ABSTRACT
cytohesin-2 is a guanine nucleotide exchange factor to activate ARF1 and ARF6, which are involved in various biological processes, including signal transduction, cell differentiation, cell structure organization, and survival. Nevertheless, there is a lack of evidence revealing the role of cytohesin-2 in osteoclast differentiation and in the development of osteoporosis. In this study, we find cytohesin-2 and ARF1 positively regulate osteoclast differentiation and function. Blocking the cytohesin-2 /ARF1 axis with SecinH3 or by genetic silencing of cytohesin-2 inhibits osteoclast formation and function in vitro. In vivo treatment with SecinH3 ameliorates ovariectomy-induced osteoporosis. Mechanistically, RNA-sequencing combined with molecular biological methodologies reveal that the regulatory function of cythohesin-2/ARF1 axis in osteoclast differentiation is mainly dependent on activating the JNK pathway. Further, in addition to the common viewpoint that JNK is activated by IRE1 via its kinase activity, we found that JNK can act upstream and regulate the endoribonuclease activity of IRE1 to promote XBP1 splicing. Both SecinH3 and silencing of cytohesin-2 inhibit JNK activation and IRE1 endoribonuclease activity, leading to the suppression of osteoclast differentiation. Taken together, our findings add new insights into the regulation between JNK and IRE1, and reveal that inhibiting the cytohesin-2/ARF1/JNK/IRE1 axis might represent a potential new strategy for the treatment of post-menopause osteoporosis.
Subject(s)
ADP-Ribosylation Factors , Osteoporosis , Humans , ADP-Ribosylation Factors/physiology , Osteoclasts/metabolism , ADP-Ribosylation Factor 6 , Osteoporosis/drug therapy , Endoribonucleases/metabolism , Protein Serine-Threonine KinasesABSTRACT
ELMOD2 is a GTPase-activating protein with uniquely broad specificity for ARF family GTPases. We previously showed that it acts with ARL2 in mitochondrial fusion and microtubule stability and with ARF6 during cytokinesis. Mouse embryonic fibroblasts deleted for ELMOD2 also displayed changes in cilia-related processes including increased ciliation, multiciliation, ciliary morphology, ciliary signaling, centrin accumulation inside cilia, and loss of rootlets at centrosomes with loss of centrosome cohesion. Increasing ARL2 activity or overexpressing Rootletin reversed these defects, revealing close functional links between the three proteins. This was further supported by the findings that deletion of Rootletin yielded similar phenotypes, which were rescued upon increasing ARL2 activity but not ELMOD2 overexpression. Thus, we propose that ARL2, ELMOD2, and Rootletin all act in a common pathway that suppresses spurious ciliation and maintains centrosome cohesion. Screening a number of markers of steps in the ciliation pathway supports a model in which ELMOD2, Rootletin, and ARL2 act downstream of TTBK2 and upstream of CP110 to prevent spurious release of CP110 and to regulate ciliary vesicle docking. These data thus provide evidence supporting roles for ELMOD2, Rootletin, and ARL2 in the regulation of ciliary licensing.
Subject(s)
Cilia/metabolism , Cytoskeletal Proteins/metabolism , ADP-Ribosylation Factors/metabolism , ADP-Ribosylation Factors/physiology , Animals , Cell Line , Centrosome/metabolism , Cilia/physiology , Cytokinesis , Cytoskeletal Proteins/physiology , Fibroblasts/metabolism , GTP-Binding Proteins/metabolism , GTPase-Activating Proteins/metabolism , Humans , Mice , Microtubules/metabolism , Mitochondria/metabolism , Mitochondrial Dynamics/physiology , Signal TransductionABSTRACT
The differentiation of cells depends on a precise control of their internal organization, which is the result of a complex dynamic interplay between the cytoskeleton, molecular motors, signaling molecules, and membranes. For example, in the developing neuron, the protein ADAP1 (ADP-ribosylation factor GTPase-activating protein [ArfGAP] with dual pleckstrin homology [PH] domains 1) has been suggested to control dendrite branching by regulating the small GTPase ARF6. Together with the motor protein KIF13B, ADAP1 is also thought to mediate delivery of the second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to the axon tip, thus contributing to PIP3 polarity. However, what defines the function of ADAP1 and how its different roles are coordinated are still not clear. Here, we studied ADAP1's functions using in vitro reconstitutions. We found that KIF13B transports ADAP1 along microtubules, but that PIP3 as well as PI(3,4)P2 act as stop signals for this transport instead of being transported. We also demonstrate that these phosphoinositides activate ADAP1's enzymatic activity to catalyze GTP hydrolysis by ARF6. Together, our results support a model for the cellular function of ADAP1, where KIF13B transports ADAP1 until it encounters high PIP3/PI(3,4)P2 concentrations in the plasma membrane. Here, ADAP1 disassociates from the motor to inactivate ARF6, promoting dendrite branching.
