Biology subjects

Missler, M.

Publications and source records attributed to Missler, M..

5 recordsLinked to original sources

bioRxiv2025

A nanobody-gold conjugate for the detection of GFP-tagged synaptic proteins by dual light-electron microscopy

To meet the constantly improving spatial resolution offered by advanced microscopy techniques to study sub-cellular structures in biology, there is a need for small, monovalent probes that label proteins of interest with high specificity and minimal distance to the target, and are compatible with various imaging modalities. In this direction, we designed a strategy to generate minimal-size probes composed of a controlled 1:1 conjugate between a small domain binder and a 1.4 nm-gold nanoparticle with direct access to fluorescent labelling for dual light-electron microscopy. Our approach was applied to the widely used single-domain antibody against GFP (GBP). The modified GBP-gold conjugate retained normal binding to purified GFP in vitro, specifically labelled COS-7 cells and neurons expressing GFP-tagged synaptic membrane proteins, and penetrated readily into tight cell-cell contacts including neuronal synapses. The optional fluorescence labelling with a second ALFA nanobody allowed dSTORM imaging, while the silver-enhanced nanogold particle detected in TEM was used to characterize the number and nanoscale organization of individual proteins in the synaptic cleft. We counted a small number of endogenous neurexins in the pre-synapse and a larger number of AMPA receptors in the post-synapse, often aligned in nanodomains. This GBP-gold probe thus emerges as a potent tool to label an ever-increasing repertoire of GFP-tagged proteins in numerous biological organisms and models.

bioengineering↗

bioRxiv2025

Optical recordings of synaptic vesicle fusion reveal diffusional dispersion

The primary unit of exocytosis, the synaptic vesicle, is replete with proteins which enable vesicle fusion. For maintaining synaptic transmission the vesicle proteins have to be rapidly cleared from the active zone. The immediate fate of the cargo post-fusion and the precise mechanism of its resorting into a readily retrievable pool (RRetP) for endocytosis remains unclear. To address this, we developed a purely presynaptic preparation, the xenapse, presynaptic boutons formed en face directly onto a glass coverslip enabling optical single vesicle recording by total internal reflection microscopy (TIRFM). Electron microscopy showed xenapses contain a few hundred synaptic vesicles (SVs), of which [~]40 SVs are primed (readily releasable pool, RRP), as revealed by single vesicle recordings using the pH-sensitive fluorescent protein pHluorin. Single fusion events synchronous with action potentials could be localized with [~]20 nm precision and rapid post-fusion diffusional dispersion of vesicular proteins observed with diffusion constants in the order of 0.1 {micro}m2/s. Unroofing of xenapses revealed numerous Clathrin-coated structures, enriched for vesicle cargo and numerically equivalent to about twice the measured RRP size. In this way SV proteins are rapidly cleared from the release site into this RRetP of pre-assembled pits to allow for rapid re-docking and re-priming at vacated release sites in synapses tuned for high-fidelity fast signaling.

neuroscience↗

bioRxiv2024

Dissecting the binding mechanisms of synaptic membrane adhesion complexes using a micropattern based cellular model

The formation of adhesive cell-cell contacts is based on the intrinsic binding properties between specific transmembrane ligand-receptor pairs. In neurons, synaptic adhesion molecules provide a physical linkage between pre- and post-synaptic compartments, but the strength and the dynamic of these complexes in their actual membrane environments remain essentially unknown. To access such information, we developed a versatile assay to measure the affinity and binding kinetics of synaptic ligand-receptor interactions, based on the immobilization of Fc-tagged ligands on micropatterned substrates combined with live imaging of fluorescently-tagged counter receptors in heterologous cells. We applied this strategy to study the heterophilic complex formed between neurexin-1{beta} (Nrx1{beta}) and neuroligin-1 (Nlg1), compared to the homophilic SynCAM1 complex. First, the control of ligand density combined to the measurement of steady-state receptor enrichment at micropatterns demonstrates the high specificity of the matching molecular interactions and allows for the quantification of the two-dimensional affinity of the interaction in a membrane environment. Second, long-term FRAP experiments performed on the two molecular complexes and fitted with analytical models, demonstrate a diffusion-limited regime for SynCAM1 and a reaction-limited regime for Nlg1. This analysis provides a very long bond lifetime of the Nrx1{beta}-Nlg1 complex, which by comparison with a monomeric mutant of Nlg1, can be attributed to the constitutive dimerization of Nlg1. Finally, we used the stable Nrx1{beta}-Nlg1 complex as a pseudo-synaptic platform to analyze the rapid binding kinetics between the scaffolding protein PSD-95 and the intracellular domain of Nlg1, dissecting the contribution of the different PDZ domains through the use of specific PSD-95 point mutants.

neuroscience↗

bioRxiv2023

Regulation of hippocampal mossy fiber-CA3 synapse function by a Bcl11b/C1ql2/Nrxn3(25b+) pathway

The transcription factor Bcl11b has been linked to neurodevelopmental and neuropsychiatric disorders associated with synaptic dysfunction. Bcl11b is highly expressed in dentate gyrus granule neurons and is required for the structural and functional integrity of mossy fiber-CA3 synapses. The underlying molecular mechanisms, however, remained unclear. We show that the synaptic organizer molecule C1ql2 is a direct functional target of Bcl11b that regulates synaptic vesicle recruitment and long-term potentiation at mossy fiber-CA3 synapses in vivo and in vitro. Furthermore, we demonstrate C1ql2 to exert its functions through direct interaction with a specific splice variant of neurexin-3, Nrxn3(25b+). Interruption of C1ql2-Nrxn3(25b+) interaction by expression of a non-binding C1ql2 mutant or by deletion of Nrxn3 in the dentate gyrus granule neurons recapitulates major parts of the Bcl11b as well as C1ql2 mutant phenotype. Together, this study identifies a novel C1ql2-Nrxn3(25b+)-dependent signaling pathway through which Bcl11b controls mossy fiber-CA3 synapse function. Thus, our findings contribute to the mechanistic understanding of neurodevelopmental disorders accompanied by synaptic dysfunction.

neuroscience↗

bioRxiv2021

PI(4,5)P2 controls slit diaphragm formation and endocytosis in Drosophila nephrocytes

Drosophila nephrocytes are an emerging model system for mammalian podocytes and podocyte-associated diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. Here we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-Kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-Kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development.

cell biology↗