ABSTRACT
A key event in the process of spermiogenesis is the formation of the flagella, which enables sperm to reach eggs for fertilization. Yeast two-hybrid studies revealed that meiosis-expressed gene 1 (MEIG1) and Parkin co-regulated gene (PACRG) interact, and that sperm-associated antigen 16, which encodes an axoneme central apparatus protein, is also a binding partner of MEIG1. In spermatocytes of wild-type mice, MEIG1 is expressed in the whole germ cell bodies, but the protein migrates to the manchette, a unique structure at the base of elongating spermatid that directs formation of the flagella. In the elongating spermatids of wild-type mice, PACRG colocalizes with α-tubulin, a marker for the manchette, whereas this localization was not changed in the few remaining elongating spermatids of Meig1-deficient mice. In addition, MEIG1 no longer localizes to the manchette in the remaining elongating spermatids of Pacrg-deficient mice, indicating that PACRG recruits MEIG1 to the manchette. PACRG is not stable in mammalian cells, but can be stabilized by MEIG1 or by inhibition of proteasome function. SPAG16L is present in the spermatocyte cytoplasm of wild-type mice, and in the manchette of elongating spermatids, but in the Meig1 or Pacrg-deficient mice, SPAG16L no longer localizes to the manchette. By contrast, MEIG1 and PACRG are still present in the manchette of Spag16L-deficient mice, indicating that SPAG16L is a downstream partner of these two proteins. Together, our studies demonstrate that MEIG1/PACRG forms a complex in the manchette and that this complex is necessary to transport cargos, such as SPAG16L, to build the sperm flagella.
Subject(s)
Cell Cycle Proteins/metabolism , Flagella/genetics , Microtubule-Associated Proteins/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Proteins/metabolism , Animals , Antibodies, Monoclonal , Blotting, Western , COS Cells , Cell Cycle Proteins/genetics , Chlorocebus aethiops , Flagella/metabolism , Fluorescent Antibody Technique , Mice , Mice, Mutant Strains , Microfilament Proteins , Microtubule-Associated Proteins/genetics , Molecular Chaperones , Nuclear Proteins/genetics , Oligonucleotide Array Sequence Analysis , Phosphoproteins/genetics , Protein Binding , Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Spermatogenesis/genetics , Spermatogenesis/physiology , Two-Hybrid System TechniquesABSTRACT
Autophagy is primarily considered a non-selective degradation process induced by starvation. Nutrient-independent basal autophagy, in contrast, imposes intracellular QC by selective disposal of aberrant protein aggregates and damaged organelles, a process critical for suppressing neurodegenerative diseases. The molecular mechanism that distinguishes these two fundamental autophagic responses, however, remains mysterious. Here, we identify the ubiquitin-binding deacetylase, histone deacetylase-6 (HDAC6), as a central component of basal autophagy that targets protein aggregates and damaged mitochondria. Surprisingly, HDAC6 is not required for autophagy activation; rather, it controls the fusion of autophagosomes to lysosomes. HDAC6 promotes autophagy by recruiting a cortactin-dependent, actin-remodelling machinery, which in turn assembles an F-actin network that stimulates autophagosome-lysosome fusion and substrate degradation. Indeed, HDAC6 deficiency leads to autophagosome maturation failure, protein aggregate build-up, and neurodegeneration. Remarkably, HDAC6 and F-actin assembly are completely dispensable for starvation-induced autophagy, uncovering the fundamental difference of these autophagic modes. Our study identifies HDAC6 and the actin cytoskeleton as critical components that define QC autophagy and uncovers a novel regulation of autophagy at the level of autophagosome-lysosome fusion.
Subject(s)
Autophagy/physiology , Histone Deacetylases/metabolism , Phagosomes/metabolism , Ubiquitin/metabolism , Actins/metabolism , Animals , Animals, Genetically Modified , Autophagy/genetics , Cell Line , Drosophila , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Histone Deacetylase 6 , Histone Deacetylases/genetics , Immunohistochemistry , Lysosomes/genetics , Lysosomes/metabolism , Mice , Microscopy, Fluorescence , Phagosomes/geneticsABSTRACT
Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD) is a dominantly inherited degenerative disorder caused by mutations in the valosin-containing protein (VCP7) gene. VCP (p97 in mouse, TER94 in Drosophila melanogaster, and CDC48 in Saccharomyces cerevisiae) is a highly conserved AAA(+) (ATPases associated with multiple cellular activities) ATPase that regulates a wide array of cellular processes. The mechanism of IBMPFD pathogenesis is unknown. To elucidate the pathogenic mechanism, we developed and characterized a Drosophila model of IBMPFD (mutant-VCP-related degeneration). Based on genetic screening of this model, we identified three RNA-binding proteins that dominantly suppressed degeneration; one of these was TBPH, the Drosophila homolog of TAR (trans-activating response region) DNA-binding protein 43 (TDP-43). Here we demonstrate that VCP and TDP-43 interact genetically and that disease-causing mutations in VCP lead to redistribution of TDP-43 to the cytoplasm in vitro and in vivo, replicating the major pathology observed in IBMPFD and other TDP-43 proteinopathies. We also demonstrate that TDP-43 redistribution from the nucleus to the cytoplasm is sufficient to induce cytotoxicity. Furthermore, we determined that a pathogenic mutation in TDP-43 promotes redistribution to the cytoplasm and enhances the genetic interaction with VCP. Together, our results show that degeneration associated with VCP mutations is mediated in part by toxic gain of function of TDP-43 in the cytoplasm. We suggest that these findings are likely relevant to the pathogenic mechanism of a broad array of TDP-43 proteinopathies, including frontotemporal lobar degeneration and amyotrophic lateral sclerosis.