Subject(s)
ADP-Ribosylation Factors/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Nerve Tissue Proteins/metabolism , Phosphatidylinositols/metabolism , ADP-Ribosylation Factor 6 , ADP-Ribosylation Factors/physiology , Adaptor Proteins, Signal Transducing/physiology , Animals , Axons/metabolism , Biological Transport/physiology , Cell Membrane/metabolism , Cytoskeleton/metabolism , GTPase-Activating Proteins/metabolism , Humans , Inositol Phosphates/metabolism , Kinesins/metabolism , Microtubules/metabolism , Nerve Tissue Proteins/physiology , Phosphatidylinositol Phosphates/metabolism , Signal TransductionABSTRACT
Interstitial axon branching is an essential step during the establishment of neuronal connectivity. However, the exact mechanisms on how the number and position of branches are determined are still not fully understood. Here, we investigated the role of Arl8B, an adaptor molecule between lysosomes and kinesins. In chick retinal ganglion cells (RGCs), downregulation of Arl8B reduces axon branch density and shifts their location more proximally, while Arl8B overexpression leads to increased density and more distal positions of branches. These alterations correlate with changes in the location and density of lysosomes and autophagosomes along the axon shaft. Diminishing autophagy directly by knock-down of atg7, a key autophagy gene, reduces branch density, while induction of autophagy by rapamycin increases axon branching, indicating that autophagy plays a prominent role in axon branch formation. In vivo, local inactivation of autophagy in the retina using a mouse conditional knock-out approach disturbs retino-collicular map formation which is dependent on the formation of interstitial axon branches. These data suggest that Arl8B plays a principal role in the positioning of axon branches by spatially controlling autophagy, thus directly controlling formation of neural connectivity in the brain.SIGNIFICANCE STATEMENT The formation of interstitial axonal branches plays a prominent role in numerous places of the developing brain during neural circuit establishment. We show here that the GTPase Arl8B controls density and location of interstitial axon branches, and at the same time controls also density and location of the autophagy machinery. Upregulation or downregulation of autophagy in vitro promotes or inhibits axon branching. Local disruption of autophagy in vivo disturbs retino-collicular mapping. Our data suggest that Arl8B controls axon branching by controlling locally autophagy. This work is one of the first reports showing a role of autophagy during early neural circuit development and suggests that autophagy in general plays a much more prominent role during brain development than previously anticipated.
Subject(s)
ADP-Ribosylation Factors/physiology , Autophagosomes/physiology , Axons/physiology , Lysosomes/physiology , ADP-Ribosylation Factors/metabolism , Animals , Autophagosomes/enzymology , Autophagosomes/ultrastructure , Autophagy/genetics , Axons/enzymology , Axons/ultrastructure , Chick Embryo , Down-Regulation , Gene Knockdown Techniques , Lysosomes/enzymology , Lysosomes/ultrastructure , Mice, Knockout , Primary Cell Culture , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/ultrastructureABSTRACT
ADP-ribosylation factor (Arf)-like 4D (Arl4D), one of the Arf-like small GTPases, functions in the regulation of cell morphology, cell migration, and actin cytoskeleton remodeling. End-binding 1 (EB1) is a microtubule (MT) plus-end tracking protein that preferentially localizes at the tips of the plus ends of growing MTs and at the centrosome. EB1 depletion results in many centrosome-related defects. Here, we report that Arl4D promotes the recruitment of EB1 to the centrosome and regulates MT nucleation. We first showed that Arl4D interacts with EB1 in a GTP-dependent manner. This interaction is dependent on the C-terminal EB homology region of EB1 and partially dependent on an SxLP motif of Arl4D. We found that Arl4D colocalized with γ-tubulin in centrosomes and the depletion of Arl4D resulted in a centrosomal MT nucleation defect. We further demonstrated that abolishing Arl4D-EB1 interaction decreased MT nucleation rate and diminished the centrosomal recruitment of EB1 without affecting MT growth rate. In addition, Arl4D binding to EB1 increased the association between the p150 subunit of dynactin and the EB1, which is important for MT stabilization. Together, our results indicate that Arl4D modulates MT nucleation through regulation of the EB1-p150 association at the centrosome.
