Image-guided MALDI mass spectrometry for high-throughput single-organelle characterization

Peptidergic dense-core vesicles are involved in packaging and releasing neuropeptides and peptide hormones—critical processes underlying brain, endocrine and exocrine function. Yet, the heterogeneity within these organelles, even for morphologically defined vesicle types, is not well characterized because of their small volumes. We present image-guided, high-throughput mass spectrometry-based protocols to chemically profile large populations of both dense-core vesicles and lucent vesicles for their lipid and peptide contents, allowing observation of the chemical heterogeneity within and between these two vesicle populations. The proteolytic processing products of four prohormones are observed within the dense-core vesicles, and the mass spectral features corresponding to the specific peptide products suggest three distinct dense-core vesicle populations. Notable differences in the lipid mass range are observed between the dense-core and lucent vesicles. These single-organelle mass spectrometry approaches are adaptable to characterize a range of subcellular structures.

Specific KIF1A–adaptor interactions control selective cargo recognition

Intracellular transport in neurons is driven by molecular motors that carry many different cargos along cytoskeletal tracks in axons and dendrites. Identifying how motors interact with specific types of transport vesicles has been challenging. Here, we use engineered motors and cargo adaptors to systematically investigate the selectivity and regulation of kinesin-3 family member KIF1A–driven transport of dense core vesicles (DCVs), lysosomes, and synaptic vesicles (SVs). We dissect the role of KIF1A domains in motor activity and show that CC1 regulates autoinhibition, CC2 regulates motor dimerization, and CC3 and PH mediate cargo binding. Furthermore, we identify that phosphorylation of KIF1A is critical for binding to vesicles. Cargo specificity is achieved by specific KIF1A adaptors; MADD/Rab3GEP links KIF1A to SVs, and Arf-like GTPase Arl8A mediates interactions with DCVs and lysosomes. We propose a model where motor dimerization, posttranslational modifications, and specific adaptors regulate selective KIF1A cargo trafficking.

Synaptotagmin-7 places dense-core vesicles at the cell membrane to promote Munc13-2- and Ca2+-dependent priming

Synaptotagmins confer calcium-dependence to the exocytosis of secretory vesicles, but how coexpressed synaptotagmins interact remains unclear. We find that synaptotagmin-1 and synaptotagmin-7 when present alone act as standalone fast and slow Ca2+-sensors for vesicle fusion in mouse chromaffin cells. When present together, synaptotagmin-1 and synaptotagmin-7 are found in largely non-overlapping clusters on dense-core vesicles. Synaptotagmin-7 stimulates Ca2+-dependent vesicle priming and inhibits depriming, and it promotes ubMunc13-2- and phorbolester-dependent priming, especially at low resting calcium concentrations. The priming effect of synaptotagmin-7 increases the number of vesicles fusing via synaptotagmin-1, while negatively affecting their fusion speed, indicating both synergistic and competitive interactions between synaptotagmins. Synaptotagmin-7 places vesicles in close membrane apposition (<6 nm); without it, vesicles accumulate out of reach of the fusion complex (20–40 nm). We suggest that a synaptotagmin-7-dependent movement toward the membrane is involved in Munc13-2/phorbolester/Ca2+-dependent priming as a prelude to fast and slow exocytosis triggering.

Simulation of a sudden drop-off in distal dense core vesicle concentration in Drosophila type II motoneuron terminals

This paper aims to investigate whether the sudden drop in the content of dense core vesicles (DCVs) reported in [J. Tao, D. Bulgari, D.L. Deitcher, E.S. Levitan, Limited distal organelles and synaptic function in extensive monoaminergic innervation, J. Cell. Sci. 130 (2017) 2520-2529] can be explained without modifying the parameters characterizing the ability of distal en passant boutons to capture and accumulate DCVs. We hypothesize that the drop in DCV content in distal boutons is due to an insufficient supply of anterogradely moving DCVs coming from the soma. As anterogradely moving DCVs are captured (and eventually destroyed) in more proximal boutons on their way to the end of the terminal, the fluxes of anterogradely moving DCVs between the boutons become increasingly smaller, and the most distal boutons are left without DCVs. We tested this hypothesis by modifying the flux of DCVs entering the terminal and found that the number of most distal boutons left unfilled increases if the DCV flux entering the terminal is decreased. The number of anterogradely moving DCVs in the axon can be increased either by the release of a portion of captured DCVs into the anterograde component or by an increase of the anterograde DCV flux into the terminal. This increase could lead to having enough anterogradely moving DCVs such that they could reach the most distal bouton and then turn around by changing molecular motors that propel them. The model suggests that this could result in an increased concentration of resident DCVs in distal boutons beginning with bouton 2. This is because in distal boutons, DCVs have a larger chance to be captured from the transiting state as they pass the boutons moving anterogradely and then again as they pass the same boutons moving retrogradely.

Ultrastructural distribution of presynaptic active zones and dense core vesicles in olfactory projection neurons of Drosophila

In Drosophila melanogaster, olfactory projection neurons (PNs) convey odor information from peripheral olfactory center, antenna lobe, to central olfactory center, mushroom body (MB), and lateral horn (LH). In MB, the mechanisms underlining the transformation from coarse-coding PNs to sparse-coding MB intrinsic Kenyon cells (KCs) remain an open question. Here, we used HRP-labeled electron microscopy (EM) to volume reconstruct 89 PN axonal boutons in a reference area of the input region, the calyx of MB. The results showed that the number of presynaptic active zones (PAZs), neurotransmitter release site, is in positive linear correlation with the surface area of PN axonal boutons, while the number of dense core vesicles (DCVs), vesicles that containing neuropeptides, monoamines, or neurotrophic factors, is weakly related to the morphology of PN axonal boutons. Further analysis illustrated that DCVs preferentially exist in PN axonal boutons labeled by GH146-GAL4, a most widely used genetic marker for studying olfactory PNs. Our data suggest that synapses are uniformly distributed on the surface of all PN boutons, thus the neurotransmission capability of a PN axonal bouton could be predicted by its size, and PN subtypes release neuropeptides, monoamines, or neurotrophic factors, as well as classical neurotransmitters, to mediate the PN-KC transformation.