RESUMO
Secreted bacterial type III secretion system (T3SS) proteins are essential for successful infection by many human pathogens. Both T3SS translocator SipC and effector SipA are critical for Salmonella infection by subversion of the host cell cytoskeleton, but the precise molecular interplay between them remains unknown. Here, using cryo-electron microscopy, we show that SipA binds along the F-actin grooves with a unique binding pattern. SipA stabilizes F-actin through charged interface residues and appears to prevent inorganic phosphate release through closure of the "back door" of adenosine 5'-triphosphate pocket. We also show that SipC enhances the binding of SipA to F-actin, thus demonstrating that a sequential presence of T3SS proteins in host cells is associated with a sequence of infection events-starting with actin nucleation, filament growth, and stabilization. Together, our data explain the coordinated interplay of a precisely tuned and highly effective mechanism during Salmonella infection and provide a blueprint for interfering with Salmonella effectors acting on actin.
Assuntos
Actinas , Infecções por Salmonella , Humanos , Actinas/metabolismo , Microscopia Crioeletrônica , Proteínas de Bactérias/metabolismo , Citoesqueleto de Actina/metabolismoRESUMO
Cellular actin dynamic is controlled by a plethora of actin binding proteins (ABPs), including actin nucleating, bundling, cross-linking, capping, and severing proteins. In this review, regulation of actin dynamics by ABPs will be introduced, and the role of the F-actin severing protein cofilin-1 and the F-actin bundling protein L-plastin in actin dynamics discussed in more detail. Since up-regulation of these proteins in different kinds of cancers is associated with malignant progression of cancer cells, we suggest the cryogenic electron microscopy (Cryo-EM) structure of F- actin with the respective ABP as template for in silico drug design to specifically disrupt the interaction of these ABPs with F-actin.
Assuntos
Actinas , Proteínas dos Microfilamentos , Actinas/metabolismo , Microscopia Crioeletrônica , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Descoberta de Drogas , Ligação ProteicaRESUMO
Tumor plasticity is essential for adaptation to changing environmental conditions, in particular during the process of metastasis. In this study, we compared morphological and biochemical differences between LAN-1 neuroblastoma (NB) cells recovered from a subcutaneous xenograft primary tumor (PT) and the corresponding three generations of bone metastasis (BM I-III). Moreover, growth behavior, as well as the response to chemotherapy and immune cells were assessed. For this purpose, F-actin was stained, mRNA and protein expression assessed, and lactate secretion analyzed. Further, we measured adhesion to collagen I, the growth rate of spheroids in the presence and absence of vincristine, and the production of IL-6 by peripheral blood mononuclear cells (PBMCs) co-incubated with PT or BM I-III. Analysis of PT and the three BM generations revealed that their growth rate decreased from PT to BM III, and accordingly, PT cells reacted most sensitively to vincristine. In addition, morphology, adaption to hypoxic conditions, as well as transcriptomes showed strong differences between the cell lines. Moreover, BM I and BM II cells exhibited a significantly different ability to stimulate human immune cells compared to PT and BM III cells. Interestingly, the differences in immune cell stimulation corresponded to the expression level of the cancer-testis antigen MAGE-A3. In conclusion, our ex vivo model allows to analyze the adaption of tumor populations to different microenvironments and clearly demonstrates the strong alteration of tumor cell populations during this process.
