RESUMO
RhoA plays a crucial role in neuronal polarization, where its action restraining axon outgrowth has been thoroughly studied. We now report that RhoA has not only an inhibitory but also a stimulatory effect on axon development depending on when and where exerts its action and the downstream effectors involved. In cultured hippocampal neurons, FRET imaging revealed that RhoA activity selectively localized in growth cones of undifferentiated neurites, whereas in developing axons it displayed a biphasic pattern, being low in nascent axons and high in elongating ones. RhoA-Rho kinase (ROCK) signaling prevented axon initiation but had no effect on elongation, whereas formin inhibition reduced axon extension without significantly altering initial outgrowth. In addition, RhoA-mDia signaling promoted axon elongation by stimulating growth cone microtubule stability and assembly, as opposed to RhoA-ROCK signaling, which restrained growth cone microtubule assembly and protrusion.
Assuntos
Axônios , Cones de Crescimento , Microtúbulos , Transdução de Sinais , Proteína rhoA de Ligação ao GTP , Microtúbulos/metabolismo , Animais , Proteína rhoA de Ligação ao GTP/metabolismo , Axônios/metabolismo , Cones de Crescimento/metabolismo , Quinases Associadas a rho/metabolismo , Hipocampo/metabolismo , Hipocampo/citologia , Ratos , Forminas/metabolismo , Células Cultivadas , Neurônios/metabolismoRESUMO
Development of the mammalian neocortex requires proper inside-out migration of developing cortical neurons from the germinal ventricular zone toward the cortical plate. The mechanics of this migration requires precise coordination of different cellular phenomena including cytoskeleton dynamics, membrane trafficking, and cell adhesion. The small GTPases play a central role in all these events. The small GTPase Rab21 regulates migration and neurite growth in developing neurons. Moreover, regulators and effectors of Rab21 have been implicated in brain pathologies with cortical malformations, suggesting a key function for the Rab21 signaling pathway in cortical development. Mechanistically, it has been posited that Rab21 influences cell migration by controlling the trafficking of endocytic vesicles containing adhesion molecules. However, direct evidence of the participation of Rab21 or its mechanism of action in the regulation of cortical migration is still incomplete. In this study, we demonstrate that Rab21 plays a critical role in the differentiation and migration of pyramidal neurons by regulating the levels of the amyloid precursor protein on the neuronal cell surface. Rab21 loss of function increased the levels of membrane-exposed APP, resulting in impaired cortical neuronal differentiation and migration. These findings further our understanding of the processes governing the development of the cerebral cortex and shed light onto the molecular mechanisms behind cortical development disorders derived from the malfunctioning of Rab21 signaling effectors.
Assuntos
GTP Fosfo-Hidrolases , Neocórtex , Animais , GTP Fosfo-Hidrolases/metabolismo , Córtex Cerebral/metabolismo , Neurônios/metabolismo , Neocórtex/metabolismo , Movimento Celular/fisiologia , Precursor de Proteína beta-Amiloide/metabolismo , Mamíferos/metabolismoRESUMO
Neurons are highly polarized cells requiring precise regulation of trafficking and targeting of membrane proteins to generate and maintain different and specialized compartments, such as axons and dendrites. Disruption of the Golgi apparatus (GA) secretory pathway in developing neurons alters axon/dendritic formation. Therefore, detailed knowledge of the mechanisms underlying vesicles exiting from the GA is crucial for understanding neuronal polarity. In this study, we analyzed the role of Brefeldin A-Ribosylated Substrate (CtBP1-S/BARS), a member of the C-terminal-binding protein family, in the regulation of neuronal morphological polarization and the exit of membrane proteins from the Trans Golgi Network. Here, we show that BARS is expressed during neuronal development in vitro and that RNAi suppression of BARS inhibits axonal and dendritic elongation in hippocampal neuronal cultures as well as largely perturbed neuronal migration and multipolar-to-bipolar transition during cortical development in situ. In addition, using plasma membrane (PM) proteins fused to GFP and engineered with reversible aggregation domains, we observed that expression of fission dominant-negative BARS delays the exit of dendritic and axonal membrane protein-containing carriers from the GA. Taken together, these data provide the first set of evidence suggesting a role for BARS in neuronal development by regulating post-Golgi membrane trafficking.
