Imaging of post-synaptic membrane trafficking in neuronal dendrites: progress, limitations, and new developments.

Membrane trafficking of post-synaptic cargo is a key determinant of synaptic transmission and synaptic plasticity. We describe here the latest developments in visualizing individual exocytosis and endocytosis events in neurons using pH-sensitive tags. We show how these tools help decipher the spatial and temporal regulation of membrane trafficking steps during synaptic plasticity.

Rational Engineering of an Improved Genetically Encoded pH Sensor Based on Superecliptic pHluorin.

Genetically encoded pH sensors based on fluorescent proteins are valuable tools for the imaging of cellular events that are associated with pH changes, such as exocytosis and endocytosis. Superecliptic pHluorin (SEP) is a pH-sensitive green fluorescent protein (GFP) variant widely used for such applications. Here, we report the rational design, development, structure, and applications of Lime, an improved SEP variant with higher fluorescence brightness and greater pH sensitivity.

Selective endocytosis of Ca-permeable AMPARs by the Alzheimer's disease risk factor CALM bidirectionally controls synaptic plasticity.

AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission, and the plastic modulation of their surface levels determines synaptic strength. AMPARs of different subunit compositions fulfill distinct roles in synaptic long-term potentiation (LTP) and depression (LTD) to enable learning. Largely unknown endocytic mechanisms mediate the subunit-selective regulation of the surface levels of GluA1-homomeric Ca-permeable (CP) versus heteromeric Ca-impermeable (CI) AMPARs.

Confocal and TIRF microscopy based approaches to visualize arrestin trafficking in living cells.

Arrestins are key proteins that serve as versatile scaffolds to control and mediate G protein coupled receptors (GPCR) activity. Arrestin control of GPCR functions involves their recruitment from the cytosol to plasma membrane-localized GPCRs and to endosomal compartments, where they mediate internalization, sorting and signaling of GPCRs. Several methods can be used to monitor trafficking of arrestins; however, live fluorescence imaging remains the method of choice to both assess arrestin recruitment to ligand-activated receptors and to monitor its dynamic subcellular localization.

Pharmacological Characterization of Low Molecular Weight Biased Agonists at the Follicle Stimulating Hormone Receptor.

Follicle-stimulating hormone receptor (FSHR) plays a key role in reproduction through the activation of multiple signaling pathways. Low molecular weight (LMW) ligands composed of biased agonist properties are highly valuable tools to decipher complex signaling mechanisms as they allow selective activation of discrete signaling cascades. However, available LMW FSHR ligands have not been fully characterized yet.