RESUMO
Mitochondria, which generate ATP through aerobic respiration, also have important noncanonical functions. Mitochondria are dynamic organelles, that engage in fission (division), fusion (joining) and translocation. They also regulate intracellular calcium homeostasis, serve as oxygen-sensors, regulate inflammation, participate in cellular and organellar quality control and regulate the cell cycle. Mitochondrial fission is mediated by the large GTPase, dynamin-related protein 1 (Drp1) which, when activated, translocates to the outer mitochondrial membrane (OMM) where it interacts with binding proteins (Fis1, MFF, MiD49 and MiD51). At a site demarcated by the endoplasmic reticulum, fission proteins create a macromolecular ring that divides the organelle. The functional consequence of fission is contextual. Physiological fission in healthy, nonproliferating cells mediates organellar quality control, eliminating dysfunctional portions of the mitochondria via mitophagy. Pathological fission in somatic cells generates reactive oxygen species and triggers cell death. In dividing cells, Drp1-mediated mitotic fission is critical to cell cycle progression, ensuring that daughter cells receive equitable distribution of mitochondria. Mitochondrial fusion is regulated by the large GTPases mitofusin-1 (Mfn1) and mitofusin-2 (Mfn2), which fuse the OMM, and optic atrophy 1 (OPA-1), which fuses the inner mitochondrial membrane. Mitochondrial fusion mediates complementation, an important mitochondrial quality control mechanism. Fusion also favors oxidative metabolism, intracellular calcium homeostasis and inhibits cell proliferation. Mitochondrial lipids, cardiolipin and phosphatidic acid, also regulate fission and fusion, respectively. Here we review the role of mitochondrial dynamics in health and disease and discuss emerging concepts in the field, such as the role of central versus peripheral fission and the potential role of dynamin 2 (DNM2) as a fission mediator. In hyperproliferative diseases, such as pulmonary arterial hypertension and cancer, Drp1 and its binding partners are upregulated and activated, positing mitochondrial fission as an emerging therapeutic target.
Assuntos
Neoplasias , Hipertensão Arterial Pulmonar , Humanos , Dinâmica Mitocondrial/fisiologia , Cálcio , Dinaminas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Ciclo CelularRESUMO
AIMS: Dynamin-2 is a large GTPase, a member of the dynamin superfamily that regulates membrane remodelling and cytoskeleton dynamics. Mutations in the dynamin-2 gene (DNM2) cause autosomal dominant centronuclear myopathy (CNM), a congenital neuromuscular disorder characterised by progressive weakness and atrophy of the skeletal muscles. Cognitive defects have been reported in some DNM2-linked CNM patients suggesting that these mutations can also affect the central nervous system (CNS). Here we studied how a dynamin-2 CNM-causing mutation influences the CNS function. METHODS: Heterozygous mice harbouring the p.R465W mutation in the dynamin-2 gene (HTZ), the most common causing autosomal dominant CNM, were used as disease model. We evaluated dendritic arborisation and spine density in hippocampal cultured neurons, analysed excitatory synaptic transmission by electrophysiological field recordings in hippocampal slices, and evaluated cognitive function by performing behavioural tests. RESULTS: HTZ hippocampal neurons exhibited reduced dendritic arborisation and lower spine density than WT neurons, which was reversed by transfecting an interference RNA against the dynamin-2 mutant allele. Additionally, HTZ mice showed defective hippocampal excitatory synaptic transmission and reduced recognition memory compared to the WT condition. CONCLUSION: Our findings suggest that the dynamin-2 p.R465W mutation perturbs the synaptic and cognitive function in a CNM mouse model and support the idea that this GTPase plays a key role in regulating neuronal morphology and excitatory synaptic transmission in the hippocampus.
