Cell Biology, Neuroscience, Biochemistry, Genetics and Molecular Biology
12
Scopus Publications
Scopus Publications
A versatile nanobody platform for live and super-resolution imaging of synaptic vesicle dynamics and plasticity in rodent and human neurons Rashi Goel, Kristina Jevdokimenko, Ronja Rehm, Jannik Hentze, Paola Agüi-Gonzalez, Momchil Ninov, Erik Maier, Yin Wu, Felix Lange, Agata Witkowska, Svenja Bolz, Francesca Pennacchietti, Martina Damenti, Natalie Kaempf, Vladimir Khayenko, Carles Calatayud, Viveka Nand Malviya, Natali L. Chanaday, Emma Scaletti Hutchinson, Hao Liu, Kirsten Weyand, Valentin Schwarze, Daniela Ivanova, Tristan P. Wallis, Christopher Small, Hans M. Maric, Merja Joensuu, Michael A. Cousin, Frédéric A. Meunier, Patrik Verstreken, Ilaria Testa, Ege T. Kavalali, Volker Haucke, Stefan Jakobs, Henning Urlaub, Nils Brose, Benjamin H. Cooper, Pål Stenmark, Felipe Opazo, Reinhard Jahn, Silvio O. Rizzoli, Eugenio F. Fornasiero Journal of Nanobiotechnology, 2026 Synaptic neurotransmission is a critical hallmark of brain activity and one of the first processes affected in neural diseases. Monitoring this process, particularly synaptic vesicle recycling, in living cells has been instrumental in revealing the mechanisms responsible for neurotransmitter release. However, currently available reporters suffer from limitations, such as large probe sizes or limited compatibility for human neurons, hampering the quantitative analysis of synaptic pathophysiology. Here, we describe the NbLumSyt1 toolkit, a panel of nanobody-based affinity probes that target the luminal domain of the synaptic vesicle protein Synaptotagmin 1 (Syt1). These new tools enable quantitative, noninvasive imaging and functional interrogation of Syt1 exo-endocytosis and trafficking in human neurons, with unprecedented precision, versatility and cost efficiency, in technologies ranging from fixed- and live-cell super-resolution imaging to electron microscopy and mass spectrometry. Overall, NbLumSyt1 nanobinders provide a valuable platform for studying synaptic physiology and pathophysiology, benefiting fundamental neuroscience and translational efforts to study and develop treatments for brain-related disorders. Graphical Abstract
Mycorrhizal C/N ratio determines plant-derived carbon and nitrogen allocation to symbiosis Rodica Pena, Sarah L. Bluhm, Silke Ammerschubert, Paola Agüi-Gonzalez, Silvio O. Rizzoli, Stefan Scheu, Andrea Polle Communications Biology, 2023 Carbon allocation of trees to ectomycorrhizas is thought to shape forest nutrient cycling, but the sink activities of different fungal taxa for host resources are unknown. Here, we investigate fungal taxon-specific differences in naturally composed ectomycorrhizal (EM) communities for plant-derived carbon and nitrogen. After aboveground dual labeling of young beech with 15N and 13C, ectomycorrhizas formed with different fungal taxa exhibit strong differences in label enrichment. Secondary Ion Mass Spectrometry (SIMS) imaging of nitrogen in cross sections of ectomycorrhizas demonstrates plant-derived 15N in both root and fungal structures. Isotope enrichment in ectomycorrhizas correlates with that in the corresponding ectomycorrhiza-attached lateral root, supporting fungal taxon-specific N and C fluxes in ectomycorrhizas. The enrichments with 13C and 15N in the symbiosis decrease with increasing C/N ratio of ectomycorrhizas, converging to zero at high C/N. The relative abundances of EM fungal species on roots are positively correlated with 13C enrichment, demonstrating higher fitness of stronger than of less C-demanding symbioses. Overall, our results support that differences among the C/N ratios in ectomycorrhizas formed with different fungal species regulate the supply of the symbioses with host-derived carbon and provide insights on functional traits of ectomycorrhizas, which are important for major ecosystem processes.
