Computational protocol: ARF6-mediated endosomal transport of Telencephalin affects dendritic filopodia-to-spine maturation

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Protocol publication

[…] Morphometric measurements were performed using Imaris image analysis software (Bitplane Scientific Software, Zurich). Neurons were selected randomly, and aspiny interneurons were excluded from the analysis. To determine the percentage of spines or filopodia, spines were defined as dendritic protrusions of 0.4–3 μm length (with or without a head) and filopodia as dendritic protrusions of 3–10 μm in length, as previously described (). The percentages of protrusions were grouped (filopodia versus spines) and averaged. Multiple individual neurons were analysed to obtain a population mean.For the analysis of the presence of endogenous TLN within the dendrite (surface versus internal), acquired images of dendritic sections (40 μm) were processed using ImageJ software (NIH) set for Auto thresholding using the Minimum Method. The obtained data reflect area percentages of the TLN distribution relative to that of fGFP. For fGFP, the percentage defines the complete dendritic area, as it is homogenously distributed and limited to the membrane periphery. For TLN, the area percentages differed, as TLN's distribution varied depending on its internalization. Multiple images of dendrites from independent neurons were analysed to obtain a population mean.To quantify TLN entrapment in RAB5Q79L endosomes acquired images were analysed using ImageJ software. The relative levels of internalized TLN (TLNΔAC) were compared by dividing its fluorescence intensity, confined to Rab5Q79L endosomes, by its total cell fluorescence. The reported differences were observed in at least three independent experiments and quantified in multiple, randomly selected cells.For the endosomal overlap analysis, vesicles that stained positive for CD63 or flotillin were scored for the presence/absence of TLN. The percentages of vesicles containing both proteins were calculated. Means from multiple individual cells were averaged to obtain a population mean.For the microdomain overlap analysis between TLN and EFA6A or flotillin2 at the cell body of neurons, acquired images were processed in ImageJ using the JACop method ().For all data analysis, the two-tailed Student's t-test was used to compare the mean±s.e.m. values. The details regarding the ARF6 knockdown experimental setting and quantification of the observed phenomena can be found in . […]

Pipeline specifications

Software tools Imaris, ImageJ, JACoP
Application Microscopic phenotype analysis
Diseases Keratitis, Dendritic, Nervous System Diseases
Chemicals Adenosine Diphosphate