Assuntos
Dinamina II , Miopatias Congênitas Estruturais , Animais , Camundongos , Modelos Animais de Doenças , Dinamina II/genética , Dinamina II/metabolismo , Músculo Esquelético/metabolismo , Mutação , Miopatias Congênitas Estruturais/genética , Neurônios/metabolismo , Transmissão SinápticaRESUMO
Collective cell migration is the coordinated movement of multiple cells connected by cadherin-based adherens junctions and is essential for physiological and pathological processes. Cadherins undergo dynamic intracellular trafficking, and their surface level is determined by a balance between endocytosis, recycling and degradation. However, the regulatory mechanism of cadherin turnover in collective cell migration remains elusive. In this study, we show that the Bin/amphiphysin/Rvs (BAR) domain protein pacsin 2 (protein kinase C and casein kinase substrate in neurons protein 2) plays an essential role in collective cell migration by regulating N-cadherin (also known as CDH2) endocytosis in human cancer cells. Pacsin 2-depleted cells formed cell-cell contacts enriched with N-cadherin and migrated in a directed manner. Furthermore, pacsin 2-depleted cells showed attenuated internalization of N-cadherin from the cell surface. Interestingly, GST pull-down assays demonstrated that the pacsin 2 SH3 domain binds to the cytoplasmic region of N-cadherin, and expression of an N-cadherin mutant defective in binding to pacsin 2 phenocopied pacsin 2 RNAi cells both in cell contact formation and N-cadherin endocytosis. These data support new insights into a novel endocytic route of N-cadherin in collective cell migration, highlighting pacsin 2 as a possible therapeutic target for cancer metastasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Caderinas , Neoplasias , Humanos , Junções Aderentes/metabolismo , Caderinas/genética , Caderinas/metabolismo , Membrana Celular/metabolismo , Movimento Celular , Endocitose/fisiologia , Neoplasias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
The specifics of cell receptor-modulated avian reovirus (ARV) entry remain unknown. By using a viral overlay protein-binding assay (VOPBA) and an in-gel digestion coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we determined that cell-surface annexin A2 (AnxA2) and adhesion G protein-coupled receptor Latrophilin-2 (ADGRL2) modulate ARV entry. Direct interaction between the ARV σC protein and AnxA2 and ADGRL2 in Vero and DF-1 cells was demonstrated in situ by proximity ligation assays. By using short hairpin RNAs (shRNAs) to silence the endogenous AnxA2 and ADGRL2 genes, ARV entry could be efficiently blocked. A significant decrease in virus yields and the intracellular specific signal for σC protein was observed in Vero cells preincubated with the specific AnxA2 and ADGRL2 monoclonal antibodies, indicating that AnxA2 and ADGRL2 are involved in modulating ARV entry. Furthermore, we found that cells pretreated with the AnxA2/S100A10 heterotetramer (A2t) inhibitor A2ti-1 suppressed ARV-mediated activation of Src and p38 mitogen-activated protein kinase (MAPK), demonstrating that Src and p38 MAPK serve as downstream molecules of cell-surface AnxA2 signaling. Our results reveal that suppression of cell-surface AnxA2 with the A2ti-1 inhibitor increased Csk-Cbp interaction, suggesting that ARV entry suppresses Cbp-mediated relocation of Csk to the membrane, thereby activating Src. Furthermore, reciprocal coimmunoprecipitation assays revealed that σC can interact with signaling molecules, lipid raft, and vimentin. The current study provides novel insights into cell-surface AnxA2- and ADGRL2-modulated cell entry of ARV which triggers Src and p38 MAPK signaling to enhance caveolin-1-, dynamin 2-, and lipid raft-dependent endocytosis. IMPORTANCE By analyzing results from VOPBA and LC-MS/MS, we have determined that cell-surface AnxA2 and ADGRL2 modulate ARV entry. After ARV binding to receptors, Src and p38 MAPK signaling were triggered and, in turn, increased the phosphorylation of caveolin-1 (Tyr14) and upregulated dynamin 2 expression to facilitate caveolin-1-mediated and dynamin 2-dependent endocytosis. In this work, we demonstrated that ARV triggers Src activation by impeding Cbp-mediated relocation of Csk to the membrane in the early stages of the life cycle. This work provides better insight into cell-surface AnxA2 and ADGRL2, which upregulate Src and p38MAPK signaling pathways to enhance ARV entry and productive infection.
