Journal: Scientific Reports

Article Title: A minimalist model to measure interactions between proteins and synaptic vesicles

doi: 10.1038/s41598-020-77887-1

Figure Lengend Snippet: Patterning strategy and SV immobilization. ( a ) Schematic representation of photopatterning of neutravidin on a glass coverslip. The glass coverslip was uniformly coated with an anti-fouling layer of PLL-g-PEG. The photoinitiator (PLPP) was added on top of the PLL-g-PEG layer and the substrate was exposed to UV light through a virtual photomask to activate the PLPP. Under UV light, the PLPP degraded the PLL-PEG layer, leaving accessible regions for neutravidin to attach. ( b ) Schematic of our strategy to attach the SVs to the substrate. The purified SVs (gray) were attached to the neutravidin (pink) functionalized glass coverslips via a biotinylated mouse anti-synaptotagmin antibody (orange). The attachment of antibodies was controlled by a secondary anti-mouse antibody labeled with Alexa Fluor 488 (green). To image the SVs, a single-domain antibody against vGLUT1, labeled with STAR635P (red) was employed. ( c ) Fluorescence micrographs of the individual steps of the vesicle immobilization strategy. The neutravidin functionalization step was assessed by fluorescently labeled neutravidin (neutravidin-FITC, left), the attachment of the biotinylated mouse anti-synaptotagmin antibody was tested with a secondary anti-mouse antibody labeled with Alexa Fluor 488 (center), and the SV attachment was tested with a single-domain antibody against vGLUT1 labeled with STAR635P (right). Note that the uniform fluorescence in the right image shows the bound SVs, whereas the small bright spots are aggregates. The images of neutravidin-FITC and anti-ms-Alexa488 were taken on two different glass coverslips. In the actual experiments, we used unlabeled neutravidin, so as to not interfere with the fluorescence of the mEGFP-tagged proteins. The scale bars are 25 μm.

Article Snippet: The excitation light came from a mercury arc lamp (X-Cite 120 PC Q, Excelitas Technologies, Waltham, USA) and was guided onto a fluorescence filter cube (filter sets available: DAPI, GFP, Cy3, TxRed and Cy5, all from AHF Analysentechnik AG).

Techniques: Purification, Labeling, Fluorescence

Journal: Scientific Reports

Article Title: A minimalist model to measure interactions between proteins and synaptic vesicles

doi: 10.1038/s41598-020-77887-1

Figure Lengend Snippet: Comparison of cell lysate and purified α-synuclein-mEGFP. ( a ) Average normalized ACF of purified α-synuclein-mEGFP (closed squares, N = 30) and α-synuclein-mEGFP from HEK cell lysate (open squares, N = 30); there was no SV pattern present in these experiments; both protein preparations show the same diffusion behavior. ( b ) The purified α-synuclein-mEGFP interacted with the SV pattern (left hand side), as shown by the fluorescence signal (right; SV pattern visualized by anti-vGlut-STAR635P). ( c ) By contrast, α-synuclein-mEGFP from the cell lysate (left) did not bind to the SV pattern (right). The images in ( b ) and ( c ) were recorded after 2 hours of incubation of the SV layer with α-synuclein-mEGFP. The scale bars are 25 μm. Note that the uniform fluorescence in the right images in ( b ) and ( c ) shows the bound SVs, whereas the small bright spots are aggregates.

Article Snippet: The excitation light came from a mercury arc lamp (X-Cite 120 PC Q, Excelitas Technologies, Waltham, USA) and was guided onto a fluorescence filter cube (filter sets available: DAPI, GFP, Cy3, TxRed and Cy5, all from AHF Analysentechnik AG).

Techniques: Comparison, Purification, Diffusion-based Assay, Fluorescence, Incubation