Complexins (Cplxs) are small synaptic proteins that cooperate with SNARE-complexes in the control of synaptic vesicle (SV) fusion. Studies involving genetic mutation, knock-down, or knock-out indicated two key functions of Cplx that are not mutually exclusive but cannot easily be reconciled, one in facilitating SV fusion, and one in "clamping" SVs to prevent premature fusion. Most studies on the role of Cplxs in mammalian synapse function have relied on cultured neurons, heterologous expression systems, or membrane fusion assays in vitro, whereas little is known about the function of Cplxs in native synapses. We therefore studied consequences of genetic ablation of Cplx1 in the mouse calyx of Held synapse, and discovered a developmentally exacerbating phenotype of reduced spontaneous and evoked transmission but excessive asynchronous release after stimulation, compatible with combined facilitating and clamping functions of Cplx1. Because action potential waveforms, Ca 2ϩ influx, readily releasable SV pool size, and quantal size were unaltered, the reduced synaptic strength in the absence of Cplx1 is most likely a consequence of a decreased release probability, which is caused, in part, by less tight coupling between Ca 2ϩ channels and docked SV. We found further that the excessive asynchronous release in Cplx1-deficient calyces triggered aberrant action potentials in their target neurons, and slowed-down the recovery of EPSCs after depleting stimuli. The augmented asynchronous release had a delayed onset and lasted hundreds of milliseconds, indicating that it predominantly represents fusion of newly recruited SVs, which remain unstable and prone to premature fusion in the absence of Cplx1.

Abstract. A family of¯uorescent styryl dyes was synthesized to apply them as probes that monitor the ion-translocating activity of the Na,K-ATPase and the SR Ca-ATPase, similar to the widely used dye RH421. All dyes had the same chromophore but they di ered in the length of the spacer between chromophore and polar head, an isothiocyanate group, and in the lengths of the two identical acyl chains, which form the tail of the dye molecules. A number of substrate-dependent partial reactions of both P-type ATPases a ected the¯uorescence intensity, and the magnitude of the¯uorescence changes was used to characterize the usefulness of the dyes for further application. The experimental results indicate that electrochromy is the major mechanism of these dyes. While in the case of the Na,K-ATPase a single dye, 5QITC, showed larger¯uorescence changes than all others, in the case of the SR Ca-ATPase all dyes tested were almost equal in their¯uorescence responses. This prominent di erence is interpreted as a hint that the position of the ion binding sites in both ion pumps may di er signi®cantly despite their otherwise closely related structural features. Quench experiments with spin-labeled lipids in various positions of their fatty acids were used to gain information on the depth of the chromophore of the di erent dyes within the membrane dielectric, however, the spatial resolution was so poor that only qualitative information on the position of the chromophore in the lipid phase could be obtained.

With YFP near the regulatory domain and CFP in the C terminus, we recorded a 6% FRET change signaling the regulatory phosphorylation event. On the other hand, when the probe was placed at the extreme N terminus, such changes were not seen, presumably due to the length and predicted flexibility of the N terminus. Substantial FRET changes were observed cotemporaneous with cell volume changes, possibly reflective of an increase in molecular crowding upon cell shrinkage.

Interferometric Photo-Activation-Localization-Microscopy (iPALM) localizes single fluorescent molecules with 20 nm lateral and 10 nm axial resolution. We present a method utilizing glass coverslip lithography for correlative imaging between iPALM and scanning electron microscopy (SEM). Using iPALM on HIV Gag-Dendra virus-like particles (VLPs) we localized the position of HIV Gag proteins. Based on these localizations we reconstructed the central cavity of the VLPs along with imperfections within the HIV Gag lattice. The SEM images and iPALM images overlap and show imaging from single VLPs immobilized on glass coverslips. The localization of many HIV proteins including accessory proteins and Gag-Pol remains unknown, we discuss how the specificity of iPALM coupled with SEM has the potential for resolving more of HIV proteins.

The ability to observe the movement of neurotransmitter receptors in and around a synapse could provide crucial new information to our understanding of synaptic plasticity, the process that likely underlies memory formation. We have developed a new class of fluorescent probes designed to target fluorophores to natively-expressed neuronal receptors. This strategy allows for receptors to be covalently-tagged and tracked in a non-perturbed state; thus allowing for visualization of complex neuronal processes. Specifically, calcium-permeable, non-NMDA glutamate receptors (CP-AMPARs) expressed in hippocampal neurons can be targeted with this novel tri-functional molecule. CP-AMPARs have recently been shown to play a role in some forms of synaptic plasticity, aiding such processes as long-term potentiation and depression, but their basal location around the synapse remains unknown. In brief, our molecular design employs a use-dependent polyamine ligand which targets only the receptors receiving glutamatergic input at the time of labeling, a promiscuous electrophile for covalent bond formation with a nucleophillc sidechain amino acid on the channel, and a fluorophore for visualization. Bioconjugation of this molecule results in stable covalent bond formation between the probe and the target receptor. An additional aspect of our first generation probes is that the ligand is connected to the remainder of the probe with a photolabile linker, thus allowing the receptor to re-enter the non-liganded and native state.