Subject(s)
Cell Cycle Proteins/genetics , DNA-Binding Proteins/metabolism , Drosophila Proteins/genetics , Frontotemporal Dementia/genetics , Mutation/genetics , Osteitis Deformans/genetics , Aminopeptidases/metabolism , Animals , Animals, Genetically Modified , Binding Sites/genetics , Cell Line, Transformed , Central Nervous System/metabolism , Cytoplasm/genetics , Cytoplasm/metabolism , DNA-Binding Proteins/genetics , Disease Models, Animal , Drosophila , Drosophila Proteins/metabolism , Frontotemporal Dementia/complications , Frontotemporal Dementia/pathology , Gene Expression Regulation/genetics , Glycoproteins/metabolism , Humans , Indoles , Models, Biological , Osteitis Deformans/complications , Promoter Regions, Genetic/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae/genetics , Transfection/methods , Valosin Containing ProteinABSTRACT
Some of the most successful gene therapy results have been obtained using recombinant viral vectors to treat animal models of inherited and acquired ocular diseases. Clinical trials using adenovirus vector systems have been initiated for two ocular diseases. Adeno-associated viruses (AAVs) represent an attractive alternative to adenoviral vector systems as they enable stable and long-term expression and can target a variety of different ocular cell types depending on the capsid serotype; recently clinical trails for congenital blindness was initiated with a vector-based AAV serotype 2. High levels of retinal gene transfer have been achieved using vectors based on AAV serotypes 1, 2, 4 and 5. This report compares the gene transfer efficacy and stability of expression of vector systems based on three novel AAV serotypes: AAV7, 8, 9, with the established vectors AAV1, 2, 5. We show here that AAV7 and 8 enable superior long-term transduction of retinal and also anterior chamber structures.
Subject(s)
Dependovirus/genetics , Gene Transfer Techniques , Genetic Vectors/genetics , Retina/metabolism , Animals , Dependovirus/classification , Erythropoietin/genetics , Genetic Therapy , Genetic Vectors/classification , Green Fluorescent Proteins/genetics , Mice , Mice, Inbred C57BL , SerotypingABSTRACT
Neovascularization leads to blindness in numerous ocular diseases, including diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, and sickle cell disease. More effective and stable treatments for ocular neovascularization are needed, yet there are major limitations in the present animal models. To develop primate models of diabetic retinopathy and choroidal neovascularization, rhesus monkeys were injected subretinally or intravitreally with an adeno-associated virus (AAV)-2 vector carrying the cDNA encoding human vascular endothelial growth factor (VEGF). Overexpression of VEGF was measured by intraocular fluid sampling over time. Neovascularization was evaluated by ophthalmoscopy through angiography, optical coherence tomography, and ultimately histopathology. Overexpression of VEGF through AAV2 results in rapid development of features of diabetic retinopathy or macular edema, depending on the targeted cell type/mode of production of VEGF and diffusion of VEGF. Nonhuman primate models will be useful in testing long-term safety and efficacy of novel therapeutic agents for blinding neovascular diseases.
Subject(s)
Gene Expression/physiology , Retinal Neovascularization/physiopathology , Vascular Endothelial Growth Factor A/physiology , Adenoviridae , Animals , Dependovirus , Diabetic Retinopathy/physiopathology , Disease Models, Animal , Female , Macaca mulatta , Retina/ultrastructure , Time Factors , Transfection , Vascular Endothelial Growth Factor A/biosynthesis , VirusesABSTRACT
Sorafenib has an antitumor activity in patients with radioactive iodine-refractory differentiated thyroid carcinoma (RAIR-DTC). Prior research has implicated signaling through the MAPK and AKT/PI3K pathways in the progression of DTC. To assess whether the activity of these pathways is predictive of response to sorafenib, we retrospectively studied molecular tumor markers from these two pathways from a phase 2 study of sorafenib in RAIR-DTC. Tumor samples from 40 of 53 DTC subjects obtained prior to initiation of sorafenib were immunostained with DAB-labeled antibodies to phospho-AKT (pAKT), phospho-ERK (pERK), and phospho-S6 (pS6). BRAFV600E genetic mutation analysis was performed on all samples. Expression levels and mutational status were compared to response and progression-free survival (PFS) for each patient. Low tumor expression of nuclear pAKT was associated with partial response to sorafenib (p < 0.01). Patients with nuclear pAKT expression that was below the median for our sample were more than three times as likely to have a partial response as patients with equal to or above median expression. There was no correlation between tumor expression of nuclear pERK or pS6 and response. Endothelial cell and pericyte expression of pERK, pAKT, and pS6 were not predictive of response. There was no correlation between BRAFV600E mutation status and partial response. No correlation was observed between either the expression of pAKT, pERK, or pS6, or the presence of the BRAFV600E mutation, and PFS. In conclusion, lower tumor expression of nuclear pAKT was associated with higher rate of response to sorafenib. This observation justifies evaluation of combination therapy with sorafenib and an inhibitor of the PI3K/AKT signaling pathway in RAIR-DTC.
