Marco Scarselli

Quantitative Photo Activated Localization Microscopy: Unraveling the Effects of Photoblinking

In this work we discuss how to use photophysical information for improved quantitative measurements using Photo Activated Localization Microscopy (PALM) imaging. We introduce a method that reliably estimates the number of photoblinking molecules present in a biological sample and gives a robust way to quantify proteins at the single-cell level from PALM images. We apply this method to determine the amount of β2 adrenergic receptor, a prototypical G Protein Coupled Receptor, expressed on the plasma membrane of HeLa cells.

Identification of clustering artifacts in photoactivated localization microscopy

Keywords: Fluorescent Protein Reference EPFL-ARTICLE-167359doi:10.1038/nmeth.1627View record in Web of Science Record created on 2011-07-04, modified on 2017-05-12

Multiple Residues in the Second Extracellular Loop Are Critical for M3 Muscarinic Acetylcholine Receptor Activation

Recent studies suggest that the second extracellular loop (o2 loop) of bovine rhodopsin and other class I G protein-coupled receptors (GPCRs) targeted by biogenic amine ligands folds deeply into the transmembrane receptor core where the binding of cis-retinal and biogenic amine ligands is known to occur. In the past, the potential role of the o2 loop in agonist-dependent activation of biogenic amine GPCRs has not been studied systematically. To address this issue, we used the M3 muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR, as a model system. Specifically, we subjected the o2 loop of the M3R to random mutagenesis and subsequently applied a novel yeast genetic screen to identity single amino acid substitutions that interfered with M3R function. This screen led to the recovery of about 20 mutant M3Rs containing single amino acid changes in the o2 loop that were inactive in yeast. In contrast, application of the same strategy to the extracellular N-terminal domain of the M3R did not yield any single point mutations that disrupted M3R function. Pharmacological characterization of many of the recovered mutant M3Rs in mammalian cells, complemented by site-directed mutagenesis studies, indicated that the presence of several o2 loop residues is important for efficient agonist-induced M3R activation. Besides the highly conserved Cys220 residue, Gln207, Gly211, Arg213, Gly218, Ile222, Phe224, Leu225, and Pro228 were found to be of particular functional importance. In general, mutational modification of these residues had little effect on agonist binding affinities. Our findings are therefore consistent with a model in which multiple o2 loop residues are involved in stabilizing the active state of the M3R. Given the high degree of structural homology found among all biogenic amine GPCRs, our findings should be of considerable general relevance.

Constitutive Internalization of G Protein-coupled Receptors and G Proteins via Clathrin-independent Endocytosis

Although agonist-dependent endocytosis of G protein-coupled receptors (GPCRs) as a means to modulate receptor signaling has been widely studied, the constitutive endocytosis of GPCRs has received little attention. Here we show that two prototypical class I GPCRs, the β2 adrenergic and M3 muscarinic receptors, enter cells constitutively by clathrin-independent endocytosis and colocalize with markers of this endosomal pathway on recycling tubular endosomes, indicating that these receptors can subsequently recycle back to the plasma membrane (PM). This constitutive endocytosis of these receptors was not blocked by antagonists, indicating that receptor signaling was not required. Interestingly, the G proteins that these receptors couple to, Gαs and Gαq, localized together with their receptors at the plasma membrane and on tubular recycling endosomes. Upon agonist stimulation, Gαs and Gαq remained associated with the PM and these endosomal membranes, whereas β2 and M3 receptors now entered cells via clathrin-dependent endocytosis. Deletion of the third intracellular loop (i3 loop), which is thought to play a role in agonist-dependent endocytosis of the M3 receptor, had no effect on the constitutive internalization of the receptor. Surprisingly, with agonist, the mutated M3 receptor still internalized and accumulated in cells but through clathrin-independent and not clathrin-dependent endocytosis. These findings demonstrate that GPCRs are versatile PM proteins that can utilize different mechanisms of internalization depending upon ligand activation.

Potent activation of dopamine D3/D2 heterodimers by the antiparkinsonian agents, S32504, pramipexole and ropinirole

Recombinant, human dopamine D3 and D2 receptors form functional heterodimers upon co‐expression in COS‐7 cells. Herein, actions of the antiparkinsonian agents, S32504, ropinirole and pramipexole, at D3/D2L heterodimers were compared to their effects at the respective monomers and at split, chimeric D3trunk/D2tail and D2trunk/D3tail receptors: the trunk incorporated transmembrane domains (TDs) I–V and the tail TDs VI and VII. In binding assays with the antagonist [3H]nemonapride, all agonists were potent ligands of D3 receptors showing, respectively, 100‐, 18‐ and 56‐fold lower affinity at D2L receptors, mimicking the selective D3 receptor antagonist, S33084 (100‐fold). At D3trunk/D2tail receptors, except for ropinirole, all drugs showed lower affinities than at D3 sites, whereas for D2trunk/D3tail receptors, affinities of all drugs were higher than at D2L sites. The proportion of high affinity binding sites recognized by S32504, pramipexole and ropinirole in membranes derived from cells co‐expressing D3 and D2L sites was higher than in an equivalent mixture of membranes from cells expressing D3 or D2L sites, consistent with the promotion of heterodimer formation. In contrast, the percentage of high and low affinity sites (biphasic isotherms) recognized by S33084 was identical. Functional actions were determined by co‐transfection of a chimeric adenylyl cyclase (AC)‐V/VI insensitive to D3 receptors. Accordingly, D3 receptor‐transfected cells were irresponsive whereas, in D2L receptor‐transfected cells, agonists suppressed forskolin‐stimulated cAMP production with modest potencies. In cells co‐transfected with D3 and D2L receptors, S32504, ropinirole and pramipexole potently suppressed AC‐V/VI with EC50s 33‐, 19‐ and 11‐fold lower than at D2L receptors, respectively. S32504 also suppressed AC‐V/VI activity at split D3trunk/D2tail and D2trunk/D3tail chimeras transfected into COS‐7 cells. In conclusion, antiparkinson agents behave as potent agonists at D3/D2‘heterodimers’, though any role in their actions in vivo remains to be demonstrated.