Assuntos
Anexina A2 , Orthoreovirus Aviário , Animais , Chlorocebus aethiops , Caveolina 1/genética , Caveolina 1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Células Vero , Orthoreovirus Aviário/metabolismo , Internalização do Vírus , Anexina A2/genética , Anexina A2/metabolismo , Dinamina II/metabolismo , Cromatografia Líquida , Espectrometria de Massas em Tandem , Endocitose , Fosforilação , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Breast cancer cells abnormally express vasopressin (AVP) and its receptors. The effect of AVP is largely orchestrated through its downstream signaling and by receptor-mediated endocytosis (RME), in which Dynamin 2 (Dyn2) plays an integral role in vesicle closure. In this work, luminal A breast cancer cells were treated with AVP, and then Dynasore (DYN) was employed to inhibit Dyn2 to explore the combined effect of AVP and Dyn2 inhibition on the survival of breast cancer cells. The results revealed that DYN alone demonstrated a concentration-dependent cytotoxic effect in AVP untreated cells. Apoptosis developed in 29.7 and 30.3% of cells treated with AVP or AVP+DYN, respectively, compared to 32.5% in cells treated with Wortmannin (Wort, a selective PI3K pathway inhibitor). More apoptosis was observed when cells were treated with DYN+Wort in presence or absence of exogenous AVP. Besides, 2 or 4- fold increases in the expression of Bax and Caspase-3, were observed in cells exposed to AVP in absence or presence of DYN, respectively. This was associated with higher levels of the autophagy marker (LC3II protein). Meanwhile, the activation of Akt protein, sequentially decreased in the same pattern. Cell's invasion decreased when they were exposed to AVP alone or combined with DYN or/and Wort. Conclusively, although many reports suggested the proliferative effect of AVP, the results predict the antiproliferative and antimetastatic effects of 100 nM AVP in luminal A breast cancer cells. However, the hormone did not enhance the cytotoxic effect of Dyn 2 or PI3K pathway inhibition. Summary of the Dynamin 2 independent AVP antiproliferative effects. Breast cancer cells expresses AVP as a Prohormone (A). At high dose of AVP, the hormone is liganded with AVP receptor (B) to initiate RME, where the endosomed complex (C) is degraded through the endosome-lysosome system, as a part of signal management. These events consume soluble Dyn2 in neck closure and vesicle fission (D). This makes the cells more substitutable to the direct apoptotic effect of DYN (E). Alternatively, at lower AVP doses the liganded AVP may initiate cAMP-mediated downstream signaling (F) and cellular proliferation. In parallel, Wort inhibits PIP2-PIP3 conversion (G) and the subsequent inhibition of PI3K/Akt/mTOR pathway leading to cell death.
Assuntos
Neoplasias da Mama , Fosfatidilinositol 3-Quinases , Humanos , Feminino , Proteínas Proto-Oncogênicas c-akt , Dinamina II , Neoplasias da Mama/tratamento farmacológico , Vasopressinas , ApoptoseRESUMO
Dominant centronuclear myopathy (CNM) is a rare form of congenital myopathy associated with a wide clinical spectrum, from severe neonatal to milder adult forms. There is no available treatment for this disease due to heterozygous mutations in the DNM2 gene encoding Dynamin 2 (DNM2). Dominant DNM2 mutations also cause rare forms of Charcot-Marie-Tooth disease and hereditary spastic paraplegia, and deleterious DNM2 overexpression was noticed in several diseases. The proof of concept for therapy by allele-specific RNA interference devoted to silence the mutated mRNA without affecting the normal allele was previously achieved in a mouse model and patient-derived cells, both expressing the most frequent DNM2 mutation in CNM. In order to have versatile small interfering RNAs (siRNAs) usable regardless of the mutation, we have developed allele-specific siRNAs against two non-pathogenic single-nucleotide polymorphisms (SNPs) frequently heterozygous in the population. In addition, allele-specific siRNAs against the p.S619L DNM2 mutation, a mutation frequently associated with severe neonatal cases, were developed. The beneficial effects of these new siRNAs are reported for a panel of defects occurring in patient-derived cell lines. The development of these new molecules allows targeting the large majority of the patients harboring DNM2 mutations or overexpression by only a few siRNAs.