Subject(s)
Antineoplastic Agents/therapeutic use , Niacinamide/analogs & derivatives , Phenylurea Compounds/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-akt/metabolism , Thyroid Neoplasms/drug therapy , Thyroid Neoplasms/metabolism , Aged , Antineoplastic Agents/pharmacology , Biomarkers, Tumor , Endothelial Cells/metabolism , Female , Gene Expression , Humans , Immunohistochemistry , Male , Middle Aged , Mutation , Neoplasm Grading , Niacinamide/pharmacology , Niacinamide/therapeutic use , Phenylurea Compounds/pharmacology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/genetics , Ribosomal Protein S6 Kinases/genetics , Ribosomal Protein S6 Kinases/metabolism , Sorafenib , Thyroid Neoplasms/mortality , Thyroid Neoplasms/pathology , Treatment OutcomeABSTRACT
Adeno-associated viral gene therapy has shown promise for the treatment of inherited and degenerative diseases in a variety of animal models. Some of the most dramatic results have been obtained in the field of ocular gene therapy, where efficacy has been tremendous in inherited and acquired retinal disorders. For the promise of this approach to be realized it will be necessary to create vectors capable of pharmacologic or physiologic regulation of the transgene. We describe in this paper a dimerizer-inducible viral expression system that is able to reproducibly drive expression of the reporter gene erythropoietin in the eyes of nonhuman primates over a period of 2.5 years. The expression profiles were characterized by minimal basal expression in the absence of inducer and dose-responsive maximal expression in the presence of inducer drug.
Subject(s)
Dependovirus/genetics , Eye/drug effects , Eye/metabolism , Gene Expression Regulation/physiology , Gene Transfer Techniques , Genetic Therapy , Animals , Erythropoietin/genetics , Erythropoietin/immunology , Erythropoietin/metabolism , Female , Genetic Vectors , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Immunosuppressive Agents/pharmacology , Macaca mulatta , Sirolimus/pharmacology , Transgenes/physiologyABSTRACT
PURPOSE: Choroideremia (CHM), an X-linked retinal disease, is caused by mutations affecting the CHM gene. This gene encodes REP-1, which functions in the covalent modifications of proteins involved in vesicle trafficking. The disease affects several cell types in the retina, but it is not known which cell types contribute directly or indirectly to disease progression. A study of the expression patterns of Chm and the related gene Chml in the mouse retina was undertaken in order to address this issue. METHODS: The expression patterns of Chm and Chml were determined by in situ hybridization. The localization of the Chm protein product, Rep-1, was determined spatially and temporally in the mouse retina by immunohistochemistry. RESULTS: Chm and Chml mRNA were found in every major layer of the retina in adult mice. During development, Rep-1 protein localization changes from a fairly diffuse pattern during embryogenesis to a more specific pattern at the time of retinal differentiation. In adulthood, Rep-1 localizes to distinct cellular compartments in multiple retinal cell types. CONCLUSIONS: Chm and Chml have the same broad expression profile in the mouse retina. In particular, the Chm transcript and corresponding protein are found in cell types other than those thought to be primarily affected in the human disease. These results have important implications for approaches with which to develop a relevant mouse model of choroideremia and for therapeutic strategies for this disease.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Alkyl and Aryl Transferases/genetics , Choroideremia/genetics , Gene Expression , RNA, Messenger/metabolism , Retina/metabolism , Animals , Blotting, Western , Fluorescent Antibody Technique, Indirect , Immunoenzyme Techniques , In Situ Hybridization , Mice , Mice, Inbred ICR , RNA Probes , Rabbits , Retina/embryologyABSTRACT
PURPOSE: Rapamycin exhibits significant antitumor/antiangiogenic activity that is coupled with a decrease in vascular endothelial growth factor (VEGF) production and a reduction in the response of vascular endothelial cells to stimulation by VEGF. VEGF plays a significant role in neovascular pathologies of the eye, thus we tested the possibility of using rapamycin to inhibit retinal and choroidal neovascularization (CNV). METHODS: CNV was induced in adult mice with laser photocoagulation. Retinal neovascularization was induced using the retinopathy of prematurity (ROP) hyperoxia/hypoxia model. Experimental animals received intraperitoneal (ip) injections of rapamycin (2 mg/kg/day or 4 mg/kg/day) for 1-2 weeks. Controls were not treated or received ip injections of phosphate buffered saline (PBS). Eyes were analyzed histologically for evidence of CNV or retinal neovascularization. ROP eyes were further analyzed for changes in VEGF and VEGF receptor (Flt-1 and Flk-1) protein content following rapamycin treatment. RESULTS: Rapamycin significantly reduced the extent of neovascularization in both the CNV and the ROP model. Immunohistochemical staining of treated and untreated ROP retina did not reveal a significant reduction in levels of VEGF protein or its receptors. Immunostaining for Flt-1 increased, while no obvious changes in Flk-1 were observed. Quantitative analysis of total protein via enzyme linked immunosorbent assay (ELISA) confirmed an increase in Flt-1 and VEGF, following drug treatment, with no effect on Flk-1. CONCLUSIONS: These results suggest rapamycin may provide an effective new treatment for ocular neovascularization.