White matter integrity in mice requires continuous myelin synthesis at the inner tongue Martin Meschkat, Anna M. Steyer, Marie-Theres Weil, Kathrin Kusch, Olaf Jahn, Lars Piepkorn, Paola Agüi-Gonzalez, Nhu Thi Ngoc Phan, Torben Ruhwedel, Boguslawa Sadowski, Silvio O. Rizzoli, Hauke B. Werner, Hannelore Ehrenreich, Klaus-Armin Nave, Wiebke Möbius Nature Communications, 2022 Myelin, the electrically insulating sheath on axons, undergoes dynamic changes over time. However, it is composed of proteins with long lifetimes. This raises the question how such a stable structure is renewed. Here, we study the integrity of myelinated tracts after experimentally preventing the formation of new myelin in the CNS of adult mice, using an inducible Mbp null allele. Oligodendrocytes survive recombination, continue to express myelin genes, but they fail to maintain compacted myelin sheaths. Using 3D electron microscopy and mass spectrometry imaging we visualize myelin-like membranes failing to incorporate adaxonally, most prominently at juxta-paranodes. Myelinoid body formation indicates degradation of existing myelin at the abaxonal side and the inner tongue of the sheath. Thinning of compact myelin and shortening of internodes result in the loss of about 50% of myelin and axonal pathology within 20 weeks post recombination. In summary, our data suggest that functional axon-myelin units require the continuous incorporation of new myelin membranes.
An iodine-containing probe as a tool for molecular detection in secondary ion mass spectrometry Selda Kabatas Glowacki, Paola Agüi-Gonzalez, Shama Sograte-Idrissi, Sebastian Jähne, Felipe Opazo, Nhu T. N. Phan, Silvio O. Rizzoli Chemical Communications, 2022 We developed here an iodine-containing probe that can be used to identify the molecules of interest in secondary ion mass spectrometry (SIMS) by simple immunolabelling procedures.
Extracellular matrix remodeling through endocytosis and resurfacing of Tenascin-R Tal M. Dankovich, Rahul Kaushik, Linda H. M. Olsthoorn, Gabriel Cassinelli Petersen, Philipp Emanuel Giro, Verena Kluever, Paola Agüi-Gonzalez, Katharina Grewe, Guobin Bao, Sabine Beuermann, Hannah Abdul Hadi, Jose Doeren, Simon Klöppner, Benjamin H. Cooper, Alexander Dityatev, Silvio O. Rizzoli Nature Communications, 2021 The brain extracellular matrix (ECM) consists of extremely long-lived proteins that assemble around neurons and synapses, to stabilize them. The ECM is thought to change only rarely, in relation to neuronal plasticity, through ECM proteolysis and renewed protein synthesis. We report here an alternative ECM remodeling mechanism, based on the recycling of ECM molecules. Using multiple ECM labeling and imaging assays, from super-resolution optical imaging to nanoscale secondary ion mass spectrometry, both in culture and in brain slices, we find that a key ECM protein, Tenascin-R, is frequently endocytosed, and later resurfaces, preferentially near synapses. The TNR molecules complete this cycle within ~3 days, in an activity-dependent fashion. Interfering with the recycling process perturbs severely neuronal function, strongly reducing synaptic vesicle exo- and endocytosis. We conclude that the neuronal ECM can be remodeled frequently through mechanisms that involve endocytosis and recycling of ECM proteins.
Gold-conjugated nanobodies for targeted imaging using high-resolution secondary ion mass spectrometry Paola Agüi-Gonzalez, Tal M. Dankovich, Silvio O. Rizzoli, Nhu T. N. Phan Nanomaterials, 2021 Nanoscale imaging with the ability to identify cellular organelles and protein complexes has been a highly challenging subject in the secondary ion mass spectrometry (SIMS) of biological samples. This is because only a few isotopic tags can be used successfully to target specific proteins or organelles. To address this, we generated gold nanoprobes, in which gold nanoparticles are conjugated to nanobodies. The nanoprobes were well suited for specific molecular imaging using NanoSIMS at subcellular resolution. They were demonstrated to be highly selective to different proteins of interest and sufficiently sensitive for SIMS detection. The nanoprobes offer the possibility of correlating the investigation of cellular isotopic turnover to the positions of specific proteins and organelles, thereby enabling an understanding of functional and structural relations that are currently obscure.