RESUMO
Endothelial fenestrae are transcellular pores divided by a diaphragm consisting of plasmalemma vesicle-associated protein (PLVAP). They function as a channel for peptide hormones and other substances. Invagination of the plasma membrane is necessary for the fenestra formation. The actin cytoskeleton is essential for scission of endocytic vesicles from the invaginated plasma membrane. Therefore, we examined the involvement of the actin cytoskeleton in fenestra formation in cultured endothelial cells isolated from the anterior lobe (AL) of the rat pituitary, using immunofluorescence and scanning electron microscopy. Inhibition of polymerization and depolymerization of the actin cytoskeleton by latrunculin A and jasplakinolide, respectively, remarkably increased the PLVAP-positive sieve plate area and number of fenestrae. Jasplakinolide significantly affected the arrangement of the fenestra on the cell surface, resulting in parallel serpentine furrows of the fenestra. These results suggest that the actin cytoskeleton not only induces fenestra formation but also regulates cell arrangement. Dynamin is a scission protein of the invaginated plasma membrane and interacts with the actin cytoskeleton. We found that dynamin2 is mainly expressed in the endothelial cells of the rat AL. We then investigated the function of dynamin2 by the treatment with dyngo-4a, a potent inhibitor of dynamin1 and dynamin2, on the fenestra formation. As a result, the PLVAP-positive area is significantly increased by the treatment. These results show that the actin-dynamin2 interaction is essential for the control of the fenestra formation in endothelial cells of rat AL. In conclusion, the actin cytoskeleton and dynamin2 function as regulators of endothelial fenestra formation.
Assuntos
Actinas , Dinamina II , Células Endoteliais , Animais , Ratos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Células Endoteliais/metabolismo , Dinamina II/metabolismo , Proteínas de Membrana/metabolismoRESUMO
Microtubule stability and dynamics regulations are essential for vital cellular processes, such as microtubule-dependent axonal transport. Dynamin is involved in many membrane fission events, such as clathrin-mediated endocytosis. The ubiquitously expressed dynamin-2 has been reported to regulate microtubule stability. However, the underlying molecular mechanisms remain unclear. This study aimed to investigate the roles of intrinsic properties of dynamin-2 on microtubule regulation by rescue experiments. A heterozygous DNM2 mutation in HeLa cells was generated, and an increase in the level of stabilized microtubules in these heterozygous cells was observed. The expression of wild-type dynamin-2 in heterozygous cells reduced stabilized microtubules. Conversely, the expression of self-assembly-defective mutants of dynamin-2 in the heterozygous cells failed to decrease stabilized microtubules. This indicated that the self-assembling ability of dynamin-2 is necessary for regulating microtubule stability. Moreover, the heterozygous cells expressing the GTPase-defective dynamin-2 mutant, K44A, reduced microtubule stabilization, similar to the cells expressing wild-type dynamin-2, suggesting that GTPase activity of dynamin-2 is not essential for regulating microtubule stability. These results showed that the mechanism of microtubule regulation by dynamin-2 is diverse from that of endocytosis.
Assuntos
Dinaminas , Endocitose , Microtúbulos , Humanos , Dinaminas/genética , Endocitose/fisiologia , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Microtúbulos/metabolismoRESUMO
Gain-of-function mutations of dynamin-2, a mechano-GTPase that remodels membrane and actin filaments, cause centronuclear myopathy (CNM), a congenital disease that mainly affects skeletal muscle tissue. Among these mutations, the variants p.A618T and p.S619L lead to a gain of function and cause a severe neonatal phenotype. By using total internal reflection fluorescence microscopy (TIRFM) in immortalized human myoblasts expressing the pH-sensitive fluorescent protein (pHluorin) fused to the insulin-responsive aminopeptidase IRAP as a reporter of the GLUT4 vesicle trafficking, we measured single pHluorin signals to investigate how p.A618T and p.S619L mutations influence exocytosis. We show here that both dynamin-2 mutations significantly reduced the number and durations of pHluorin signals induced by 10 µM ionomycin, indicating that in addition to impairing exocytosis, they also affect the fusion pore dynamics. These mutations also disrupt the formation of actin filaments, a process that reportedly favors exocytosis. This altered exocytosis might importantly disturb the plasmalemma expression of functional proteins such as the glucose transporter GLUT4 in skeletal muscle cells, impacting the physiology of the skeletal muscle tissue and contributing to the CNM disease.