Assuntos
Complexo de Golgi , Neurônios , Axônios/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/fisiologia , Rede trans-Golgi/metabolismoRESUMO
Actin dynamics in dendritic spines can be associated with the neurobiological mechanisms supporting the comorbidity between stress exposure and cocaine increase rewards. The actin cytoskeleton remodeling in the nucleus accumbens (NA) has been implicated in the expression of stress-induced cross-sensitization with cocaine. The present study evaluates the involvement of cofilin, a direct regulator of actin dynamics, in the impact of stress on vulnerability to cocaine addiction. We assess whether the neurobiological mechanisms that modulate repeated-cocaine administration also occur in a chronic restraint stress-induced cocaine self-administration model. We also determine if chronic stress induces alterations in dendritic spines through dysregulation of cofilin activity in the NA core. Here, we show that the inhibition of cofilin expression in the NA core using viral short-hairpin RNA is sufficient to prevent the cocaine sensitization induced by chronic stress. The reduced cofilin levels also impede a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor surface expression enhancement and promote the reduction of head diameter in animals pre-exposed to stress after a cocaine challenge in the NA core. Moreover, downregulation of cofilin expression prevents facilitation of the acquisition of cocaine self-administration (SA) in male rats pre-exposed to chronic stress without modifying performance in sucrose SA. These findings reveal a novel, crucial role for cofilin in the neurobiological mechanisms underpinning the comorbidity between stress exposure and addiction-related disorders.
RESUMO
BACKGROUND INFORMATION: The endo-lysosomal system (ELS) comprises a set of membranous organelles responsible for transporting intracellular and extracellular components within cells. Defects in lysosomal proteins usually affect a large variety of processes and underlie many diseases, most of them with a strong neuronal impact. Mutations in the endoplasmic reticulum-resident CLN8 protein cause CLN8 disease. This condition is one of the 14 known neuronal ceroid lipofuscinoses (NCLs), a group of inherited diseases characterised by accumulation of lipofuscin-like pigments within lysosomes. Besides mediating the transport of soluble lysosomal proteins, recent research suggested a role for CLN8 in the transport of vesicles and lipids, and autophagy. However, the consequences of CLN8 deficiency on ELS structure and activity, as well as the potential impact on neuronal development, remain poorly characterised. Therefore, we performed CLN8 knockdown in neuronal and non-neuronal cell models to analyse structural, dynamic and functional changes in the ELS and to assess the impact of CLN8 deficiency on axodendritic development. RESULTS: CLN8 knockdown increased the size of the Golgi apparatus, the number of mobile vesicles and the speed of endo-lysosomes. Using the fluorescent fusion protein mApple-LAMP1-pHluorin, we detected significant lysosomal alkalisation in CLN8-deficient cells. In turn, experiments in primary rat hippocampal neurons showed that CLN8 deficiency decreased the complexity and size of the somatodendritic compartment. CONCLUSIONS: Our results suggest the participation of CLN8 in vesicular distribution, lysosomal pH and normal development of the dendritic tree. We speculate that the defects triggered by CLN8 deficiency on ELS structure and dynamics underlie morphological alterations in neurons, which ultimately lead to the characteristic neurodegeneration observed in this NCL. SIGNIFICANCE: This is, to our knowledge, the first characterisation of the effects of CLN8 dysfunction on the structure and dynamics of the ELS. Moreover, our findings suggest a novel role for CLN8 in somatodendritic development, which may account at least in part for the neuropathological manifestations associated with CLN8 disease.
