Lateral diffusion and exocytosis of membrane proteins in cultured neurons assessed using fluorescence recovery and fluorescence-loss photobleaching.

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins. Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins(1-2). At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)(3-5). Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination(6-10). FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application(8,10). We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines(10), and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

Measuring membrane protein dynamics in neurons using fluorescence recovery after photobleach.

The use of genetically encoded fluorescent tags such as green fluorescent protein (GFP) as reporters to monitor processes in living cells has transformed cell biology. One major application for these tools has been to analyze protein dynamics in neurons. In particular, fluorescence recovery after photobleach (FRAP) of surface expressed fluorophore-tagged proteins has been instrumental to addressing outstanding questions about how neurons orchestrate the synaptic delivery of proteins. Here, we provide an overview of the methodology, equipment, and analysis required to perform, analyze, and interpret these experiments.

Dynamin-dependent membrane drift recruits AMPA receptors to dendritic spines.

The surface expression and localization of AMPA receptors (AMPARs) at dendritic spines are tightly controlled to regulate synaptic transmission. Here we show that de novo exocytosis of the GluR2 AMPAR subunit occurs at the dendritic shaft and that new AMPARs diffuse into spines by lateral diffusion in the membrane. However, membrane topology restricts this lateral diffusion. We therefore investigated which mechanisms recruit AMPARs to spines from the shaft and demonstrated that inhibition of dynamin GTPase activity reduced lateral diffusion of membrane-anchored green fluorescent protein and super-ecliptic pHluorin (SEP)-GluR2 into spines. In addition, the activation of synaptic N-methyl-d-aspartate (NMDA) receptors enhanced lateral diffusion of SEP-GluR2 and increased the number of endogenous AMPARs in spines. The NMDA-invoked effects were prevented by dynamin inhibition, suggesting that activity-dependent dynamin-mediated endocytosis within spines generates a net inward membrane drift that overrides lateral diffusion barriers to enhance membrane protein delivery into spines. These results provide a novel mechanistic explanation of how AMPARs and other membrane proteins are recruited to spines by synaptic activity.

Human GluR6c, a functional splicing variants of GluR6, is mainly expressed in non-nervous cells.

Kainate-type glutamate receptors (KARs) are receptor channels with a variety of distinct physiological functions in synaptic transmission, depending on their sub-cellular location in functional neuronal compartments. The kainate receptor subunit GluR6 presents different splice variants involving the C-terminal domain, namely GluR6a, GluR6b and GluR6c. In this study, we report the analysis of the three human splicing isoforms and in particular of the uncharacterized hGluR6c. When expressed in COS-7 cells, hGluR6a receptor subunit was highly present on the surface of the plasma membrane, whereas hGluR6b and hGluRc were poorly transported to the membrane. Electrophysiological studies of homomeric receptors showed that hGluR6c subunit can generate functional receptors with characteristics similar to the GluR6b variant. mRNA expression analysis demonstrated that hGluR6c variant is mainly expressed in non-neuronal cells and barely expressed in neuronal ones. Interestingly, undifferentiated NT2 cells expressing only the hGluR6c isoform, during neuronal differentiation induced by retinoic acid, increased the expression level of the neuronal form hGluR6a with a parallel decreased of hGluR6c. Overall, our data indicate that hGluR6c might have unique properties in non-nervous cells and in the first stages of CNS development.

Retaining synaptic AMPARs.

Endocytosis, exocytosis, and lateral diffusion are key mechanisms for AMPA receptor trafficking. Endocytosis of AMPARs and other postsynaptic proteins has been proposed to occur at specific endocytic zones (EZs), but the mechanisms that regulate this process are not at all clear. In this issue of Neuron, Lu et al. show that correct synaptic EZ positioning requires links between the GTPase dynamin-3 and the Homer/Shank complex.

Fast regulation of axonal growth cone motility by electrical activity.

Axonal growth cones are responsible for the correct guidance of developing axons and the establishment of functional neural networks. They are highly motile because of fast and continuous rearrangements of their actin-rich cytoskeleton. Here we have used live imaging of axonal growth cones of hippocampal neurons in culture and quantified their motility with a temporal resolution of 2 s. Using novel methods of analysis of growth cone dynamics, we show that transient activation of kainate receptors by bath-applied kainate induced a fast and reversible growth cone stalling. This effect depends on electrical activity and can be mimicked by the transient discharge of action potentials elicited in the neuron by intracellular current injections at the somatic level through a patch pipette. Growth cone stalling induced by electrical stimulation is mediated by calcium entry from the extracellular medium as well as by calcium release from intracellular stores that define spatially restricted microdomains directly affecting cytoskeletal dynamics. We propose that growth cone motility is dynamically controlled by transient bursts of spontaneous electrical activity, which constitutes a prominent feature of developing neural networks in vivo.

Co-assembly of two GluR6 kainate receptor splice variants within a functional protein complex.