Secondary Ion Mass Spectrometry Imaging Reveals Changes in the Lipid Structure of the Plasma Membranes of Hippocampal Neurons following Drugs Affecting Neuronal Activity Paola Agüi-Gonzalez, Bao Guobin, Maria A. Gomes de Castro, Silvio O. Rizzoli, Nhu T. N. Phan ACS Chemical Neuroscience, 2021 The cellular functions of lipids in the neuronal plasma membranes have been increasingly acknowledged, particularly their association to neuronal processes and synaptic plasticity. However, the knowledge of their regulatory mechanisms in neuronal cells remains sparse. To address this, we investigated the lipid organization of the plasma membranes of hippocampal neurons in relation to neuronal activity using secondary ion mass spectrometry imaging. The neurons were treated with drugs, particularly tetrodotoxin (TTX) and bicuculline (BIC), to induce chronic activation and silencing. Distinct lipid organization was found in the plasma membrane of the cell body and the neurites. Moreover, significant alterations of the levels of the membrane lipids, especially ceramides, phosphatidylserines, phosphatidic acids, and triacylglycerols, were observed under the TTX and BIC treatments. We suggest that many types of membrane lipids are affected by, and may be involved in, the regulation of neuronal function.
Correlative fluorescence microscopy, transmission electron microscopy and secondary ion mass spectrometry (CLEM-SIMS) for cellular imaging Felix Lange, Paola Agüi-Gonzalez, Dietmar Riedel, Nhu T. N. Phan, Stefan Jakobs, Silvio O. Rizzoli Plos One, 2021 Electron microscopy (EM) has been employed for decades to analyze cell structure. To also analyze the positions and functions of specific proteins, one typically relies on immuno-EM or on a correlation with fluorescence microscopy, in the form of correlated light and electron microscopy (CLEM). Nevertheless, neither of these procedures is able to also address the isotopic composition of cells. To solve this, a correlation with secondary ion mass spectrometry (SIMS) would be necessary. SIMS has been correlated in the past to EM or to fluorescence microscopy in biological samples, but not to CLEM. We achieved this here, using a protocol based on transmission EM, conventional epifluorescence microscopy and nanoSIMS. The protocol is easily applied, and enables the use of all three technologies at high performance parameters. We suggest that CLEM-SIMS will provide substantial information that is currently beyond the scope of conventional correlative approaches.
Presynaptic activity and protein turnover are correlated at the single-synapse level Sebastian Jähne, Fabian Mikulasch, Helge G.H. Heuer, Sven Truckenbrodt, Paola Agüi-Gonzalez, Katharina Grewe, Angela Vogts, Silvio O. Rizzoli, Viola Priesemann Cell Reports, 2021 Synaptic transmission relies on the continual exocytosis and recycling of synaptic vesicles. Aged vesicle proteins are prevented from recycling and are eventually degraded. This implies that active synapses would lose vesicles and vesicle-associated proteins over time, unless the supply correlates to activity, to balance the losses. To test this hypothesis, we first model the quantitative relation between presynaptic spike rate and vesicle turnover. The model predicts that the vesicle supply needs to increase with the spike rate. To follow up this prediction, we measure protein turnover in individual synapses of cultured hippocampal neurons by combining nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorescence microscopy. We find that turnover correlates with activity at the single-synapse level, but not with other parameters such as the abundance of synaptic vesicles or postsynaptic density proteins. We therefore suggest that the supply of newly synthesized proteins to synapses is closely connected to synaptic activity.
SIMS imaging in neurobiology and cell biology Paola Agüi-Gonzalez, Sebastian Jähne, Nhu T. N. Phan Journal of Analytical Atomic Spectrometry, 2019 Secondary ion mass spectrometry (SIMS) has been increasingly recognized as a powerful technique for visualizing molecular architectures in the fields of neurobiology and cell biology.