Assuntos
Lipofuscinoses Ceroides Neuronais , Animais , Retículo Endoplasmático , Complexo de Golgi , Lisossomos , Proteínas de Membrana/genética , Lipofuscinoses Ceroides Neuronais/genética , RatosRESUMO
Fluorescent nanoscopy approaches have been used to characterize the periodic organization of actin, spectrin and associated proteins in neuronal axons and dendrites. This membrane-associated periodic skeleton (MPS) is conserved across animals, suggesting it is a fundamental component of neuronal extensions. The nanoscale architecture of the arrangement (190 nm) is below the resolution limit of conventional fluorescent microscopy. Fluorescent nanoscopy, on the other hand, requires costly equipment and special analysis routines, which remain inaccessible to most research groups. This report aims to resolve this issue by using protein-retention expansion microscopy (pro-ExM) to reveal the MPS of axons. ExM uses reagents and equipment that are readily accessible in most neurobiology laboratories. We first explore means to accurately estimate the expansion factors of protein structures within cells. We then describe the protocol that produces an expanded specimen that can be examined with any fluorescent microscopy allowing quantitative nanoscale characterization of the MPS. We validate ExM results by direct comparison to stimulated emission depletion (STED) nanoscopy. We conclude that ExM facilitates three-dimensional, multicolor and quantitative characterization of the MPS using accessible reagents and conventional fluorescent microscopes.
Assuntos
Axônios/metabolismo , Microscopia de Fluorescência/métodos , Espectrina/metabolismo , Animais , Calibragem , Membrana Celular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Ratos Wistar , Reprodutibilidade dos TestesRESUMO
Neurons are the most extensive and polarized cells that display a unique single long axon and multiple dendrites, which are compartments exhibiting structural and functional differences. Polarity occurs early in neuronal development and it is maintained by complex subcellular mechanisms throughout cell life. A well-defined and controlled spatio-temporal program of cellular and molecular events strictly regulates the formation of the axon and dendrites from a non-polarized cell. This event is critical for an adequate neuronal wiring and therefore for the normal functioning of the nervous system. Neuronal polarity is very sensitive to the harmful effects of different factors present in the environment. In this regard, rotenone is a crystalline, colorless and odorless isoflavone used as insecticide, piscicide and broad spectrum pesticide commonly used earlier in agriculture. In the present review we will summarize the toxicity mechanism caused by this pesticide in different neuronal cell types, focusing on a particular biological mechanism whereby rotenone could impair neuronal polarization in cultured hippocampal neurons. Recent advances suggest that the inhibition of axonogenesis produced by rotenone could be related with its effect on microtubule dynamics, the actin cytoskeleton and their regulatory pathways, particularly affecting the small RhoGTPase RhoA. Unveiling the mechanism by which rotenone produces neurotoxicity will be instrumental to understand the cellular mechanisms involved in neurodegenerative diseases influenced by this environmental pollutant, which may lead to research focused on the design of new therapeutic strategies.
RESUMO
Rotenone, a broad-spectrum insecticide, piscicide and pesticide, produces a complete and selective suppression of axonogenesis in cultured hippocampal neurons. This effect is associated with an inhibition of actin dynamics through activation of Ras homology member A (RhoA) activity. However, the upstream signaling mechanisms involved in rotenone-induced RhoA activation were unknown. We hypothesized that rotenone might inhibit axon growth by the activation of RhoA/ROCK pathway because of the changes in microtubule (MT) dynamics and the concomitant release of Lfc, a MT-associated Guanine Nucleotide Exchange Factor (GEF) for RhoA. In this study, we demonstrate that rotenone decreases MT stability in morphologically unpolarized neurons. Taxol (3 nM), a drug that stabilizes MT, attenuates the inhibitory effect of rotenone (0.1 µM) on axon formation. Radiometric Forster Resonance Energy Transfer, revealed that this effect is associated with inhibition of rotenone-induced RhoA and ROCK activation. Interestingly, silencing of Lfc, but not of the RhoA GEF ArhGEF1, prevents the inhibitory effect of rotenone on axon formation. Our results suggest that rotenone-induced MT de-stabilization releases Lfc from MT thereby promoting RhoA and ROCK activities and the consequent inhibition of axon growth. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Inseticidas/uso terapêutico , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Rotenona/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Células Cultivadas , Embrião de Mamíferos , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Hipocampo/citologia , Fosforilação/efeitos dos fármacos , Gravidez , Ratos , Transdução Genética , Tubulina (Proteína)/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Proteínas tau/metabolismoRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMO
Axonal degeneration occurs in the developing nervous system for the appropriate establishment of mature circuits, and is also a hallmark of diverse neurodegenerative diseases. Despite recent interest in the field, little is known about the changes (and possible role) of the cytoskeleton during axonal degeneration. We studied the actin cytoskeleton in an in vitro model of developmental pruning induced by trophic factor withdrawal (TFW). We found that F-actin decrease and growth cone collapse (GCC) occur early after TFW; however, treatments that prevent axonal fragmentation failed to prevent GCC, suggesting independent pathways. Using super-resolution (STED) microscopy we found that the axonal actin/spectrin membrane-associated periodic skeleton (MPS) abundance and organization drop shortly after deprivation, remaining low until fragmentation. Fragmented axons lack MPS (while maintaining microtubules) and acute pharmacological treatments that stabilize actin filaments prevent MPS loss and protect from axonal fragmentation, suggesting that MPS destruction is required for axon fragmentation to proceed.