Subject(s)
Choroidal Neovascularization/drug therapy , Immunosuppressive Agents/therapeutic use , Retinal Neovascularization/drug therapy , Sirolimus/therapeutic use , Animals , Animals, Newborn , Choroid/drug effects , Choroid/metabolism , Choroid/pathology , Choroidal Neovascularization/metabolism , Choroidal Neovascularization/pathology , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Extracellular Matrix Proteins , Female , Fluorescent Antibody Technique, Indirect , Injections, Intraperitoneal , Mice , Mice, Inbred C57BL , Myosin Heavy Chains , Nonmuscle Myosin Type IIB , Proteins/metabolism , Retina/drug effects , Retina/metabolism , Retina/pathology , Retinal Neovascularization/metabolism , Retinal Neovascularization/pathology , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-1 , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
PURPOSE: Pigment epithelium derived factor (PEDF) is a secreted protein with demonstrated anti-angiogenic properties, and with potential application for the treatment of neovascular disease. Delivery of pigment epithelium derived factor to the retina via virus mediated gene transfer has been shown to inhibit neovascularization in a number of experimental models. While pigment epithelium derived factor is endogenously expressed in the retina, its role in guiding normal vessel development and growth is not yet known. This study aimed to determine whether over-expression of pigment epithelium derived factor alters the normal pattern of retinal vessel development. METHODS: Neonatal (age postnatal day 2 (P2)) CD1 mice were injected subretinally unilaterally with AAV2/1.CMV.PEDF while contralateral eyes were injected subretinally with AAV2/1.CMV.EGFP as control. Cohorts of animals were sacrificed at P7 to P21 and the retinal vasculature was co-labeled through fluorescein-dextran perfusion and immunohistochemistry. Vascular size, localization, and structure were analyzed using light and confocal microscopy. Additional cohorts were use to obtain quantitative levels of pigment epithelium derived factor protein through ELISA. RESULTS: The extent of vessel growth from the optic disk to periphery over time (i.e., the radius of retinal vasculature), and the area of expansion of the neural retina were unaffected by over-expression of pigment epithelium derived factor to levels at least 3.5 fold higher than endogenous levels. The thicknesses of the various retinal layers were similar in AAV2/1.CMV.PEDF treated and control injected eyes. Three dimensional analysis of confocal images shows a slight delay in the rate of growth of vasculature into the deeper layers of the retina in pigment epithelium derived factor treated eyes compared to EGFP treated control eyes. However, the normal differentiation of vessels into arterioles, and venules, and the formation of a capillary network continued to occur, achieving normal and complete maturation of vascular structure by P21. CONCLUSIONS: Over-expression of pigment epithelium derived factor in the developing retina exerted no marked or permanent effects on retinal vessel growth and differentiation. The findings are relevant to the safety of the potential therapeutic use of pigment epithelium derived factor in human retinal disease.