Subject(s)
ADP-Ribosylation Factors/metabolism , Centrosome/metabolism , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , ADP-Ribosylation Factors/physiology , Animals , COS Cells , Chlorocebus aethiops/metabolism , Chlorocebus aethiops/physiology , Humans , Mice , Microtubule-Associated Proteins/physiologyABSTRACT
Staphylococcus aureus, a pathogen most frequently found in diabetic foot ulcer infection, was recently suggested as an intracellular pathogen. Autophagy in professional phagocytes like macrophages allows selective destruction of intracellular pathogens, and its dysfunction can increase the survival of internalized pathogens, causing infections to worsen and spread. Previous works have shown that S. aureus infections in diabetes appeared more severe and invasive, and coincided with the suppressed autophagy in dermal tissues of diabetic rat, but the exact mechanisms are unclear. Here, we demonstrated that accumulation of advanced glycation end products (AGEs) contributed to the diminished autophagy-mediated clearance of S. aureus in the macrophages differentiated from PMA-treated human monocytic cell line THP-1. Importantly, infected macrophages showed increased S. aureus containing autophagosome, but the subsequent fusion of S. aureus containing autophagosome and lysosome was suppressed in AGEs-pretreated cells, suggesting AGEs blocked the autophagic flux and enabled S. aureus survival and escape. At the molecular level, elevated lysosomal ARL8 expression in AGEs-treated macrophages was required for AGEs-mediated inhibition of autophagosome-lysosome fusion. Silencing ARL8 in AGEs-treated macrophages restored autophagic flux and increased S. aureus clearance. Our results therefore demonstrate a new mechanism, in which AGEs accelerate S. aureus immune evasion in macrophages by ARL8-dependent suppression of autophagosome-lysosome fusion and bactericidal capability.
Subject(s)
ADP-Ribosylation Factors/physiology , Glycation End Products, Advanced/physiology , Lysosomes/physiology , Macrophages/immunology , Phagocytosis , Staphylococcus aureus/immunology , Autophagosomes/physiology , Humans , Immune Evasion , THP-1 Cells , Up-RegulationABSTRACT
Coordinated assembly and disassembly of integrin-mediated focal adhesions (FAs) is essential for cell migration. Many studies have shown that FA disassembly requires Ca2+ influx, however our understanding of this process remains incomplete. Here, we show that Ca2+ influx via STIM1/Orai1 calcium channels, which cluster near FAs, leads to activation of the GTPase Arf5 via the Ca2+-activated GEF IQSec1, and that both IQSec1 and Arf5 activation are essential for adhesion disassembly. We further show that IQSec1 forms a complex with the lipid transfer protein ORP3, and that Ca2+ influx triggers PKC-dependent translocation of this complex to ER/plasma membrane (PM) contact sites adjacent to FAs. In addition to allosterically activating IQSec1, ORP3 also extracts PI4P from the PM, in exchange for phosphatidylcholine. ORP3-mediated lipid exchange is also important for FA turnover. Together, these findings identify a new pathway that links calcium influx to FA turnover during cell migration.