RESUMO
Expression of Ins(1,4,5)P3-kinase-A (ITPKA), the neuronal isoform of Ins(1,4,5)P3-kinases, is up-regulated in many tumor types. In particular, in lung cancer cells this up-regulation is associated with bad prognosis and it has been shown that a high level of ITPKA increases migration and invasion of lung cancer cell lines. However, since ITPKA exhibits actin bundling and Ins(1,4,5)P3-kinase activity, it was not clear which of these activities account for ITPKA-promoted migration and invasion of cancer cells. To address this issue, we inhibited endogenous actin bundling activity of ITPKA in lung cancer H1299 cells by overexpressing the dominant negative mutant ITPKAL34P. Analysis of actin dynamics in filopodia as well as wound-healing migration revealed that ITPKAL34P inhibited both processes. Moreover, the formation of invasive protrusions into collagen I was strongly blocked in cells overexpressing ITPKAL34P. Furthermore, we found that ATP stimulation slightly but significantly (by 13%) increased migration of cells overexpressing ITPKA while under basal conditions up-regulation of ITPKA had no effect. In accordance with these results, overexpression of a catalytic inactive ITPKA mutant did not affect migration, and the Ins(1,4,5)P3-kinase-inhibitor GNF362 reversed the stimulating effect of ITPKA overexpression on migration. In summary, we demonstrate that under basal conditions the actin bundling activity controls ITPKA-facilitated migration and invasion and in presence of ATP the Ins(1,4,5)P3-kinase activity slightly enhances this effect.
Assuntos
Actinas , Neoplasias Pulmonares , Fosfotransferases (Aceptor do Grupo Álcool) , Humanos , Actinas/genética , Actinas/metabolismo , Trifosfato de Adenosina , Linhagem Celular Tumoral , Neoplasias Pulmonares/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
Breast cancer (BC) is the second-most common cause of brain metastases (BM) and BCBM patients have a reduced quality of life and a poor prognosis. Hyaluronan (HA), and in particular the hyaluronidase Hyal-1, has been already linked to the development of BCBM, and therefore presents an interesting opportunity to develop new effective therapeutic options. HA metabolism was further discovered by the CRISPR/Cas9-mediated knockout of HYAL1 and the shRNA-mediated down-regulation of HA-receptor CD44 in the brain-seeking triple-negative breast cancer (TNBC) cell line MDA-MB-231-BR. Therefore, the impact of Hyal-1 on adhesion, disruption, and invasion through the brain endothelium, both in vitro and in vivo, was studied. Our analysis points out a key role of Hyal-1 and low-molecular-weight HA (LMW-HA) in the formation of a pericellular HA-coat in BC cells, which in turn promotes tumor cell adhesion, disruption, and migration through the brain endothelium in vitro as well as the extent of BM in vivo. CD44 knockdown in MDA-MB-231-BR significantly reduced the pericellular HA-coat on these cells, and, consequently, tumor cell adhesion and invasion through the brain endothelium. Thus, the interaction between Hyal-1-generated LMW-HA fragments and the HA-receptor CD44 might represent a potential target for future therapeutic options in BC patients with a high risk of cerebral metastases formation.
Assuntos
Neoplasias Encefálicas , Ácido Hialurônico , Hialuronoglucosaminidase , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundário , Linhagem Celular Tumoral , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/genética , Hialuronoglucosaminidase/metabolismo , Qualidade de Vida , RNA Interferente Pequeno , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The chemotherapeutic agent paclitaxel (PTX) selectively binds to and stabilizes microtubule (MTs). Also, the activated formin Diaphanous Related Formin 1 (DIAPH1) binds to MTs and increases its stability. In a recent study, we found that high DIAPH1 levels correlated with increased survival of ovarian cancer (Ovca) patients. A possible explanation for this finding is that Ovca cells with high DIAPH1 levels are more sensitive to PTX. To examine this assumption, in this study the effect of DIAPH1 depletion on PTX-mediated cytotoxicity of OVCAR8 and OAW42 cells was analyzed. Our data showed that down-regulation of DIAPH1 expression decreased PTX sensitivity in both cell lines by reducing apoptosis or necrosis. Analysis of MT stability by Western blotting revealed a decreased concentration of stable, detyrosinated MTs in PTX-treated DIAPH1 knock-down compared to control cells. Also, in fixed metaphase cells the level of stable, detyrosinated spindle MTs decreased in cells with reduced DIAPH1 expression. In vitro analysis with recombinant DIAPH1 protein showed that PTX and DIAPH1 exhibited additive effects on MT-polymerization, showing that also in a cell-free system DIAPH1 increased the effect of PTX on MT-stability. Together, our data strongly indicate that DIAPH1 increases the response of Ovca cells to PTX by enhancing PTX-mediated MT-stability.
