Biology subjects

Dresbach, T.

Publications and source records attributed to Dresbach, T..

7 recordsLinked to original sources

bioRxiv2025

3D MINFLUX combined with DNA-PAINT resolves the arrangement of Bassoon at active zones

Neurotransmitter release and membrane retrieval at active zones require precise spatial and temporal coordination relying on an intricate molecular machinery. However, the exact nano-structural organization of this machinery is not yet fully elucidated. Here, we used 3D MINFLUX combined with both spectral demixing and DNA-PAINT to analyze the positioning of the scaffolding protein Bassoon at presynaptic active zones of glutamatergic spine synapses of hippocampal neurons, achieving a localization precision of 5 nm in 3D. This approach allowed us to visualize directly the distribution of N-terminal and the C-terminal regions of Bassoon, and demonstrates that Bassoon exhibits an orientation at the active zone, where the C-terminal region is directed toward the synaptic cleft and the N-terminal region towards synaptic vesicles. Having demonstrated this spatial configuration for endogenous and recombinant Bassoon molecules, our study paves the way towards molecular-scale resolution analysis of other prominent proteins of the presynaptic release machinery.

neuroscience↗

bioRxiv2025

A correlative workflow streamlines synaptic imaging by cryo-electron tomography

Despite decades of intense research, the molecular organization of the synapse is not well understood. To address this issue, we sought to develop a method for systematic imaging of synapses by cryo-electron tomography (cryo-ET), a technology capable of mapping cellular architecture at molecular resolution. Thinning of cellular samples by cryo-focused ion beam milling is a prerequisite for high-quality cryo-ET imaging, but this process needs to be guided to the structures of interest. To allow robust synaptic targeting, we established a correlative cryo-light/electron microscopy approach by which synapses are fluorescently labeled in a minimally invasive manner, using a synthetic binder of the postsynaptic scaffold PSD-95 and antibodies against the presynaptic protein Synaptotagmin-1. Cryo-ET imaging at sites of colocalization consistently revealed excitatory synapses. Our method allows structural studies of synaptic protein complexes in situ, facilitating investigations of the molecular architecture of synapses.

neuroscience↗

bioRxiv2024

Establishing synthetic ribbon-type active zones in a heterologous expression system

Encoding of several sensory modalities into neural signals is mediated by ribbon synapses. The synaptic ribbon tethers synaptic vesicles at the presynaptic active zone (AZ) and may act as a super-scaffold organizing AZ topography. Here we employed a synthetic biology approach to reconstitute structures mimicking ribbon-type AZs in HEK293 cells for probing minimal molecular requirements and studying presynaptic Ca2+ channel clustering. Co-expressing a membrane-targeted version of the AZ-protein Bassoon and the ribbon core protein RIBEYE, we observed structures recapitulating basic aspects of ribbon-type AZs, which we call synthetic ribbons or SyRibbons. Super-resolution STED microscopy and cryo-correlative electron tomography revealed SyRibbons were similar to native ribbons at AZs of cochlear inner hair cells in shape and size. SyRibbons with Ca2+ channel clusters formed upon additional expression of CaV1.3 Ca2+ channels and RIM-binding protein 2 (RBP2). CaV1.3 Ca2+ channel clusters associated with SyRibbons were larger than ribbonless CaV1.3 Ca2+ channels clusters and functional analysis by Ca2+-imaging in combination with patch clamp showed partial confinement of the Ca2+ signal at SyRibbons. In summary, we identify Ca2+ channels, RBP, membrane-anchored Bassoon, and RIBEYE as minimal components for reconstituting a basic ribbon-type AZ. SyRibbons might complement animal studies on molecular interactions of AZ proteins. Significance StatementEncoding of sensory information in our eyes and ears builds on specialized ribbon synapses of sensory cells. Elucidating the molecular underpinning of their fascinating structure and function is an ongoing effort to which we add a bottom-up reconstitution approach in cultured cells. Aiming to recapitulate basic properties of ribbon-type presynaptic active zones of cochlear inner hair cells, we identified a minimal set of proteins that assemble in cellular nanodomains, structurally and functionally alike active zones. While not yet reconstituting synaptic vesicle exocytosis, we consider the established synthetic ribbon-type active zones a valuable platform for studying molecular interactions of active zone proteins. We expect the approach to complement and reduce experiments on native ribbon synapses asserted from animals.

neuroscience↗

bioRxiv2022

The synaptic vesicle protein Mover/TPRG1L is associated with lipid droplets in astrocytes

Crucial brain functions such as neurotransmission, myelination, and signaling pose a high demand for lipids. Lipid dysregulation is associated with neuroinflammation and neurodegeneration. Astrocytes protect neurons from lipid induced damage by accumulating and metabolizing toxic lipids in organelles called lipid droplets (LDs). LDs have long been considered as lipid storage compartments in adipocytes, but less is known about their biogenesis and composition in the brain. In particular, proteins covering the LD surface are not yet fully identified. Here, we report that the synaptic vesicle protein Mover/TPRG1L, which regulates the probability of neurotransmitter release in neurons, is a component of the LD coat in astrocytes. Using conventional and super resolution microscopy, we demonstrate that Mover surrounds naive and oleic acid induced astrocytic LDs. We confirm the identity of astrocytic LDs using the neutral lipid stains Bodipy and LipidTox, as well as immunofluorescence for perilipin-2, a known component of the LD coat. In astrocytes, recombinant Mover was sufficient to induce an accumulation of LDs. Furthermore, we identified point mutations that abolish targeting to LDs and show similarities in the required binding sequences for association to the presynapse and LDs. Our results show that Mover is not only a presynaptic protein but also a candidate for LD regulation. This highlights the dual role of Mover in synaptic transmission and regulation of astrocytic LDs, which may be particularly important in the context of lipid-related neurological disorders. Main points- Mover is a lipid droplet protein - Overexpression of Mover drives lipid droplet accumulation - The F206 is important for LD targeting

neuroscience↗

bioRxiv2021

Nanoscopical analysis reveals an orderly arrangement of the presynaptic scaffold protein Bassoon at the Golgi-apparatus