Assuntos
Dinamina II , Miopatias Congênitas Estruturais , Dinamina II/genética , Dinamina II/metabolismo , Exocitose , Mutação com Ganho de Função , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Humanos , Ionomicina , Músculo Esquelético/metabolismo , Mutação , Mioblastos/metabolismo , Miopatias Congênitas Estruturais/metabolismoRESUMO
BACKGROUND: Over the past few decades, numerous clinical and experimental studies have confirmed that oxidative stress is enhanced in heart failure (HF). We recently found that inhibition of highly expressed dynamin2 can protect myocardial ischemia-reperfusion injury in mice and inhibit oxidative stress in ischemic cardiomyocytes. However, the specific mechanisms are still not fully understood. In this study, we hypothesized that oxidative stress induces cardiomyocyte apoptosis through IGF2BP2 regulation, which is regulated through the dynamin2 expression. METHODS: H2O2-treated cardiomyocytes were observed for the regulatory effect of reactive oxygen species (ROS) on IGF2BP2 and the effect of IGF2BP2 on dynamin2 gene expression was determined by lentiviral-mediated IGF2BP2 overexpression. Then, siRNA knockdown of dynamin2 was used to observe whether it can alter the effect of IGF2BP2 on myocardial cells. Finally, IGF2BP2 was knocked down in ischemic rats using shRNA to determine the effects of IGF2BP2 on myocardial ischemia. RESULTS: ROS can promote dynamin2 expression by inducing IGF2BP2 expression and dynamin2 knockdown could reduce the injury of IGF2BP2 to cardiomyocytes. Inhibition of IGF2BP2 expression in myocardial ischemic rats ameliorated cardiac fibrosis in ischemic myocardium. CONCLUSION: Oxidative stress can induce cardiomyocyte apoptosis through the IGF2BP2-dynamin2 pathway. Inhibition of IGF2BP2 expression significantly improves the fibrosis and remodeling that occurs in ischemic myocardium.
Assuntos
Peróxido de Hidrogênio , Miócitos Cardíacos , Animais , Apoptose , Metabolismo Energético , Peróxido de Hidrogênio/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , RNA Interferente Pequeno/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Human adenovirus type 26 (HAdV26) has been recognized as a promising platform for vaccine vector development, and very recently vaccine against COVID-19 based on HAdV26 was authorized for emergency use. Nevertheless, basic biology of this virus, namely, pathway which HAdV26 uses to enter the cell, is still insufficiently known. We have shown here that HAdV26 infection of human epithelial cells expressing low amount of αvß3 integrin involves clathrin and is caveolin-1-independent, while HAdV26 infection of cells with high amount of αvß3 integrin does not involve clathrin but is caveolin-1-dependent. Thus, this study demonstrates that caveolin-1 is limiting factor in αvß3 integrin-mediated HAdV26 infection. Regardless of αvß3 integrin expression, HAdV26 infection involves dynamin-2. Our data provide for the first-time description of HAdV26 cell entry pathway, hence increase our knowledge of HAdV26 infection. Knowing that functionality of adenovirus vector is influenced by its cell entry pathway and intracellular trafficking, our results will contribute to better understanding of HAdV26 immunogenicity and antigen presentation when used as vaccine vector. IMPORTANCE In order to fulfill its role as a vector, adenovirus needs to successfully deliver its DNA genome to the host nucleus, a process highly influenced by adenovirus intracellular translocation. Thus, cell entry pathway and intracellular trafficking determine functionality of human adenovirus-based vectors. Endocytosis of HAdV26, currently extensively studied as a vaccine vector, has not been described so far. We present here that HAdV26 infection of human epithelial cells with high expression of αvß3 integrin, one of the putative HAdV26 receptors, is caveolin-1- and partially dynamin-2-dependent. Since caveolin containing domains provide a unique environment for specific signaling events and participate in inflammatory signaling one can imagine that directing HAdV26 cell entry toward caveolin-1-mediate pathway might play role in immunogenicity of this virus. Therefore, our results contribute to better understanding of HAdV26 infection pathway, hence, can be helpful in explaining induction of immune response and antigen presentation by HAdV26-based vaccine vector.
