Silvio O. Rizzoli

Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins

High-definition view of the synapse Individual neurons communicate with one another via their synapses, so to understand the nervous system, we need to understand in detail how the synapses are organized. Wilhelm et al. present a quantitative molecular-scale image of the “average” synapse populated with realistic renditions of each of the protein components that contribute to the inner workings of neurons. Science, this issue p. 1023 A model of the “average” synapse shows ~300,000 proteins and all organelles in the correct copy numbers, sizes, and positions. Synaptic vesicle recycling has long served as a model for the general mechanisms of cellular trafficking. We used an integrative approach, combining quantitative immunoblotting and mass spectrometry to determine protein numbers; electron microscopy to measure organelle numbers, sizes, and positions; and super-resolution fluorescence microscopy to localize the proteins. Using these data, we generated a three-dimensional model of an “average” synapse, displaying 300,000 proteins in atomic detail. The copy numbers of proteins involved in the same step of synaptic vesicle recycling correlated closely. In contrast, copy numbers varied over more than three orders of magnitude between steps, from about 150 copies for the endosomal fusion proteins to more than 20,000 for the exocytotic ones.

Synaptic vesicle recycling: steps and principles

Synaptic vesicle recycling is one of the best‐studied cellular pathways. Many of the proteins involved are known, and their interactions are becoming increasingly clear. However, as for many other pathways, it is still difficult to understand synaptic vesicle recycling as a whole. While it is generally possible to point out how synaptic reactions take place, it is not always easy to understand what triggers or controls them. Also, it is often difficult to understand how the availability of the reaction partners is controlled: how the reaction partners manage to find each other in the right place, at the right time. I present here an overview of synaptic vesicle recycling, discussing the mechanisms that trigger different reactions, and those that ensure the availability of reaction partners. A central argument is that synaptic vesicles bind soluble cofactor proteins, with low affinity, and thus control their availability in the synapse, forming a buffer for cofactor proteins. The availability of cofactor proteins, in turn, regulates the different synaptic reactions. Similar mechanisms, in which one of the reaction partners buffers another, may apply to many other processes, from the biogenesis to the degradation of the synaptic vesicle.

Endosomal sorting of readily releasable synaptic vesicles

Neurotransmitter release is achieved through the fusion of synaptic vesicles with the neuronal plasma membrane (exocytosis). Vesicles are then retrieved from the plasma membrane (endocytosis). It was hypothesized more than 3 decades ago that endosomes participate in vesicle recycling, constituting a slow endocytosis pathway required especially after prolonged stimulation. This recycling model predicts that newly endocytosed vesicles fuse with an endosome, which sorts (organizes) the molecules and buds exocytosis-competent vesicles. We analyzed here the endosome function using hippocampal neurons, isolated nerve terminals (synaptosomes), and PC12 cells by stimulated emission depletion microscopy, photooxidation EM, and several conventional microscopy assays. Surprisingly, we found that endosomal sorting is a rapid pathway, which appeared to be involved in the recycling of the initial vesicles to be released on stimulation, the readily releasable pool. In agreement with the endosomal model, the vesicle composition changed after endocytosis, with the newly formed vesicles being enriched in plasma membrane proteins. Vesicle proteins were organized in clusters both in the plasma membrane (on exocytosis) and in the endosome. In the latter compartment, they segregated from plasma membrane components in a process that is likely important for sorting/budding of newly developed vesicles from the endosome.

Multi-protein assemblies underlie the mesoscale organization of the plasma membrane

Most proteins have uneven distributions in the plasma membrane. Broadly speaking, this may be caused by mechanisms specific to each protein, or may be a consequence of a general pattern that affects the distribution of all membrane proteins. The latter hypothesis has been difficult to test in the past. Here, we introduce several approaches based on click chemistry, through which we study the distribution of membrane proteins in living cells, as well as in membrane sheets. We found that the plasma membrane proteins form multi-protein assemblies that are long lived (minutes), and in which protein diffusion is restricted. The formation of the assemblies is dependent on cholesterol. They are separated and anchored by the actin cytoskeleton. Specific proteins are preferentially located in different regions of the assemblies, from their cores to their edges. We conclude that the assemblies constitute a basic mesoscale feature of the membrane, which affects the patterning of most membrane proteins, and possibly also their activity.

Aptamers as potential tools for super-resolution microscopy

(a) The TfnR aptamer c2, the EGFR aptamer E07 and their respective control aptamers (random sequences) were incubated at 37°C with human A431 cells as described in Supplementary Methods. Similarly, HeLa cells stably transfected with a human PSMA construct were incubated with the PSMA A9 aptamer or its random control. The pairs of images (control and aptamer) are equally scaled to allow a direct visual comparison. The insets in the control images correspond to the same images, scaled to a level where autofluorescence can be visualized. Scale bar, 10 μm. (b) Colocalization of the different aptamers with endosomal labels. We co-incubated the cells (same as above) with aptamers against TfnR (c2) or PSMA (A9) and Alexa488-transferrin (Invitrogen), since the latter constitutes an ideal marker for early endosomes.

The Aspergillus nidulans MAPK module AnSte11-Ste50-Ste7-Fus3 controls development and secondary metabolism.

