Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min-1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.

• MeSH
• barvení a značení MeSH
• buněčná adheze fyziologie   MeSH
• časové faktory MeSH
• fibroblasty fyziologie   MeSH
• krysa rodu rattus MeSH
• mikroskopie metody   MeSH
• myši MeSH
• paxilin chemie   genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The fusion protein Bcr-Abl, which is the molecular cause of chronic myelogenous leukemia (CML) interacts in multiple points with signaling pathways regulating the cellular adhesivity and cytoskeleton architecture and dynamics. We explored the effects of imatinib mesylate, an inhibitor of Bcr-Abl protein used in front-line CML therapy, on the adhesivity of JURL-MK1 cells to fibronectin and searched for underlying changes in the cell proteome. As imatinib induces apoptosis of JURL-MK1 cells, we used three different caspase inhibitors to discriminate between direct consequences of Bcr-Abl inhibition and secondary changes related to the apoptosis. Imatinib treatment caused a transient increase in JURL-MK1 cell adhesivity to fibronectin, possibly due to the switch off of Bcr-Abl activity. Subsequently, we observed a number of changes including a decrease in cell adhesivity, F-actin decomposition, reduction of integrin β1, CD44, and paxillin expression levels and a marked increase in cofilin phophorylation at Ser3. These events were generally related to the proceeding apoptosis but they differed in their sensitivity to the individual caspase inhibitors.

• MeSH
• 2D gelová elektroforéza MeSH
• antitumorózní látky farmakologie   MeSH
• apoptóza účinky léků   MeSH
• buněčná adheze účinky léků   MeSH
• chronická myeloidní leukemie metabolismus   MeSH
• fibronektiny metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• paxilin metabolismus   MeSH
• piperaziny farmakologie   MeSH
• průtoková cytometrie MeSH
• pyrimidiny farmakologie   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• tropomyosin metabolismus   MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) which is being introduced into clinic for the treatment of hematological diseases. We studied the effect of this compound on six human hematopoietic cell lines (JURL-MK1, K562, CML-T1, Karpas-299, HL-60, and ML-2) as well as on normal human lymphocytes and on leukemic primary cells. SAHA induced dose-dependent and cell type-dependent cell death which displayed apoptotic features (caspase-3 activation and apoptotic DNA fragmentation) in most cell types including the normal lymphocytes. At subtoxic concentrations (0.5-1 microM), SAHA increased the cell adhesivity to fibronectin (FN) in all leukemia/lymphoma-derived cell lines but not in normal lymphocytes. This increase was accompanied by an enhanced expression of integrin beta1 and paxillin, an essential constituent of focal adhesion complexes, both at the protein and mRNA level. On the other hand, the inhibition of ROCK protein, an important regulator of cytoskeleton structure, had no consistent effect on SAHA-induced increase in the cell adhesivity. The promotion of cell adhesivity to FN seems to be specific for SAHA as we observed no such effects with other HDAC inhibitors (trichostatin A and sodium butyrate).

• MeSH
• antigeny CD29 MeSH
• antitumorózní látky farmakologie   MeSH
• apoptóza MeSH
• buněčná adheze účinky léků   MeSH
• fibronektiny metabolismus   MeSH
• kyseliny hydroxamové farmakologie   MeSH
• leukemie metabolismus   MeSH
• lidé MeSH
• lymfocyty metabolismus   účinky léků   MeSH
• nádorové buněčné linie MeSH
• paxilin metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• průtoková cytometrie MeSH
• separace buněk MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH