RESUMEN
We describe a simple fabrication technique - targeted towards non-specialists - that allows for the production of leak-proof polydimethylsiloxane (PDMS) microfluidic devices that are compatible with live-cell microscopy. Thin PDMS base membranes were spin-coated onto a glass-bottom cell culture dish and then partially cured via microwave irradiation. PDMS chips were generated using a replica molding technique, and then sealed to the PDMS base membrane by microwave irradiation. Once a mold was generated, devices could be rapidly fabricated within hours. Fibronectin pre-treatment of the PDMS improved cell attachment. Coupling the device to programmable pumps allowed application of precise fluid flow rates through the channels. The transparency and minimal thickness of the device enabled compatibility with inverted light microscopy techniques (e.g. phase-contrast, fluorescence imaging, etc.). The key benefits of this technique are the use of standard laboratory equipment during fabrication and ease of implementation, helping to extend applications in live-cell microfluidics for scientists outside the engineering and core microdevice communities.
Asunto(s)
Dispositivos Laboratorio en un Chip , Microscopía/instrumentación , Células 3T3 , Animales , Supervivencia Celular , Dimetilpolisiloxanos , Diseño de Equipo , Hidrodinámica , Ratones , NylonsRESUMEN
Vacuolar-type H(+)-ATPases (V-ATPases) are located in lysosomes and at the ruffled border in osteoclasts. We showed previously that the R740S mutation is dominant negative for V-ATPase activity, uncouples proton transport from ATP hydrolysis and causes osteopetrosis in heterozygous mice (+/R740S). Here we show mice homozygous for R740S (R740S/R740S) have more severe osteopetrosis and die by postnatal day 14. Although R740S/R740S osteoclasts express wild-type levels of a3, it is mislocalized. Acridine orange staining of R740S/R740S osteoclasts grown on a Corning resorptive surface reveals no resorption and no acidification of intracellular compartments. Whereas osteoblast and osteocyte apoptosis is normal, R740S/R740S osteoclasts exhibit increased apoptosis compared with wild-type osteoclasts. Localization of the enzyme tartrate-resistant acid phosphatase (TRAP) is also aberrant. Transmission electron microscopy reveals that R740S/R740S osteoclasts do not polarize, lack ruffled borders, and contain fewer autophagosomes. Consistent with an early stage defect in autophagy, expression of LC3II is reduced and expression of p62 is increased in R740S/R740S compared to wild-type osteoclasts. These results indicate the importance of intracellular acidification for the early stages of autophagy as well as for osteoclast survival, maturation, and polarization with appropriate cytoplasmic distribution of key osteoclast enzymes such as TRAP.
Asunto(s)
Osteoclastos/citología , Osteopetrosis/genética , Subunidades de Proteína/genética , ATPasas de Translocación de Protón Vacuolares/genética , Animales , Apoptosis/genética , Autofagia/genética , Diferenciación Celular/genética , Citoplasma/genética , Lisosomas/genética , Lisosomas/metabolismo , Ratones , Mutación , Osteoclastos/metabolismo , Osteopetrosis/enzimología , Osteopetrosis/patología , Subunidades de Proteína/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
Vacuolar H(+) -ATPase (V-ATPase), a multisubunit enzyme located at the ruffled border and in lysosomes of osteoclasts, is necessary for bone resorption. We previously showed that heterozygous mice with an R740S mutation in the a3 subunit of V-ATPase (+/R740S) have mild osteopetrosis resulting from an â¼90% reduction in proton translocation across osteoclast membranes. Here we show that lysosomal pH is also higher in +/R740S compared with wild-type (+/+) osteoclasts. Both osteoclast number and size were decreased in cultures of +/R740S compared with +/+ bone marrow cells, with concomitant decreased expression of key osteoclast markers (TRAP, cathepsin K, OSCAR, DC-STAMP, and NFATc1), suggesting that low lysosomal pH plays an important role in osteoclastogenesis. To elucidate the molecular mechanism of this inhibition, NFATc1 activation was assessed. NFATc1 nuclear translocation was significantly reduced in +/R740S compared with +/+ cells; however, this was not because of impaired enzymatic activity of calcineurin, the phosphatase responsible for NFATc1 dephosphorylation. Protein and RNA expression levels of regulator of calcineurin 1 (RCAN1), an endogenous inhibitor of NFATc1 activation and a protein degraded in lysosomes, were not significantly different between +/R740S and +/+ osteoclasts, but the RCAN1/NFATc1 ratio was significantly higher in +/R740S versus +/+ cells. The lysosomal inhibitor chloroquine significantly increased RCAN1 accumulation in +/+ cells, consistent with the hypothesis that higher lysosomal pH impairs RCAN1 degradation, leading to a higher RCAN1/NFATc1 ratio and consequently NFATc1 inhibition. Our data indicate that increased lysosomal pH in osteoclasts leads to decreased NFATc1 signaling and nuclear translocation, resulting in a cell autonomous impairment of osteoclastogenesis in vitro.
