Browsing by MeSH term "Microscopy, Electron, Transmission"
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Publication Combined deficiency of Notch1 and Notch3 causes pericyte dysfunction, models CADASIL, and results in arteriovenous malformations.(Nature Publishing Group, 2015-11-13) Kofler, Natalie M; Cuervo, Henar; Uh, Minji K; Murtomäki, Aino; Kitajewski, JanPericytes regulate vessel stability and pericyte dysfunction contributes to retinopathies, stroke, and cancer. Here we define Notch as a key regulator of pericyte function during angiogenesis. In Notch1(+/-); Notch3(-/-) mice, combined deficiency of Notch1 and Notch3 altered pericyte interaction with the endothelium and reduced pericyte coverage of the retinal vasculature. Notch1 and Notch3 were shown to cooperate to promote proper vascular basement membrane formation and contribute to endothelial cell quiescence. Accordingly, loss of pericyte function due to Notch deficiency exacerbates endothelial cell activation caused by Notch1 haploinsufficiency. Mice mutant for Notch1 and Notch3 develop arteriovenous malformations and display hallmarks of the ischemic stroke disease CADASIL. Thus, Notch deficiency compromises pericyte function and contributes to vascular pathologies.Publication Energetics and geometry of FtsZ polymers: nucleated self-assembly of single protofilaments.(Cell Press, 2008-03-01) Huecas, Sonia; Llorca, Oscar; Boskovic, Jasminka; Martín-Benito, Jaime; Valpuesta, José María; Andreu, José ManuelEssential cell division protein FtsZ is an assembling GTPase which directs the cytokinetic ring formation in dividing bacterial cells. FtsZ shares the structural fold of eukaryotic tubulin and assembles forming tubulin-like protofilaments, but does not form microtubules. Two puzzling problems in FtsZ assembly are the nature of protofilament association and a possible mechanism for nucleated self-assembly of single-stranded protofilaments above a critical FtsZ concentration. We assembled two-dimensional arrays of FtsZ on carbon supports, studied linear polymers of FtsZ with cryo-electron microscopy of vitrified unsupported solutions, and formulated possible polymerization models. Nucleated self-assembly of FtsZ from Escherichia coli with GTP and magnesium produces flexible filaments 4-6 nm-wide, only compatible with a single protofilament. This agrees with previous scanning transmission electron microscopy results and is supported by recent cryo-electron tomography studies of two bacterial cells. Observations of double-stranded FtsZ filaments in negative stain may come from protofilament accretion on the carbon support. Preferential protofilament cyclization does not apply to FtsZ assembly. The apparently cooperative polymerization of a single protofilament with identical intermonomer contacts is explained by the switching of one inactive monomer into the active structure preceding association of the next, creating a dimer nucleus. FtsZ behaves as a cooperative linear assembly machine.Publication IIIG9 inhibition in adult ependymal cells changes adherens junctions structure and induces cellular detachment(Springer, 2021-09-17) Baeza, Victor; Cifuentes, Manuel; Martínez, Fernando; Ramírez, Eder; Nualart, Francisco; Ferrada, Luciano; Oviedo, María José; De Lima, Isabelle; Troncoso, Ninoschka; Saldivia, Natalia; Salazar, Katterine; [Baeza,V; Martínez,F; Ramírez,E; Nualart,F; Oviedo,MJ; De Lima,I; Troncoso,N; Saldivia,N; Salazar,K] Laboratory of Neurobiology and Stem Cells, NeuroCellT, Department of Cellular Biology, Faculty of Biological Sciences, University of Concepcion, Concepcion, Chile. [Nualart,F; Ferrada,L; Salazar,K] Faculty of Biological Sciences, Center for Advanced Microscopy CMA BIOBIO, University of Concepcion, Concepcion, Chile. [Cifuentes,M] Department of Cell Biology, Genetics and Physiology, University of Malaga, IBIMA, Malaga, Spain. [Cifuentes,M] Andalusian Center for Nanomedicine and Biotechnology, BIONAND, Malaga, Spain. [Cifuentes,M] Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, Malaga, Spain.Ependymal cells have multiple apical cilia that line the ventricular surfaces and the central canal of spinal cord. In cancer, the loss of ependymal cell polarity promotes the formation of different types of tumors, such as supratentorial anaplastic ependymomas, which are highly aggressive in children. IIIG9 (PPP1R32) is a protein restricted to adult ependymal cells located in cilia and in the apical cytoplasm and has unknown function. In this work, we studied the expression and localization of IIIG9 in the adherens junctions (cadherin/β-catenin-positive junctions) of adult brain ependymal cells using confocal and transmission electron