Structure Of Synaptic Vesicles Research Articles

High-Precision Mapping of Membrane Proteins on Synaptic Vesicles using Spectrally Encoded Super-Resolution Imaging.

The spatial resolution of single-molecule localization microscopy is limited by the photon number of a single switching event because of the difficulty of correlating switching events dispersed in time. Here we overcome this limitation by developing a new class of photoswitching semiconducting polymer dots (Pdots) with structured and highly dispersed single-particle spectra. We imaged the Pdots at the first and the second vibronic emission peaks and used the ratio of peak intensities as a spectral coding. By correlating switching events using the spectral coding and performing 4-9 frame binning, we achieved a 2-3 fold experimental resolution improvement versus conventional superresolution imaging. We applied this method to count and map SV2 and proton ATPase proteins on synaptic vesicles (SVs). The results reveal that these proteins are trafficked and organized with high precision, showing unprecedented level of detail about the composition and structure of SVs.

High‐Precision Mapping of Membrane Proteins on Synaptic Vesicles using Spectrally Encoded Super‐Resolution Imaging

AbstractThe spatial resolution of single‐molecule localization microscopy is limited by the photon number of a single switching event because of the difficulty of correlating switching events dispersed in time. Here we overcome this limitation by developing a new class of photoswitching semiconducting polymer dots (Pdots) with structured and highly dispersed single‐particle spectra. We imaged the Pdots at the first and the second vibronic emission peaks and used the ratio of peak intensities as a spectral coding. By correlating switching events using the spectral coding and performing 4–9 frame binning, we achieved a 2–3 fold experimental resolution improvement versus conventional superresolution imaging. We applied this method to count and map SV2 and proton ATPase proteins on synaptic vesicles (SVs). The results reveal that these proteins are trafficked and organized with high precision, showing unprecedented level of detail about the composition and structure of SVs.

Overexpression of Mitochondrial Ligases Reverses Rotenone-Induced Effects in a Drosophila Model of Parkinson's Disease.

Mul1 and Park are two major mitochondrial ligases responsible for mitophagy. Damaged mitochondria that cannot be removed are a source of an increased level of free radicals, which in turn can destructively affect other cell organelles as well as entire cells. One of the toxins that damages mitochondria is rotenone, a neurotoxin that after exposure displays symptoms typical of Parkinson’s disease. In the present study, we showed that overexpressing genes encoding mitochondrial ligases protects neurons during treatment with rotenone. Drosophila strains with overexpressed mul1 or park show a significantly reduced degeneration of dopaminergic neurons, as well as normal motor activity during exposure to rotenone. In the nervous system, rotenone affected synaptic proteins, including Synapsin, Synaptotagmin and Disk Large1, as well as the structure of synaptic vesicles, while high levels of Mul1 or Park suppressed degenerative events at synapses. We concluded that increased levels of mitochondrial ligases are neuroprotective and could be considered in developing new therapies for Parkinson’s disease.

Structure Analysis of Synaptic Vesicles by Solution Small-Angle Scattering of X-Rays

The release of neurotransmitters from neurons, in response to stimulation, forms the basis of communication in the nervous system. Neurotransmitters are stored in small membraneous organelles, synaptic vesicles (SVs), within the presynaptic terminal. These vesicles undergo an elaborate cycle of fusion with the plasma membrane (releasing neurotransmitter), followed by retrieval and reformation and transport back to the plasma membrane for further rounds of fusion [1].In recent years there has been enormous progress in our knowledge of the molecular composition and structure of synaptic vesicles [2]. However, we still lack a detailed view of the physical properties of this trafficking organelle as it proceeds through its life-cycle.Here we use small-angle x-ray scattering (SAXS) to determine the average radial density profile rho(r) and the size polydispersity of SVs [3]. We show that SAXS can be used to study the supra-molecular structure of an entire functional organelle under physiological conditions. The profile rho(r) of SVs including structural parameters of the protein layers, as well as the polydispersity function p(R), are derived with no free prefactors on an absolute scale. The measured SV structure on length scales between the constituent biomolecules and the SV size confirms the main aspects of recent numerical modeling [2], which was based on the crystal structures of the constituent proteins and stoichiometric knowledge from biochemical studies. In addition, we present first evidence of a laterally anisotropic structure, indicative of larger protein clusters.[1] T. Sudhof, Annu. Rev. Neurosci. 27 (2004) 509.[2] S. Takamori et al, Cell 127 (2006) 831.[3] S. Castorph et al, in preparation.

New structural features of synapses in the anteroventral cochlear nucleus prepared by direct freezing and freeze-substitution.

The rostral anteroventral cochlear nucleus (AVCN) of the chinchilla provides a preparation in which neuronal cell bodies and synapses in the mammalian central nervous system can be examined after direct freezing and freeze-substitution of rapidly excised brain stem slices. Cell bodies and synapses in the freeze-substituted AVCN differed from those in perfusion-fixed AVCN in several interesting respects. Despite of these differences, four types of synaptic terminal were distinguished in freeze-substituted AVCN and correlated with the four well-known types of perfusion-fixed terminal. Since the transmitter at each of the four types of terminal has been tentatively identified, the structure of synaptic vesicles and junctions in the freeze-substituted terminals could be related to transmitter type. Synaptic vesicles were uniformly round, but their diameters, deployment, and related cytoskeletal elements near the synaptic junction differed in each chemical type of synapse; the synapses thought to be cholinergic, for instance, had only a few vesicles clustered at their presynaptic junctions while the rest of the vesicles were separated from the junction by a network of fine filaments. Two types of filamentous components, short vertical projections from the postsynaptic membrane and thin filaments protruding from these projections, comprised the basic structure of the postsynaptic specialization, but their sizes and distribution differed at each chemical type of terminal. For instance, the postsynaptic specialization at the glycine terminal was distinguished by numerous thin filaments which curved sideways to run parallel to the plasmalemma. Thus, freeze-substitution gives new information about structural differences between chemically different types of synapses, which may reflect differences in their transmitter storage, release, and reception. In addition, the AVCN preparation is of general interest in making cell bodies in the mammalian central nervous system available to the various structural and analytical techniques which depend on direct, rapid freezing.

Enzymatic activities of coated vesicle fractions from bovine and rat cerebrums

The changes in the enzymatic properties of coated vesicle fractions obtained from bovine cerebrums and rat forebrains were investigated as the preparation procedures were modified. Because Mg(2+)-dependent ATPase activity in the coated vesicle fractions that were prepared by conventional centrifugation methods appeared to derive from contaminating particulates, the activity was examined after further purification by column chromatography and confirmed by histochemical technique. Both the coat proteins and the vesicles enclosed in the coat networks failed to show the activity. Since plain synaptic vesicles are known to have ATPase activities, the results may indicate that the membrane structure of synaptic vesicles is modified between the coated vesicle stage and the plain vesicle stage of vesicle recycling as Heuser and Reese proposed (J. Cell Biol.57, 315-344, 1973). The comparison of the specific activity change in ATPase with those of acetylcholinesterase and NADPH-cytochrome c reductase suggested that there were two types of microsomes contaminating coated vesicle fractions that were prepared conventionally.