Subject(s)
Eye Proteins/physiology , Gene Expression , Nerve Growth Factors/physiology , Retinal Vessels/growth & development , Serpins/physiology , Animals , Animals, Newborn , Cell Differentiation , Dependovirus/genetics , Endothelium, Vascular/metabolism , Enzyme-Linked Immunosorbent Assay , Genetic Vectors , Mice , Microscopy, Confocal , Microscopy, Fluorescence , Optic Disk/blood supply , RNA, Messenger/metabolism , Retinal Vessels/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transgenes , Vascular Endothelial Growth Factor A/metabolismABSTRACT
PURPOSE: RNA interference mediated by small interfering RNAs (siRNAs) is a powerful technology allowing the silencing of mamalian genes with great specificity and potency. The purpose of this study was to demonstrate the feasibility of RNA interference mediated by siRNA in retinal cells in vitro and in the murine retina in vivo. METHODS: siRNAs specific for enhanced green fluorescent protein (EGFP) and murine and human vascular endothelial growth factor (VEGF) were designed. In vitro studies in human cell lines entailed modulation of endogenous VEGF levels through chemically induced hypoxia. Effects of siRNA treatment on these levels were measured by ELISA. In vivo studies evaluating effects of siRNA on levels of EGFP and VEGF were performed by co-injecting recombinant viruses carrying EGFP or hVEGF cDNAs along with the appropriate siRNAs subretinally in mice. Additional studies aimed at blocking production of endogenous mVEGF were performed using laser-induced choroidal neovascularization (CNV) in mice. Effects of in vivo treatments were evaluated ophthalmoscopically. Retinal/choroidal flat mounts were evaluated after perfusion with dextran-fluorescein. Alternatively, retinas were evaluated in histological sections or VEGF levels were measured in intact eyes using ELISA. RESULTS: Successful delivery of siRNA to the subretinal space was confirmed by observing significantly reduced levels of EGFP in eyes treated with Ad.CMV.EGFP plus EGFP-directed siRNA. siRNAs directed against hVEGF effectively and specifically inhibit hypoxia-induced VEGF levels in human cell lines and after adenoviral induced hVEGF transgene expression in vivo. In addition, subretinal delivery of siRNA directed against murine Vegf significantly inhibited CNV after laser photocoagulation. CONCLUSIONS: Delivery of siRNA can be used in vitro and in vivo to target specific RNAs and to reduce the levels of the specific protein product in the targeted cells. This work suggests that RNA interference has potential for application to studies of retinal biology and for the treatment of a variety of retinal diseases, including those involving abnormal blood vessel growth.
Subject(s)
Choroidal Neovascularization/prevention & control , Endothelial Growth Factors/genetics , Gene Silencing , Gene Targeting , Intercellular Signaling Peptides and Proteins/genetics , Lymphokines/genetics , RNA, Small Interfering/genetics , Adenoviruses, Human/genetics , Animals , Choroid/blood supply , Choroid/surgery , Choroidal Neovascularization/metabolism , Defective Viruses , Disease Models, Animal , Down-Regulation , Endothelial Growth Factors/metabolism , Enzyme-Linked Immunosorbent Assay , Green Fluorescent Proteins , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Laser Coagulation , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Lymphokines/metabolism , Mice , Mice, Inbred C57BL , Pigment Epithelium of Eye/metabolism , Pigment Epithelium of Eye/virology , Transfection , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
SPAG6, an axoneme central apparatus protein, is essential for function of ependymal cell cilia and sperm flagella. A significant number of Spag6-deficient mice die with hydrocephalus, and surviving males are sterile because of sperm motility defects. In further exploring the ciliary dysfunction in Spag6-null mice, we discovered that cilia beat frequency was significantly reduced in tracheal epithelial cells, and that the beat was not synchronized. There was also a significant reduction in cilia density in both brain ependymal and trachea epithelial cells, and cilia arrays were disorganized. The orientation of basal feet, which determines the direction of axoneme orientation, was apparently random in Spag6-deficient mice, and there were reduced numbers of basal feet, consistent with reduced cilia density. The polarized epithelial cell morphology and distribution of intracellular mucin, α-tubulin, and the planar cell polarity protein, Vangl2, were lost in Spag6-deficient tracheal epithelial cells. Polarized epithelial cell morphology and polarized distribution of α-tubulin in tracheal epithelial cells was observed in one-week old wild-type mice, but not in the Spag6-deficient mice of the same age. Thus, the cilia and polarity defects appear prior to 7 days post-partum. These findings suggest that SPAG6 not only regulates cilia/flagellar motility, but that in its absence, ciliogenesis, axoneme orientation, and tracheal epithelial cell polarity are altered.