Assuntos
Adenovírus Humanos , COVID-19 , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Vacinas contra COVID-19 , Caveolina 1/genética , Caveolina 1/metabolismo , Clatrina/metabolismo , Dinamina II/metabolismo , Humanos , Integrinas/metabolismo , Internalização do VírusRESUMO
Dynamin 2 mutations are associated with Charcot-Marie-Tooth neuropathy. We report two siblings with a novel missense heterozygous point mutation (c.1609 G>A) in the highly conserved pleckstrin homology domain in exon 15 of Dynamin 2 presenting with progressive length-dependent sensorimotor polyneuropathy with mixed demyelinating and axonal features on electrodiagnostic studies. The previously unrecognized missense point mutation, which was inherited from their symptomatic but previously undiagnosed mother, was determined to be likely pathogenic based on a non-conservative amino acid substitution (p.Gly537Ser) that is predicted to damage secondary protein structure or function. This report emphasizes the importance of recognizing inherited neuropathies in clinical practice and evaluating suspected pathogenic gene variants initially classified to be of undetermined clinical significance in family cohorts. These cases add to the spectrum of pathogenic Dynamin 2 mutations associated with dominant-intermediate Charcot-Marie-Tooth neuropathy.
Assuntos
Doença de Charcot-Marie-Tooth , Dinamina II , Doença de Charcot-Marie-Tooth/diagnóstico , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Dinamina II/genética , Humanos , Mutação , Mutação de Sentido IncorretoRESUMO
Dominant dynamin 2 (DNM2) mutations are responsible for the autosomal dominant centronuclear myopathy (AD-CNM), a rare progressive neuromuscular disorder ranging from severe neonatal to mild adult forms. We previously demonstrated that mutant-specific RNA interference is an efficient therapeutic strategy to rescue the muscle phenotype at the onset of the symptoms in the AD-CNM knockin-Dnm2 R465W/+ mouse model. Our objective was to evaluate the long-term benefit of the treatment along with the disease time course. We demonstrate here that the complete rescue of the muscle phenotype is maintained for at least 1 year after a single injection of adeno-associated virus expressing the mutant-specific short hairpin RNA (shRNA). This was achieved by a maintained reduction of the mutant Dnm2 transcript. Moreover, this long-term study uncovers a pathological accumulation of DNM2 protein occurring with age in the mouse model and prevented by the treatment. Conversely, a physiological DNM2 protein decrease with age was observed in muscles from wild-type mice. Therefore, this study highlights a new potential pathophysiological mechanism linked to mutant protein accumulation and underlines the importance of DNM2 protein expression level for proper muscle function. Overall, these results strengthen the allele-specific silencing approach as a robust, safe, and efficient therapy for AD-CNM.
RESUMO
The TSH receptor (TSHR) is the major regulator of thyroid hormone biosynthesis in human thyrocytes by regulating the transcription of a number of genes including thyroglobulin (TG) and thyroperoxidase (TPO). Until recently, it was thought that TSHR initiated signal transduction pathways only at the cell-surface and that internalization was primarily involved in TSHR desensitization and downregulation. Studies primarily in mouse cells showed that TSHR internalization regulates gene transcription at an intracellular site also. However, this has not been shown for genes involved in thyroid hormone biosynthesis in human thyrocytes. We used human thyrocytes in primary culture. In these cells, the dose-response to TSH for gene expression is biphasic with low doses upregulating gene expression and higher doses decreasing gene expression. We used two approaches to inhibit internalization. In the first, we used inhibitors of dynamins, dynasore and dyngo-4a. Pretreatment with dynasore or dyngo-4a markedly inhibited TSH upregulation of TG and TPO mRNAs, as well as TG secretion. In the second, we used knockdown of dynamin 2, which is the most abundant dynamin in human thyrocytes. We showed that dynamin 2 knockdown inhibited TSHR internalization and decreased the TSH-stimulated levels of TG and TPO mRNAs and proteins. Lastly, we showed that the level of the activatory transcription factor phosphorylated cAMP response element binding protein (pCREB) in the cell nuclei was reduced by 68% when internalization was inhibited. We conclude that upregulation of genes involved in thyroid hormone synthesis in human thyrocytes is, in part, dependent on internalization leading to nuclear localization of an activated transcription factor(s).