Kainate receptors (KAR) are composed of several distinct subunits and splice variants, but the functional relevance of this diversity remains largely unclear. Here we show that two splice variants of the GluR6 subunit, GluR6a and GluR6b, which differ in their C-terminal domains, do not show distinct functional properties, but coassemble as heteromers in vitro and in vivo. Using a proteomic approach combining affinity purification and MALDI-TOF mass spectrometry, we found that GluR6a and GluR6b interact with two distinct subsets of cytosolic proteins mainly involved in Ca(2+) regulation of channel function and intracellular trafficking. Guided by these results, we provide evidence that the regulation of native KAR function by NMDA receptors depends on the heteromerization of GluR6a and GluR6b and interaction of calcineurin with GluR6b. Thus, GluR6a and GluR6b bring in close proximity two separate subsets of interacting proteins that contribute to the fine regulation of KAR trafficking and function.

An automated method to quantify and visualize colocalized fluorescent signals.

The most commonly used method to analyze colocalization of fluorescent signal in paired images is based on superimposition of images ("merging") and visual inspection. A method based on the comparison of the mean deviation of fluorescent signal intensity has recently been proposed to quantify colocalization within a user-defined area [Li Q, Lau A, Morris TJ, Guo L, Fordyce CB, Stanley EF. A syntaxin 1, Galpha(o), and N-type calcium channel complex at a presynaptic nerve terminal: analysis by quantitative immunocolocalization. J Neurosci 2004;24:4070-81]. Unfortunately, the latter quantification method does not provide a spatial representation of the correlation between the two fluorescent signals. Here we propose a new method that combines quantification and imaging of colocalization. We describe an algorithm based on edge detection and calculation of signal intensity deviation. The method is illustrated and validated on both simulated images and experimental data. This new and automated method calculates a correlation index (I(corr)) and generates an image of the correlated signals from the two original images. In addition to help in comparing and quantifying colocalization between two fluorescent stainings, this method can be adapted to measure the distribution of ions, proteins, organelles and cells in a large array of techniques.

Differential trafficking of GluR7 kainate receptor subunit splice variants.

Kainate receptors (KARs) are heteromeric ionotropic glutamate receptors that play a variety of roles in the regulation of synaptic network activity. The function of glutamate receptors (GluRs) is highly dependent on their surface density in specific neuronal domains. Alternative splicing is known to regulate surface expression of GluR5 and GluR6 subunits. The KAR subunit GluR7 exists under different splice variant isoforms in the C-terminal domain (GluR7a and GluR7b). Here we have studied the trafficking of GluR7 splice variants in cultured hippocampal neurons from wild-type and KAR mutant mice. We have found that alternative splicing regulates surface expression of GluR7-containing KARs. GluR7a and GluR7b differentially traffic from the ER to the plasma membrane. GluR7a is highly expressed at the plasma membrane, and its trafficking is dependent on a stretch of positively charged amino acids also found in GluR6a. In contrast, GluR7b is detected at the plasma membrane at a low level and retained mostly in the endoplasmic reticulum (ER). The RXR motif of GluR7b does not act as an ER retention motif, at variance with other receptors and ion channels, but might be involved during the assembly process. Like GluR6a, GluR7a promotes surface expression of ER-retained subunit splice variants when assembled in heteromeric KARs. However, our results also suggest that this positive regulation of KAR trafficking is limited by the ability of different combinations of subunits to form heteromeric receptor assemblies. These data further define the complex rules that govern membrane delivery and subcellular distribution of KARs.

Subcellular localization and trafficking of kainate receptors.

Glutamate receptors of the kainate type have been identified recently as key players in the modulation of neuronal-network activity. The role of kainate receptors depends on their precise subcellular localization in presynaptic, postsynaptic and extrasynaptic domains. Subcellular localization of kainate receptors has been inferred mainly from electrophysiological studies with the help of selective pharmacological tools and kainate receptor mutant mice. These studies, combined with recent ultrastructural data, highlight the diversity of subcellular localizations of kainate receptors. It is important to understand the molecular mechanisms that underlie the polarized trafficking of kainate receptors in distinct neuronal domains. In this article, we review recent data that shed light on the trafficking and membrane delivery of kainate receptor isoforms, and on the identification of proteins that interact with kainate receptors and might regulate this trafficking.

Subunit composition and alternative splicing regulate membrane delivery of kainate receptors.

Kainate receptors (KARs) are heteromeric ionotropic glutamate receptors (GluRs) that play various roles in the regulation of synaptic transmission. The KAR subunits GluR5 and GluR6 exist under different splice variant isoforms in the C-terminal domain (GluR5a, GluR5b, GluR5c, GluR6a, GluR6b). The differential role of KAR subunit splice variants is presently unknown. In transfected COS-7 cells and neurons from wild-type and GluR5 x GluR6 mice, we have found that the subcellular localization and membrane delivery differed between these splice variants. GluR6a was highly expressed at the plasma membrane. GluR6b, GluR5a, and GluR5b were detected at lower levels in the plasma membrane and mainly colocalized with calreticulin in the endoplasmic reticulum (ER). GluR5c was strongly retained in the ER by an RXR motif. GluR6a acted as a key subunit splice variant promoting surface expression of ER-retained subunit splice variants when assembled in heteromeric KARs. Surface expression of GluR6a was independent of its PDZ (postsynaptic density-95/discs large/zona occludens-1) binding motif and was promoted by a stretch of four basic amino acid residues at its C terminus. Overall, splice variants and subunit composition of KARs regulate receptor trafficking from the endoplasmic reticulum to the plasma membrane.