| Popis: |
The mechanisms by which sonoporation increases the native plasma membrane permeability are still unknown but various hypotheses have been suggested including pore formation and endocytosis. We have shown recently that caveolae-mediated endocytosis plays a major role during sonoporation. In addition, some studies have reported the clear participation of actin in mammalian cell endocytosis. Thus, this study aims to investigate the effect of sonoporation on actin microfilaments and microtubules and to identify the role of both cytoskeletons on sonoporation-mediated membrane permeabilization. Adherent U-87 MG cells were insonated at 1MHz, 1 W/cm2, 20% duty cycle for 60 s, in the presence of BR14® microbubbles. SYTOX® Green was used to assess the membrane permeabilization, by flow cytometry. The cells were incubated with phalloidin-TRITC to stain actin microfilaments and tubulin antibody Alexa Fluor® 555 to stain tubulin. The ultrastructural changes of plasma membrane were monitored by scanning electron microscopy. To inhibit the polymerization of actin and tubulin cytoskeleton, the cells were treated with cytochalasin D (cytoD) and nocodazole (Noco), respectively. Immunofluorescence results show alteration of actin and tubulin cytoskeleton, immediately after sonoporation while control cells present a filamentous cytoskeleton with polygonal shape. However, the disorganization of the cytoskeleton network is reversible since 60 min post-sonoporation, only few cells show a tubulin (8%) and actin (25%) cytoskeleton disruption. Moreover based on SEM study reveals that the treatment of the cells with both cytoD and Noco induced a strong decrease in the number of TPS (transient and permeant structures): 98.5 ± 0.2% and 96 ± 0.6%, respectively. Moreover, flow cytometry results showed that cytoD and Noco lead to a decrease in the membrane permeabilization rate: 58% and 87%, respectively. In conclusion, this study demonstrates the transient alteration of actin and tubulin cytoskeleton following sonoporation. It suggests that cytoskeleton plays a role during sonoporation, as cytoskeleton inhibitors provoke a decrease in the cell permeabilization rate. Its implication could occur during both the entry and transport of endocytosed molecules. |
