RESUMO
Epithelial tight junctions form a barrier against passive paracellular flux. This barrier is regulated by complex physiologic and pathophysiologic signals that acutely fine-tune tight junction permeability. Although actomyosin contraction and myosin light chain phosphorylation are clearly involved in some forms of tight junction regulation, the contributions of other signaling events and the role of myosin light chain phosphorylation in this response are poorly understood. Here we ask if activation of myosin light chain kinase alone is sufficient to induce downstream tight junction regulation. We use a confluent polarized intestinal epithelial cell model system in which constitutively active myosin light chain kinase, tMLCK, is expressed using an inducible promoter. tMLCK expression increases myosin light chain phosphorylation, reorganizes perijunctional F-actin, and increases tight junction permeability. TJ proteins ZO-1 and occludin are markedly redistributed, morphologically and biochemically, but effects on claudin-1 and claudin-2 are limited. tMLCK inhibition prevents changes in barrier function and tight junction organization induced by tMLCK expression, suggesting that these events both require myosin light chain phosphorylation. We conclude that myosin light chain phosphorylation alone is sufficient to induce tight junction regulation and provide new insights into the molecular mechanisms that mediate this regulation.
Assuntos
Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Junções Íntimas/metabolismo , Actinas/metabolismo , Células CACO-2 , Permeabilidade da Membrana Celular/fisiologia , Claudina-1 , Claudinas , Indução Enzimática , Epitélio/enzimologia , Epitélio/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Quinase de Cadeia Leve de Miosina/biossíntese , Ocludina , Fosfoproteínas/metabolismo , Fosforilação , Junções Íntimas/enzimologia , Junções Íntimas/ultraestrutura , Proteína da Zônula de Oclusão-1RESUMO
We report the first high-precision interferometer using large sapphire mirrors, and we present the first direct, broadband measurements of the fundamental thermal noise in these mirrors. Our results agree well with the thermoelastic-damping noise predictions of Braginsky, et al. and Cerdonio et al., which have been used to predict the astrophysical reach of advanced interferometric gravitational wave detectors.