Subject(s)
Calcium/metabolism , Fatty Acid-Binding Proteins/physiology , Focal Adhesions/physiology , Guanine Nucleotide Exchange Factors/physiology , ADP-Ribosylation Factors/physiology , Cell Membrane/metabolism , Cells, Cultured , Humans , Lipid Metabolism , Phosphatidylcholines/metabolism , Phosphatidylinositols/physiologyABSTRACT
ADP ribosylation factors (ARFs) are a family of small GTPases composed of six members (ARF1-6) that control various cellular functions, including membrane trafficking and actin cytoskeletal rearrangement, in eukaryotic cells. Among them, ARF1 and ARF6 are the most studied in neurons, particularly at glutamatergic synapses, but their roles at GABAergic synapses have not been investigated. Here, we show that a subset of ARF6 protein is localized at GABAergic synapses in cultured hippocampal neurons. In addition, we found that knockdown (KD) of ARF6, but not ARF1, triggered a reduction in the number of GABAergic synaptic puncta in mature cultured neurons in an ARF activity-dependent manner. ARF6 KD also reduced GABAergic synaptic density in the mouse hippocampal dentate gyrus (DG) region. Furthermore, ARF6 KD in the DG increased seizure susceptibility in an induced epilepsy model. Viewed together, our results suggest that modulating ARF6 and its regulators could be a therapeutic strategy against brain pathologies involving hippocampal network dysfunction, such as epilepsy.
Subject(s)
ADP-Ribosylation Factors/physiology , GABAergic Neurons/physiology , Synapses/metabolism , ADP-Ribosylation Factor 1/physiology , ADP-Ribosylation Factor 6 , ADP-Ribosylation Factors/antagonists & inhibitors , ADP-Ribosylation Factors/genetics , Animals , Cells, Cultured , GABAergic Neurons/ultrastructure , Gene Knockdown Techniques , Genetic Vectors/genetics , Genetic Vectors/therapeutic use , Hippocampus/cytology , Hippocampus/embryology , Humans , Kainic Acid/toxicity , Male , Mice, Inbred C57BL , Point Mutation , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacology , Rats , Recombinant Proteins/metabolism , Seizures/chemically induced , Seizures/genetics , Seizures/physiopathology , Seizures/prevention & controlABSTRACT
Photoreceptor neurons are modified primary cilia with an extended ciliary compartment known as the outer segment (OS). The mechanism behind the elaboration of photoreceptor cilia and OS morphogenesis remains poorly understood. In this review, we discuss the role of ADP-ribosylation factor-like GTPase 13B (ARL13B), a small GTPase in OS morphogenesis and its impact on photoreceptor health and biology.
Subject(s)
ADP-Ribosylation Factors/physiology , Cilia/physiology , Photoreceptor Cells, Vertebrate/physiology , Humans , Retina/cytology , Retina/growth & developmentABSTRACT
ADP-ribosylation factors (ARFs) are a family of small monomeric GTPases comprising six members categorized into three classes: class I (ARF1, 2, and 3), class II (ARF4 and 5), and class III (ARF6). In contrast to class I and III ARFs, which are the key regulators in vesicular membrane trafficking, the cellular function of class II ARFs remains unclear. In the present study, we generated class II ARF-deficient mice and found that ARF4+/-/ARF5-/- mice exhibited essential tremor (ET)-like behaviors. In vivo electrophysiological recordings revealed that ARF4+/-/ARF5-/- mice of both sexes exhibited abnormal brain activity when moving, raising the possibility of abnormal cerebellar excitability. Slice patch-clamp experiments demonstrated the reduced excitability of the cerebellar Purkinje cells (PCs) in ARF4+/-/ARF5-/- mice. Immunohistochemical and electrophysiological analyses revealed a severe and selective decrease of pore-forming voltage-dependent Na+ channel subunit Nav1.6, important for maintaining repetitive action potential firing, in the axon initial segment (AIS) of PCs. Importantly, this decrease in Nav1.6 protein localized in the AIS and the consequent tremors in ARF4+/-/ARF5-/- mice could be alleviated by the PC-specific expression of ARF5 using adeno-associated virus vectors. Together, our data demonstrate that the decreased expression of the class II ARF proteins in ARF4+/-/ARF5-/- mice, leading to a haploinsufficiency of ARF4 in the absence of ARF5, impairs the localization of Nav1.6 to the AIS and hence reduces the membrane excitability in PCs, resulting in the ET-like movement disorder. We suggest that class II ARFs function in localizing specific proteins, such as Nav1.6, to the AIS.SIGNIFICANCE STATEMENT We found that decreasing the expression of class II ARF proteins, through the generation of ARF4+/-/ARF5-/- mice, impairs Nav1.6 distribution to the axon initial segment (AIS) of cerebellar Purkinje cells (PCs), thereby resulting in the impairment of action potential firing of PCs. The ARF4+/-/ARF5-/- mutant mice exhibited movement-associated essential tremor (ET)-like behavior with pharmacological profiles similar to those in ET patients. The exogenous expression of ARF5 reduced the tremor phenotype and restored the localization of Nav1.6 immunoreactivity to the AIS in ARF4+/-/ARF5-/- mice. Thus, our results suggest that class II ARFs are involved in the localization of Nav1.6 to the AISs in cerebellar PCs and that the reduction of class II ARF activity leads to ET-like movement disorder.