Assuntos
Actinas/metabolismo , Axônios/patologia , Membrana Celular/metabolismo , Cones de Crescimento/patologia , Plasticidade Neuronal , Degeneração Retrógrada , Espectrina/metabolismo , Citoesqueleto de Actina , Animais , Axônios/metabolismo , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , Ratos , Ratos WistarRESUMO
Here, will review current evidence regarding the signaling pathways and mechanisms underlying membrane addition at sites of active growth during axon formation. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 170-180, 2018.
Assuntos
Axônios/metabolismo , Membrana Celular/metabolismo , Animais , Crescimento Celular , Transdução de SinaisRESUMO
Prion diseases include a number of progressive neuropathies involving conformational changes in cellular prion protein (PrPc) that may be fatal sporadic, familial or infectious. Pathological evidence indicated that neurons affected in prion diseases follow a dying-back pattern of degeneration. However, specific cellular processes affected by PrPc that explain such a pattern have not yet been identified. Results from cell biological and pharmacological experiments in isolated squid axoplasm and primary cultured neurons reveal inhibition of fast axonal transport (FAT) as a novel toxic effect elicited by PrPc. Pharmacological, biochemical and cell biological experiments further indicate this toxic effect involves casein kinase 2 (CK2) activation, providing a molecular basis for the toxic effect of PrPc on FAT. CK2 was found to phosphorylate and inhibit light chain subunits of the major motor protein conventional kinesin. Collectively, these findings suggest CK2 as a novel therapeutic target to prevent the gradual loss of neuronal connectivity that characterizes prion diseases.
Assuntos
Transporte Axonal/fisiologia , Axônios/metabolismo , Caseína Quinase II/metabolismo , Proteínas Priônicas/fisiologia , Animais , Células Cultivadas , Hipocampo/citologia , Hipocampo/metabolismo , Cinesinas/metabolismo , Camundongos , Mitocôndrias/metabolismo , FosforilaçãoRESUMO
Here we describe the use of confocal microscopy in combination with antibodies specific to Golgi proteins to visualize dendritic Golgi outposts (GOPs) in cultured hippocampal pyramidal neurons. We also describe the use of spinning disk confocal microscopy, in combination with ectopically expressed glycosyltransferases fused to GFP variants, to visualize GOPs in living neurons.
Assuntos
Anticorpos/química , Complexo de Golgi/metabolismo , Células Piramidais/citologia , Células Piramidais/metabolismo , Animais , Humanos , Microscopia Confocal/métodosRESUMO
The neuronal Golgi apparatus (GA) localizes to the perinuclear region and dendrites as tubulo-vesicular structures designated Golgi outposts (GOPs). Current evidence suggests that GOPs shape dendrite morphology and serve as platforms for the local delivery of synaptic receptors. However, the mechanisms underlying GOP formation remain a mystery. Using live-cell imaging and confocal microscopy in cultured hippocampal neurons, we now show that GOPs destined to major "apical" dendrites are generated from the somatic GA by a sequence of events involving: (1) generation of a GA-derived tubule; (2) tubule elongation and deployment into the dendrite; (3) tubule fission; and (4) transport and condensation of the fissioned tubule. A RhoA-Rock signaling pathway involving LIMK1, PKD1, slingshot, cofilin, and dynamin regulates polarized GOP formation by controlling the tubule fission. Our observations identify a mechanism underlying polarized GOP biogenesis and provide new insights regarding involvement of RhoA in dendritic development and polarization.