Assuntos
Antineoplásicos Fitogênicos/toxicidade , Apoptose/efeitos dos fármacos , Forminas/biossíntese , Neoplasias Ovarianas/metabolismo , Paclitaxel/toxicidade , Apoptose/fisiologia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológicoRESUMO
LIN28A overexpression has been identified in malignant brain tumors called embryonal tumors with multilayered rosettes (ETMR) but its specific role during brain development remains largely unknown. Radial glia cells of the ventricular zone (VZ) are proposed as a cell of origin for ETMR. We asked whether an overexpression of LIN28A in such cells might affect brain development or result in the formation of brain tumors.Constitutive overexpression of LIN28A in hGFAP-cre::lsl-Lin28A (GL) mice led to a transient increase of proliferation in the cortical VZ at embryonic stages but no postnatal brain tumor formation. Postnatally, GL mice displayed a pyramidal cell layer dispersion of the hippocampus and altered spine and dendrite morphology, including reduced dendritic spine densities in the hippocampus and cortex. GL mice displayed hyperkinetic activity and differential quantitative MS-based proteomics revealed altered time dependent molecular functions regarding mRNA processing and spine morphogenesis. Phosphoproteomic analyses indicated a downregulation of mTOR pathway modulated proteins such as Map1b being involved in microtubule dynamics.In conclusion, we show that Lin28A overexpression transiently increases proliferation of neural precursor cells but it is not sufficient to drive brain tumors in vivo. In contrast, Lin28A impacts on protein abundancy patterns related to spine morphogenesis and phosphorylation levels of proteins involved in microtubule dynamics, resulting in decreased spine densities of neurons in the hippocampus and cortex as well as in altered behavior. Our work provides new insights into the role of LIN28A for neuronal morphogenesis and development and may reveal future targets for treatment of ETMR patients.
Assuntos
Neoplasias Encefálicas/patologia , Neoplasias Embrionárias de Células Germinativas/metabolismo , Células-Tronco Neurais/metabolismo , Proteínas de Ligação a RNA/biossíntese , Medula Espinal/patologia , Animais , Proliferação de Células , Córtex Cerebral/patologia , Hipocampo/patologia , Humanos , Camundongos , Camundongos Transgênicos , Microtúbulos/patologia , Microtúbulos/ultraestrutura , Neoplasias Embrionárias de Células Germinativas/patologia , Células-Tronco Neurais/patologia , Proteômica , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Cancer testis antigen Melanoma associated antigen A3 (MAGE-A3) has been subject of research for many years. Being expressed in various tumor types and influencing proliferation, metastasis, and tumor pathogenicity, MAGE-A3 is an attractive target for cancer therapy, particularly because in healthy tissues, MAGE-A3 is only expressed in testes and placenta. MAGE-A3 acts as a cellular master regulator by stimulating E3 ubiquitin ligase tripartite motif-containing protein 28 (TRIM28), resulting in regulation of various cellular targets. These include tumor suppressor protein p53 and cellular energy sensor AMP-activated protein kinase (AMPK). The restricted expression of MAGE-A3 in tumor cells makes MAGE-A3 an attractive target for vaccine-based immune therapy. However, although phase I and phase II clinical trials involving MAGE-A3-specific immunotherapeutic interventions were promising, large phase III studies failed. This article gives an overview about the role of MAGE-A3 as a cellular master switch and discusses approaches to improve MAGE-A3-based immunotherapies.