Subject(s)
ADP-Ribosylation Factors/physiology , Axons/metabolism , Movement Disorders/etiology , NAV1.6 Voltage-Gated Sodium Channel/physiology , Purkinje Cells/metabolism , Tremor/etiology , ADP-Ribosylation Factors/deficiency , ADP-Ribosylation Factors/genetics , Action Potentials , Animals , Dependovirus/genetics , Electroencephalography , Electromyography , Genetic Vectors/genetics , Genetic Vectors/therapeutic use , Genotype , Head Movements , Mice , Mice, Inbred C57BL , Mice, Knockout , Movement Disorders/metabolism , Movement Disorders/physiopathology , NAV1.6 Voltage-Gated Sodium Channel/deficiency , Patch-Clamp Techniques , Protein Transport , Purkinje Cells/physiology , Rotarod Performance Test , Single-Blind Method , Tremor/metabolism , Tremor/physiopathologyABSTRACT
Lysosomes are acidic organelles responsible for degrading both exogenous and endogenous materials. The small GTPase Arl8 localizes primarily to lysosomes and is involved in lysosomal function. In the present study, using Arl8b gene-trapped mutant (Arl8b-/- ) mice, we show that Arl8b is required for the development of dorsal structures of the neural tube, including the thalamus and hippocampus. In embryonic day (E) 10.5 Arl8b-/- embryos, Sox1 (a neuroepithelium marker) was ectopically expressed in the roof plate, whereas the expression of Gdf7 and Msx1 (roof plate markers) was reduced in the dorsal midline of the midbrain. Ectopic expression of Sox1 in Arl8b-/- embryos was detected also at E9.0 in the neural fold, which gives rise to the roof plate. In addition, the levels of Bmp receptor IA and phosphorylated Smad 1/5/8 (downstream of BMP signaling) were increased in the neural fold of E9.0 Arl8b-/- embryos. These results suggest that Arl8b is involved in the development of the neural fold and the subsequently formed roof plate, possibly via control of BMP signaling.
Subject(s)
ADP-Ribosylation Factors/genetics , ADP-Ribosylation Factors/physiology , Neural Crest/embryology , Animals , Gene Expression Regulation, Developmental/genetics , Lysosomes/genetics , Lysosomes/physiology , Mice/embryology , Mice, Inbred C57BL , Monomeric GTP-Binding Proteins/metabolism , Neural Crest/metabolism , Neural Tube/embryology , Neural Tube/metabolism , SOXB1 Transcription Factors/physiology , Signal TransductionABSTRACT
Abiotic stress poses constant challenges for plant survival and is a serious problem for global agricultural productivity. On a molecular level, stress conditions result in elevation of reactive oxygen species (ROS) production causing oxidative stress associated with oxidation of proteins and nucleic acids as well as impairment of membrane functions. Adaptation of root growth to ROS accumulation is facilitated through modification of auxin and cytokinin hormone homeostasis. Here, we report that in Arabidopsis root meristem, ROS-induced changes of auxin levels correspond to decreased abundance of PIN auxin efflux carriers at the plasma membrane (PM). Specifically, increase in H2O2 levels affects PIN2 endocytic recycling. We show that the PIN2 intracellular trafficking during adaptation to oxidative stress requires the function of the ADP-ribosylation factor (ARF)-guanine-nucleotide exchange factor (GEF) BEN1, an actin-associated regulator of the trafficking from the PM to early endosomes and, presumably, indirectly, trafficking to the vacuoles. We propose that H2O2 levels affect the actin dynamics thus modulating ARF-GEF-dependent trafficking of PIN2. This mechanism provides a way how root growth acclimates to stress and adapts to a changing environment.