Assuntos
Iodeto Peroxidase , Tireoglobulina , Animais , Humanos , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Camundongos , Receptores da Tireotropina/genética , Receptores da Tireotropina/metabolismo , Tireoglobulina/genética , Tireoglobulina/metabolismo , Tireotropina/genética , Tireotropina/farmacologia , Transcrição GênicaRESUMO
PURPOSE: Spatiotemporal regulation of cell membrane dynamics is a major process that promotes cancer cell invasion by acting as a driving force for cell migration. Beta-Pix (ßPix), a guanine nucleotide exchange factor for Rac1, has been reported to be involved in actin-mediated cellular processes, such as cell migration, by interacting with various proteins. As yet, however, the molecular mechanisms underlying ßPix-mediated cancer cell invasion remain unclear. METHODS: The clinical significance of ßPix was analyzed in patients with colorectal cancer (CRC) using public clinical databases. Pull-down and immunoprecipitation assays were employed to identify novel binding partners for ßPix. Additionally, various cell biological assays including immunocytochemistry and time-lapse video microscopy were performed to assess the effects of ßPix on CRC progression. A ßPix-SH3 antibody delivery system was used to determine the effects of the ßPix-Dyn2 complex in CRC cells. RESULTS: We found that the Src homology 3 (SH3) domain of ßPix interacts with the proline-rich domain of Dynamin 2 (Dyn2), a large GTPase. The ßPix-Dyn2 interaction promoted lamellipodia formation, along with plasma membrane localization of membrane-type 1 matrix metalloproteinase (MT1-MMP). Furthermore, we found that Src kinase-mediated phosphorylation of the tyrosine residue at position 442 of ßPix enhanced ßPix-Dyn2 complex formation. Disruption of the ßPix-Dyn2 complex by ßPix-SH3 antibodies targeting intracellular ßPix inhibited CRC cell invasion. CONCLUSIONS: Our data indicate that spatiotemporal regulation of the Src-ßPix-Dyn2 axis is crucial for CRC cell invasion by promoting membrane dynamics and MT1-MMP recruitment into the leading edge. The development of inhibitors that disrupt the ßPix-Dyn2 complex may be a useful therapeutic strategy for CRC.
Assuntos
Membrana Celular/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Progressão da Doença , Dinamina II/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Movimento Celular/genética , Dinamina II/química , Regulação Neoplásica da Expressão Gênica , Ouro/química , Células HEK293 , Humanos , Metaloproteinase 14 da Matriz/metabolismo , Nanopartículas Metálicas/química , Invasividade Neoplásica , Fosforilação , Fosfotirosina/metabolismo , Ligação Proteica , Pseudópodes/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/química , Regulação para Cima , Proteínas rac1 de Ligação ao GTP/metabolismo , Domínios de Homologia de srcRESUMO
Dynamin 2 (DNM2) is an ubiquitously expressed large GTPase well known for its role in vesicle formation in endocytosis and intracellular membrane trafficking also acting as a regulator of cytoskeletons. During the last two decades, DNM2 involvement, through mutations or overexpression, emerged in an increasing number of cancers and often associated with poor prognosis. A wide panel of DNM2-dependent processes was described in cancer cells which explains DNM2 contribution to cancer pathomechanisms. First, DNM2 dysfunction may promote cell migration, invasion and metastasis. Second, DNM2 acts on intracellular signaling pathways fostering tumor cell proliferation and survival. Relative to these roles, DNM2 was demonstrated as a therapeutic target able to reduce cell proliferation, induce apoptosis, and reduce the invasive phenotype in a wide range of cancer cells in vitro. Moreover, proofs of concept of therapy by modulation of DNM2 expression was also achieved in vivo in several animal models. Consequently, DNM2 appears as a promising molecular target for the development of anti-invasive agents and the already provided proofs of concept in animal models represent an important step of preclinical development.