Assuntos
Antígenos de Neoplasias/imunologia , Carcinogênese/imunologia , Imunoterapia/métodos , Proteínas de Neoplasias/imunologia , Neoplasias/imunologia , Células Neoplásicas Circulantes/imunologia , Proteínas Quinases Ativadas por AMP/imunologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Carcinogênese/metabolismo , Humanos , Imunoterapia/tendências , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , Células Neoplásicas Circulantes/metabolismo , Estrutura Terciária de ProteínaRESUMO
Overexpression of the neuronal InsP3kinase-A increases malignancy of different tumor types. Since InsP3kinase-A highly selectively binds Ins(1,4,5)P3, small molecules competing with Ins(1,4,5)P3 provide a promising approach for the therapeutic targeting of InsP3kinase-A. Based on this consideration, we analyzed the binding mechanism of BIP-4 (2-[3,5-dimethyl-1-(4-nitrophenyl)-1H-pyrazol-4-yl]-5, 8-dinitro-1H-benzo[de]isoquinoline-1,3(2H)-dione), a known competitive small-molecule inhibitor of Ins(1,4,5)P3. We tested a total of 80 BIP-4 related compounds in biochemical assays. The results of these experiments revealed that neither the nitrophenyl nor the benzisochinoline group inhibited InsP3kinase-A activity. Moreover, none of the BIP-4 related compounds competed for Ins(1,4,5)P3, demonstrating the high selectivity of BIP-4. To analyze the inhibition mechanism of BIP-4, mutagenesis experiments were performed. The results of these experiments suggest that the nitro groups attached to the benzisochinoline ring compete for binding of Ins(1,4,5)P3 while the nitrophenyl group is associated with amino acids of the ATP-binding pocket. Our results now offer the possibility to optimize BIP-4 to design specific InsP3Kinase-A inhibitors suitable for therapeutic targeting of the enzyme.
Assuntos
Trifosfato de Adenosina/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Naftalimidas/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Pirazóis/farmacologia , Ligação Competitiva , Domínio Catalítico , Desenho Assistido por Computador , Inibidores Enzimáticos/química , Cinética , Simulação de Acoplamento Molecular , Estrutura Molecular , Mutação , Naftalimidas/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Pirazóis/química , Relação Estrutura-AtividadeRESUMO
Chromosomal instability (CIN) is a hallmark of cancer, resulting from misalignment and missegregation of chromosomes during meta- and anaphase, due to non-precise regulation of spindle-MT dynamics. Diaphanous Related Formin 1 (DIAPH1) is an actin nucleator and also binds microtubule (MT) with high affinity. In this study, we analyzed the role of DIAPH1 in regulation of spindle MT-dynamics and CIN in HT29 and HCT-116 colorectal cancer (CRC) cells. Our data show that down-regulation of DIAPH1 in these cell lines decreased spindle-MT speed by 50 % and the fraction of cells with misaligned and missegregated chromosomes was significantly increased. Furthermore, in HCT-116 DIAPH1 depleted cells deviation of chromosome number was elevated and the number of cells with micronuclei and cytosolic DNA was increased in both DIAPH1-knock down cell lines. In line with these results, database analysis revealed a significant correlation with low DIAPH1 mRNA expression and aneuploidy. Thus, DIAPH1 is substantially involved in the control of CIN in CRC cells. Since in vitro, DIAPH1 directly increased MT-polymerization, we assume that DIAPH1 controls CIN by regulating spindle-MT dynamics.
Assuntos
Neoplasias do Colo/metabolismo , Forminas/metabolismo , Microtúbulos/metabolismo , Linhagem Celular Tumoral , Instabilidade Cromossômica/genética , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , HumanosRESUMO
T cell activation starts with formation of second messengers that release Ca2+ from the endoplasmic reticulum (ER) and thereby activate store-operated Ca2+ entry (SOCE), one of the essential signals for T cell activation. Recently, the steroidal 2-methoxyestradiol was shown to inhibit nuclear translocation of the nuclear factor of activated T cells (NFAT). We therefore investigated 2-methoxyestradiol for inhibition of Ca2+ entry in T cells, screened a library of 2-methoxyestradiol analogues, and characterized the derivative 2-ethyl-3-sulfamoyloxy-17ß-cyanomethylestra-1,3,5(10)-triene (STX564) as a novel, potent and specific SOCE inhibitor. STX564 inhibits Ca2+ entry via SOCE without affecting other ion channels and pumps involved in Ca2+ signaling in T cells. Downstream effects such as cytokine expression and cell proliferation were also inhibited by both 2-methoxyestradiol and STX564, which has potential as a new chemical biology tool.