Subject(s)
Alcohol Oxidoreductases/metabolism , Arabidopsis Proteins/metabolism , Hydrogen Peroxide/metabolism , Oxidative Stress , Plant Roots/metabolism , ADP-Ribosylation Factors/metabolism , ADP-Ribosylation Factors/physiology , Actins/metabolism , Adaptation, Physiological , Alcohol Oxidoreductases/physiology , Arabidopsis/metabolism , Arabidopsis/physiology , Arabidopsis Proteins/physiology , Cytoskeleton/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Guanine Nucleotide Exchange Factors/physiology , Plant Roots/physiology , Reactive Oxygen Species/metabolismABSTRACT
The small GTPase Arl8 is known to be involved in the periphery-directed motility of lysosomes. However, the overall importance of moving these vesicles is still poorly understood. Here we show that Drosophila Arl8 is required not only for the proper distribution of lysosomes, but also for autophagosome-lysosome fusion in starved fat cells, endosome-lysosome fusion in garland nephrocytes, and developmentally programmed secretory granule degradation (crinophagy) in salivary gland cells. Moreover, proper Arl8 localization to lysosomes depends on the shared subunits of the BLOC-1 and BORC complexes, which also promote autophagy and crinophagy. In conclusion, we demonstrate that Arl8 is responsible not only for positioning lysosomes but also acts as a general lysosomal fusion factor.
Subject(s)
ADP-Ribosylation Factors/physiology , Drosophila Proteins/physiology , Lysosomes/physiology , ADP-Ribosylation Factors/genetics , ADP-Ribosylation Factors/metabolism , Animals , Autophagosomes/physiology , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/physiology , Drosophila melanogaster/ultrastructure , Lysosomes/metabolism , Lysosomes/ultrastructure , Membrane Fusion , Protein Subunits/physiology , rab GTP-Binding Proteins/physiologyABSTRACT
Mutations in the Joubert syndrome-associated small GTPase ARL13B are linked to photoreceptor impairment and vision loss. To determine the role of ARL13B in the development, function, and maintenance of ciliated photoreceptors, we generated a pan-retina knock-out (Six3-Cre) and a rod photoreceptor-specific inducible conditional knock-out (Pde6g-CreERT2) of ARL13B using murine models. Embryonic deletion of ARL13B led to defects in retinal development with reduced cell proliferation. In the absence of ARL13B, photoreceptors failed to develop outer segment (OS) membranous discs and axonemes, resulting in loss of function and rapid degeneration. Additionally, the majority of photoreceptor basal bodies did not dock properly at the apical edge of the inner segments. The removal of ARL13B in adult rod photoreceptor cells after maturation of OS resulted in loss of photoresponse and vesiculation in the OS. Before changes in photoresponse, removal of ARL13B led to mislocalization of rhodopsin, prenylated phosphodiesterase-6 (PDE6), and intraflagellar transport protein-88 (IFT88). Our findings show that ARL13B is required at multiple stages of retinogenesis, including early postnatal proliferation of retinal progenitor cells, development of photoreceptor cilia, and morphogenesis of photoreceptor OS discs regardless of sex. Last, our results establish a need for ARL13B in photoreceptor maintenance and protein trafficking.SIGNIFICANCE STATEMENT The normal development of photoreceptor cilia is essential to create functional, organized outer segments with stacked membrane discs that house the phototransduction proteins necessary for sight. Our study identifies a complex role for ARL13B, a small GTPase linked to Joubert syndrome and visual impairment, at various stages of photoreceptor development. Loss of ARL13B led to defects in retinal proliferation, altered placement of basal bodies crucial for components of the cilium (transition zone) to emanate, and absence of photoreceptor-stacked discs. These defects led to extinguished visual response and dysregulated protein trafficking. Our findings show the complex role ARL13B plays in photoreceptor development, viability, and function. Our study accounts for the severe retinal impairment observed in ARL13B-linked Joubert syndrome patients.
