Cell-type-specific 2 adrenergic receptor clusters identified using photo-activated localization microscopy are not lipid raft related, but depend on actin cytoskeleton integrity

Recent developments in the field of optical super-resolution techniques allow both a 10-fold increase in resolution as well as an increased ability to quantify the number of labeled molecules visualized in the fluorescence measurement. By using photoactivated localization microscopy (PALM) and an experimental approach based on the systematic comparison with a nonclustering peptide as a negative control, we found that the prototypical G protein-coupled receptor beta 2-adrenergic receptor is partially preassociated in nanoscale-sized clusters only in the cardiomyocytes, such as H9C2 cells, but not in other cell lines, such as HeLa and Chinese hamster ovary (CHO). The addition of the agonist for very short times or the addition of the inverse agonist did not significantly affect the organization of receptor assembly. To investigate the mechanism governing cluster formation, we altered plasma membrane properties with cholesterol removal and actin microfilament disruption. Although cholesterol is an essential component of cell membranes and it is supposed to be enriched in the lipid rafts, its sequestration and removal did not affect receptor clustering, whereas the inhibition of actin polymerization did decrease the number of clusters. Our findings are therefore consistent with a model in which beta 2 receptor clustering is influenced by the actin cytoskeleton, but it does not rely on lipid raft integrity, thus ruling out the possibility that cell type-specific beta 2 receptor clustering is associated with the raft.

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

Miniaturized bioanalytics to probe the function of membrane proteins

Pedro Pascoal

G-protein coupled receptors (GPCRs) are the most abundant class of proteins in the cell body. Such receptors are of major interest as potential therapeutic targets. Downscaling and parallelization of bioanalytics opens novel routes to rapidly screen and identify potential drugs with a decrease in regard to the costs, and elucidate novel functions of signaling networks under physiological conditions. Native vesicles are small autonomous biological containers, which are efficiently produced from all cell lines. They are composed of their mother cell plasma membrane and enclose part of their cytoplasm. Membrane receptors are then exposed at their surface and already demonstrate to induce cellular signaling when exposed to receptor ligands. Native vesicles were investigated in this present work, as novel possibilities to downscale receptor investigation in live cells, using neurokinin 1 receptor (NK1R) as a representative model. Here native vesicle production and purification was optimized. The influence of the cell cycle on the production efficiency was demonstrated. Biological proteins were downregulated in order to produce blebbing cells. Native vesicle characterization was achieved. The receptors also demonstrate to be efficiently labeled by agonist and antagonist, allowing to access information about the binding kinetics as well as KD values. The results are in agreement with those obtained in live cells. Native vesicles also demonstrate to internalize agonist after application, demonstrating receptor desensitization and signaling performance similar as live cells. Confocal microscopy shows that cells expressing the NK1R-CFP have two binding affinities for their main agonist, substance P. Similar results could be observed with flow cytometry. The high affinity binding is related to cholesterol content in the cell membrane and was abolished by cholesterol depletion with methyl-β-cyclodextrin. Micro-contact printing (µCP) was used to (bio)functionalize surfaces with proteins, polymers or functionalized nanoparticles. Precise sample positioning by micro-contact printing shows improves nuclear magnetic resonance excitation and detection, when performed with a planar microcoil probe. µCP was used to produce native vesicle arrays by two procedures, and fluorescent binding assay shows the binding of fluorescent ligands to the receptor. Laser tweezers allow manipulating cell membranes without requiring the use of polystyrene beads. From pulled membranes, native vesicles were produced. In addition from pulled membranes, large tethers were produced and artificial connections were established with neighboring cells. Intercellular communication was investigated by whole-cell patch clamp in dissociated primary dorsal root ganglion neurons after optical induced connection, as well as in HEK cells expressing Cx36. The lab-on-chip assay development demonstrates: the high production of native vesicles in microchannels; the efficient purification obtained by negative dielectrophoresis depletion in MEMS chips; perfect trapping and thus immobilization of native vesicles in a new optical multi-tweezer array. Fluorescence labeling was performed with native vesicles trapped in a multi-tweezer array inside microfluidic channel in the presence of two laminar flows. Optical multi-tweezer array setup shows to be the fastest and more efficient technique in order to perform immobilization and labeling of native vesicles in the microfluidic channel. It is presently the only technique to perform fluorescence measurements when maintaining objects trapped.

EPFL2008

Investigating the Impact of Single Molecule Fluorescence Dynamics on Photo Activated Localization Microscopy Experiments

Paolo Annibale

When fluorophores are densely packed in a biological sample, localizing them one at a time is one of the paths to optical super-resolution. Photo Activated Localization Microscopy (PALM) is one of the methods of this burgeoning field that presents a large promise for addressing physical and biological questions requiring non-invasive, high-resolution optical imaging at the nanoscale. Since this work began when PALM was only two years old, the thesis contains a significant portion of research on the technique itself, and has therefore a marked methodological cut. The principal, and recurring, theme is the investigation of the single molecule fluorescence dynamics of the fluorophores used, that in the end determine to a large extent the ensemble imaging performance as well as the interpretation of the data. A few assumptions reported in the literature are questioned, and, beginning with a discussion of the properties of a bright fluorescent protein, mEos2, we propose an original approach based on the use of the temporal information besides the spatial one when treating PALM data. This allows both a more accurate quantification of the number of fluorophores activated in the sample as well as the correct identification of relevant biological structures, such as clusters, on the plasma membrane of cells. The second theme is the application of PALM to probe the functional arrangement of an important class of cell membrane proteins, through the study of the prototypical G protein-coupled receptor [beta]2-Adrenergic Receptor. First, the well studied biological properties of this family of signaling proteins are used to validate the potential of this approach. Then, PALM is used to gain new insight on the basal arrangement of [beta]2-AR in a relevant biological context using tools from spatial point pattern analysis. Provided that appropriate control experiments are performed, PALM is shown to have the potential to be included in the palette of the available techniques to study membrane protein organization. An important finding is the quantification of a cell-type specific clustering of this receptor in cardiomyocite-like cells. Finally, some technical issues relevant to dual color imaging, in particular concerning the axial stability of the microscope, are addressed as a third, perspective theme, together with a detailed first-time characterization of three representative fluorescent protein pairs currently available for dual color PALM imaging.

EPFL2012