1.Bassoon is a large, 440 kDa, scaffold protein of the specialized sites mediating neurotransmitter release from presynaptic nerve terminals, called active zones. At active zones of the brain, Bassoon is arranged with its C-terminus facing the plasma membrane. In young, cultured neurons Bassoon is associated with the Golgi-apparatus, where it may assemble active zone precursors structures, but whether or not Bassoon is an extended protein at the Golgi-apparatus and whether it is arranged in an orderly fashion is unknown. Understanding the topology of this large scaffold protein is important for models of active zone biogenesis. Here, we generated recombinant Bassoon constructs for expression in neurons, with tags positioned to allow for the specific detection of N- and C-terminal regions of Bassoon by single-domain antibodies, called nanobodies. Using stimulated emission depletion (STED) nanoscopy in cultured hippocampal neurons, we confirmed that recombinant Bassoon is oriented with its C-terminus towards the active zone plasma membrane at synapses. Focusing on the soma, we found that an intramolecular tag located immediately downstream of amino acid 97 of Bassoon, showed prominent colocalization with markers of the trans Golgi network, including TGN38 and syntaxin-6. In contrast, tags located immediately downstream of the C-terminal amino acid 3938 of Bassoon showed significantly less colocalization with these Golgi-markers. The intramolecular N-terminal tag was located between 48 and 69 nm away from TGN38, while C-terminal tags were located between 100 and 115 nm away from TGN38. Sequences within the first 95 amino acids of Bassoon, but not its N-myristoylation sequence, were required for this arrangement. Our results indicate that at the Golgi-apparatus Bassoon is oriented with its N-terminus towards and its C-terminus away from the trans Golgi network membrane. Moreover, they suggest that Bassoon is an extended molecule at the trans Golgi network with the distance between amino acids 97 and 3938 estimated to be between 46 and 52 nm. Our data are consistent with a model, in which the N-terminus of Bassoon binds to the membranes of the trans-Golgi network, while the C-terminus associates with active zone components, thus reflecting the topographic arrangement characteristic of synapses also at the Golgi-apparatus.

neuroscience↗

bioRxiv2019

Increased synaptic facilitation and exploratory behavior in mice lacking the presynaptic protein Mover

In vertebrates and invertebrates, neurotransmitter release relies on a highly conserved molecular machinery. A surprisingly small number of presynaptic proteins evolved specifically in vertebrates. How they expand the power or versatility of the conserved core machinery is unclear. One of these vertebrate-specific proteins, called Mover / TPRGL / SVAP30, is heterogeneously expressed throughout the brain, suggesting that it adds special functions to subtypes of presynaptic terminals. In this study we generated Mover knockout mice to investigate the role of Mover from synaptic transmission to behavior. Deletion of Mover did not affect synaptic transmission at CA3 to CA1 synapses. In contrast, Mover deficient mice had strongly increased short-term facilitation at mossy fiber to CA3 synapses. This increase included frequency facilitation, a hallmark of mossy fiber terminal function. The effect was age- and Ca2+-dependent, and relied on the Kainate receptor/cAMP pathway in the mossy fiber terminals. Despite this change in presynaptic plasticity, the absence of Mover did not affect long-term spatial reference memory or working memory, but led to reduced anxiety. These discoveries suggest that Mover has distinct roles at different synapses. At mossy-fiber terminals, it acts to constrain the extent of presynaptic facilitation. Its role in activity-dependent neurotransmission could be necessary for normal anxiety responses. Significance StatementThe enormous increase in the complexity of brains during evolution is accompanied by a remarkably small number of new, vertebrate-specific presynaptic proteins. These proteins are unlikely to be essential for transmitter release, because invertebrate synapses do not need them. But what functions do they fulfill? We show that the vertebrate-specific protein Mover is involved in constraining the release of neurotransmitters in some synapses in the hippocampus, while not affecting others. We further demonstrate that the absence of this protein leads to decreased anxiety levels. Understanding the function of such a protein can help us further understand synaptic transmission, the specializations that are brought about in vertebrate synapses, and how this can help or hinder neurological or psychiatric disorders.

neuroscience↗

bioRxiv2018

Neuroligins mediate presynaptic maturation through brain-derived neurotrophic factor signaling

Maturation is a process that allows synapses to acquire full functionality; failures in synaptic maturation may contribute to neurodevelopmental disorders. Neuroligins are postsynaptic cell-adhesion molecules essential for synapse maturation, but the underlying pathways are incompletely understood. Here, we show that maturational increases in active zone stability and synaptic vesicle recycling rely on the joint action of Neuroligins and brain-derived neurotrophic factor (BDNF). Applying BDNF to neuronal cultures mimicked the maturation-promoting effects of overexpressing the Neuroligin isoforms NL1 or NL2. Inhibiting BDNF signaling reduced the effects of NL1 and NL2 on presynaptic maturation and of NL2 on synapse formation. Applying BDNF to NL1-knockout mouse cultures rescued defective presynaptic maturation, indicating that BDNF acts downstream of NL1 and can restore presynaptic maturation at late stages of network development. Our data introduce BDNF as a novel and essential component in a transsynaptic pathway linking Neuroligin-mediated cell adhesion, neurotrophin action and presynaptic maturation.

neuroscience↗