Biology subjects

Georgiev, S. V.

Publications and source records attributed to Georgiev, S. V..

5 recordsLinked to original sources

bioRxiv2024

PHluorin-conjugated secondary nanobodies as a tool for measuring synaptic vesicle exo- and endocytosis

Neuronal communication relies on synaptic vesicle recycling, which has long been investigated by live imaging approaches. Synapto-pHluorins, genetically encoded reporters that incorporate a pH-sensitive variant of GFP within the lumen of the synaptic vesicle, have been especially popular. However, they require genetic manipulation, implying that a tool combining their excellent reporter properties with the ease of use of classical immunolabeling would be desirable. We introduce this tool here, relying on primary antibodies against the luminal domain of synaptotagmin 1, decorated with secondary single-domain antibodies (nanobodies) carrying a pHluorin moiety. The application of the antibodies and nanobodies to cultured neurons results in labeling their recycling vesicles, without the need for any additional manipulations. The labeled vesicles respond to stimulation, in the expected fashion, and the pHluorin signals enable the quantification of both exo- and endocytosis. We conclude that pHluorin-conjugated secondary nanobodies are a convenient tool for the analysis of vesicle recycling.

neuroscience↗

bioRxiv2024

Power-law adaptation in the presynaptic vesicle cycle

After synaptic transmission, fused synaptic vesicles are recycled, enabling the synapse to recover its capacity for renewed release. The recovery steps, which range from endocytosis to vesicle docking and priming, have been studied individually, but it is not clear what their impact on the overall dynamics of synaptic recycling is, and how they influence signal transmission. Here we model the dynamics of vesicle recycling and find that the multiple timescales of the recycling steps are reflected in synaptic recovery. This leads to multi-timescale synapse dynamics, which can be described by a simplified synaptic model with power-law adaptation. Using cultured hippocampal neurons, we test this model experimentally, and show that the duration of synaptic exhaustion changes the effective synaptic recovery timescale, as predicted by the model. Finally, we show that this adaptation could implement a specific function in the hippocampus, namely enabling efficient communication between neurons through the temporal whitening of hippocampal spike trains.

neuroscience↗

bioRxiv2024

Calcium-triggered (de)ubiquitination events in synapses

Neuronal communication relies on neurotransmitter release from synaptic vesicles (SVs), whose dynamics are controlled by calcium-dependent pathways, as many thoroughly studied phosphorylation cascades. However, little is known about other post-translational modifications, as ubiquitination. To address this, we analysed resting and stimulated synaptosomes (isolated synapses) by quantitative mass spectrometry. We identified more than 5,000 ubiquitination sites on [~]2,000 proteins, the majority of which participate in SV recycling processes. Several proteins showed significant changes in ubiquitination in response to calcium influx, with the most pronounced changes in CaMKII and the clathrin adaptor protein AP180. To validate this finding, we generated a CaMKII mutant lacking the ubiquitination target site (K291) and analysed it both in neurons and non-neuronal cells. K291 ubiquitination influences CaMKII activity and synaptic function by modulating its autophosphorylation at a functionally important site (T286). We suggest that ubiquitination in response to synaptic activity is an important regulator of synaptic function.

neuroscience↗

bioRxiv2022

Expansion microscopy at one nanometer resolution

Fluorescence imaging is one of the most versatile and widely-used tools in biology1. Although techniques to overcome the diffraction barrier were introduced more than two decades ago, and the nominal attainable resolution kept improving2, 3, fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context4, 5. Here we report a solution to this problem, in the form of one-step nanoscale expansion (ONE) microscopy. We combined the 10-fold axial expansion of the specimen (1000-fold by volume) with a fluorescence fluctuation analysis6, 7 to enable the description of cultured cells, tissues, viral particles, molecular complexes and single proteins. At the cellular level, using immunostaining, our technology revealed detailed nanoscale arrangements of synaptic proteins, including a quasi-regular organisation of PSD95 clusters. At the single molecule level, upon main chain fluorescent labelling, we could visualise the shape of individual membrane and soluble proteins. Moreover, conformational changes undergone by the [~]17 kDa protein calmodulin upon Ca2+ binding were readily observable. We also imaged and classified molecular aggregates in cerebrospinal fluid samples from Parkinsons Disease (PD) patients, which represents a promising new development towards improved PD diagnosis. ONE microscopy is compatible with conventional microscopes and can be performed with the software we provide here as a free, open-source package. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, and provides a new avenue for discoveries in biology and medicine.

biophysics↗

bioRxiv2019

Autophagy-dependent filopodial kinetics restrict synaptic partner choice during Drosophila brain wiring

Brain wiring is remarkably precise, yet most neurons readily form synapses with incorrect partners when given the opportunity. Dynamic axon-dendritic positioning can restrict synaptogenic encounters, but the spatiotemporal interaction kinetics and their regulation remain essentially unknown inside developing brains. Here we show that the kinetics of axonal filopodia restrict synapse formation and partner choice for neurons that are not otherwise prevented from making incorrect synapses. Using 4D imaging in developing Drosophila brains, we show that filopodial kinetics are regulated by autophagy, a prevalent degradation mechanism whose role in brain development remains poorly understood. With surprising specificity, autophagosomes form in synaptogenic filopodia, followed by filopodial collapse. Altered autophagic degradation of synaptic building material quantitatively regulates synapse formation as shown by computational modeling and genetic experiments. Increased filopodial stability enables incorrect synaptic partnerships. Hence, filopodial autophagy restricts inappropriate partner choice through a process of kinetic exclusion that critically contributes to wiring specificity.

neuroscience↗