Assuntos
2-Metoxiestradiol/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Estrenos/farmacologia , Fatores de Transcrição NFATC/metabolismo , Linfócitos T/citologia , 2-Metoxiestradiol/análogos & derivados , Animais , Cálcio/metabolismo , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Estrenos/síntese química , Estrenos/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ratos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
BACKGROUND: The survival rate is poor in breast cancer patients with brain metastases. Thus, new concepts for therapeutic approaches are required. During metastasis, the cytoskeleton of cancer cells is highly dynamic and therefore cytoskeleton-associated proteins are interesting targets for tumour therapy. METHODS: Screening for genes showing a significant correlation with brain metastasis formation was performed based on microarray data from breast cancer patients with long-term follow up information. Validation of the most interesting target was performed by MTT-, Scratch- and Transwell-assay. In addition, intracellular trafficking was analyzed by live-cell imaging for secretory vesicles, early endosomes and multiple vesicular bodies (MVB) generating extracellular vesicles (EVs). EVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), Western blotting, mass spectrometry, and ingenuity pathway analysis (IPA). Effect of EVs on the blood-brain-barrier (BBB) was examined by incubating endothelial cells of the BBB (hCMEC/D3) with EVs, and permeability as well as adhesion of breast cancer cells were analyzed. Clinical data of a breast cancer cohort was evaluated by χ2-tests, Kaplan-Meier-Analysis, and log-rank tests while for experimental data Student's T-test was performed. RESULTS: Among those genes exhibiting a significant association with cerebral metastasis development, the only gene coding for a cytoskeleton-associated protein was Tubulin Tyrosine Ligase Like 4 (TTLL4). Overexpression of TTLL4 (TTLL4plus) in MDA-MB231 and MDA-MB468 breast cancer cells (TTLL4plus cells) significantly increased polyglutamylation of ß-tubulin. Moreover, trafficking of secretory vesicles and MVBs was increased in TTLL4plus cells. EVs derived from TTLL4plus cells promote adhesion of MDA-MB231 and MDA-MB468 cells to hCMEC/D3 cells and increase permeability of hCMEC/D3 cell layer. CONCLUSIONS: These data suggest that TTLL4-mediated microtubule polyglutamylation alters exosome homeostasis by regulating trafficking of MVBs. The TTLL4plus-derived EVs may provide a pre-metastatic niche for breast cancer cells by manipulating endothelial cells of the BBB.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias da Mama/genética , Exossomos/genética , Peptídeo Sintases/genética , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/secundário , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Citoesqueleto/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Vesículas Extracelulares/genética , Feminino , Humanos , Metástase Neoplásica , Peptídeos/genéticaRESUMO
Inositol-1,4,5-trisphosphate 3-kinase-A (ITPKA) is the neuronal isoform of ITPKs and exhibits both actin bundling and InsP3kinase activity. In addition to neurons, ITPKA is ectopically expressed in tumor cells, where its oncogenic activity increases tumor cell malignancy. In order to analyze the physiological relevance of ITPKA, here we performed a broad phenotypic screening of itpka deficient mice. Our data show that among the neurobehavioral tests analyzed, itpka deficient mice reacted faster to a hotplate, prepulse inhibition was impaired and the accelerating rotarod test showed decreased latency of itpka deficient mice to fall. These data indicate that ITPKA is involved in the regulation of nociceptive pathways, sensorimotor gating and motor learning. Analysis of extracerebral functions in control and itpka deficient mice revealed significantly reduced glucose, lactate, and triglyceride plasma concentrations in itpka deficient mice. Based on this finding, expression of ITPKA was analyzed in extracerebral tissues and the highest level was found in the small intestine. However, functional studies on CaCo-2 control and ITPKA depleted cells showed that glucose, as well as triglyceride uptake, were not significantly different between the cell lines. Altogether, these data show that ITPKA exhibits distinct functions in the central nervous system and reveal an involvement of ITPKA in energy metabolism.