Assuntos
Dinamina II/metabolismo , Neoplasias/genética , Animais , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Humanos , Camundongos , Metástase Neoplásica , Neoplasias/mortalidade , Análise de SobrevidaRESUMO
Podosomes are actin-rich adhesion structures formed in a variety of cell types, such as monocytic cells or cancer cells, to facilitate attachment to and degradation of the extracellular matrix (ECM). Previous studies showed that dynamin 2, a large GTPase involved in membrane remodeling and actin organization, is required for podosome function. However, precise roles of dynamin 2 at the podosomes remain to be elucidated. In this study, we identified a BAR (Bin-Amphiphysin-Rvs167) domain protein pacsin 2 as a functional partner of dynamin 2 at podosomes. Dynamin 2 and pacsin 2 interact and co-localize to podosomes in Src-transformed NIH 3T3 (NIH-Src) cells. RNAi of either dynamin 2 or pacsin 2 in NIH-Src cells inhibited podosome formation and maturation, suggesting essential and related roles at podosomes. Consistently, RNAi of pacsin 2 prevented dynamin 2 localization to podosomes, and reciprocal RNAi of dynamin 2 prevented pacsin 2 localization to podosomes. Taking these results together, we conclude that dynamin 2 and pacsin 2 co-operatively regulate organization of podosomes in NIH-Src cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Dinamina II/metabolismo , Podossomos/metabolismo , Animais , Células Cultivadas , Humanos , CamundongosRESUMO
The patient, a boy born in 1991, showed pronounced polyostotic fibrous dysplasia due to McCune-Albright syndrome, as well as Gilbert syndrome and Charcot-Marie-Tooth neuropathy caused by a DNM2 mutation. In addition, the patient, his sister, mother and maternal grandfather had intermittently increased plasma arginine and lysine levels, most probably due to heterozygosity for a novel pathogenic SLC7A2 variant.
Assuntos
Doença de Charcot-Marie-Tooth , Colestase , Displasia Fibrosa Poliostótica , Doenças do Recém-Nascido , Adulto , Humanos , Recém-Nascido , Masculino , MutaçãoRESUMO
Membrane remodeling is required for dynamic cellular processes such as cell division, polarization, and motility. BAR domain proteins and dynamins are key molecules in membrane remodeling that work together for membrane deformation and fission. In striated muscles, sarcolemmal invaginations termed T-tubules are required for excitation-contraction coupling. BIN1 and DNM2, which encode a BAR domain protein BIN1 and dynamin 2, respectively, have been reported to be causative genes of centronuclear myopathy (CNM), a hereditary degenerative disease of skeletal muscle, and deformation of T-tubules is often observed in the CNM patients. However, it remains unclear how BIN1 and dynamin 2 are implicated in T-tubule biogenesis and how mutations in these molecules cause CNM to develop. Here, using an in cellulo reconstitution assay, we demonstrate that dynamin 2 is required for stabilization of membranous structures equivalent to T-tubules. GTPase activity of wild-type dynamin 2 is suppressed through interaction with BIN1, whereas that of the disease-associated mutant dynamin 2 remains active due to lack of the BIN1-mediated regulation, thus causing aberrant membrane remodeling. Finally, we show that in cellulo aberrant membrane remodeling by mutant dynamin 2 variants is correlated with their enhanced membrane fission activities, and the results can explain severity of the symptoms in patients. Thus, this study provides molecular insights into dysregulated membrane remodeling triggering the pathogenesis of DNM2-related CNM.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Dinamina II/metabolismo , Músculo Esquelético/metabolismo , Miopatias Congênitas Estruturais/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Western Blotting , Dinamina II/genética , Células HEK293 , Humanos , Imunoprecipitação , Microscopia de Fluorescência , Miopatias Congênitas Estruturais/genética , Nanotubos/química , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Dynamin 2 is a GTPase protein that has been implicated in cancer progression through its various roles such as endocytosis, morphogenesis, epithelial-mesenchymal transition (EMT), cellular contractions, and focal adhesion maturation. The increased expression levels of this molecule have been demonstrated with the development of several cancers such as prostate, pancreas, and bladder. However, its clinical significance in breast cancer is unclear yet. In the present study, the membranous, cytoplasmic, and nuclear expression levels of dynamin 2 molecule were evaluated for the first time, using immunohistochemistry (IHC) on tissue microarray (TMA) slides in 113 invasive breast cancer tissues. Moreover, afterward, the association between the dynamin 2 expression and clinicopathological features was determined. Our finding showed that, a higher nuclear expression of dynamin 2 is significantly associated with an increase in tumor stage (P = 0.05), histological grade (P = 0.001), and age of the patients (P = 0.03). In addition, analysis of the cytoplasmic expression levels of this molecule revealed that, there was a statistically significant difference between the expression levels of dynamin 2 among the different breast cancer subtypes (P = 0.003). Moreover, a significant association was found between the increased expression of dynamin 2 membranous and vascular invasion (VI) (P = 0.02). We showed that dynamin 2 protein expression has an association with more aggressive tumor behavior and more advanced disease in the patients with breast cancer; therefore, dynamin 2 molecule could be considered as an indicator of disease progression and aggressiveness.