Subject(s)
Cilia/physiology , Microtubule Proteins/metabolism , Animals , Axoneme/metabolism , Brain/pathology , Cell Polarity , Ependyma/metabolism , Ependyma/pathology , Epithelial Cells/metabolism , Epithelial Cells/pathology , Male , Mice , Mice, Knockout , Microscopy, Electron, Scanning , Microscopy, Video , Microtubule Proteins/deficiency , Microtubule Proteins/genetics , Trachea/pathologyABSTRACT
The functional significance of decreased RAP1GAP protein expression in human tumors is unclear. To identify targets of RAP1GAP downregulation in the thyroid gland, RAP1 and RAP2 protein expression in human thyroid cells and in primary thyroid tumors were analyzed. RAP1GAP and RAP2 were co-expressed in normal thyroid follicular cells. Intriguingly, RAP1 was not detected in normal thyroid cells, although it was detected in papillary thyroid carcinomas, which also expressed RAP2. Both RAP proteins were detected at the membrane in papillary thyroid tumors, suggesting that they are activated when RAP1GAP is downregulated. To explore the functional significance of RAP1GAP depletion, RAP1GAP was transiently expressed at the lowest level that is sufficient to block endogenous RAP2 activity in papillary and anaplastic thyroid carcinoma cell lines. RAP1GAP impaired the ability of cells to spread and migrate on collagen. Although RAP1GAP had no effect on protein tyrosine phosphorylation in growing cells, RAP1GAP impaired phosphorylation of focal adhesion kinase and paxillin at sites phosphorylated by SRC in cells acutely plated on collagen. SRC activity was increased in suspended cells, where it was inhibited by RAP1GAP. Inhibition of SRC kinase activity impaired cell spreading and motility. These findings identify SRC as a target of RAP1GAP depletion and suggest that the downregulation of RAP1GAP in thyroid tumors enhances SRC-dependent signals that regulate cellular architecture and motility.
Subject(s)
Carcinoma, Papillary/pathology , Cell Movement , Cytoskeleton/physiology , GTPase-Activating Proteins/physiology , Thyroid Neoplasms/pathology , Carcinoma, Papillary/genetics , Carcinoma, Papillary/metabolism , Cell Line, Tumor , Cell Movement/genetics , Cell Movement/physiology , Cytoskeleton/metabolism , Down-Regulation/genetics , GTPase-Activating Proteins/genetics , GTPase-Activating Proteins/metabolism , Gene Expression Regulation, Neoplastic , HT29 Cells , Humans , Proto-Oncogene Proteins pp60(c-src)/genetics , Proto-Oncogene Proteins pp60(c-src)/metabolism , Proto-Oncogene Proteins pp60(c-src)/physiology , Thyroid Carcinoma, Anaplastic , Thyroid Neoplasms/genetics , Thyroid Neoplasms/metabolism , Transfection , rap GTP-Binding Proteins/genetics , rap GTP-Binding Proteins/metabolism , rap1 GTP-Binding Proteins/genetics , rap1 GTP-Binding Proteins/metabolismABSTRACT
The constant self renewal and differentiation of adult intestinal stem cells maintains a functional intestinal mucosa for a lifetime. However, the molecular mechanisms that regulate intestinal stem cell division and epithelial homeostasis are largely undefined. We report here that the small GTPases Cdc42 and Rab8a are critical regulators of these processes in mice. Conditional ablation of Cdc42 in the mouse intestinal epithelium resulted in the formation of large intracellular vacuolar structures containing microvilli (microvillus inclusion bodies) in epithelial enterocytes, a phenotype reminiscent of human microvillus inclusion disease (MVID), a devastating congenital intestinal disorder that results in severe nutrient deprivation. Further analysis revealed that Cdc42-deficient stem cells had cell division defects, reduced capacity for clonal expansion and differentiation into Paneth cells, and increased apoptosis. Cdc42 deficiency impaired Rab8a activation and its association with multiple effectors, and prevented trafficking of Rab8a vesicles to the midbody. This impeded cytokinesis, triggering crypt apoptosis and disrupting epithelial morphogenesis. Rab8a was also required for Cdc42-GTP activity in the intestinal epithelium, where continued cell division takes place. Furthermore, mice haploinsufficient for both Cdc42 and Rab8a in the intestine demonstrated abnormal crypt morphogenesis and epithelial transporter physiology, further supporting their functional interaction. These data suggest that defects of the stem cell niche can cause MVID. This hypothesis represents a conceptual departure from the conventional view of this disease, which has focused on the affected enterocytes, and suggests stem cell-based approaches could be beneficial to infants with this often lethal condition.
Subject(s)
Gene Expression Regulation , Intestines/cytology , Stem Cells/cytology , cdc42 GTP-Binding Protein/metabolism , rab GTP-Binding Proteins/metabolism , Animals , Biological Transport , Cell Cycle , Cell Differentiation , Cell Survival , HeLa Cells , Humans , Intestinal Mucosa/metabolism , Mice , Models, Biological , Models, Genetic , PhenotypeABSTRACT
Rap1GAP expression is decreased in human tumors. The significance of its downregulation is unknown. We show that Rap1GAP expression is decreased in primary colorectal carcinomas. To elucidate the advantages conferred on tumor cells by loss of Rap1GAP, Rap1GAP expression was silenced in human colon carcinoma cells. Suppressing Rap1GAP induced profound alterations in cell adhesion. Rap1GAP-depleted cells exhibited defects in cell/cell adhesion that included an aberrant distribution of adherens junction proteins. Depletion of Rap1GAP enhanced adhesion and spreading on collagen. Silencing of Rap expression normalized spreading and restored E-cadherin, beta-catenin, and p120-catenin to cell/cell contacts, indicating that unrestrained Rap activity underlies the alterations in cell adhesion. The defects in adherens junction protein distribution required integrin signaling as E-cadherin and p120-catenin were restored at cell/cell contacts when cells were plated on poly-l-lysine. Unexpectedly, Src activity was increased in Rap1GAP-depleted cells. Inhibition of Src impaired spreading and restored E-cadherin at cell/cell contacts. These findings provide the first evidence that Rap1GAP contributes to cell/cell adhesion and highlight a role for Rap1GAP in regulating cell/matrix and cell/cell adhesion. The frequent downregulation of Rap1GAP in epithelial tumors where alterations in cell/cell and cell/matrix adhesion are early steps in tumor dissemination supports a role for Rap1GAP depletion in tumor progression.