Asunto(s)
Sustitución de Aminoácidos/genética , Lisosomas/metabolismo , Mutación/genética , Factores de Transcripción NFATC/metabolismo , Osteoclastos/metabolismo , Osteogénesis , ATPasas de Translocación de Protón Vacuolares/genética , Animales , Biomarcadores/metabolismo , Calcineurina/metabolismo , Proteínas de Unión al Calcio , Regulación de la Expresión Génica , Concentración de Iones de Hidrógeno , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , Proteínas Musculares/metabolismo , Factores de Transcripción NFATC/antagonistas & inhibidores , Osteoclastos/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Proteínas Tirosina Quinasas/metabolismo , Quinasas DyrKRESUMEN
A mouse founder with high bone mineral density and an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) screen. It was found to carry a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit of the vacuolar type H(+)-ATPase (V-ATPase), resulting in replacement of a highly conserved amino acid (R740S). The +/R740S mice have normal appearance, size, and weight but exhibit high bone density. Osteoblast parameters are unaffected in bones of +/R740S mice, whereas osteoclast number and marker expression are increased, concomitant with a decrease in the number of apoptotic osteoclasts. Consistent with reduced osteoclast apoptosis, expression of Rankl and Bcl2 is elevated, whereas Casp3 is reduced. Transmission electron microscopy revealed that unlike other known mutations in the a3 subunit of V-ATPase, polarization and ruffled border formation appear normal in +/R740S osteoclasts. However, V-ATPases from +/R740S osteoclast membranes have severely reduced proton transport, whereas ATP hydrolysis is not significantly affected. We show for the first time that a point mutation within the a3 subunit, R740S, which is dominant negative for proton pumping and bone resorption, also uncouples proton pumping from ATP hydrolysis but has no effect on ruffled border formation or polarization of osteoclasts. These results suggest that the V(0) complex has proton-pumping-independent functions in mammalian cells.
Asunto(s)
Sustitución de Aminoácidos/genética , Genes Dominantes/genética , Mutación Missense/genética , Osteopetrosis/genética , Subunidades de Proteína/genética , ATPasas de Translocación de Protón Vacuolares/genética , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Secuencia de Bases , Transporte Biológico , Recuento de Células , Forma de la Célula , Análisis Mutacional de ADN , Femenino , Fémur/diagnóstico por imagen , Fémur/patología , Heterocigoto , Hidrólisis , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Osteoblastos/metabolismo , Osteoblastos/patología , Osteoclastos/metabolismo , Osteoclastos/patología , Osteopetrosis/patología , Protones , Microtomografía por Rayos XRESUMEN
Vacuolar H(+)-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor kappaB-ligand (RANKL). We found that Receptor Activator of Nuclear Factor kappaB (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.
Asunto(s)
Microglía/metabolismo , Ligando RANK/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Actinas/metabolismo , Animales , Línea Celular , Ratones , Ratones Endogámicos , Microglía/efectos de los fármacos , Ligando RANK/farmacología , RatasRESUMEN
V-ATPases are multimeric proton pumps. The 100-kDa "a" subunit is encoded by four isoforms (a1-a4) in mammals and two (Vph1p and Stv1p) in yeast. a3 is enriched in osteoclasts and is essential for bone resorption, whereas a4 is expressed in the distal nephron and acidifies urine. Mutations in human a3 and a4 result in osteopetrosis and distal renal tubular acidosis, respectively. Human a3 (G405R and R444L) and a4 (P524L and G820R) mutations were recreated in the yeast ortholog Vph1p, a3 (G424R and R462L), and a4 (W520L and G812R). Mutations in a3 resulted in wild type vacuolar acidification and growth on media containing 4 mM ZnCl2, 200 mM CaCl2, or buffered to pH 7.5 with V-ATPase hydrolytic and pumping activity decreased by 30-35%. Immunoblots confirmed wild type levels for V-ATPase a, A, and B subunits on vacuolar membranes. a4 G812R resulted in defective growth on selective media with V-ATPase hydrolytic and pumping activity decreased by 83-85% yet with wild type levels of a, A, and B subunits on vacuolar membranes. The a4 W520L mutation had defective growth on selective media with no detectable V-ATPase activity and reduced expression of a, A, and B subunits. The a4 W520L mutation phenotypes were dominant negative, as overexpression of wild type yeast a isoforms, Vph1p, or Stv1p, did not restore growth. However, deletion of endoplasmic reticulum assembly factors (Vma12p, Vma21p, and Vma22p) partially restored a and B expression. That a4 W520L affects both Vo and V1 subunits is a unique phenotype for any V-ATPase subunit mutation and supports the concerted pathway for V-ATPase assembly in vivo.