Assuntos
Polaridade Celular/fisiologia , Dendritos/metabolismo , Complexo de Golgi/metabolismo , Hipocampo/citologia , Neurônios/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Células Cultivadas , Humanos , Microscopia Confocal , Neurônios/citologia , Transdução de Sinais/fisiologiaRESUMO
The early branching Giardia lamblia has highly polarized vacuoles, located underneath the plasma membrane, which have at least some of the characteristics of endosomes and of lysosomes. These peripheral vacuoles (PVs) are necessary for nutrient uptake and the maintenance of plasma membrane composition, but whether they carry out sorting and segregation of receptors and ligands is a matter of debate. Here, we showed that the internalization of low-density lipoprotein (LDL) to the PVs is highly dynamic in trophozoites with a rate similar to the internalization of the low-density lipoprotein receptor-related protein 1. Moreover, by analyzing receptor-mediated and fluid-phase endocytosis in living cells, we showed that after endocytosis LDL but not dextran moved laterally between the PVs. We speculate on PV functional heterogeneity and maturation in this parasite.
Assuntos
Endocitose , Endossomos/metabolismo , Giardia lamblia/fisiologia , Lisossomos/metabolismo , Vacúolos/metabolismo , Dextranos/metabolismo , Giardia lamblia/metabolismo , Lipoproteínas LDL/metabolismoRESUMO
Cultured neurons obtained from MAP1B-deficient mice have a delay in axon outgrowth and a reduced rate of axonal elongation compared with neurons from wild-type mice. Here we show that MAP1B deficiency results in a significant decrease in Rac1 and cdc42 activity and a significant increase in Rho activity. We found that MAP1B interacted with Tiam1, a guanosine nucleotide exchange factor for Rac1. The decrease in Rac1/cdc42 activity was paralleled by decreases in the phosphorylation of the downstream effectors of these proteins, such as LIMK-1 and cofilin. The expression of a constitutively active form of Rac1, cdc42, or Tiam1 rescued the axon growth defect of MAP1B-deficient neurons. Taken together, these observations define a new and crucial function of MAP1B that we show to be required for efficient cross-talk between microtubules and the actin cytoskeleton during neuronal polarization.
Assuntos
Axônios/enzimologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Cinética , Quinases Lim/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/deficiência , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The parasite Giardia lamblia possesses PVs (peripheral vacuoles) that function as both endosomes and lysosomes and are implicated in the adaptation, differentiation and survival of the parasite in different environments. The mechanisms by which Giardia traffics essential proteins to these organelles and regulates their secretion have important implications in the control of parasite dissemination. In the present study, we describe the participation of the heterotetrameric clathrin-adaptor protein gAP2 (Giardia adaptor protein 2) complex in lysosomal protein trafficking. A specific monoclonal antibody against the medium subunit (gmu2) of gAP2 showed localization of this complex to the PVs, cytoplasm and plasma membrane in the growing trophozoites. gAP2 also co-localized with clathrin in the PVs, suggesting its involvement in endocytosis. Uptake experiments using standard molecules for the study of endocytosis revealed that gAP2 specifically participated in the endocytosis of LDL (low-density lipoprotein). Targeted down-regulation of the gene encoding gmu2 in growing and encysting trophozoites resulted in a large decrease in the amount of cell growth and cyst wall formation, suggesting a distinct mechanism in which gAP2 is directly involved in both endocytosis and vesicular trafficking.