Subject(s)
Cell Adhesion/physiology , Cell Line, Tumor/metabolism , Colorectal Neoplasms/metabolism , Extracellular Matrix/metabolism , GTPase-Activating Proteins/metabolism , Adherens Junctions/metabolism , Animals , Cadherins/genetics , Cadherins/metabolism , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , GTPase-Activating Proteins/genetics , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , beta Catenin/genetics , beta Catenin/metabolism , src-Family Kinases/genetics , src-Family Kinases/metabolismABSTRACT
SPAG6 and SPAG16L are proteins localized to the "9+2" axoneme central apparatus. Both are essential for sperm motility and male fertility. These two proteins are also expressed in other tissues containing ciliated cells, such as brain and lung. To study the effects of combined deficiency of these two proteins, a double mutant mouse model was created. The double mutant mice displayed a more profound phenotype of growth retardation and hydrocephalus compared to mice nullizygous for SPAG6 and SPAG16L alone. The double mutant mice died younger, and mortality was significantly higher than in single mutant mice. In addition, the double mutant mice demonstrated pneumonia and its complications, including hemorrhage, edema, and atelectasis, phenotypes not observed in mice nullizygous for mutations in the individual genes. No other cilia-related phenotypic change was detected in double mutant mice including lateralization defects. The ultrastructure of cilia in both the brain and lung of the double mutant mice appeared normal. This model of combined SPAG6 and SPAG16L deficiency provides a new platform to study primary ciliary dyskinesia. The findings also demonstrate that SPAG6 and SPAG16L have related roles in controlling the function of cilia in the brain and lung.
Subject(s)
Hydrocephalus/mortality , Microtubule Proteins/deficiency , Microtubule Proteins/physiology , Microtubule-Associated Proteins/deficiency , Microtubule-Associated Proteins/physiology , Pneumonia/mortality , Animals , Brain/diagnostic imaging , Brain/ultrastructure , Cilia/genetics , Cilia/ultrastructure , Heterozygote , Hydrocephalus/genetics , Lung/diagnostic imaging , Lung/ultrastructure , Magnetic Resonance Imaging , Male , Mice , Mice, Knockout , Mice, Mutant Strains , Microtubule Proteins/genetics , Microtubule-Associated Proteins/genetics , Pneumonia/genetics , RadiographyABSTRACT
Congenital hearing deficits can be caused by a variety of genetic and acquired conditions. Complete reversal of deficits in the peripheral auditory system may require delivery of corrective genes to cochlear progenitor cells. We tested delivery of lentivirus and an array of recombinant adeno-associated viral (AAV) serotypes for efficiency and cellular specificity of transgene expression after in utero delivery to the developing mouse otocyst. Stability of expression and safety with respect to auditory function were then tested in those vectors that had the most favorable in utero cochlear transduction characteristics (AAV2/1, AAV2/8, and lentivirus). AAV2/1 was found to be the optimal vector for in utero cochlear gene transfer. It efficiently transduced progenitors giving rise to both inner and outer hair cells and supporting cells and had no adverse effect on cochlear cell differentiation. Further, it had no pathological effect on differentiated hair cells or the integrity of the auditory nerve or brain-stem nuclei as measured by auditory brain-stem response testing. AAV2/1 promises to be useful in further studies evaluating differentiation pathways of cochlear cells in health and disease and for developing gene-based therapies for congenital and acquired forms of peripheral hearing loss.
