Cross-linking mass spectrometry uncovers protein interactions and functional assemblies in synaptic vesicle membranes

Synaptic vesicles are storage organelles for neurotransmitters. They pass through a trafficking cycle and fuse with the pre-synaptic membrane when an action potential arrives at the nerve terminal. While molecular components and biophysical parameters of synaptic vesicles have been determined, our knowledge on the protein interactions in their membranes is limited. Here, we apply cross-linking mass spectrometry to study interactions of synaptic vesicle proteins in an unbiased approach without the need for specific antibodies or detergent-solubilisation. Our large-scale analysis delivers a protein network of vesicle sub-populations and functional assemblies including an active and an inactive conformation of the vesicular ATPase complex as well as non-conventional arrangements of the luminal loops of SV2A, Synaptophysin and structurally related proteins. Based on this network, we specifically target Synaptobrevin-2, which connects with many proteins, in different approaches. Our results allow distinction of interactions caused by ‘crowding’ in the vesicle membrane from stable interaction modules

Neurotransmitter uptake of synaptic vesicles studied by X-ray diffraction

The size, polydispersity, and electron density profile of synaptic vesicles (SVs) can be studied by small-angle X-ray scattering (SAXS), i.e. by X-ray diffraction from purified SV suspensions in solution. Here we show that size and shape transformations, as they appear in the functional context of these important synaptic organelles, can also be monitored by SAXS. In particular, we have investigated the active uptake of neurotransmitters, and find a mean vesicle radius increase of about 12% after the uptake of glutamate, which indicates an unusually large extensibility of the vesicle surface, likely to be accompanied by conformational changes of membrane proteins and rearrangements of the bilayer. Changes in the electron density profile (EDP) give first indications for such a rearrangement. Details of the protein structure are screened, however, by SVs polydispersity. To overcome the limitations of large ensemble averages and heterogeneous structures, we therefore propose serial X-ray diffraction by single free electron laser pulses. Using simulated data for realistic parameters, we show that this is in principle feasible, and that even spatial distances between vesicle proteins could be assessed by this approach

In vitro fusion of single synaptic and dense core vesicles reproduces key physiological properties.

Regulated exocytosis of synaptic vesicles is substantially faster than of endocrine dense core vesicles despite similar molecular machineries. The reasons for this difference are unknown and could be due to different regulatory proteins, different spatial arrangements, different vesicle sizes, or other factors. To address these questions, we take a reconstitution approach and compare regulated SNARE-mediated fusion of purified synaptic and dense core chromaffin and insulin vesicles using a single vesicle-supported membrane fusion assay. In all cases, Munc18 and complexin are required to restrict fusion in the absence of calcium. Calcium triggers fusion of all docked vesicles. Munc13 (C1C2MUN domain) is required for synaptic and enhanced insulin vesicle fusion, but not for chromaffin vesicles, correlating inversely with the presence of CAPS protein on purified vesicles. Striking disparities in calcium-triggered fusion rates are observed, increasing with curvature with time constants 0.23 s (synaptic vesicles), 3.3 s (chromaffin vesicles), and 9.1 s (insulin vesicles) and correlating with rate differences in cells

A importância da comunicação durante o período de recuperação pós-operatória

Este estudo teve como objetivo conhecer a importância da comunicação durante as orientações pós-operatórias fornecidas pela equipe de enfermagem a pacientes e/ou familiares de uma instituição privada e apreender a percepção destes indivíduos acerca das orientações recebidas. Foi um estudo transversal, descritivo, com abordagem qualitativa, que utilizou o referencial teórico da Comunicação Interpessoal e o referencial metodológico da Análise de Conteúdo. Participaram do estudo 16 pacientes entrevistados no período pós-operatório mediato. Os resultados evidenciaram que a equipe de enfermagem focaliza as orientações nas técnicas instrumentais da profissão, não abordando o indivíduo de forma holística. Também foi possível perceber que, quando o profissional enfermeiro permanece afastado do paciente e/ou não presta informações adequadas gera sentimentos de ansiedade, medo, insegurança e sensação de falta de cuidado. Por outro lado, quando a equipe de enfermagem se fez presente com cuidado e informações coerentes, os clientes relataram um alto nível de satisfação e a sensação de ser bem cuidado

Colocalization of different neurotransmitter transporters on synaptic vesicles is sparse except for VGLUT1 and ZnT3

Vesicular transporters (VTs) define the type of neurotransmitter that synaptic vesicles (SVs) store and release. While certain mammalian neurons release multiple transmitters, it is not clear whether the release occurs from the same or distinct vesicle pools at the synapse. Using quantitative single-vesicle imaging, we show that a vast majority of SVs in the rodent brain contain only one type of VT, indicating specificity for a single neurotransmitter. Interestingly, SVs containing dual transporters are highly diverse (27 types) but small in proportion (2% of all SVs), excluding the largest pool that carries VGLUT1 and ZnT3 (34%). Using VGLUT1-ZnT3 SVs, we demonstrate that the transporter colocalization influences the SV content and synaptic quantal size. Thus, the presence of diverse transporters on the same vesicle is bona fide, and depending on the VT types, this may act to regulate neurotransmitter type, content, and release in space and time

Estudo da adsorção de dispersante à base de poliacrilato em um sistema varistor comercial: caracterização físico-química

Um dos grandes desafios no processamento cerâmico é a obtenção de dispersões de partículas estáveis e homogêneas contendo materiais com diferentes características físicas e químicas, como densidade, área de superfície específica, distribuição de tamanho de partículas e ponto isoelétrico. O comportamento de suspensões obtidas a partir da composição de um sistema varistor à base de ZnO (aditivos: Bi2O3, Sb2O3, Co3O4, Mn2O3, NiO, Cr2O3) em meio aquoso, utilizando o poliacrilato de amônio como dispersante foi avaliada para determinar a influência dos diferentes óxidos no processamento. Os óxidos foram caracterizados a partir das técnicas mobilidade eletroforética dinâmica, espectroscopia no infravermelho, distribuição granulométrica e área de superfície específica. Os óxidos aditivos apresentam características de superfície, como ponto isoelétricos (pie), distintos com relação ao óxido base (ZnO - pie 9.4). Os dois mais importantes, devido às fortes diferenças de pie foram o Sb2O3 e o Cr2O3, pie = 3,4 e 4,4, respectivamente. Esta diferença de pie entre o ZnO e os aditivos promovem uma mudança no pH quando em dispersão aquosa, que passa a ser de aproximadamente 8,0 e que pode comprometer a estabilidade da dispersão em algumas etapas do processo de dispersão

Vesicle tethering on the surface of phase-separated active zone condensates

Tethering of synaptic vesicles (SVs) to the active zone determines synaptic strength, although the molecular basis governing SV tethering is elusive. Here, we discover that small unilamellar vesicles (SUVs) and SVs from rat brains coat on the surface of condensed liquid droplets formed by active zone proteins RIM, RIM-BP, and ELKS via phase separation. Remarkably, SUV-coated RIM/RIM-BP condensates are encapsulated by synapsin/SUV condensates, forming two distinct SUV pools reminiscent of the reserve and tethered SV pools that exist in presynaptic boutons. The SUV-coated RIM/RIM-BP condensates can further cluster Ca2+ channels anchored on membranes. Thus, we reconstitute a presynaptic bouton-like structure mimicking the SV-tethered active zone with its one side attached to the presynaptic membrane and the other side connected to the synapsin-clustered SV condensates. The distinct interaction modes between membraneless protein condensates and membrane-based organelles revealed here have general implications in cellular processes, including vesicular formation and trafficking, organelle biogenesis, and autophagy