microscopy. Through in vivo loss-of-function studies, ependymal denudation (single-dose injection experiments of inhibitory adenovirus) was observed, inducing the formation of ependymal cells with a "balloon-like" morphology. These cells had reduced cadherin expression (and/or delocalization) and cleavage of the cell death marker caspase-3, with "cilia rigidity" morphology (probably vibrational beating activity) and ventriculomegaly occurring prior to these events. Finally, after performing continuous infusions of adenovirus for 14 days, we observed total cell denudation and reactive parenchymal astrogliosis. Our data confirmed that IIIG9 is essential for the maintenance of adherens junctions of polarized ependymal cells. Eventually, altered levels of this protein in ependymal cell differentiation may increase ventricular pathologies, such as hydrocephalus or neoplastic transformation.Publication Inactivation of the Thymidylate Synthase thyA in Non-typeable Haemophilus influenzae Modulates Antibiotic Resistance and Has a Strong Impact on Its Interplay with the Host Airways(Frontiers Media, 2017-06) Rodríguez-Arce, Irene; Martí, Sara; Euba, Begoña; Fernández-Calvet, Ariadna; Moleres, Javier; López-López, Nahikari; Barberán, Montserrat; Ramos-Vivas, José; Tubau, Fe; Losa, Carmen; Ardanuy, Carmen; Leiva, José; Yuste, Jose Enrique; Garmendia, Junkal; Ministerio de Economía y Competitividad (España); Comunidad Foral de Navarra (España); Sociedad Española de Neumología y Cirugía TorácicaAntibacterial treatment with cotrimoxazol (TxS), a combination of trimethoprim and sulfamethoxazole, generates resistance by, among others, acquisition of thymidine auxotrophy associated with mutations in the thymidylate synthase gene thyA, which can modify the biology of infection. The opportunistic pathogen non-typeable Haemophilus influenzae (NTHi) is frequently encountered in the lower airways of chronic obstructive pulmonary disease (COPD) patients, and associated with acute exacerbation of COPD symptoms. Increasing resistance of NTHi to TxS limits its suitability as initial antibacterial against COPD exacerbation, although its relationship with thymidine auxotrophy is unknown. In this study, the analysis of 2,542 NTHi isolates recovered at Bellvitge University Hospital (Spain) in the period 2010-2014 revealed 119 strains forming slow-growing colonies on the thymidine low concentration medium Mueller Hinton Fastidious, including one strain isolated from a COPD patient undergoing TxS therapy that was a reversible thymidine auxotroph. To assess the impact of thymidine auxotrophy in the NTHi-host interplay during respiratory infection, thyA mutants were generated in both the clinical isolate NTHi375 and the reference strain RdKW20. Inactivation of the thyA gene increased TxS resistance, but also promoted morphological changes consistent with elongation and impaired bacterial division, which altered H. influenzae self-aggregation, phosphorylcholine level, C3b deposition, and airway epithelial infection patterns. Availability of external thymidine contributed to overcome such auxotrophy and TxS effect, potentially facilitated by the nucleoside transporter nupC. Although, thyA inactivation resulted in bacterial attenuation in a lung infection mouse model, it also rendered a lower clearance upon a TxS challenge in vivo. Thus, our results show that thymidine auxotrophy modulates both the NTHi host airway interplay and antibiotic resistance, which should be considered at the clinical setting for the consequences of TxS administration.Publication Plk1 regulates contraction of postmitotic smooth muscle cells and is required for vascular homeostasis(Nature Publishing Group, 2017-08) de Carcer Diez, Guillermo; Wachowicz, Paulina; Martinez-Martinez, Sara; Oller, Jorge; Mendez-Barbero, Nerea; Escobar, Beatriz; González-Loyola, Alejandra; Takaki, Tohru; El Bakkali, Aicha; Cámara, Juan A; Jimenez-Borreguero, Luis J.; Bustelo, Xosé R; Cañamero, Marta; Mulero, Francisca; de Los Ángeles Sevilla, María; Montero, María Jose; Redondo, Juan Miguel; Malumbres Martinez, Marcos; Unión Europea. Comisión Europea; Ministerio de Economía y Competitividad (España); Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF); Fundación La Marató TV3; Junta de Castilla y León (España); Fundación Ramón Areces; Fundación Solorzano; Comunidad de Madrid (España); Worldwide Cancer Research; Fundación ProCNICPolo-like kinase 1 (PLK1), an essential regulator of cell division, is currently undergoing clinical evaluation as a target for cancer therapy. We report an unexpected function of Plk1 in sustaining cardiovascular homeostasis. Plk1 haploinsufficiency in mice did not