Subject(s)
Cochlea/metabolism , Dependovirus/genetics , Genetic Therapy/methods , Hearing Loss, Sensorineural/therapy , Animals , Cell Differentiation , Female , Fetus , Genetic Vectors/genetics , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Hair Cells, Auditory, Inner/cytology , Hair Cells, Auditory, Inner/metabolism , Hair Cells, Auditory, Outer/cytology , Hair Cells, Auditory, Outer/metabolism , Lentivirus/genetics , Mice , Microinjections , Transduction, Genetic/methods , UterusABSTRACT
The axonemes of cilia and flagella contain a "9+2" structure of microtubules and associated proteins. Proteins associated with the central doublet pair have been identified in Chlamydomonas that result in motility defects when mutated. The murine orthologue of the Chlamydomonas PF20 gene, sperm-associated antigen 16 (Spag16), encodes two proteins of M(r) approximately 71 x 10(3) (SPAG16L) and M(r) approximately 35 x 10(3) (SPAG16S). In sperm, SPAG16L is found in the central apparatus of the axoneme. To determine the function of SPAG16L, gene targeting was used to generate mice lacking this protein but still expressing SPAG16S. Mutant animals were viable and showed no evidence of hydrocephalus, lateralization defects, sinusitis, bronchial infection, or cystic kidneys-symptoms typically associated with ciliary defects. However, males were infertile with a lower than normal sperm count. The sperm had marked motility defects, even though ultrastructural abnormalities of the axoneme were not evident. In addition, the testes of some nullizygous animals showed a spermatogenetic defect, which consisted of degenerated germ cells in the seminiferous tubules. We conclude that SPAG16L is essential for sperm flagellar function. The sperm defect is consistent with the motility phenotype of the Pf20 mutants of Chlamydomonas, but morphologically different in that the mutant algal axoneme lacks the central apparatus.
Subject(s)
Infertility, Male/etiology , Microtubule-Associated Proteins/deficiency , Sperm Motility , Animals , Female , Fertility , Germ Cells/transplantation , Infertility, Male/physiopathology , Male , Mice , Microtubule-Associated Proteins/genetics , Mutagenesis, Insertional , Spermatogenesis , Spermatozoa/ultrastructure , Testis/anatomy & histologyABSTRACT
Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) promotes cell-to-cell spread at basolateral surfaces of epithelial cells, but its activity in neurons is less clear. We used the mouse retina infection model and neuronal cell cultures to define the spread phenotype of gE mutant viruses. Wild-type (WT) and gE-null (NS-gEnull) viruses both infected retina ganglion cell neurons; however, NS-gEnull viral antigens failed to reach the optic nerve, which indicates a defect in axonal localization. We evaluated two Fc receptor-negative gE mutant viruses containing four amino acid inserts in the gE ectodomain. One mutant virus failed to spread from the retina into the optic nerve, while the other spread normally. Therefore, the gE ectodomain is involved in axonal localization, and the Fc receptor and neuronal spread are mediated by overlapping but distinct gE domains. In the retina infection model, virus can travel to the brain via the optic nerve from presynaptic to postsynaptic neurons (anterograde direction) or via nerves that innervate the iris and ciliary body from postsynaptic to presynaptic neurons (retrograde direction). WT virus infected the brain by anterograde and retrograde routes, whereas NS-gEnull virus failed to travel by either pathway. The site of the defect in retrograde spread remains to be determined; however, infection of rat superior cervical ganglia neurons in vitro indicates that gE is required to target virion components to the axon initial segment. The requirement for gE in axonal targeting and retrograde spread highlights intriguing similarities and differences between HSV-1 and pseudorabies virus gE.
Subject(s)
Axonal Transport , Capsid/metabolism , Herpes Simplex/virology , Simplexvirus/physiology , Viral Envelope Proteins/metabolism , Viral Envelope Proteins/physiology , Viral Structural Proteins/metabolism , Animals , Brain/virology , Cells, Cultured , Disease Models, Animal , Mice , Mutation , Optic Nerve/virology , Rats , Receptors, Fc/genetics , Retina/virology , Simplexvirus/metabolism , Simplexvirus/pathogenicity , Superior Cervical Ganglion/virology , Viral Envelope Proteins/genetics , Virulence , Virus ReplicationABSTRACT
The axoneme central apparatus is thought to control flagellar/ciliary waveform and maintain the structural integrity of the axoneme, but proteins involved in these processes have not been fully elucidated. Moreover the network of interactions among them that allows these events to take place in a compact space has not been defined. PF6, a component of the Chlamydomonas central apparatus, is localized to the 1a projection of the C1 microtubule. Mutations in the Chlamydomonas PF6 gene result in flagellar paralysis. We characterized human and murine orthologues of PF6. The murine Pf6 gene is expressed in a pattern consistent with a role in flagella and cilia, and the PF6 protein is indeed localized to the central apparatus of the sperm flagellar axoneme. We discovered that a portion of PF6 associates with the mammalian orthologue of Chlamydomonas PF16 (sperm-associated antigen 6 (SPAG6)), another central apparatus protein that is localized to the C1 microtubule in algae. A fragment of PF6 corresponding to the PF6 domain that interacts with SPAG6 in yeast two-hybrid assays and colocalizes with SPAG6 in transfected cells was missing from epididymal sperm of SPAG6-deficient mice. SPAG6 binds to the mammalian orthologue of PF20, which in Chlamydomonas is located in bridges connecting the C2 and C1 microtubules. Thus, PF6, SPAG6, and PF20 form a newly identified network that links together components of the axoneme central apparatus and presumably participates in its dynamic regulation of ciliary and flagellar beat.