Assuntos
Neurônios/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Inibição Pré-Pulso/fisiologia , Animais , Células CACO-2 , Feminino , Humanos , Isoenzimas/deficiência , Isoenzimas/genética , Masculino , Camundongos , Camundongos Knockout , Fosfotransferases (Aceptor do Grupo Álcool)/genéticaRESUMO
The majority of cancer-related deaths are due to hematogenous metastases, and the bone marrow (BM) represents one of the most frequent metastatic sites. To study BM metastasis formation in vivo, the most efficient approach is based on intracardiac injection of human tumor cells into immunodeficient mice. However, such a procedure circumvents the early steps of the metastatic cascade. Here we describe the development of xenograft mouse models (balb/c rag2-/- and severe combined immunodeficient (SCID)), in which BM metastases are spontaneously derived from subcutaneous (s.c.) primary tumors (PTs). As verified by histology, the described methodology including ex vivo bioluminescence imaging (BLI) even enabled the detection of micrometastases in the BM. Furthermore, we established sublines from xenograft primary tumors (PTs) and corresponding BM (BM) metastases using LAN-1 neuroblastoma xenografts as a first example. In vitro "metastasis" assays (viability, proliferation, transmigration, invasion, colony formation) partially indicated pro-metastatic features of the LAN-1-BM compared to the LAN-1-PT subline. Unexpectedly, after s.c. re-injection into mice, LAN-1-BM xenografts developed spontaneous BM metastases less frequently than LAN-1-PT xenografts. This study provides a novel methodologic approach for modelling the spontaneous metastatic cascade of human BM metastasis formation in mice. Moreover, our data indicate that putative bone-metastatic features get rapidly lost upon routine cell culture.
RESUMO
SHIP1 is an inositol 5-phosphatase which is well established for its tumour suppressor potential in leukaemia. Enzymatically, two SHIP1 substrates, PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4 have been identified to date. Additional substrates were found for the homologue SHIP2. In this study, we identified new inositol phosphate (InsP) substrates of SHIP1 by metal dye detection high-performance liquid chromatography and compared the substrate profiles of SHIP1 and SHIP2. We were able to verify Ins(1,3,4,5)P4 as a substrate of SHIP1 and interestingly found Ins(1,2,3,4,5)P5 and Ins(2,3,4,5)P4 to be preferably used as substrates and Ins(1,4,5,6)P4 and Ins(2,4,5,6)P4 to be weak substrates. All of those except Ins(2,3,4,5)P4 are also known substrates of SHIP2 indicating a possible exclusive role of Ins(2,3,4,5)P4 hydrolysis for SHIP1 but not SHIP2 function.