Assuntos
Complexo 2 de Proteínas Adaptadoras/metabolismo , Endocitose/fisiologia , Giardia lamblia/crescimento & desenvolvimento , Proteínas de Protozoários/metabolismo , Complexo 2 de Proteínas Adaptadoras/química , Complexo 2 de Proteínas Adaptadoras/genética , Animais , Anticorpos Monoclonais/imunologia , Membrana Celular/metabolismo , Regulação para Baixo , Endossomos/metabolismo , Imunofluorescência , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Conformação Proteica , Proteínas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genéticaRESUMO
Axonal elongation is one of the hallmarks of neuronal polarization. This phenomenon requires axonal membrane growth by exocytosis of plasmalemmal precursor vesicles (PPVs) at the nerve growth cone, a process regulated by IGF-1 activation of the PI3K (phosphatidylinositol-3 kinase) pathway. Few details are known, however, about the targeting mechanisms for PPVs. Here, we show, in cultured hippocampal pyramidal neurons and growth cones isolated from fetal rat brain, that IGF-1 activates the GTP-binding protein TC10, which triggers translocation to the plasma membrane of the exocyst component exo70 in the distal axon and growth cone. We also show that TC10 and exo70 function are necessary for addition of new membrane and, thus, axon elongation stimulated by IGF-1. Moreover, expression silencing of either TC10 or exo70 inhibit the establishment of neuronal polarity by hindering the insertion of IGF-1 receptor in one of the undifferentiated neurites. We conclude that, in hippocampal pyramidal neurons in culture, (1) membrane expansion at the axonal growth cone is regulated by IGF-1 via a cascade involving TC10 and the exocyst complex, (2) TC10 and exo70 are essential for the polarized externalization of IGF-1 receptor, and (3) this process is necessary for axon specification.
Assuntos
Axônios/fisiologia , Axônios/ultraestrutura , Fator de Crescimento Insulin-Like I/farmacologia , Células Piramidais/citologia , Proteínas de Transporte Vesicular/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Axônios/efeitos dos fármacos , Células Cultivadas , Estruturas Celulares/efeitos dos fármacos , Estruturas Celulares/metabolismo , Cromonas/farmacologia , Embrião de Mamíferos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Morfolinas/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Receptor IGF Tipo 1/fisiologia , Fatores de Tempo , Transfecção/métodosRESUMO
In this study, we have used a combination of biochemical and molecular biology techniques to demonstrate that the C-terminal tail domain of KIF4 directly interacts with P0, a major protein component of ribosomes. Besides, in dorsal root ganglion neurons, KIF4 and P0, as well as other ribosomal constituents, colocalize in clusters distributed along axons and neuritic tips. RNA interference suppression of KIF4 or expression of KIF4 variants lacking the tail domain or mutations of the ATP-binding site result in accumulation of P0 and other ribosomal proteins at the cell body and in their disappearance from axons. Our results also show one additional function for KIF4 involving an Ezrin-Radixin-Moesin-like domain in the second coiled-coiled region of KIF4. Expression of a KIF4 mutant lacking this domain abolishes the clustering of ribosomal constituents and prevents the anterograde translocation of the cell adhesion molecule L1. Taken together, the present results suggest that by binding to P0 through its tail domain and by using its motor activity, KIF4 is involved in the anterograde trafficking of ribosomal constituents to axons and that by means of its Ezrin-Radixin-Moesin-like domain interacts and transports L1.
Assuntos
Transporte Axonal , Axônios/metabolismo , Cinesinas/metabolismo , Ribossomos/metabolismo , Animais , Células Cultivadas , Feminino , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Cinesinas/genética , Masculino , Camundongos , Mutação/genética , Ligação Proteica , Transporte Proteico , Interferência de RNA , RatosRESUMO
In non-neuronal cells, inactivation of protein kinase D (PKD) blocks fission of trans-Golgi network (TGN) transport carriers, inducing the appearance of long tubules filled with cargo. We now report on the function of PKD1 in neuronal protein trafficking. In cultured hippocampal pyramidal cells, the transferrin receptor (TfR) and the low-density receptor-related protein (LRP) are predominantly transported to dendrites and excluded from axons. Expression of kinase-inactive PKD1 or its depletion by RNA interference treatment dramatically and selectively alter the intracellular trafficking and membrane delivery of TfR- and LRP-containing vesicles, without inhibiting exit from the TGN or inducing Golgi tubulation. After PKD1 suppression, dendritic membrane proteins are mispackaged into carriers that transport VAMP2; these vesicles are distributed to both axons and dendrites, but are rapidly endocytosed from dendrites and preferentially delivered to the axonal membrane. A kinase-defective mutant of PKD1 lacking the ability to bind diacylglycerol and hence its Golgi localization does not cause missorting of TfR or LRP. These results suggest that in neurons PKD1 regulates TGN-derived sorting of dendritic proteins and hence has a role in neuronal polarity.