A minimalist model to measure interactions between proteins and synaptic vesicles

Perego, Eleonora; Reshetniak, Sofiia; Lorenz, Charlotta; Hoffmann, Christian; Milovanović, Dragomir; Rizzoli, Silvio O.; Köster, Sarah

doi:10.1038/s41598-020-77887-1

Cited by 11 publications

(12 citation statements)

References 36 publications

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“…After two washes with lysis buffer and one wash with PBS (ice-cold), the bound proteins were eluted by adding the ALFA peptide. The purified protein was analyzed by Coomassie gel imaging (published in 77 ).…”

Section: Purified Proteinsmentioning

confidence: 99%

Visualizing proteins by expansion microscopy

Shaib

Chouaib

Chowdhury

et al. 2022

Preprint

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Fluorescence imaging is one of the most versatile and widely-used tools in biology. Although techniques to overcome the diffraction barrier were introduced more than two decades ago, and the nominal attainable resolution kept improving to reach single-digit nm, fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context. Here we report a solution to this problem, in the form of one-nanometer expansion (ONE) microscopy. We combined the 10-fold axial expansion of the specimen (1000-fold by volume) with a fluorescence fluctuation analysis to achieve resolutions down to 1 nm or better. We have successfully applied ONE microscopy to image 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 could also image and classify molecular aggregates in cerebrospinal fluid samples from Parkinson's Disease (PD) patients, which represents a promising new development towards an 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.

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Section: Purified Proteinsmentioning

confidence: 99%

Visualizing proteins by expansion microscopy

Shaib

Chouaib

Chowdhury

et al. 2022

Preprint

Self Cite

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“…It is bound by the ALFA-Nb with an ultra-high affinity of K d = 26 pM and has been successfully used in various extra-and intracellular applications. [5] For these reasons, the ALFA system consisting of the ALFA-tag and the ALFA-Nb is being broadly used and adopted to numerous applications, e. g. in immunofluorescence [15] or super-resolution microscopy, [5] immunoblotting [5,16] and protein purification, [5,17] and was the prime candidate for us to develop a photobody against a short epitope tag.…”

Section: Introductionmentioning

confidence: 99%

A Light‐Activatable Photocaged Variant of the Ultra‐High Affinity ALFA‐Tag Nanobody

Jedlitzke

Mootz

2022

ChemBioChem

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Nanobodies against short linear peptide‐epitopes are widely used to detect and bind proteins of interest (POI) in fusion constructs. Engineered nanobodies that can be controlled by light have found very recent attention for various extra‐ and intracellular applications. We here report the design of a photocaged variant of the ultra‐high affinity ALFA‐tag nanobody, also termed ALFA‐tag photobody. ortho‐Nitrobenzyl tyrosine was incorporated into the paratope region of the nanobody by genetic code expansion technology and resulted in a ≥9,200 to 100,000‐fold impairment of the binding affinity. Irradiation with light (365 nm) leads to decaging and reconstitutes the native nanobody. We show the light‐dependent binding of the ALFA‐tag photobody to HeLa cells presenting the ALFA‐tag. The generation of the first photobody directed against a short peptide epitope underlines the generality of our photobody design concept. We envision that this photobody will be useful for the spatiotemporal control of proteins in many applications using cultured cells.

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“…There, detailed organelle and protein compositions have been available for years, 3 , 4 , 5 and the dynamics of many presynaptic proteins have been investigated. 6 , 7 , 8 Taken together, these studies indicate that the behavior of the majority of proteins is largely affected by their interaction with the synaptic vesicle cluster, 9 as many of presynaptic trafficking proteins are known to bind to the synaptic vesicles and other components of the cluster. 4 , 7 , 9 , 10 , 11 …”

Section: Introductionmentioning

confidence: 86%