Assuntos
Fosfatos de Inositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Ensaios Enzimáticos , Humanos , Fosfatos de Inositol/química , Cinética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Dendritic arborization is highly regulated and requires tight control of dendritic growth, branching, cytoskeletal dynamics, and ion channel expression to ensure proper function. Abnormal dendritic development can result in altered network connectivity, which has been linked to neurodevelopmental disorders, including autism spectrum disorders (ASDs). How neuronal growth control programs tune dendritic arborization to ensure function is still not fully understood. Using Drosophila dendritic arborization (da) neurons as a model, we identified the conserved Ste20-like kinase Tao as a negative regulator of dendritic arborization. We show that Tao kinase activity regulates cytoskeletal dynamics and sensory channel localization required for proper sensory function in both male and female flies. We further provide evidence for functional conservation of Tao kinase, showing that its ASD-linked human ortholog, Tao kinase 2 (Taok2), could replace Drosophila Tao and rescue dendritic branching, dynamic microtubule alterations, and behavioral defects. However, several ASD-linked Taok2 variants displayed impaired rescue activity, suggesting that Tao/Taok2 mutations can disrupt sensory neuron development and function. Consistently, we show that Tao kinase activity is required in developing and as well as adult stages for maintaining normal dendritic arborization and sensory function to regulate escape and social behavior. Our data suggest an important role for Tao kinase signaling in cytoskeletal organization to maintain proper dendritic arborization and sensory function, providing a strong link between developmental sensory aberrations and behavioral abnormalities relevant for Taok2-dependent ASDs.SIGNIFICANCE STATEMENT Autism spectrum disorders (ASDs) are linked to abnormal dendritic arbors. However, the mechanisms of how dendritic arbors develop to promote functional and proper behavior are unclear. We identified Drosophila Tao kinase, the ortholog of the ASD risk gene Taok2, as a regulator of dendritic arborization in sensory neurons. We show that Tao kinase regulates cytoskeletal dynamics, controls sensory ion channel localization, and is required to maintain somatosensory function in vivo Interestingly, ASD-linked human Taok2 mutations rendered it nonfunctional, whereas its WT form could restore neuronal morphology and function in Drosophila lacking endogenous Tao. Our findings provide evidence for a conserved role of Tao kinase in dendritic development and function of sensory neurons, suggesting that aberrant sensory function might be a common feature of ASDs.
Assuntos
Citoesqueleto/fisiologia , Dendritos/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Sensação/fisiologia , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Citoesqueleto/ultraestrutura , Dendritos/ultraestrutura , Drosophila , Reação de Fuga , Feminino , Humanos , Masculino , Mecanorreceptores/fisiologia , Mutação/genética , Comportamento SocialRESUMO
Although inactivating mutations of PLS3, encoding the actin-bundling protein plastin-3, have been identified to cause X-linked osteoporosis, the cellular and molecular influence of PLS3 on bone remodeling is poorly defined. Moreover, although a previous study has demonstrated moderate osteopenia in 12 week-old Pls3-deficient mice based on µCT scanning, there is no reported analysis of such a model on the basis of undecalcified histology and bone-specific histomorphometry. To fill this knowledge gap we applied a deep phenotyping approach and studied Pls3-deficient mice at different ages. Surprisingly, we did not detect significant differences between wildtype and Pls3-deficient littermates with respect to trabecular bone mass, and the same was the case for all histomorphometric parameters determined at 12 weeks of age. Remarkably however, the cortical thickness in both, tibia and femur, was significantly reduced in Pls3-deficient mice in all age groups. We additionally studied the ex vivo behavior of Pls3-deficient primary osteoblasts, which displayed moderately impaired mineralization capacity. Of note, while most osteoblastogenesis markers were not differentially expressed between wildtype and Pls3-deficient cultures, the expression of Sfrp4 was significantly reduced in the latter, a potentially relevant finding, since Sfrp4 inactivation, in mice and humans, specifically causes cortical thinning. We finally addressed the question, if Pls3-deficiency would impair the osteoanabolic influence of parathyroid hormone (PTH). For this purpose we applied daily injection of PTH into wildtype and Pls3-deficient mice and found a similar response regardless of the genotype. Taken together, our data reveal that Pls3-deficiency in mice only recapitulates the cortical bone phenotype of individuals with X-linked osteoporosis by negatively affecting the early stage of cortical bone acquisition.