Subject(s)
ADP-Ribosylation Factors/physiology , Retina/metabolism , Rod Cell Outer Segment/metabolism , ADP-Ribosylation Factors/deficiency , ADP-Ribosylation Factors/genetics , Aging/metabolism , Animals , Axoneme/metabolism , Axoneme/ultrastructure , Cilia/metabolism , Cilia/ultrastructure , Eye Proteins/metabolism , Female , Gene Expression Regulation, Developmental , Gene Knockout Techniques , Male , Mice , Mice, Inbred C57BL , Organelle Biogenesis , Protein Transport/physiology , Retina/abnormalities , Retina/embryology , Retina/growth & development , Rod Cell Outer Segment/radiation effects , Sensory Rhodopsins/metabolismABSTRACT
The activation of the small GTPase ARF6 has been implicated in promoting several pathological processes related to vascular instability and tumor formation, growth, and metastasis. ARF6 also plays a vital role during embryonic development. Recent studies have suggested that ARF6 carries out these disparate functions primarily by controlling protein trafficking within the cell. ARF6 helps direct proteins to intracellular or extracellular locations where they function in normal cellular responses during development and in pathological processes later in life. This transport of proteins is accomplished through a variety of mechanisms, including endocytosis and recycling, microvesicle release, and as yet uncharacterized processes. This Commentary will explore the functions of ARF6, while focusing on the role of this small GTPase in development and postnatal physiology, regulating barrier function and diseases associated with its loss, and tumor formation, growth, and metastasis.
Subject(s)
ADP-Ribosylation Factors/physiology , ADP-Ribosylation Factor 6 , Animals , Embryonic Development , Endothelium, Vascular/physiology , Humans , Neoplasms/enzymology , Neoplasms/pathology , Protein TransportABSTRACT
Primary cilia are tiny microtubule-based signaling devices that regulate a variety of physiological functions, including metabolism and cell division. Defects in primary cilia lead to a myriad of diseases in humans such as obesity and cancers. In the mature brain, both neurons and astrocytes contain a single primary cilium. Although neuronal primary cilia are not directly involved in synaptic communication, their pathophysiological impacts on obesity and mental disorders are well recognized. In contrast, research on astrocytic primary cilia lags far behind. Currently, little is known about their functions and molecular pathways in the mature brain. Unlike neurons, postnatal astrocytes retain the capacity of cell division and can become reactive and proliferate in response to various brain insults such as epilepsy, ischemia, traumatic brain injury, and neurodegenerative ß-amyloid plaques. Since primary cilia derive from the mother centrioles, astrocyte proliferation must occur in coordination with the dismantling and ciliogenesis of astrocyte cilia. In this regard, the functions, signal pathways, and structural dynamics of neuronal and astrocytic primary cilia are fundamentally different. Here we discuss and compare the current understanding of neuronal and astrocytic primary cilia.
Subject(s)
Astrocytes/physiology , Brain/physiology , Cilia/physiology , Neurons/physiology , ADP-Ribosylation Factors/physiology , Abnormalities, Multiple/physiopathology , Adenylyl Cyclases/physiology , Animals , Humans , Mental Disorders/physiopathology , Obesity/physiopathologyABSTRACT
UNC119 and PDEδ are lipid-binding proteins and are thought to form diffusible complexes with transducin-α and prenylated OS proteins, respectively, to mediate their trafficking to photoreceptor outer segments. Here, we investigate mechanisms of trafficking which are controlled by Arf-like protein 3 (Arl3), a small GTPase. The activity of ARL3 is regulated by a GEF (ARL13b) and a GAP (RP2). In a mouse germline knockout of RP2, ARL3-GTP is abundant as its intrinsic GTPase activity is extremely low. High levels of ARL3-GTP impair binding and trafficking of cargo to the outer segment. Germline knockout of ARL3 is embryonically lethal generating a syndromic ciliopathy-like phenotype. Retina- and rod-specific knockout of ARL3 allow to determine the precise mechanisms leading to photoreceptor degeneration. The knockouts reveal binary functions of ARL3-GTP as a key molecule in late-stage photoreceptor ciliogenesis and cargo displacement factor.
