RESUMO
Keratin filaments (KFs) comprise the intermediate filaments of epithelial cells and are well known for their cytoprotective properties and their mechanical resilience. Although, several studies have demonstrated KFs' remarkable tensile properties relatively little is known about acute implications of mechanical stretch on KFs in living cells. This includes structural effects on the KFs and their higher level assembly structures as well as posttranslational response mechanisms to possibly modify KF's properties. We subjected simple epithelial A549 lung cells to 30% unidirectional stretch and already after 10 seconds we observed morphological changes of the KF-network as well as structural effects on their desmosomal anchor sites-both apparently caused by the tensile strain. Interestingly, the effect on the desmosomes was attenuated after 30 seconds of cell stretch with a concomitant increase in phosphorylation of keratin8-S432, keratin18-S53, and keratin18-S34 without an apparent increase in keratin solubility. When mimicking the phosphorylation of keratin18-S34 the stretch-induced effect on the desmosomes could be diminished and probing the cell surface with atomic force microscopy showed a lowered elastic modulus. We conclude that the stretch-induced KF phosphorylation affects KF's tensile properties, probably to lower the mechanical load on strained desmosomal cell-cell contacts, and hence, preserve epithelial integrity.
Assuntos
Queratinas/metabolismo , Pulmão/metabolismo , Células A549 , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Humanos , Filamentos Intermediários/metabolismo , Fosforilação/fisiologiaRESUMO
Exocytosis is the intracellular trafficking step where a secretory vesicle fuses with the plasma membrane to release vesicle content. Actin and microtubules both play a role in exocytosis; however, their interplay is not understood. Here we study the interaction of actin and microtubules during exocytosis in lung alveolar type II (ATII) cells that secrete surfactant from large secretory vesicles. Surfactant extrusion is facilitated by an actin coat that forms on the vesicle shortly after fusion pore opening. Actin coat compression allows hydrophobic surfactant to be released from the vesicle. We show that microtubules are localized close to actin coats and stay close to the coats during their compression. Inhibition of microtubule polymerization by colchicine and nocodazole affected the kinetics of actin coat formation and the extent of actin polymerisation on fused vesicles. In addition, microtubule and actin cross-linking protein IQGAP1 localized to fused secretory vesicles and IQGAP1 silencing influenced actin polymerisation after vesicle fusion. This study demonstrates that microtubules can influence actin coat formation and actin polymerization on secretory vesicles during exocytosis.