The sexual Fus3 MAP kinase module of yeast is highly conserved in eukaryotes and transmits external signals from the plasma membrane to the nucleus. We show here that the module of the filamentous fungus Aspergillus nidulans (An) consists of the AnFus3 MAP kinase, the upstream kinases AnSte7 and AnSte11, and the AnSte50 adaptor. The fungal MAPK module controls the coordination of fungal development and secondary metabolite production. It lacks the membrane docking yeast Ste5 scaffold homolog; but, similar to yeast, the entire MAPK modules proteins interact with each other at the plasma membrane. AnFus3 is the only subunit with the potential to enter the nucleus from the nuclear envelope. AnFus3 interacts with the conserved nuclear transcription factor AnSte12 to initiate sexual development and phosphorylates VeA, which is a major regulatory protein required for sexual development and coordinated secondary metabolite production. Our data suggest that not only Fus3, but even the entire MAPK module complex of four physically interacting proteins, can migrate from plasma membrane to nuclear envelope.

The same synaptic vesicles drive active and spontaneous release

Synaptic vesicles release neurotransmitter both actively (on stimulation) and spontaneously (at rest). It has been assumed that identical vesicles use both modes of release; however, recent evidence has challenged this view. Using several assays (FM dye imaging, pHluorin imaging and antibody-labeling of synaptotagmin) in neuromuscular preparations from Drosophila, frog and mouse, as well as rat cultured neurons, we found that the same vesicles participate in active and spontaneous release.

A small pool of vesicles maintains synaptic activity in vivo

Chemical synapses contain substantial numbers of neurotransmitter-filled synaptic vesicles, ranging from approximately 100 to many thousands. The vesicles fuse with the plasma membrane to release neurotransmitter and are subsequently reformed and recycled. Stimulation of synapses in vitro generally causes the majority of the synaptic vesicles to release neurotransmitter, leading to the assumption that synapses contain numerous vesicles to sustain transmission during high activity. We tested this assumption by an approach we termed cellular ethology, monitoring vesicle function in behaving animals (10 animal models, nematodes to mammals). Using FM dye photooxidation, pHluorin imaging, and HRP uptake we found that only approximately 1–5% of the vesicles recycled over several hours, in both CNS synapses and neuromuscular junctions. These vesicles recycle repeatedly, intermixing slowly (over hours) with the reserve vesicles. The latter can eventually release when recycling is inhibited in vivo but do not seem to participate under normal activity. Vesicle recycling increased only to ≈5% in animals subjected to an extreme stress situation (frog predation on locusts). Synapsin, a molecule binding both vesicles and the cytoskeleton, may be a marker for the reserve vesicles: the proportion of vesicles recycling in vivo increased to 30% in synapsin-null Drosophila. We conclude that synapses do not require numerous reserve vesicles to sustain neurotransmitter release and thus may use them for other purposes, examined in the accompanying paper.

An RNA Alternative to Human Transferrin: A New Tool for Targeting Human Cells

The transferrin receptor, CD71, is an attractive target for drug development because of its high expression on a number of cancer cell lines and the blood brain barrier. To generate serum-stabilized aptamers that recognize the human transferrin receptor, we have modified the traditional aptamer selection protocol by employing a functional selection step that enriches for RNA molecules which bind the target receptor and are internalized by cells. Selected aptamers were specific for the human receptor, rapidly endocytosed by cells and shared a common core structure. A minimized variant was found to compete with the natural ligand, transferrin, for receptor binding and cell uptake, but performed ~twofold better than it in competition experiments. Using this molecule, we generated aptamer-targeted siRNA-laden liposomes. Aptamer targeting enhanced both uptake and target gene knockdown in cells grown in culture when compared to nonmodified or nontargeted liposomes. The aptamer should prove useful as a surrogate for transferrin in many applications including cell imaging and targeted drug delivery.

The reserve pool of synaptic vesicles acts as a buffer for proteins involved in synaptic vesicle recycling

Presynaptic nerve terminals contain between several hundred vesicles (for example in small CNS synapses) and several tens of thousands (as in neuromuscular junctions). Although it has long been assumed that such high numbers of vesicles are required to sustain neurotransmission during conditions of high demand, we found that activity in vivo requires the recycling of only a few percent of the vesicles. However, the maintenance of large amounts of reserve vesicles in many evolutionarily distinct species suggests that they are relevant for synaptic function. We suggest here that these vesicles constitute buffers for soluble accessory proteins involved in vesicle recycling, preventing their loss into the axon. Supporting this hypothesis, we found that vesicle clusters contain a large variety of proteins needed for vesicle recycling, but without an obvious function within the clusters. Disrupting the clusters by application of black widow spider venom resulted in the diffusion of numerous soluble proteins into the axons. Prolonged stimulation and ionomycin application had a similar effect, suggesting that calcium influx causes the unbinding of soluble proteins from vesicles. Confirming this hypothesis, we found that isolated synaptic vesicles in vitro sequestered soluble proteins from the cytosol in a process that was inhibited by calcium addition. We conclude that the reserve vesicles support neurotransmission indirectly, ensuring that soluble recycling proteins are delivered upon demand during synaptic activity.