体(BLOC)所组成, 被分别命名为BLOC-1、BLOC-2 和 BLOC-3。越来越多的证据表明这些复合体与以前认识较清楚的 AP3 和 HOPS 复合体共同在胞内体运输中起重要作用。这些复合体之间的相互作用构成了以胞内体和细胞骨架为连接纽带的参与蛋白质运输的复杂网络。该网络中的每个节点的相互作用可区分为复合体内和复合体外相互作用两大类。复合体之间的联系可以是来自不同复合体亚基间的直接相互作用, 也可以通过耦联的节点联结不同的复合体。解析这一复杂网络有助于进一步了解参与蛋白质和膜运输这一动态而精细网络的结构与功能。一旦该网络结构得到破坏, 则可能导致如 HPS 这类囊泡运输或细胞器发生障碍性疾病。     

Protein kinase Balpha is required for vesicle trafficking and class II presentation of IgA Fc receptor (CD89)-targeted antigen

Ag presentation stimulates Ag-specific adaptive immune responses. FcalphaR (CD89)-mediated capture of IgA-bound exogenous Ag leads to efficient MHC class II Ag presentation by APCs. CD89 signaling is required for trafficking of internalized Ag to specialized multivesicular bodies known as MHC class II compartments (MIIC) and subsequent class II presentation. In the present study, we tested the hypothesis that the vesicle trafficking regulator protein kinase Balpha (PKBalpha) is required for CD89-mediated trafficking to MIIC and Ag presentation. We observed by two independent methods (chemical inhibitors and specific RNA interference) that PKBalpha was required for CD89 trafficking to MIIC and class II Ag presentation. Expression of constitutively active PKBalpha in APCs expressing a mutant CD89 accessory signaling molecule (deficient in CD89/Ag trafficking, processing, and presentation) induced trafficking of CD89 to lamp1-containing late endocytic vesicles, but not class II-containing vesicles (MIIC), or class II Ag presentation. These studies show for the first time that PKBalpha is required for receptor-mediated Ag presentation and suggest the mechanism of action includes regulation of vesicle trafficking.     

Crossroads between membrane trafficking machinery and copper homeostasis in the nerve system

Imbalanced copper homeostasis and perturbation of membrane trafficking are two common symptoms that have been associated with the pathogenesis of neurodegenerative and neurodevelopmental diseases. Accumulating evidence from biophysical, cellular and in vivo studies suggest that membrane trafficking orchestrates both copper homeostasis and neural functions—however, a systematic review of how copper homeostasis and membrane trafficking interplays in neurons remains lacking. Here, we summarize current knowledge of the general trafficking itineraries for copper transporters and highlight several critical membrane trafficking regulators in maintaining copper homeostasis. We discuss how membrane trafficking regulators may alter copper transporter distribution in different membrane compartments to regulate intracellular copper homeostasis. Using Parkinson''s disease and MEDNIK as examples, we further elaborate how misregulated trafficking regulators may interplay parallelly or synergistically with copper dyshomeostasis in devastating pathogenesis in neurodegenerative diseases. Finally, we explore multiple unsolved questions and highlight the existing challenges to understand how copper homeostasis is modulated through membrane trafficking.     

Protein kinase C promotes N-methyl-D-aspartate (NMDA) receptor trafficking by indirectly triggering calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation

Regulation of neuronal NMDA receptor (NMDAR) is critical in synaptic transmission and plasticity. Protein kinase C (PKC) promotes NMDAR trafficking to the cell surface via interaction with NMDAR-associated proteins (NAPs). Little is known, however, about the NAPs that are critical to PKC-induced NMDAR trafficking. Here, we showed that calcium/calmodulin-dependent protein kinase II (CaMKII) could be a NAP that mediates the potentiation of NMDAR trafficking by PKC. PKC activation promoted the level of autophosphorylated CaMKII and increased association with NMDARs, accompanied by functional NMDAR insertion, at postsynaptic sites. This potentiation, along with PKC-induced long term potentiation of the AMPA receptor-mediated response, was abolished by CaMKII antagonist or by disturbing the interaction between CaMKII and NR2A or NR2B. Further mutual occlusion experiments demonstrated that PKC and CaMKII share a common signaling pathway in the potentiation of NMDAR trafficking and long-term potentiation (LTP) induction. Our results revealed that PKC promotes NMDA receptor trafficking and induces synaptic plasticity through indirectly triggering CaMKII autophosphorylation and subsequent increased association with NMDARs.