Assuntos
Densidade Óssea , Osteoporose , Animais , Densidade Óssea/genética , Osso Cortical/diagnóstico por imagem , Glicoproteínas de Membrana , Camundongos , Proteínas dos Microfilamentos/genética , Osteoporose/genéticaRESUMO
Leukemia-initiating cells reside within the bone marrow in specialized niches where they undergo complex interactions with their surrounding stromal cells. We have identified the actin-binding protein Plastin-3 (PLS3) as potential player within the leukemic bone marrow niche and investigated its functional role in acute myeloid leukemia. High expression of PLS3 was associated with a poor overall and event-free survival for AML patients. These findings were supported by functional in vitro and in vivo experiments. AML cells with a PLS3 knockdown showed significantly reduced colony numbers in vitro while the PLS3 overexpression variants resulted in significantly enhanced colony numbers compared to their respective controls. Furthermore, the survival of NSG mice transplanted with the PLS3 knockdown cells showed a significantly prolonged survival in comparison to mice transplanted with the control AML cells. Further studies should focus on the underlying leukemia-promoting mechanisms and investigate PLS3 as therapeutic target.
RESUMO
The centrosome is thought to be the major neuronal microtubule-organizing center (MTOC) in early neuronal development, producing microtubules with a radial organization. In addition, albeit in vitro, recent work showed that isolated centrosomes could serve as an actin-organizing center, raising the possibility that neuronal development may, in addition, require a centrosome-based actin radial organization. Here, we report, using super-resolution microscopy and live-cell imaging of cultured rodent neurons, F-actin organization around the centrosome with dynamic F-actin aster-like structures with F-actin fibers extending and retracting actively. Photoactivation/photoconversion experiments and molecular manipulations of F-actin stability reveal a robust flux of somatic F-actin toward the cell periphery. Finally, we show that somatic F-actin intermingles with centrosomal PCM-1 (pericentriolar material 1 protein) satellites. Knockdown of PCM-1 and disruption of centrosomal activity not only affect F-actin dynamics near the centrosome but also in distal growth cones. Collectively, the data show a radial F-actin organization during early neuronal development, which might be a cellular mechanism for providing peripheral regions with a fast and continuous source of actin polymers, hence sustaining initial neuronal development.
Assuntos
Actinas/metabolismo , Cones de Crescimento/metabolismo , Neurogênese , Animais , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Centrossomo/metabolismo , Hipocampo/citologia , Hipocampo/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , RatosRESUMO
In this study, we analyzed the functional role of the formin Drosophila Homologue of Diaphanous2 (Diaph2) in colorectal cancer cells. We show that stable down-regulation of Diaph2 expression in HT29 cells decreased chromosome alignment and the velocity of chromosome movement during M-phase, thus reducing the proliferation rate and colony formation. In interphase cells, Diaph2 was diffusely distributed in the cytosol, while in metaphase cells the protein was located to spindle microtubules (MTs). Diaph2-depletion increased the concentration of stable spindle MTs, showing that the formin is required to control spindle MT-dynamics. Our cellular data indicate that Diaph2-controls spindle MT-dynamics independent of Cdc42 activity and our in vitro results reveal that bacterially produced full-length (FL) Diaph2 strongly altered MT-dynamics in absence of Cdc42, where its actin-nucleating activity is auto-inhibited. FL-Diaph2 mediates a 10-fold increase in MT-polymerization compared to the Diaph2-FH2-domain. Interestingly, a Diaph2-mutant lacking the FH2-domain (ΔFH2) increased MT-polymerization to a similar extent as the FH2-domain, indicating the existence of a second MT-binding domain. However, in contrast to FL-Diaph2 and the FH2-domain, ΔFH2 did not alter the density of taxol-stabilized MTs. Thus, the FH2-domain and the second Diaph2-binding domain appear to control MT-dynamics by different mechanisms. In summary, our data indicate that Diaph2 controls M-phase progression under basal conditions by regulating spindle MT-dynamics. In addition, a region outside of the canonical MT-regulating FH2-domain is involved in Diaph2-mediated control of MT-dynamics.