Expansion microscopy for the analysis of centrioles and cilia.

Centrioles are vital cellular structures that organise centrosomes and cilia. Due to their subresolutional size, centriole ultrastructural features have been traditionally analysed by electron microscopy. Here we present an adaptation of magnified analysis of the proteome expansion microscopy method, to be used for a robust analysis of centriole number, duplication status, length, structural abnormalities and ciliation by conventional optical microscopes. The method allows the analysis of centriole's structural features from large populations of adherent and nonadherent cells and multiciliated cultures. We validate the method using EM and superresolution microscopy and show that it can be used as an affordable and reliable alternative to electron microscopy in the analysis of centrioles and cilia in various cell cultures. LAY DESCRIPTION: Centrioles are microtubule-based structures organised as ninefold symmetrical cylinders which are, in human cells, ∼500 nm long and ∼230 nm wide. Centrioles assemble dozens of proteins around them forming centrosomes, which nucleate microtubules and organise spindle poles in mitosis. Centrioles, in addition, assemble cilia and flagella, two critically important organelles for signalling and motility. Due to centriole small size, electron microscopy has been a major imaging technique for the analysis of their ultrastructural features. However, being technically demanding, electron microscopy it is not easily available to the researchers and it is rarely used to collect large datasets. Expansion microscopy is an emerging approach in which biological specimens are embedded in a swellable polymer and isotopically expanded several fold. Physical separation of cellular structures allows the analysis of, otherwise unresolvable, structures by conventional optical microscopes. We present an adaptation of expansion microscopy approach, specifically developed for a robust analysis of centrioles and cilia. Our protocol can be used for the analysis of centriole number, duplication status, length, localisation of various centrosomal components and ciliation from large populations of cultured adherent and nonadherent cells and multiciliated cultures. We validate the method against electron microscopy and superresolution microscopy and demonstrate that it can be used as an accessible and reliable alternative to electron microscopy.

Predictive-focus illumination for reducing photodamage in live-cell microscopy.

Due to photobleaching and phototoxicity induced by high-intensity excitation light, the number of fluorescence images that can be obtained in live cells is always limited. This limitation becomes particularly prominent in multidimensional recordings when multiple Z-planes are captured at every time point. Here we present a simple technique, termed predictive-focus illumination (PFI), which helps to minimize cells' exposure to light by decreasing the number of Z-planes that need to be captured in live-cell 3D time-lapse recordings. PFI utilizes computer tracking to predict positions of objects of interest (OOIs) and restricts image acquisition to small dynamic Z-regions centred on each OOI. Importantly, PFI does not require hardware modifications and it can be easily implemented on standard wide-field and spinning-disc confocal microscopes.

Bacterial O6-methylguanine-DNA methyltransferase reduces N-methyl-N'-nitro-N-nitrosoguanidine induction of plasminogen activator in Mer- human glioblastoma A1235 cell line.

The alkylation repair deficient (Mer- phenotype) cells produce high levels of proteolytic enzyme plasminogen activator (PA) after treatment with alkylation agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Both, in Escherichia coli and in mammalian cells O6-methylguanine (O6-MeG) is repaired by analogues O6-methylguanine-DNA methyltransferase (MGMT). In E. coli MGMT is product of ada gene. To investigate the effect of bacterial MGMT expression on the induction of PA activity in human cells, we have transfected ada-alkB operon into Mer- human A1235 cells that are known to produce high levels of PA after MNNG treatment. We have shown here that A4 and A8 transformants that harbour ada gene become resistant to killing by MNNG. In addition, MNNG produced induction of extracellular PA activity was much less pronounced in A4 and A8 transformants (induction ratio 3.42 and 3.74, respectively) than in control A1235 and Aneo-1 cells (induction ratio 11.04 and 9.11, respectively). However, changes of intracellular PA activity were not significant. It appears, therefore, that induction of extracellular PA activity is inversely related to the cell capacity to repair the DNA lesions induced by alkylation agents.