induce obvious cell proliferation defects but did result in arterial structural alterations, which frequently led to aortic rupture and death. Specific ablation of Plk1 in vascular smooth muscle cells (VSMCs) led to reduced arterial elasticity, hypotension, and an impaired arterial response to angiotensin II in vivo. Mechanistically, we found that Plk1 regulated angiotensin II-dependent activation of RhoA and actomyosin dynamics in VSMCs in a mitosis-independent manner. This regulation depended on Plk1 kinase activity, and the administration of small-molecule Plk1 inhibitors to angiotensin II-treated mice led to reduced arterial fitness and an elevated risk of aneurysm and aortic rupture. We thus conclude that a partial reduction of Plk1 activity that does not block cell division can nevertheless impair aortic homeostasis. Our findings have potentially important implications for current approaches aimed at PLK1 inhibition for cancer therapy.Publication RAB7 counteracts PI3K-driven macropinocytosis activated at early stages of melanoma development.(Impact Journals, 2015-05-20) Osterloh, Lisa; Martínez-Herranz, Raúl; Riveiro-Falkenbach, Erica; Romero, Pablo-Ortiz; Rodríguez-Peralto, José Luis; Pastor Fernandez, Joaquin; Soengas, MS; Ministerio de Economía y Competitividad (España); Instituto de Salud Carlos III; Fundación La Caixa; Asociación Española Contra el Cáncer; Melanoma Research AllianceDerailed endolysosomal trafficking is emerging as a widespread feature of aggressive neoplasms. However, the oncogenic signals that alter membrane homeostasis and their specific contribution to cancer progression remain unclear. Understanding the upstream drivers and downstream regulators of aberrant vesicular trafficking is distinctly important in melanoma. This disease is notorious for its inter- and intra-tumoral heterogeneity. Nevertheless, melanomas uniformly overexpress a cluster of endolysosomal genes, being particularly addicted to the membrane traffic regulator RAB7. Still, the underlying mechanisms and temporal determinants of this dependency have yet to be defined. Here we addressed these questions by combining electron microscopy, real time imaging and mechanistic analyses of vesicular trafficking in normal and malignant human melanocytic cells. This strategy revealed Class I PI3K as the key trigger of a hyperactive influx of macropinosomes that melanoma cells counteract via RAB7-mediated lysosomal degradation. In addition, gain- and loss-of-function in vitro studies followed by histopathological validation in clinical biopsies and genetically-engineered mouse models, traced back the requirement of RAB7 to the suppression of premature cellular senescence traits elicited in melanocytes by PI3K-inducing oncogenes. Together, these results provide new insight into the regulators and modes of action of RAB7, broadening the impact of endosomal fitness on melanoma development.Publication Treatment of skeletal and non-skeletal alterations of Mucopolysaccharidosis type IVA by AAV-mediated gene therapy.(Nature Publishing Group, 2021-09-09) Bertolin, Joan; Sánchez, Víctor; Ribera, Albert; Jaén, Maria Luisa; Garcia, Miquel; Pujol, Anna; Sánchez, Xavier; Muñoz, Sergio; Marcó, Sara; Pérez, Jennifer; Elias, Gemma; León, Xavier; Roca, Carles; Jimenez, Veronica; Otaegui, Pedro; Navarro, Marc; Ruberte, Jesús; Bosch, Fatima; Mulero, Francisca; Ministerio de Ciencia, Innovación y Universidades (España); Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF); Government of Catalonia (España)Mucopolysaccharidosis type IVA (MPSIVA) or Morquio A disease, a lysosomal storage disorder, is caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency, resulting in keratan sulfate (KS) and chondroitin-6-sulfate accumulation. Patients develop severe skeletal dysplasia, early cartilage deterioration and life-threatening heart and tracheal complications. There is no cure and enzyme replacement therapy cannot correct skeletal abnormalities. Here, using CRISPR/Cas9 technology, we generate the first MPSIVA rat model recapitulating all skeletal and non-skeletal alterations experienced by patients. Treatment of MPSIVA rats with adeno-associated viral vector serotype 9 encoding Galns (AAV9-Galns) results in widespread transduction of bones, cartilage and peripheral tissues. This led to long-term (1 year) increase of GALNS activity and whole-body correction of KS levels, thus preventing body size reduction and severe alterations of bones, teeth, joints, trachea and heart. This study demonstrates the potential of AAV9-Galns gene therapy to correct the disabling MPSIVA pathology, providing strong rationale for future clinical translation to MPSIVA patients.