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GFP nanobodies reveal recently-exocytosed pHluorin molecules.

ABSTRACT: Neurotransmitter release requires vesicle recycling, which consists of exocytosis, endocytosis and the reformation of new fusion-competent vesicles. One poorly understood aspect in this cycle is the fate of the vesicle proteins after exocytosis, when they are left on the plasma membrane. Such proteins are often visualized by coupling to pH-sensitive GFP moieties (pHluorins). However, pHluorin imaging is typically limited by diffraction to spots several-fold larger than the vesicles. Here we show that pHuorin-tagged vesicle proteins can be easily detected using single-domain antibodies (nanobodies) raised against GFP. By coupling the nanobodies to chemical fluorophores that were optimal for super-resolution imaging, we could analyze the size and intensity of the groups of pHluorin-tagged proteins under a variety of conditions, in a fashion that would have been impossible based solely on the pHluorin fluorescence. We conclude that nanobody-based pHluorin detection is a promising tool for investigating post-exocytosis events in neurons.

SUBMITTER: Seitz KJ

PROVIDER: S-EPMC6533288 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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GFP nanobodies reveal recently-exocytosed pHluorin molecules.

Seitz Katharina J KJ Rizzoli Silvio O SO

Scientific reports 20190523 1

Neurotransmitter release requires vesicle recycling, which consists of exocytosis, endocytosis and the reformation of new fusion-competent vesicles. One poorly understood aspect in this cycle is the fate of the vesicle proteins after exocytosis, when they are left on the plasma membrane. Such proteins are often visualized by coupling to pH-sensitive GFP moieties (pHluorins). However, pHluorin imaging is typically limited by diffraction to spots several-fold larger than the vesicles. Here we show ...[more]

PMID: 31123313

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Project description:Norovirus is the leading cause of gastroenteritis worldwide. Despite recent developments in norovirus propagation in cell culture, these viruses are still challenging to grow routinely. Moreover, little is known on how norovirus infects the host cells, except that histo-blood group antigens (HBGAs) are important binding factors for infection and cell entry. Antibodies that bind at the HBGA pocket and block attachment to HBGAs are believed to neutralize the virus. However, additional neutralization epitopes elsewhere on the capsid likely exist and impeding the intrinsic structural dynamics of the capsid could be equally important. In the current study, we investigated a panel of Nanobodies in order to probe functional epitopes that could trigger capsid rearrangement and/ or interfere with HBGA binding interactions. The precise binding sites of six Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42) were identified using X-ray crystallography. We showed that these Nanobodies bound on the top, side, and bottom of the norovirus protruding domain. The impact of Nanobody binding on norovirus capsid morphology was analyzed using electron microscopy and dynamic light scattering. We discovered that distinct Nanobody epitopes were associated with varied changes in particle structural integrity and assembly. Interestingly, certain Nanobody-induced capsid morphological changes lead to the capsid protein degradation and viral RNA exposure. Moreover, Nanobodies employed multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, which included steric obstruction (Nano-14), allosteric interference (Nano-32), and violation of normal capsid morphology (Nano-26 and Nano-85). Finally, we showed that two Nanobodies (Nano-26 and Nano-85) not only compromised capsid integrity and inhibited VLPs attachment to HBGAs, but also recognized a broad panel of norovirus genotypes with high affinities. Consequently, Nano-26 and Nano-85 have a great potential to function as novel therapeutic agents against human noroviruses.

Project description:G-protein-coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signalling effectors such as G proteins and ?-arrestins. It is well known that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (‘efficacy’). Furthermore, increasing biophysical evidence, primarily using the ?2-adrenergic receptor (?2AR) as a model system, supports the existence of multiple active and inactive conformational states. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct ?2AR conformations using single domain camelid antibodies (nanobodies)—a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60). We show that Nb60 stabilizes a previously unappreciated low-affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoprenaline has a 15,000-fold higher affinity for ?2AR in the presence of Nb80 compared to the affinity of isoprenaline for ?2AR in the presence of Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the ?2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the inactive, Nb60-bound state (R60) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states.

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GFP nanobodies reveal recently-exocytosed pHluorin molecules.

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GFP nanobodies reveal recently-exocytosed pHluorin molecules.

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