Subject(s)
ADP-Ribosylation Factors/physiology , Protein Transport/physiology , ADP-Ribosylation Factors/deficiency , ADP-Ribosylation Factors/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Ciliopathies/genetics , Ciliopathies/metabolism , Ciliopathies/pathology , Cone-Rod Dystrophies/genetics , Cone-Rod Dystrophies/metabolism , Cone-Rod Dystrophies/pathology , Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , GTP-Binding Proteins , Genes, Lethal , Guanosine Triphosphate/metabolism , Lipoproteins/metabolism , Membrane Proteins/metabolism , Mice , Mice, Knockout , Organ Specificity , Protein Prenylation , Pyrophosphatases/deficiency , Pyrophosphatases/physiology , Rod Cell Outer Segment/metabolismABSTRACT
Vascular smooth muscle cells (VSMC) can exhibit a contractile or a synthetic phenotype depending on the extracellular stimuli present and the composition of the extracellular matrix. Uncontrolled activation of the synthetic VSMC phenotype is however associated with the development of cardiovascular diseases. Here, we aimed to elucidate the role of the ARF GTPases in the regulation of VSMC dedifferentiation. First, we observed that the inhibition of the activation of ARF proteins with SecinH3, a blocker of the cytohesin ARF GEF family, reduced the ability of the cells to migrate and proliferate. In addition, this inhibitor also blocked expression of sm22α and αSMA, two contractile markers, at the transcription level impairing cell contractility. Specific knockdown of ARF1 and ARF6 showed that both isoforms were required for migration and proliferation, but ARF1 only regulated contractility through sm22α and αSMA expression. Expression of these VSMC markers was correlated with the degree of actin polymerization. VSMC treatment with SecinH3 as well as ARF1 depletion was both able to block the formation of stress fibres and focal adhesions, demonstrating the role of this GTPase in actin filament formation. Consequently, we observed that both treatments increased the ratio of G-actin to F-actin in these cells. The elevated amounts of cytoplasmic G-actin, acting as a signaling intermediate, blocked the recruitment of the Mkl1 (MRTF-A) transcription factor in the nucleus, demonstrating its involvement in the regulation of contractile protein expression. Altogether, these findings show for the first time that ARF GTPases are actively involved in VSMC phenotypic switching through the regulation of actin function in migration and proliferation, and the control of actin dependent gene regulation.
Subject(s)
ADP-Ribosylation Factor 1/physiology , ADP-Ribosylation Factors/physiology , Actins/metabolism , Microfilament Proteins/metabolism , Muscle Proteins/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , ADP-Ribosylation Factor 6 , Actin Cytoskeleton/metabolism , Animals , Cell Adhesion , Cell Differentiation , Gene Expression Regulation , Muscle Contraction , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/cytology , Phenotype , Rats , Signal Transduction , Stress Fibers/metabolism , Transcription Factors/metabolism , Triazoles/pharmacologyABSTRACT
OBJECTIVES: ARL15 is a novel susceptibility gene identified in a recent GWAS in a north Indian rheumatoid arthritis (RA) cohort. However, the role of ARL15 or ARF family genes in RA aetiology remains unknown. Therefore, we aimed to i) establish the expression of ARL15 in rheumatoid arthritis synovial fibroblasts (RASF) and ii) its functional characterisation by assessing its effects on major inflammatory cytokines and interacting partners using a knockdown approach. METHODS: RASF were cultured from synovial tissue obtained from RA patients (n=5) and osteoarthritis (OA) patients (n=3) serving as controls. Expression of ARL15, ARF1 and ARF6 in RASF was checked by semi-quantitative PCR and western blots; and altered expression of ARL15, if any, by induction of RASF with TNF using real-time PCR. The effect of ARL15 on the expression of adiponectin, adiponectin receptor I, IL6 and GAPDH and on cell mobility by invasion and migration assays were assessed by siRNA mediated gene knockdown. RESULTS: Expression of ARL15, ARF1 and ARF6 was confirmed in RASF and OASF samples but ARL15 expression remained unaltered on TNF induction. Notably, ARL15 knockdown resulted in downregulation of IL6 and GAPDH, upregulation of adiponectin and adiponectin receptor I genes; and significant reduction in migration and invasion of RASF. Genemania showed significant interactions of ARL15 with genes responsible for insulin resistance and phospholipase D. CONCLUSIONS: This first report on ARL15 expression in RASF and its likely role in inflammation and metabolic syndromes through a TNF independent pathway, encourages hypothesis-free studies to identify additional pathways underlying RA disease biology.