In Situ Characterization of Bak Clusters Responsible for Cell Death Using Single Molecule Localization Microscopy

Apoptosis plays a pivotal role in development and tissue homeostasis in multicellular organisms. Clustering of Bak proteins on the mitochondrial outer membrane is responsible for the induction of apoptosis by evoking a release of pro-apoptotic proteins from mitochondria into cytosol. However, how the protein cluster permeabilizes the mitochondrial membrane remains unclear because elucidation of the cluster characteristics such as size and protein density has been hampered by the diffraction-limited resolution of light microscopy. Here, we describe an approach to quantitatively characterize Bak clusters in situ based on single molecule localization. We showed that Bak proteins form densely packed clusters at the nanoscale on mitochondria during apoptosis. Quantitative analysis based on the localization of each Bak protein revealed that the density of Bak protein is uniform among clusters although the cluster size is highly heterogeneous. Our approach provides unprecedented information on the size and protein density of Bak clusters possibly critical for the permeabilization and is applicable for the analysis of different cluster formations.

Enlightening G-protein-coupled receptors on the plasma membrane using super-resolution photoactivated localization microscopy

Paolo Annibale, Aleksandra Radenovic, Marco Scarselli

The possibility to visualize and image the arrangement of proteins within the cell at the molecular level has always been an attraction for scientists in biological research. In particular, for signalling molecules such as GPCRs (G-protein-coupled receptors), the existence of protein aggregates such as oligomers or clusters has been the topic of extensive debate. One of the reasons for this lively argument is that the molecular size is below the diffraction-limited resolution of the conventional microscopy, precluding the direct visualization of protein super-structures. On the other hand, new super-resolution microscopy techniques, such as the PALM (photoactivated localization microscopy), allow the limit of the resolution power of conventional optics to be broken and the localization of single molecules to be determined with a precision of 10-20 nm, close to their molecular size. The application of super-resolution microscopy to study the spatial and temporal organization of GPCRs has brought new insights into receptor arrangement on the plasma membrane. Furthermore, the use of this powerful microscopy technique as a quantitative tool opens up the possibility for investigating and quantifying the number of molecules in biological assemblies and determining the protein stoichiometry in signalling complexes.

Portland Press2013

Chromatin organization, transcription factor dynamics and gene expression in higher eukaryotes with superresolution imaging and single molecule tracking

Aleksandr Benke

Gene expression is dynamic and heterogeneous across all cell types. It serves several fundamental functions such as adaptation for changing environment and developing tissue specific phenotype and functionality during development and in adult organism. Control of gene expression mostly occurs at the transcription stage. Two main factors that regulate transcription in high eukaryotes are chromatin organization and transcription factor (TF) protein dynamics. Until recently they were mostly studied by methods that are performed in vitro or/and require ensemble averaging. We have been contributing to a development of new single molecule imaging methods that allow quantifying chromatin organization and TF dynamics in vivo and at single cell/single molecule level. Here we present our methodological developments and apply them to study how chromatin organization and TF dynamics regulate transcription. On the technical side of the study we, first, developed a method of imaging DNA with enhanced resolution in living cells. Super-resolution (SR) point localization based microscopy allows visualizing sub-diffraction organization of cellular structures and their dynamics in vivo. However, until recently almost exclusively proteins were imaged in SR microscopy. We found photoswitching conditions to image with stochastic optical reconstruction microscopy (STORM) DNA directly using the site-specific DNA –binding dye Picogreen (Chapter 2). We achieved a resolution of 50-70 nm which is ~5 fold better than conventional microscopy. Due to the excellent dye preservation we were able to do time lapse SR imaging. This study was a first demonstration of using a site-specific dye for STORM SR imaging in living cells. Photoswitching principle used in SR microscopy can be applied to single molecule (SM) tracking. It provides up to 1000x increase in density of trajectories compared to classic SM tracking. Initially, photoactivatable (PA) fusion proteins were used for high density SR based tracking. However, PA proteins are relatively dim and there is a limited choice of them. We demonstrated a new approach to SR based high density tracking by using organic dyes’ photoswitching to track proteins (Chapter 4). We showed that different dyes can be photoswitched in similar live-cell compatible media on membrane and in different organelles. Together with orthogonal protein labelling schemes this allows us to perform multicolour high density tracking across different compartments. This approach gives flexibility of labelling and increased track length compared to PA-FP SR based tracking. In a separate study, using tracking we developed algorithm which can account for long-living molecules in live cell SR imaging and allows us to correct clustering artefacts (Chapter 5). [...]

EPFL2015

Analytical Methods, Correlative Microscopy and Software Tools for Quantitative Single Molecule Localization Microscopy

Arun Shivanandan

Single Molecule Localization Microscopy (SMLM) techniques, such as PhotoActivated Localization Microscopy (PALM) and STochastic Optical Reconstruction Microscopy(STORM), can get around the diffraction limited resolution of conventional fluorescent microscopy (FM). However, there are a number of possible sources of imaging errors in SMLM which can significantly impact such studies. They include labeling artifacts, a limited detection efficiency of label molecules(of about 40-60% in PALM, e.g.,) and an uncertainty in localization in the range of 20-50nm. This thesis describes a rigorous review of such sources of error. Also, the SMLM readout is different from FM, the tools used in FM may not be directly applicable to SMLM. Accurate and precise quantitative imaging with SMLM requires analytical, experimental and software tools that address such issues. We describe analytical methods that accounts for two major sources of errors in analysis of membrane protein organization with SMLM. We model limited detection efficiency as independent subsampling of the set of label molecules. We then use a theoretical property of commonly used second order properties in quantitative SMLM, such as Ripleyâs K-function, L(r) - r function and the Pair Correlation Function (PCF), to show that they are invariant to such subsampling. We derive expressions for their stochastic estimators due to subsampling, and characterize the errors. The results can be extended to co-localization analysis as well. We then describe a method that estimate the true locations of points given the observed ones in clusters. We characterize the relative Mean Squared Error of the combined approach, and find that it can significantly reduce the errors in quantification. We apply these methods on data on clustering due to photoblinking of individual fluorophores, and data with redundant labeling. We then study the theoretical properties of a function that has been proposed as an estimator of cluster size. We also describe a method to identify the cluster model from data. SMLM provides single molecule resolution images of specific molecular species. Atomic Force Microscopy ( AFM), on the other hand, provides nonspecific, high resolution spatial profile information. Correlative AFM-SMLM can provide not only validation of SMLM, but also the complementary information so obtained can be used to design innovative experiments. We describe in vitro imaging of actin filaments with an AFM-SMLM correlative tool, that could provide information about sources of imaging inhomogeneity in SMLM. The tool were also used to image live mammalian cells, and can be used to obtain nanoscale information about mechanical properties of cells and also as a tool for nanomanipulation. Co-localization is a common spatial interaction quantification method used in biological imaging. It is possible to use tools from spatial statistics to obtain better statistical power on tests of spatial interaction, compared to conventional co-localization measures. Such tools can also handle SMLM data better, since they work on point patterns rather than images. We describe an ImageJ/Fiji plugin that implements a spatial statistics framework that extends the concept of co-localization, by means of a model based on Gibbs function of interaction potentials. We describe the application of this software on both confocal microscopy data of virus-endosomes, and SMLM data on GPCR protein-clathrin.

EPFL2015

Chromatin organization, transcription factor dynamics and gene expression in higher eukaryotes with superresolution imaging and single molecule tracking

Aleksandr Benke

EPFL2015

Analytical Methods, Correlative Microscopy and Software Tools for Quantitative Single Molecule Localization Microscopy

Arun Shivanandan

EPFL2015