RESUMEN
Gametophyte development is a pre-requisite for plant reproduction and seed yield; therefore, studies of gametophyte development help us understand fundamental biological questions and have potential applications in agriculture. The biogenesis and dynamics of endomembrane compartments are critical for cell survival, and their regulatory mechanisms are just beginning to be revealed. Here, we report that the Arabidopsis thaliana SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) protein YKT61 is essential for both male and female gametogenesis. By using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based genome editing, we demonstrated that male and female gametophytes carrying YKT61 loss-of-function alleles do not survive. Specifically, loss of YKT61 function resulted in the arrest of male gametophytic development at pollen mitosis I and the degeneration of female gametophytes. A three-base-pair deletion in YKT61 in the ykt61-3 mutant resulted in a single-amino acid deletion in the longin domain of YKT61; the resulting mutant protein does not interact with multiple SNAREs and showed substantially reduced membrane association, suggesting that the N-terminal longin domain of YKT61 plays multiple roles in its function. This study demonstrates that Arabidopsis YKT61 is essential for male and female gametogenesis and sets an example for functional characterization of essential genes with the combination of Cas9-mediated editing and expression from a Cas9-resistant transgene.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Polen/metabolismo , Proteínas R-SNARE/metabolismo , Alelos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Polen/genética , Tubo Polínico/genética , Tubo Polínico/metabolismo , Proteínas R-SNARE/genéticaRESUMEN
Increasing evidences reveal that autophagy inhibitor could enhance the effect of chemotherapy to cancer. However, few autophagy inhibitors are currently approved for clinical application in humans. Berbamine (BBM) is a natural compound extracted from traditional Chinese medicine that is widely used for treatment of a variety of diseases without any obvious side effects. Here we found that BBM is a novel auophagy inhibitor, which potently induced the accumulation of autophagosomes by inhibiting autophagosome-lysosome fusion in human breast cancer cells. Mechanistically, we found that BBM blocked autophagosome-lysosome fusion by inhibiting the interaction of SNAP29 and VAMP8. Furthermore, BBM induced upregulation of BNIP3 and the interaction between SNAP29 and BNIP3. BNIP3 depletion or SNAP29 overexpression abrogated BBM-mediated blockade of autophagosome-lysosome fusion through the interaction between SNAP29 and VAMP8, whereas BNIP3 overexpression blocked autophagosome-lysosome fusion through inhibition of the interaction between SNAP29 and VAMP8. These findings suggest that upregulation of BNIP3 and interaction between BNIP3 and SNAP29 could be involved in BBM-mediated blockade of autophagosome-lysosome fusion through inhibition of the interaction between SNAP29 and VAMP8. Our findings identify the critical role of BNIP3 in blockade of autophagosome-lysosome fusion mediated by BBM, and suggest that BBM could potentially be further developed as a novel autophagy inhibitor, which could enhance the effect of chemotherapy to cancer.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Autofagia/efectos de los fármacos , Bencilisoquinolinas/farmacología , Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genética , Proteínas R-SNARE/genética , Células A549 , Autofagosomas/metabolismo , Autofagosomas/virología , Autofagia/genética , Línea Celular Tumoral , Regulación de la Expresión Génica , Humanos , Lisosomas/metabolismo , Lisosomas/virología , Células MCF-7 , Fusión de Membrana/efectos de los fármacos , Proteínas de la Membrana/agonistas , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Proto-Oncogénicas/agonistas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND/AIMS: Electroconvulsive therapy (ECT) is an effective treatment modality for severe psychiatric disorders. Many studies have suggested that the therapeutic efficacy of ECT can be attributed to the structural and functional readjustment of the brain cells, which is mediated by differential gene expression in the brain. The aim of this study is to understand the molecular mechanism of ECT. METHODS: We used microarray-based gene expression profiling technology and real-time quantitative PCR (RT-qPCR) to screen differentially expressed genes in the brain in a rat model of ECT. RESULTS: Four upregulated and three downregulated genes were identified in this study. The 4 upregulated genes are S100 protein, beta polypeptide (S100b), S100 calcium binding protein A13_predicted (S100a13_predicted), diazepam-binding inhibitor (Dbi), and YKT6 homolog (S. Cerevisiae) (Ykt6), respectively; while the 3 downregulated genes are basigin (Bsg), histidine triad nucleotide binding protein 1(Hint 1), and neural precursor cell expressed, developmentally downregulated gene 8 (Nedd8), respectively. CONCLUSION: In view of the neurobiological function of these genes and their relevance to mental disorders, repeated ECS can affect gene expression involved in the neurotransmission and synaptic plasticity, which may account for the clinical effects of ECT.
Asunto(s)
Terapia Electroconvulsiva , Lóbulo Frontal/metabolismo , Regulación de la Expresión Génica , Animales , Basigina/genética , Basigina/metabolismo , Inhibidor de la Unión a Diazepam/metabolismo , Regulación hacia Abajo , Perfilación de la Expresión Génica , Masculino , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas S100/genética , Proteínas S100/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , Regulación hacia ArribaRESUMEN
Although much is known regarding the exocytic responses of mast cells following allergen/IgE-mediated activation, little is currently known of the fate of the activating allergens, many of which are particles. We have found that IgE-bound particulate allergens were phagocytosed by activated mast cells in a lipid raft-dependent manner. The nascent allergen-containing phagosomes were found to transform into granule compartments by acquiring VAMP7 and serotonin and exhibited the capacity to empty their contents upon mast cell activation. When allergen-harboring mast cells were stimulated, the intracellular allergens were expelled intact and shown to activate adjacent mast cells. This capacity of mast cells to phagocytose and retain whole and antigenically intact allergens could potentially contribute to the course of inflammatory diseases such as asthma.