Cell Type-specific β2-Adrenergic Receptor Clusters Identified Using Photoactivated Localization Microscopy Are Not Lipid Raft Related, but Depend on Actin Cytoskeleton Integrity

Background: The direct measurement of diffraction-limited structures, such as clusters, is outside the resolution of the available microscopy techniques. Results: β2-Adrenergic receptor clusters identified using PALM are cell-type specific. Conclusion: PALM has successfully allowed the quantitative determination of GPCR clusters. Significance: The application of this powerful microscopy technique opens up the possibility to quantify the number of molecules in biological assemblies. 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 β2-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 β2 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 β2 receptor clustering is associated with the raft.

Rapid identification of functionally critical amino acids in a G protein-coupled receptor.

G protein–coupled receptors (GPCRs) comprise one of the largest protein families found in nature. Here we describe a new experimental strategy that allows rapid identification of functionally critical amino acids in the rat M3 muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR. This approach involves low-frequency random mutagenesis of the entire M3R coding sequence, followed by the application of a new yeast genetic screen that allows the recovery of inactivating M3R single point mutations. The vast majority of recovered mutant M3Rs also showed substantial functional impairments in transfected mammalian (COS-7) cells. A subset of mutant receptors, however, behaved differently in yeast and mammalian cells, probably because of the specific features of the yeast expression system used. The screening strategy described here should be applicable to all GPCRs that can be expressed functionally in yeast.

The Paired Activation of the Two Components of the Muscarinic M3 Receptor Dimer Is Required for Induction of ERK1/2 Phosphorylation

Muscarinic M3 receptors stimulate ERK1/2, the mitogen-activated protein kinase pathway. A mutant of the muscarinic M3 receptor in which most of the third intracellular (i3) loop had been deleted (M3-short) completely lost the ability to stimulate the ERK1/2 phosphorylation in COS-7 cells. This loss was evident despite the fact that the receptor was able to couple efficiently to the phospholipase C second messenger pathway. In co-transfected cells, M3-short greatly reduced the ability of M3 to activate ERK1/2. In another set of experiments we tested the ability of a mutant M3/M2(16aa) receptor, in which the first 16 amino acids of the i3 loop of the M3 receptor were replaced with the corresponding segment of the muscarinic M2 receptor to stimulate ERK1/2 phosphorylation. This mutant is not coupled to Gαq, but it is weakly coupled to Gαi. Despite its coupling modification this receptor was able to stimulate ERK1/2 phosphorylation. Again, M3-short greatly reduced the ability of M3/M2(16aa) to activate ERK1/2 in co-transfected cells. Similar results were obtained in stable-transfected Chinese hamster ovary (CHO) cells lines. In CHO M3 cells carbachol induced a biphasic increase of ERK1/2 phosphorylation; a first increase at doses as low as 0.1 μm and a second increase starting from 10 μm. In CHO M3-short and in double-transfected CHO M3/M3-short cells we observed only the lower doses increase of ERK1/2 phosphorylation; no further increase was observed up to 1 mm carbachol. This suggests that in double-transfected CHO cells M3-short prevents the effect of the higher doses of carbachol on the M3 receptor. In a final experiment we tested the ability of co-transfected chimeric α2/M3 and M3/α2 receptors to activate the ERK1/2 pathway. When given alone, carbachol and, to a lesser extent, clonidine, stimulated the coupling of the co-transfected chimeric receptors to the phospholipase C second messenger pathway, but they were unable to stimulate ERK1/2 phosphorylation. On the contrary, a strong stimulation of ERK1/2 phosphorylation was observed when the two agonists were given together despite the fact that the overall increase in phosphatidylinositol hydrolysis was not dissimilar from that observed in cells treated with carbachol alone. Our data suggest that the activation of the ERK1/2 pathway requires the coincident activation of the two components of a receptor dimer.

Challenges in quantitative single molecule localization microscopy

Single molecule localization microscopy (SMLM), which can provide up to an order of magnitude improvement in spatial resolution over conventional fluorescence microscopy, has the potential to be a highly useful tool for quantitative biological experiments. It has already been used for this purpose in varied fields in biology, ranging from molecular biology to neuroscience. In this review article, we briefly review the applications of SMLM in quantitative biology, and also the challenges involved and some of the solutions that have been proposed. Due to its advantages in labeling specificity and the relatively low overcounting caused by photoblinking when photo‐activable fluorescent proteins (PA‐FPs) are used as labels, we focus specifically on Photo‐Activated Localization Microscopy (PALM), even though the ideas presented might be applicable to SMLM in general. Also, we focus on the following three quantitative measurements: single molecule counting, analysis of protein spatial distribution heterogeneity and co‐localization analysis.

Identification of the factors affecting co-localization precision for quantitative multicolor localization microscopy

This work explores the potential of multi-color Photoactivated Localization Microscopy (PALM) imaging to probe sub-diffraction limit interactions between proteins with spectrally separated labels. Using a PALM setup built around a commercial microscope axially stabilized to nm-level, we determined the ultimate registration accuracy that could be achieved (10 nm) and compared the performance of three different pairs of fluorescent proteins that can be used in dual color PALM. Fusion constructs were cloned and imaged either in vitro or at the cell plasma membrane, allowing to identify a current limit to co-localization precision of approximately 30–40 nm. We identified the better performing pair and present a concluding perspective application to a co-clustering study.